eDAQ Wiki - User contributions [en]

Cleaning and Polishing Voltammetric Electrodes

2025-04-23T07:08:38Z

Nick:

=== Safety ===
Polishing powders comprise micron and submicron sized particles which can be dangerous, especially if inhaled. Please follow your local safety guidelines and especially work in a well-ventilated environment.

=== Materials ===
Use a suitable polishing kit such as product number [http://www.edaq.com/ET030 ET030] from eDAQ, that uses abrasive alumina polishing powders.

Local lapidary supply companies may have equivalent products. Diamond, silica, or silicon carbide powders of similar particle size can also be used. A polishing pad that has a pressure sensitive adhesive (PSA) backing can be affixed to a glass plate to ensure a flat polishing surface.

A dry cloth can scratch the electrode and can cause the cloth to tear, always wet a new cloth with abrasive powder suspension, or deionised water before use. Use each cloth only with a powder of single particle size. Rinse cloths with deionised water after each polishing cycle, but reserve them for use only with that particle size polish in the future. Rather than adding dry powder to a polishing pad, it is often better to use a prepared polishing suspension. If you have a source of dry powder suspend it in a wash bottle containing ethanol, and then add a small amount of the suspension (shake well before use) to the pad.

=== General Procedure ===

These procedures are recommended for [http://www.edaq.com/Disk%20Electrodes eDAQ] or other voltammetric disk electrodes.

Resist the temptation to over-polish the electrodes. In most cases the electrode remains clean after the experiment, or could be washed clean with suitable organic solvent to remove greasy organic materials, or dilute acid or base (0.1 mol/L HCl, HNO<sub>3</sub>, NaOH) to remove inorganics. Protein deposits can be hydrolysed with an appropriate commercial enzyme-based cleaner.

Mercury films on platinum or glassy carbon substrates can be removed by gently wiping the electrode tip with lab tissue (dispose of tissue in mercury waste container) any residual mercury can be removed by holding the electrode at an oxidizing potential (eg +1 V) in dilute acid solution (0.1 mol/L).

Only if there is an intractable coating electrodeposited on the electrode, or after extended use, will the electrode need any polishing at all. Polishing should only be done if there is visually obvious surface contamination (use a magnifying glass) or where a trial cyclic voltammogram in clean solvent indicates that there has been surface contamination. Standard maintenance polishing should use a 0.05 micron polishing powder, only a badly scratched electrode, or one with an extremely intractable coating would need to use coarser (larger particle size) powders. Some frequent users like to do a maintenance polish every day, however others may go many days without any surface degradation of the electrode.

Automatic polishing machines are useful if large numbers of electrodes have to be polished on a regular basis, however, manual polishing is more usual in a research or teaching lab.

When hand polishing, grip the electrode low down (near its tip) and hold it in a vertical position while making figure-8 motions on the polishing pad, which is moistened with deionized water and to which has been added a small amount of polishing powder suspension. After three or four of the figure-8 motions, turn the electrode through 90 degrees in your grip and repeat the figure-8 movement. The aim is to ensure even wear on the electrode tip so that a flat surface is maintained.

If you have previously used glass body electrodes then be aware that PEEK or Teflon body electrodes need significantly less downwards pressure to be applied while polishing. Also note that coarse powders (more than 0.05 micron) will cause extensive abrasion of the electrode surface which then takes a lot more polishing with 0.05 micron alumina powder to obtain a mirror smooth finish.

After polishing remove any adherent material with a soft cloth or tissue. Some users prefer immersion of the electrode tip in an ultrasonic bath for a few seconds to remove adherent polish. eDAQ PEEK body working electrodes will withstand sonication but only immerse the tips of the electrode. Do NOT immerse the electrical contacts.

==== Summary ====

#Only polish when necessary.
#Use only light downwards pressure when polishing. When polishing, grip the electrode at its base, ie the end where it is being polished.
#Only use 0.05 micron powder for routine polishing.
#Only use coarser powders if absolutely necessary for very contaminated electrodes.

=== SAM on Gold Electrodes ===
Biosensor electrodes that have a self assembled monolayer (SAM) on gold substrate electrode usually employ an anchoring sulfur atom which binds strongly to the gold surface. It is possible to gently remove the SAM by electrochemical desorption, usually by applying a sufficiently large anodic (oxidising) potential for a short period of time. See:

Formation and removal of alkylthiolate self-assembled monolayers on gold in aqueous solutions. Christie A. Canaria, Jonathan So, James R. Maloney, C. J. Yu, Jeffrey O. Smith, Michael L. Roukes, Scott E. Fraser and Rusty Lansford, Lab on Chip, 6, 289–295, 2006. [http://dx.doi.org/10.1039/b510661c doi:10.1039/b510661c]. You can download this paper [http://authors.library.caltech.edu/4961/1/CANloac06.pdf here].

If the SAM is on a traditional disk electrode then you can also remove the coating by polishing as above. You may need to reins the polishing pad an reapply the abrasive powered several time to was away any organosulfur compounds which have a strong affinity for gold surfaces.

=== Glassy Carbon Electrodes ===

[http://wikipedia.org/wiki/Glassy_carbon Glassy carbon] is a unique material often used for electrodes. In most cases the general cleaning procedure, above, will give satisfactory results. However, a variety of subsequent surface pretreatments have been reported to optimise electrode behaviour in specific instances. An IUPAC review article is [https://publications.iupac.org/pac-2007/1984/pdf/5608x1095.pdf available].

=== [http://www.edaq.com/ET069-1 ET069] and [http://www.edaq.com/ET072-1 ET072] Leakless Reference Electrodes ===

The tip of these reference electrodes can become fouled with use in which case it can be washed or lightly polished (like a working electrode) to remove intractable adhering material. Do not try to get a mirror finish, the surface will remain dull. In extreme cases you can even use a scalpel, or razor blade, to shave a thin slice from the tip, which exposes fresh polymer junction (no need to polish).

Cleaning and Polishing Voltammetric Electrodes

2025-04-23T07:07:16Z

Nick:

=== Safety ===
Polishing powders comprise micron and submicron sized particles which can be dangerous, especially if inhaled. Please follow your local safety guidelines and especially work in a well-ventilated environment.

=== Materials ===
Use a suitable polishing kit such as product number [http://www.edaq.com/ET030 ET030] from eDAQ, that uses abrasive alumina polishing powders.

Local lapidary supply companies may have equivalent products. Diamond, silica, or silicon carbide powders of similar particle size can also be used. A polishing pad that has a pressure sensitive adhesive (PSA) backing can be affixed to a glass plate to ensure a flat polishing surface.

A dry cloth can scratch the electrode and can cause the cloth to tear, always wet a new cloth with abrasive powder suspension, or deionised water before use. Use each cloth only with a powder of single particle size. Rinse cloths with deionised water after each polishing cycle, but reserve them for use only with that particle size polish in the future. Rather than adding dry powder to a polishing pad, it is often better to use a prepared polishing suspension. If you have a source of dry powder suspend it in a wash bottle containing ethanol, and then add a small amount of the suspension (shake well before use) to the pad.

=== General Procedure ===

These procedures are recommended for [http://www.edaq.com/Disk%20Electrodes eDAQ] or other voltammetric disk electrodes.

Resist the temptation to over-polish the electrodes. In most cases the electrode remains clean after the experiment, or could be washed clean with suitable organic solvent to remove greasy organic materials, or dilute acid or base (0.1 mol/L HCl, HNO<sub>3</sub>, NaOH) to remove inorganics. Protein deposits can be hydrolysed with an appropriate commercial enzyme-based cleaner.

Mercury films on platinum or glassy carbon substrates can be removed by gently wiping the electrode tip with lab tissue (dispose of tissue in mercury waste container) any residual mercury can be removed by holding the electrode at an oxidizing potential (eg +1 V) in dilute acid solution (0.1 mol/L).

Only if there is an intractable coating electrodeposited on the electrode, or after extended use, will the electrode need any polishing at all. Polishing should only be done if there is visually obvious surface contamination (use a magnifying glass) or where a trial cyclic voltammogram in clean solvent indicates that there has been surface contamination. Standard maintenance polishing should use a 0.05 micron polishing powder, only a badly scratched electrode, or one with an extremely intractable coating would need to use coarser (larger particle size) powders. Some frequent users like to do a maintenance polish every day, however others may go many days without any surface degradation of the electrode.

Automatic polishing machines are useful if large numbers of electrodes have to be polished on a regular basis, however, manual polishing is more usual in a research or teaching lab.

When hand polishing, grip the electrode low down (near its tip) and hold it in a vertical position while making figure-8 motions on the polishing pad, which is moistened with deionized water and to which has been added a small amount of polishing powder suspension. After three or four of the figure-8 motions, turn the electrode through 90 degrees in your grip and repeat the figure-8 movement. The aim is to ensure even wear on the electrode tip so that a flat surface is maintained.

If you have previously used glass body electrodes then be aware that PEEK or Teflon body electrodes need significantly less downwards pressure to be applied while polishing. Also note that coarse powders (more than 0.05 micron) will cause extensive abrasion of the electrode surface which then takes a lot more polishing with 0.05 micron alumina powder to obtain a mirror smooth finish.

After polishing remove any adherent material with a soft cloth or tissue. Some users prefer immersion of the electrode tip in an ultrasonic bath for a few seconds to remove adherent polish. eDAQ PEEK body working electrodes will withstand sonication but only immerse the tips of the electrode. Do NOT immerse the electrical contacts.

==== Summary ====

#Only polish when necessary.
#Use only light downwards pressure when polishing. When polishing, grip the electrode at its base, ie the end where it is being polished.
#Only use 0.05 micron powder for routine polishing.
#Only use coarser powders if absolutely necessary for very contaminated electrodes.

=== SAM on Gold Electrodes ===
Biosensor electrodes that have a self assembled monolayer (SAM) on gold substrate electrode usually employ an anchoring sulfur atom which binds strongly to the gold surface. It is possible to gently remove the SAM by electrochemical desorption, usually by applying a sufficiently large anodic (oxidising) potential for a short period of time. See:

Formation and removal of alkylthiolate self-assembled monolayers on gold in aqueous solutions. Christie A. Canaria, Jonathan So, James R. Maloney, C. J. Yu, Jeffrey O. Smith, Michael L. Roukes, Scott E. Fraser and Rusty Lansford, Lab on Chip, 6, 289–295, 2006. [http://dx.doi.org/10.1039/b510661c doi:10.1039/b510661c]. You can download this paper [http://authors.library.caltech.edu/4961/1/CANloac06.pdf here].

If the SAM is on a traditional disk electrode then you can also remove the coating by polishing as above. You may need to reins the polishing pad an reapply the abrasive powered several time to was away any organosulfur compounds which have a strong affinity for gold surfaces.

=== Glassy Carbon Electrodes ===

[http://wikipedia.org/wiki/Glassy_carbon Glassy carbon] is a unique material often used for electrodes. In most cases the general cleaning procedure, above, will give satisfactory results. However, a variety of subsequent surface pretreatments have been reported to optimise electrode behaviour in specific instances. An IUPAC review article is [chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://publications.iupac.org/pac-2007/1984/pdf/5608x1095.pdf available].

=== [http://www.edaq.com/ET069-1 ET069] and [http://www.edaq.com/ET072-1 ET072] Leakless Reference Electrodes ===

The tip of these reference electrodes can become fouled with use in which case it can be washed or lightly polished (like a working electrode) to remove intractable adhering material. Do not try to get a mirror finish, the surface will remain dull. In extreme cases you can even use a scalpel, or razor blade, to shave a thin slice from the tip, which exposes fresh polymer junction (no need to polish).

Rapid Determination Method for Total Number of Pathogenic Vibrio Colonies in Shrimp Aquaculture Water

2024-04-02T04:36:16Z

Nick: Created page with "1 Scope This standard specifies the method for determining the total number of pathogenic Vibrio colonies in shrimp aquaculture water. 2 Normative references This standard..."

1 Scope

This standard specifies the method for determining the total number of pathogenic Vibrio colonies in shrimp aquaculture water.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth process of pathogenic Vibrio, the microorganism metabolizes large molecules with weak electrical conductivity, such as amino acids, into small molecules and ions with better conductivity in the culture medium (i.e., thiosulfate-citrate-bile-sucrose agar liquid selective culture medium), causing changes in the conductivity of the liquid medium system. This change can be continuously monitored online in real-time using an electronic microbial growth analyser based on the capacitively-coupled non-contact conductance principle. The corresponding measurable time (Dt) exhibits an inverse relationship with the initial concentration (C0) of the cultivable bacteria, which can be established as a quantitative measurement curve. Therefore, the number and concentration of pathogenic Vibrio colonies in shrimp aquaculture water can be determined by automatically measuring Dt using the electronic microbial growth analyser.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, the following equipment and materials are required:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 Sterile glass bottles with a capacity of 100 mL specifically designed for microbiological applications

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flask: Capacity of 250 mL, 500 mL

4.8 Sterile culture dish: Diameter of 90 mm

4.9 Sterile centrifuge tubes: Capacity of 2 mL, capacity of 15 mL

4.10 Thermal insulation container

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:3 application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A 100 mL water sample was aseptically collected at random from the shrimp culture pond and transferred into a sterile microbiological bottle. The sample was then immediately transported to the laboratory under controlled temperature conditions using an incubator.

[[File:shrimp culture pond.jpg]]

7.1.2 Aseptic procedures were employed to aseptically transfer 2 mL of the mixed sample into a sterile 50 mL centrifuge tube. Subsequently, 18 mL of liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium was added to the centrifuge tube and mixed thoroughly to prepare a 10-fold diluted sample suspension denoted as F1.

7.1.3 The F1 sample suspension was diluted using liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium to prepare six different concentration gradients (10^-2, 10^-3, 10^-4, 10^-5, 10^-6), designated as F1, F2, F3, F4, F5, and F6. These samples were prepared as standard test samples for subsequent analysis.

7.1.4 3 mL of each standard test sample (F1-F6) were aseptically transferred using a 5 mL micropipette into three separate sterile culture plates, with 1 mL of TCBS liquid culture medium used as a negative control. Approximately 15-20 mL of TCBS agar, previously cooled to around 45℃ (maintained at 45℃±1℃ using a constant-temperature water bath), was poured into each plate. The plates were gently rotated to ensure uniform mixing of the agar and the test samples (F1-F6) and negative control (TCBS liquid culture medium). After the agar had solidified, 4 mL of liquid TCBS agar was added onto the surface of each plate, which was then gently swirled to distribute the agar evenly. The plates were incubated at 35℃±1℃ for 24 hours, and bacterial colonies that ranged from 30 to 300 colony-forming units (CFU) were selected and counted to determine the total number of Vibrio parahaemolyticus colonies. The average number of bacterial colonies was calculated, and the bacterial concentration (CFU/mL) of each standard test sample (F1-F6) was determined based on the dilution factor.

[[File:3 lab.jpg]]

7.1.5 A micropipette was used to extract 6 mL of the standard samples F1, F2, F3, F4, F5, and F6 for each of the three parallel measurements. These samples were then evenly distributed into three test tubes. Additionally, 6 mL of sterile TCBS liquid culture medium was added into three test tubes as blank controls, denoted as R1, R2, and R3. The 21 test tubes were inserted into the detection channel of an electronic microbial growth analyser, and the instrument software was opened. The temperature was set to 35℃, and the recording period for the conductivity σ was set to 1 min. The instrument was then run to generate real-time conductivity changes Δσ1-Δσ18 and ΔσR1-ΔσR3, as well as the relationship curves Q1-Q18 and QR1-QR3 between the conductivity changes and the cultivation time t. The inflection points Dt1-Dt18 and DtR1-DtR3 on the curves corresponded to the recognizable times for bacterial proliferation in each test tube. The recognizable time mean values for the initial inoculation concentrations C1-C6 were obtained by averaging the inflection points for each group. The recognizable time mean value for the initial inoculation concentration of 0 was obtained by averaging the inflection points for the blank controls. The recognizable time mean values were then inputted into an Excel spreadsheet and used to obtain the linear working function relationship formula Dt (min) = -k Lg(C) + m and the correlation coefficient R2 between the recognizable time mean value and the initial pathogenic vibrio colony total concentration in the analysed shrimp culture water.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Use a micropipette to individually extract 2 mL of each on-machine sample to be tested, n1, n2, …… nx, and transfer them into separate test tubes. Use 2 mL of sterile TCBS liquid medium as a blank control. Insert the test tubes into the detection channel of an electronic microbial growth analyzer in order. Open the instrument software, set the temperature to 35℃, and set the recording period of conductivity σ to 1 min. Then, run the test for 12 hours to obtain the values of Dt1, Dt2, …… Dtn and Dtr on each curve.

7.2.3 Data acquisition: Substitute Dt1, Dt2, …… Dtn into the working function relationship formula Dt (min) = -k Lg (C) + m to obtain the corresponding values of Lg (C). This value multiplied by 10 Lg(C) is the total number of pathogenic Vibrio colonies in the shrimp aquaculture water being tested, reported in CFU/mL.

7.2.4 Result reporting: When the total number of pathogenic Vibrio colonies in the tested sample is <100, it is reported using two significant figures. When it is ≥100, the third digit is rounded using "rounding off" and then replaced with zeros to represent the result. The result can also be expressed in exponential form using 10 as the base.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 10^1-10^5 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Experimental Data Support
Experimental Procedure:
Preparation of standard curve samples:

1 Sampling location: No. 10 workshop shrimp farming pond, Nan section of Binhai West Road, Haiyang City, Huanghai Aquatic Products Co., Ltd. 121°9′26.9028″E; 36°40′49.9152″N. Two parallel samples A1 and A2 were taken from the surface water and placed in sterile microbiological bottles. (Note: The above water samples to be tested are placed in a thermos box with ice packs.)

2 The water samples were brought back to the laboratory and left at room temperature. The YSI multi-parameter water quality detector was used to measure the following results:

[[File:table 1.jpg]]

3 Under aseptic conditions, 2 mL of the mixed sample to be tested was taken and placed in a sterile centrifuge tube of 50 mL. Then, 18 mL of TCBS liquid culture medium was added and mixed thoroughly to prepare a diluted 10-fold sample homogenate (F1).

4 The F1 sample homogenate was diluted with LB liquid culture medium to prepare 6 concentrations of standard test samples with dilutions of 0.5x10^-1, 10^-2, 0.5x10^-2, 10^-3, 10^-4. These dilutions were labelled as F1, F2, F3, F4, F5, and F6.

5 Using a 5 mL micropipette, 3 mL of each standard test sample (F1, F2, F3, F4, F5, and F6) were drawn and inoculated into 3 sterile culture dishes each. 1 mL of sterile TCBS was used as a blank control. About 15-20 mL of TCBS agar medium cooled to around 45℃ (which can be placed in a constant temperature water bath at 45℃±1℃ for insulation) was carefully poured into each culture dish. The culture dishes were gently rotated to mix the medium with the standard test samples (F1, F2, F3, F4, F5, and F6) and the liquid TCBS used as a blank control. After the agar solidified, 4 mL of unset TCBS agar was added to cover the surface of each plate using a micropipette. The plates were rotated and incubated at 35℃±1℃ for 24 hours. The plates with colony counts between 30-300 CFU were selected, and the number of colonies on each plate was counted to obtain the average number of colonies.

6 From the plate count results, the bacterial concentration of the sample can be obtained, which indicates the initial bacterial concentration of each sample in the standard curve. The detection time (Dt) of the sample with different bacterial concentrations that can reach a detectable change in the reaction can be obtained from the microbial growth curve. Based on the relationship between Dt and bacterial concentration, a standard curve can be constructed.

Prepare test samples:

1 A water sample was taken from the shrimp culture pool in Workshop 10 of Huanghai Aquatic Products Co., Ltd. Aseptically, 2 mL of the mixed sample was taken and placed in a 50 mL sterile centrifuge tube. 18 mL of TCBS liquid culture medium was added, mixed well, and prepared into a uniform solution of the test sample (Y1, Y2, Y3, Y4, Y5, Y6, Y7) with 10^-1 concentration.

2 Take 2 mL of each sample homogenate and transfer it to a test tube. Prepare two replicates for each sample. Place the test tubes, along with the standard curve samples, into a 32-channel microbiological growth sensor and incubate at 35°C for 12 hours. This will generate a microbial growth curve for the test samples.

3 The detection sample is subjected to plate counting as follows: the detection sample solution is serially diluted 10 times using TCBS, resulting in three dilutions: 10^-2, 10^-3, and 10^-4. Each dilution is inoculated onto 3 sterile culture dishes, with 1 mL per dish, and 1 mL of TCBS is added to a sterile culture dish as a blank control. Then, 15-20 mL of cooled (approximately 45℃) plate counting agar (which can be kept warm in a constant temperature water bath at 45℃±1℃) is carefully poured into each culture dish. The culture dishes are rotated gently to mix the agar with the sample solution, and allowed to solidify. Afterward, 3-4 mL of TCBS is added to cover the surface of each plate, and the plates are rotated and incubated at 35℃±1℃ for 24 hours. The plates with a bacterial count between 30 and 300 CFU are selected, and the number of bacterial colonies on each plate is counted. The total concentration of pathogenic vibrio bacteria in the detection sample is obtained by plate counting.

Data processing:

Plot the microbial growth curve based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor. Obtain the quantitative working function Dt (min) = -k Lg(C) + m based on the relationship between the standard bacterial liquid concentration and detection time. Substitute the detection time Dt obtained when the 10^-1 test sample reaches detectable changes into the above function to obtain the corresponding Lg(C) value. The value of 10 Lg(C) is the total number of colonies in the 10^-1 test sample, which, when multiplied by 10, gives the total number of pathogenic Vibrio colonies in the test water sample (reported as CFU/mL).

From Table 2, we can see that the plate counting results of the standard curve sample is 2.8×10^5 CFU/mL, which means that the total number of pathogenic Vibrio colonies in the standard curve diluted samples tested by the machine are 2.8×10^4, 5.6×10^3, 2.8×10^3, 5.6×10^2, 2.8×10^2, and 2.8×10^1 CFU/mL, respectively. Based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor of the standard curve diluted samples, the microbial growth curve can be plotted (Figure 2), and the detection time when the total number of pathogenic Vibrio colonies of different concentrations reached detectable changes can be obtained (Table 3). The relationship between the total number of pathogenic Vibrio colonies and detection time in shrimp culture water can be obtained by plotting the curve based on Table 3 (Figure 3), which is Dt = -121.63 Lg(C) + 795.48, R² = 0.9736. Then, based on the microbial growth curve of the test sample, the detection time T1~T7 when the sample reached detectable changes can be obtained, and the total number of pathogenic Vibrio colonies in the test sample can be obtained by substituting the time into the above relationship.

[[File:table 2.jpg]]

[[File:figure 1.jpg]]

[[File:table 3.jpg]]

[[File:figure 2.jpg]]

[[File:figure 3.jpg]]

[[File:table 4.jpg]]

Table 4 demonstrates that the total number of pathogenic Vibrio bacterial colonies detected using microbial growth sensors in various shrimp aquaculture water samples is within the same order of magnitude as the results obtained through plate counting, indicating the method's ability to meet the detection requirements. Furthermore, this approach enables the acquisition of microbial growth curves for deeper research and provides a rapid and efficient means of determining the total number of pathogenic Vibrio bacterial colonies in shrimp aquaculture water samples.

Application Notes

2023-05-26T04:47:42Z

Nick: /* C4D Conductivity Detector */

== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]

Application Notes

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== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''ER832 ACCS Bacterial Growth Analyser Application Notes'''
* [[Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration]]
* [[Method for Counting the Total Number of Colonies in Ice Cream]]
* [[Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams]]
* [[Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water]]

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]

Application Notes

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File:Table 2.jpg

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Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water

2023-03-14T03:38:20Z

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File:3 lab.jpg

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Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water

2023-03-14T03:07:43Z

Nick:

1 Scope

This standard specifies the method for determining the total number of pathogenic Vibrio colonies in shrimp aquaculture water.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth process of pathogenic Vibrio, the microorganism metabolizes large molecules with weak electrical conductivity, such as amino acids, into small molecules and ions with better conductivity in the culture medium (i.e., thiosulfate-citrate-bile-sucrose agar liquid selective culture medium), causing changes in the conductivity of the liquid medium system. This change can be continuously monitored online in real-time using an electronic microbial growth analyser based on the capacitively-coupled non-contact conductance principle. The corresponding measurable time (Dt) exhibits an inverse relationship with the initial concentration (C0) of the cultivable bacteria, which can be established as a quantitative measurement curve. Therefore, the number and concentration of pathogenic Vibrio colonies in shrimp aquaculture water can be determined by automatically measuring Dt using the electronic microbial growth analyser.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, the following equipment and materials are required:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 Sterile glass bottles with a capacity of 100 mL specifically designed for microbiological applications

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flask: Capacity of 250 mL, 500 mL

4.8 Sterile culture dish: Diameter of 90 mm

4.9 Sterile centrifuge tubes: Capacity of 2 mL, capacity of 15 mL

4.10 Thermal insulation container

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:3 application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A 100 mL water sample was aseptically collected at random from the shrimp culture pond and transferred into a sterile microbiological bottle. The sample was then immediately transported to the laboratory under controlled temperature conditions using an incubator.

[[File:shrimp culture pond.jpg]]

7.1.2 Aseptic procedures were employed to aseptically transfer 2 mL of the mixed sample into a sterile 50 mL centrifuge tube. Subsequently, 18 mL of liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium was added to the centrifuge tube and mixed thoroughly to prepare a 10-fold diluted sample suspension denoted as F1.

7.1.3 The F1 sample suspension was diluted using liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium to prepare six different concentration gradients (10^-2, 10^-3, 10^-4, 10^-5, 10^-6), designated as F1, F2, F3, F4, F5, and F6. These samples were prepared as standard test samples for subsequent analysis.

7.1.4 3 mL of each standard test sample (F1-F6) were aseptically transferred using a 5 mL micropipette into three separate sterile culture plates, with 1 mL of TCBS liquid culture medium used as a negative control. Approximately 15-20 mL of TCBS agar, previously cooled to around 45℃ (maintained at 45℃±1℃ using a constant-temperature water bath), was poured into each plate. The plates were gently rotated to ensure uniform mixing of the agar and the test samples (F1-F6) and negative control (TCBS liquid culture medium). After the agar had solidified, 4 mL of liquid TCBS agar was added onto the surface of each plate, which was then gently swirled to distribute the agar evenly. The plates were incubated at 35℃±1℃ for 24 hours, and bacterial colonies that ranged from 30 to 300 colony-forming units (CFU) were selected and counted to determine the total number of Vibrio parahaemolyticus colonies. The average number of bacterial colonies was calculated, and the bacterial concentration (CFU/mL) of each standard test sample (F1-F6) was determined based on the dilution factor.

[[File:3 lab.jpg]]

7.1.5 A micropipette was used to extract 6 mL of the standard samples F1, F2, F3, F4, F5, and F6 for each of the three parallel measurements. These samples were then evenly distributed into three test tubes. Additionally, 6 mL of sterile TCBS liquid culture medium was added into three test tubes as blank controls, denoted as R1, R2, and R3. The 21 test tubes were inserted into the detection channel of an electronic microbial growth analyser, and the instrument software was opened. The temperature was set to 35℃, and the recording period for the conductivity σ was set to 1 min. The instrument was then run to generate real-time conductivity changes Δσ1-Δσ18 and ΔσR1-ΔσR3, as well as the relationship curves Q1-Q18 and QR1-QR3 between the conductivity changes and the cultivation time t. The inflection points Dt1-Dt18 and DtR1-DtR3 on the curves corresponded to the recognizable times for bacterial proliferation in each test tube. The recognizable time mean values for the initial inoculation concentrations C1-C6 were obtained by averaging the inflection points for each group. The recognizable time mean value for the initial inoculation concentration of 0 was obtained by averaging the inflection points for the blank controls. The recognizable time mean values were then inputted into an Excel spreadsheet and used to obtain the linear working function relationship formula Dt (min) = -k Lg(C) + m and the correlation coefficient R2 between the recognizable time mean value and the initial pathogenic vibrio colony total concentration in the analysed shrimp culture water.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Use a micropipette to individually extract 2 mL of each on-machine sample to be tested, n1, n2, …… nx, and transfer them into separate test tubes. Use 2 mL of sterile TCBS liquid medium as a blank control. Insert the test tubes into the detection channel of an electronic microbial growth analyzer in order. Open the instrument software, set the temperature to 35℃, and set the recording period of conductivity σ to 1 min. Then, run the test for 12 hours to obtain the values of Dt1, Dt2, …… Dtn and Dtr on each curve.

7.2.3 Data acquisition: Substitute Dt1, Dt2, …… Dtn into the working function relationship formula Dt (min) = -k Lg (C) + m to obtain the corresponding values of Lg (C). This value multiplied by 10 Lg(C) is the total number of pathogenic Vibrio colonies in the shrimp aquaculture water being tested, reported in CFU/mL.

7.2.4 Result reporting: When the total number of pathogenic Vibrio colonies in the tested sample is <100, it is reported using two significant figures. When it is ≥100, the third digit is rounded using "rounding off" and then replaced with zeros to represent the result. The result can also be expressed in exponential form using 10 as the base.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 10^1-10^5 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Experimental Data Support
Experimental Procedure:
Preparation of standard curve samples:

1 Sampling location: No. 10 workshop shrimp farming pond, Nan section of Binhai West Road, Haiyang City, Huanghai Aquatic Products Co., Ltd. 121°9′26.9028″E; 36°40′49.9152″N. Two parallel samples A1 and A2 were taken from the surface water and placed in sterile microbiological bottles. (Note: The above water samples to be tested are placed in a thermos box with ice packs.)
2 The water samples were brought back to the laboratory and left at room temperature. The YSI multi-parameter water quality detector was used to measure the following results:

Table 1 Water Sample Parameters
Temperature 18.2℃
Humidity 17.5% RH
DO 7.96 mg/L
SAL 29.22
pH 7.34
SPC 45.54 ms/cm

3 Under aseptic conditions, 2 mL of the mixed sample to be tested was taken and placed in a sterile centrifuge tube of 50 mL. Then, 18 mL of TCBS liquid culture medium was added and mixed thoroughly to prepare a diluted 10-fold sample homogenate (F1).

4 The F1 sample homogenate was diluted with LB liquid culture medium to prepare 6 concentrations of standard test samples with dilutions of 0.5x10^-1, 10^-2, 0.5x10^-2, 10^-3, 10^-4. These dilutions were labelled as F1, F2, F3, F4, F5, and F6.

5 Using a 5 mL micropipette, 3 mL of each standard test sample (F1, F2, F3, F4, F5, and F6) were drawn and inoculated into 3 sterile culture dishes each. 1 mL of sterile TCBS was used as a blank control. About 15-20 mL of TCBS agar medium cooled to around 45℃ (which can be placed in a constant temperature water bath at 45℃±1℃ for insulation) was carefully poured into each culture dish. The culture dishes were gently rotated to mix the medium with the standard test samples (F1, F2, F3, F4, F5, and F6) and the liquid TCBS used as a blank control. After the agar solidified, 4 mL of unset TCBS agar was added to cover the surface of each plate using a micropipette. The plates were rotated and incubated at 35℃±1℃ for 24 hours. The plates with colony counts between 30-300 CFU were selected, and the number of colonies on each plate was counted to obtain the average number of colonies.

6 From the plate count results, the bacterial concentration of the sample can be obtained, which indicates the initial bacterial concentration of each sample in the standard curve. The detection time (Dt) of the sample with different bacterial concentrations that can reach a detectable change in the reaction can be obtained from the microbial growth curve. Based on the relationship between Dt and bacterial concentration, a standard curve can be constructed.

Prepare test samples:

1 A water sample was taken from the shrimp culture pool in Workshop 10 of Huanghai Aquatic Products Co., Ltd. Aseptically, 2 mL of the mixed sample was taken and placed in a 50 mL sterile centrifuge tube. 18 mL of TCBS liquid culture medium was added, mixed well, and prepared into a uniform solution of the test sample (Y1, Y2, Y3, Y4, Y5, Y6, Y7) with 10^-1 concentration.

2 Take 2 mL of each sample homogenate and transfer it to a test tube. Prepare two replicates for each sample. Place the test tubes, along with the standard curve samples, into a 32-channel microbiological growth sensor and incubate at 35°C for 12 hours. This will generate a microbial growth curve for the test samples.

3 The detection sample is subjected to plate counting as follows: the detection sample solution is serially diluted 10 times using TCBS, resulting in three dilutions: 10^-2, 10^-3, and 10^-4. Each dilution is inoculated onto 3 sterile culture dishes, with 1 mL per dish, and 1 mL of TCBS is added to a sterile culture dish as a blank control. Then, 15-20 mL of cooled (approximately 45℃) plate counting agar (which can be kept warm in a constant temperature water bath at 45℃±1℃) is carefully poured into each culture dish. The culture dishes are rotated gently to mix the agar with the sample solution, and allowed to solidify. Afterward, 3-4 mL of TCBS is added to cover the surface of each plate, and the plates are rotated and incubated at 35℃±1℃ for 24 hours. The plates with a bacterial count between 30 and 300 CFU are selected, and the number of bacterial colonies on each plate is counted. The total concentration of pathogenic vibrio bacteria in the detection sample is obtained by plate counting.

Data processing:

Plot the microbial growth curve based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor. Obtain the quantitative working function Dt (min) = -k Lg(C) + m based on the relationship between the standard bacterial liquid concentration and detection time. Substitute the detection time Dt obtained when the 10^-1 test sample reaches detectable changes into the above function to obtain the corresponding Lg(C) value. The value of 10 Lg(C) is the total number of colonies in the 10^-1 test sample, which, when multiplied by 10, gives the total number of pathogenic Vibrio colonies in the test water sample (reported as CFU/mL).

From Table 2, we can see that the plate counting results of the standard curve sample is 2.8×10^5 CFU/mL, which means that the total number of pathogenic Vibrio colonies in the standard curve diluted samples tested by the machine are 2.8×10^4, 5.6×10^3, 2.8×10^3, 5.6×10^2, 2.8×10^2, and 2.8×10^1 CFU/mL, respectively. Based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor of the standard curve diluted samples, the microbial growth curve can be plotted (Figure 2), and the detection time when the total number of pathogenic Vibrio colonies of different concentrations reached detectable changes can be obtained (Table 3). The relationship between the total number of pathogenic Vibrio colonies and detection time in shrimp culture water can be obtained by plotting the curve based on Table 3 (Figure 3), which is Dt = -121.63 Lg(C) + 795.48, R² = 0.9736. Then, based on the microbial growth curve of the test sample, the detection time T1~T7 when the sample reached detectable changes can be obtained, and the total number of pathogenic Vibrio colonies in the test sample can be obtained by substituting the time into the above relationship.

Table 2 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture（CFU/mL）
The results of plate counting（CFU/mL）
Standard curve samples 2.8×105
F1 2.8×104
F2 5.6×103
F3 2.8×103
F4 5.6×102
F5 2.8×102
F6 2.8×101

Figure 1 Microbial growth curve
Table 3 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture, and the detection time T for observable changes in the samples
F1 F2 F3 F4 F5 F6
Total number of pathogenic Vibrio bacteria in aquaculture water (CFU/mL) 2.8×104 5.6×103 2.8×103 5.6×102 2.8×102 2.8×101
The logarithmic (lg) (CFU/mL) value of the total number of pathogenic Vibrio bacteria in aquaculture water 4.447 3.748 3.447 2.748 2.447 1.447
The time required to detect observable changes（min） 238 330 402 456 525 598

Figure 2 The curve depicting the relationship between the total number of pathogenic Vibrio bacteria in aquaculture water and the detection time

Figure 3 The growth curves of the water samples to be tested

Table 4 The quantity of pathogenic Vibrio bacteria present in the water sample under analysis

Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained by the plate counting method Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained via the growth curves
Y1 1620 1395
Y2 2310 1853
Y3 1620 1222
Y4 2310 2326
Y5 2310 2156
Y6 1620 1343
Y7 2310 1853

Table 4 demonstrates that the total number of pathogenic Vibrio bacterial colonies detected using microbial growth sensors in various shrimp aquaculture water samples is within the same order of magnitude as the results obtained through plate counting, indicating the method's ability to meet the detection requirements. Furthermore, this approach enables the acquisition of microbial growth curves for deeper research and provides a rapid and efficient means of determining the total number of pathogenic Vibrio bacterial colonies in shrimp aquaculture water samples.

File:Shrimp culture pond.jpg

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Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water

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Nick:

1 Scope

This standard specifies the method for determining the total number of pathogenic Vibrio colonies in shrimp aquaculture water.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth process of pathogenic Vibrio, the microorganism metabolizes large molecules with weak electrical conductivity, such as amino acids, into small molecules and ions with better conductivity in the culture medium (i.e., thiosulfate-citrate-bile-sucrose agar liquid selective culture medium), causing changes in the conductivity of the liquid medium system. This change can be continuously monitored online in real-time using an electronic microbial growth analyser based on the capacitively-coupled non-contact conductance principle. The corresponding measurable time (Dt) exhibits an inverse relationship with the initial concentration (C0) of the cultivable bacteria, which can be established as a quantitative measurement curve. Therefore, the number and concentration of pathogenic Vibrio colonies in shrimp aquaculture water can be determined by automatically measuring Dt using the electronic microbial growth analyser.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, the following equipment and materials are required:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 Sterile glass bottles with a capacity of 100 mL specifically designed for microbiological applications

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flask: Capacity of 250 mL, 500 mL

4.8 Sterile culture dish: Diameter of 90 mm

4.9 Sterile centrifuge tubes: Capacity of 2 mL, capacity of 15 mL

4.10 Thermal insulation container

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:3 application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A 100 mL water sample was aseptically collected at random from the shrimp culture pond and transferred into a sterile microbiological bottle. The sample was then immediately transported to the laboratory under controlled temperature conditions using an incubator.

[[File:shrimp culture pond.jpg]]

7.1.2 Aseptic procedures were employed to aseptically transfer 2 mL of the mixed sample into a sterile 50 mL centrifuge tube. Subsequently, 18 mL of liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium was added to the centrifuge tube and mixed thoroughly to prepare a 10-fold diluted sample suspension denoted as F1.

7.1.3 The F1 sample suspension was diluted using liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium to prepare six different concentration gradients (10^-2, 10^-3, 10^-4, 10^-5, 10^-6), designated as F1, F2, F3, F4, F5, and F6. These samples were prepared as standard test samples for subsequent analysis.

7.1.4 3 mL of each standard test sample (F1-F6) were aseptically transferred using a 5 mL micropipette into three separate sterile culture plates, with 1 mL of TCBS liquid culture medium used as a negative control. Approximately 15-20 mL of TCBS agar, previously cooled to around 45℃ (maintained at 45℃±1℃ using a constant-temperature water bath), was poured into each plate. The plates were gently rotated to ensure uniform mixing of the agar and the test samples (F1-F6) and negative control (TCBS liquid culture medium). After the agar had solidified, 4 mL of liquid TCBS agar was added onto the surface of each plate, which was then gently swirled to distribute the agar evenly. The plates were incubated at 35℃±1℃ for 24 hours, and bacterial colonies that ranged from 30 to 300 colony-forming units (CFU) were selected and counted to determine the total number of Vibrio parahaemolyticus colonies. The average number of bacterial colonies was calculated, and the bacterial concentration (CFU/mL) of each standard test sample (F1-F6) was determined based on the dilution factor.

7.1.5 A micropipette was used to extract 6 mL of the standard samples F1, F2, F3, F4, F5, and F6 for each of the three parallel measurements. These samples were then evenly distributed into three test tubes. Additionally, 6 mL of sterile TCBS liquid culture medium was added into three test tubes as blank controls, denoted as R1, R2, and R3. The 21 test tubes were inserted into the detection channel of an electronic microbial growth analyser, and the instrument software was opened. The temperature was set to 35℃, and the recording period for the conductivity σ was set to 1 min. The instrument was then run to generate real-time conductivity changes Δσ1-Δσ18 and ΔσR1-ΔσR3, as well as the relationship curves Q1-Q18 and QR1-QR3 between the conductivity changes and the cultivation time t. The inflection points Dt1-Dt18 and DtR1-DtR3 on the curves corresponded to the recognizable times for bacterial proliferation in each test tube. The recognizable time mean values for the initial inoculation concentrations C1-C6 were obtained by averaging the inflection points for each group. The recognizable time mean value for the initial inoculation concentration of 0 was obtained by averaging the inflection points for the blank controls. The recognizable time mean values were then inputted into an Excel spreadsheet and used to obtain the linear working function relationship formula Dt (min) = -k Lg(C) + m and the correlation coefficient R2 between the recognizable time mean value and the initial pathogenic vibrio colony total concentration in the analysed shrimp culture water.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Use a micropipette to individually extract 2 mL of each on-machine sample to be tested, n1, n2, …… nx, and transfer them into separate test tubes. Use 2 mL of sterile TCBS liquid medium as a blank control. Insert the test tubes into the detection channel of an electronic microbial growth analyzer in order. Open the instrument software, set the temperature to 35℃, and set the recording period of conductivity σ to 1 min. Then, run the test for 12 hours to obtain the values of Dt1, Dt2, …… Dtn and Dtr on each curve.

7.2.3 Data acquisition: Substitute Dt1, Dt2, …… Dtn into the working function relationship formula Dt (min) = -k Lg (C) + m to obtain the corresponding values of Lg (C). This value multiplied by 10 Lg(C) is the total number of pathogenic Vibrio colonies in the shrimp aquaculture water being tested, reported in CFU/mL.

7.2.4 Result reporting: When the total number of pathogenic Vibrio colonies in the tested sample is <100, it is reported using two significant figures. When it is ≥100, the third digit is rounded using "rounding off" and then replaced with zeros to represent the result. The result can also be expressed in exponential form using 10 as the base.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 10^1-10^5 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Experimental Data Support
Experimental Procedure:
Preparation of standard curve samples:

1 Sampling location: No. 10 workshop shrimp farming pond, Nan section of Binhai West Road, Haiyang City, Huanghai Aquatic Products Co., Ltd. 121°9′26.9028″E; 36°40′49.9152″N. Two parallel samples A1 and A2 were taken from the surface water and placed in sterile microbiological bottles. (Note: The above water samples to be tested are placed in a thermos box with ice packs.)
2 The water samples were brought back to the laboratory and left at room temperature. The YSI multi-parameter water quality detector was used to measure the following results:

Table 1 Water Sample Parameters
Temperature 18.2℃
Humidity 17.5% RH
DO 7.96 mg/L
SAL 29.22
pH 7.34
SPC 45.54 ms/cm

3 Under aseptic conditions, 2 mL of the mixed sample to be tested was taken and placed in a sterile centrifuge tube of 50 mL. Then, 18 mL of TCBS liquid culture medium was added and mixed thoroughly to prepare a diluted 10-fold sample homogenate (F1).

4 The F1 sample homogenate was diluted with LB liquid culture medium to prepare 6 concentrations of standard test samples with dilutions of 0.5x10^-1, 10^-2, 0.5x10^-2, 10^-3, 10^-4. These dilutions were labelled as F1, F2, F3, F4, F5, and F6.

5 Using a 5 mL micropipette, 3 mL of each standard test sample (F1, F2, F3, F4, F5, and F6) were drawn and inoculated into 3 sterile culture dishes each. 1 mL of sterile TCBS was used as a blank control. About 15-20 mL of TCBS agar medium cooled to around 45℃ (which can be placed in a constant temperature water bath at 45℃±1℃ for insulation) was carefully poured into each culture dish. The culture dishes were gently rotated to mix the medium with the standard test samples (F1, F2, F3, F4, F5, and F6) and the liquid TCBS used as a blank control. After the agar solidified, 4 mL of unset TCBS agar was added to cover the surface of each plate using a micropipette. The plates were rotated and incubated at 35℃±1℃ for 24 hours. The plates with colony counts between 30-300 CFU were selected, and the number of colonies on each plate was counted to obtain the average number of colonies.

6 From the plate count results, the bacterial concentration of the sample can be obtained, which indicates the initial bacterial concentration of each sample in the standard curve. The detection time (Dt) of the sample with different bacterial concentrations that can reach a detectable change in the reaction can be obtained from the microbial growth curve. Based on the relationship between Dt and bacterial concentration, a standard curve can be constructed.

Prepare test samples:

1 A water sample was taken from the shrimp culture pool in Workshop 10 of Huanghai Aquatic Products Co., Ltd. Aseptically, 2 mL of the mixed sample was taken and placed in a 50 mL sterile centrifuge tube. 18 mL of TCBS liquid culture medium was added, mixed well, and prepared into a uniform solution of the test sample (Y1, Y2, Y3, Y4, Y5, Y6, Y7) with 10^-1 concentration.

2 Take 2 mL of each sample homogenate and transfer it to a test tube. Prepare two replicates for each sample. Place the test tubes, along with the standard curve samples, into a 32-channel microbiological growth sensor and incubate at 35°C for 12 hours. This will generate a microbial growth curve for the test samples.

3 The detection sample is subjected to plate counting as follows: the detection sample solution is serially diluted 10 times using TCBS, resulting in three dilutions: 10^-2, 10^-3, and 10^-4. Each dilution is inoculated onto 3 sterile culture dishes, with 1 mL per dish, and 1 mL of TCBS is added to a sterile culture dish as a blank control. Then, 15-20 mL of cooled (approximately 45℃) plate counting agar (which can be kept warm in a constant temperature water bath at 45℃±1℃) is carefully poured into each culture dish. The culture dishes are rotated gently to mix the agar with the sample solution, and allowed to solidify. Afterward, 3-4 mL of TCBS is added to cover the surface of each plate, and the plates are rotated and incubated at 35℃±1℃ for 24 hours. The plates with a bacterial count between 30 and 300 CFU are selected, and the number of bacterial colonies on each plate is counted. The total concentration of pathogenic vibrio bacteria in the detection sample is obtained by plate counting.

Data processing:

Plot the microbial growth curve based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor. Obtain the quantitative working function Dt (min) = -k Lg(C) + m based on the relationship between the standard bacterial liquid concentration and detection time. Substitute the detection time Dt obtained when the 10^-1 test sample reaches detectable changes into the above function to obtain the corresponding Lg(C) value. The value of 10 Lg(C) is the total number of colonies in the 10^-1 test sample, which, when multiplied by 10, gives the total number of pathogenic Vibrio colonies in the test water sample (reported as CFU/mL).

From Table 2, we can see that the plate counting results of the standard curve sample is 2.8×10^5 CFU/mL, which means that the total number of pathogenic Vibrio colonies in the standard curve diluted samples tested by the machine are 2.8×10^4, 5.6×10^3, 2.8×10^3, 5.6×10^2, 2.8×10^2, and 2.8×10^1 CFU/mL, respectively. Based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor of the standard curve diluted samples, the microbial growth curve can be plotted (Figure 2), and the detection time when the total number of pathogenic Vibrio colonies of different concentrations reached detectable changes can be obtained (Table 3). The relationship between the total number of pathogenic Vibrio colonies and detection time in shrimp culture water can be obtained by plotting the curve based on Table 3 (Figure 3), which is Dt = -121.63 Lg(C) + 795.48, R² = 0.9736. Then, based on the microbial growth curve of the test sample, the detection time T1~T7 when the sample reached detectable changes can be obtained, and the total number of pathogenic Vibrio colonies in the test sample can be obtained by substituting the time into the above relationship.

Table 2 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture（CFU/mL）
The results of plate counting（CFU/mL）
Standard curve samples 2.8×105
F1 2.8×104
F2 5.6×103
F3 2.8×103
F4 5.6×102
F5 2.8×102
F6 2.8×101

Figure 1 Microbial growth curve
Table 3 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture, and the detection time T for observable changes in the samples
F1 F2 F3 F4 F5 F6
Total number of pathogenic Vibrio bacteria in aquaculture water (CFU/mL) 2.8×104 5.6×103 2.8×103 5.6×102 2.8×102 2.8×101
The logarithmic (lg) (CFU/mL) value of the total number of pathogenic Vibrio bacteria in aquaculture water 4.447 3.748 3.447 2.748 2.447 1.447
The time required to detect observable changes（min） 238 330 402 456 525 598

Figure 2 The curve depicting the relationship between the total number of pathogenic Vibrio bacteria in aquaculture water and the detection time

Figure 3 The growth curves of the water samples to be tested

Table 4 The quantity of pathogenic Vibrio bacteria present in the water sample under analysis

Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained by the plate counting method Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained via the growth curves
Y1 1620 1395
Y2 2310 1853
Y3 1620 1222
Y4 2310 2326
Y5 2310 2156
Y6 1620 1343
Y7 2310 1853

Table 4 demonstrates that the total number of pathogenic Vibrio bacterial colonies detected using microbial growth sensors in various shrimp aquaculture water samples is within the same order of magnitude as the results obtained through plate counting, indicating the method's ability to meet the detection requirements. Furthermore, this approach enables the acquisition of microbial growth curves for deeper research and provides a rapid and efficient means of determining the total number of pathogenic Vibrio bacterial colonies in shrimp aquaculture water samples.

File:3 application block diagram.jpg

2023-03-14T01:06:02Z

Nick:

Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water

2023-03-14T01:05:36Z

1 Scope

This standard specifies the method for determining the total number of pathogenic Vibrio colonies in shrimp aquaculture water.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth process of pathogenic Vibrio, the microorganism metabolizes large molecules with weak electrical conductivity, such as amino acids, into small molecules and ions with better conductivity in the culture medium (i.e., thiosulfate-citrate-bile-sucrose agar liquid selective culture medium), causing changes in the conductivity of the liquid medium system. This change can be continuously monitored online in real-time using an electronic microbial growth analyser based on the capacitively-coupled non-contact conductance principle. The corresponding measurable time (Dt) exhibits an inverse relationship with the initial concentration (C0) of the cultivable bacteria, which can be established as a quantitative measurement curve. Therefore, the number and concentration of pathogenic Vibrio colonies in shrimp aquaculture water can be determined by automatically measuring Dt using the electronic microbial growth analyser.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, the following equipment and materials are required:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 Sterile glass bottles with a capacity of 100 mL specifically designed for microbiological applications

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flask: Capacity of 250 mL, 500 mL

4.8 Sterile culture dish: Diameter of 90 mm

4.9 Sterile centrifuge tubes: Capacity of 2 mL, capacity of 15 mL

4.10

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:3 application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A 100 mL water sample was aseptically collected at random from the shrimp culture pond and transferred into a sterile microbiological bottle. The sample was then immediately transported to the laboratory under controlled temperature conditions using an incubator.

7.1.2 Aseptic procedures were employed to aseptically transfer 2 mL of the mixed sample into a sterile 50 mL centrifuge tube. Subsequently, 18 mL of liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium was added to the centrifuge tube and mixed thoroughly to prepare a 10-fold diluted sample suspension denoted as F1.

7.1.3 The F1 sample suspension was diluted using liquid thiosulfate-citrate-bile-sucrose (TCBS) culture medium to prepare six different concentration gradients (10^-2, 10^-3, 10^-4, 10^-5, 10^-6), designated as F1, F2, F3, F4, F5, and F6. These samples were prepared as standard test samples for subsequent analysis.

7.1.4 3 mL of each standard test sample (F1-F6) were aseptically transferred using a 5 mL micropipette into three separate sterile culture plates, with 1 mL of TCBS liquid culture medium used as a negative control. Approximately 15-20 mL of TCBS agar, previously cooled to around 45℃ (maintained at 45℃±1℃ using a constant-temperature water bath), was poured into each plate. The plates were gently rotated to ensure uniform mixing of the agar and the test samples (F1-F6) and negative control (TCBS liquid culture medium). After the agar had solidified, 4 mL of liquid TCBS agar was added onto the surface of each plate, which was then gently swirled to distribute the agar evenly. The plates were incubated at 35℃±1℃ for 24 hours, and bacterial colonies that ranged from 30 to 300 colony-forming units (CFU) were selected and counted to determine the total number of Vibrio parahaemolyticus colonies. The average number of bacterial colonies was calculated, and the bacterial concentration (CFU/mL) of each standard test sample (F1-F6) was determined based on the dilution factor.

7.1.5 A micropipette was used to extract 6 mL of the standard samples F1, F2, F3, F4, F5, and F6 for each of the three parallel measurements. These samples were then evenly distributed into three test tubes. Additionally, 6 mL of sterile TCBS liquid culture medium was added into three test tubes as blank controls, denoted as R1, R2, and R3. The 21 test tubes were inserted into the detection channel of an electronic microbial growth analyser, and the instrument software was opened. The temperature was set to 35℃, and the recording period for the conductivity σ was set to 1 min. The instrument was then run to generate real-time conductivity changes Δσ1-Δσ18 and ΔσR1-ΔσR3, as well as the relationship curves Q1-Q18 and QR1-QR3 between the conductivity changes and the cultivation time t. The inflection points Dt1-Dt18 and DtR1-DtR3 on the curves corresponded to the recognizable times for bacterial proliferation in each test tube. The recognizable time mean values for the initial inoculation concentrations C1-C6 were obtained by averaging the inflection points for each group. The recognizable time mean value for the initial inoculation concentration of 0 was obtained by averaging the inflection points for the blank controls. The recognizable time mean values were then inputted into an Excel spreadsheet and used to obtain the linear working function relationship formula Dt (min) = -k Lg(C) + m and the correlation coefficient R2 between the recognizable time mean value and the initial pathogenic vibrio colony total concentration in the analysed shrimp culture water.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Use a micropipette to individually extract 2 mL of each on-machine sample to be tested, n1, n2, …… nx, and transfer them into separate test tubes. Use 2 mL of sterile TCBS liquid medium as a blank control. Insert the test tubes into the detection channel of an electronic microbial growth analyzer in order. Open the instrument software, set the temperature to 35℃, and set the recording period of conductivity σ to 1 min. Then, run the test for 12 hours to obtain the values of Dt1, Dt2, …… Dtn and Dtr on each curve.

7.2.3 Data acquisition: Substitute Dt1, Dt2, …… Dtn into the working function relationship formula Dt (min) = -k Lg (C) + m to obtain the corresponding values of Lg (C). This value multiplied by 10 Lg(C) is the total number of pathogenic Vibrio colonies in the shrimp aquaculture water being tested, reported in CFU/mL.

7.2.4 Result reporting: When the total number of pathogenic Vibrio colonies in the tested sample is <100, it is reported using two significant figures. When it is ≥100, the third digit is rounded using "rounding off" and then replaced with zeros to represent the result. The result can also be expressed in exponential form using 10 as the base.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 10^1-10^5 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Experimental Data Support
Experimental Procedure:
Preparation of standard curve samples:

1 Sampling location: No. 10 workshop shrimp farming pond, Nan section of Binhai West Road, Haiyang City, Huanghai Aquatic Products Co., Ltd. 121°9′26.9028″E; 36°40′49.9152″N. Two parallel samples A1 and A2 were taken from the surface water and placed in sterile microbiological bottles. (Note: The above water samples to be tested are placed in a thermos box with ice packs.)
2 The water samples were brought back to the laboratory and left at room temperature. The YSI multi-parameter water quality detector was used to measure the following results:

Table 1 Water Sample Parameters
Temperature 18.2℃
Humidity 17.5% RH
DO 7.96 mg/L
SAL 29.22
pH 7.34
SPC 45.54 ms/cm

3 Under aseptic conditions, 2 mL of the mixed sample to be tested was taken and placed in a sterile centrifuge tube of 50 mL. Then, 18 mL of TCBS liquid culture medium was added and mixed thoroughly to prepare a diluted 10-fold sample homogenate (F1).

4 The F1 sample homogenate was diluted with LB liquid culture medium to prepare 6 concentrations of standard test samples with dilutions of 0.5x10^-1, 10^-2, 0.5x10^-2, 10^-3, 10^-4. These dilutions were labelled as F1, F2, F3, F4, F5, and F6.

5 Using a 5 mL micropipette, 3 mL of each standard test sample (F1, F2, F3, F4, F5, and F6) were drawn and inoculated into 3 sterile culture dishes each. 1 mL of sterile TCBS was used as a blank control. About 15-20 mL of TCBS agar medium cooled to around 45℃ (which can be placed in a constant temperature water bath at 45℃±1℃ for insulation) was carefully poured into each culture dish. The culture dishes were gently rotated to mix the medium with the standard test samples (F1, F2, F3, F4, F5, and F6) and the liquid TCBS used as a blank control. After the agar solidified, 4 mL of unset TCBS agar was added to cover the surface of each plate using a micropipette. The plates were rotated and incubated at 35℃±1℃ for 24 hours. The plates with colony counts between 30-300 CFU were selected, and the number of colonies on each plate was counted to obtain the average number of colonies.

6 From the plate count results, the bacterial concentration of the sample can be obtained, which indicates the initial bacterial concentration of each sample in the standard curve. The detection time (Dt) of the sample with different bacterial concentrations that can reach a detectable change in the reaction can be obtained from the microbial growth curve. Based on the relationship between Dt and bacterial concentration, a standard curve can be constructed.

Prepare test samples:

1 A water sample was taken from the shrimp culture pool in Workshop 10 of Huanghai Aquatic Products Co., Ltd. Aseptically, 2 mL of the mixed sample was taken and placed in a 50 mL sterile centrifuge tube. 18 mL of TCBS liquid culture medium was added, mixed well, and prepared into a uniform solution of the test sample (Y1, Y2, Y3, Y4, Y5, Y6, Y7) with 10^-1 concentration.

2 Take 2 mL of each sample homogenate and transfer it to a test tube. Prepare two replicates for each sample. Place the test tubes, along with the standard curve samples, into a 32-channel microbiological growth sensor and incubate at 35°C for 12 hours. This will generate a microbial growth curve for the test samples.

3 The detection sample is subjected to plate counting as follows: the detection sample solution is serially diluted 10 times using TCBS, resulting in three dilutions: 10^-2, 10^-3, and 10^-4. Each dilution is inoculated onto 3 sterile culture dishes, with 1 mL per dish, and 1 mL of TCBS is added to a sterile culture dish as a blank control. Then, 15-20 mL of cooled (approximately 45℃) plate counting agar (which can be kept warm in a constant temperature water bath at 45℃±1℃) is carefully poured into each culture dish. The culture dishes are rotated gently to mix the agar with the sample solution, and allowed to solidify. Afterward, 3-4 mL of TCBS is added to cover the surface of each plate, and the plates are rotated and incubated at 35℃±1℃ for 24 hours. The plates with a bacterial count between 30 and 300 CFU are selected, and the number of bacterial colonies on each plate is counted. The total concentration of pathogenic vibrio bacteria in the detection sample is obtained by plate counting.

Data processing:

Plot the microbial growth curve based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor. Obtain the quantitative working function Dt (min) = -k Lg(C) + m based on the relationship between the standard bacterial liquid concentration and detection time. Substitute the detection time Dt obtained when the 10^-1 test sample reaches detectable changes into the above function to obtain the corresponding Lg(C) value. The value of 10 Lg(C) is the total number of colonies in the 10^-1 test sample, which, when multiplied by 10, gives the total number of pathogenic Vibrio colonies in the test water sample (reported as CFU/mL).

From Table 2, we can see that the plate counting results of the standard curve sample is 2.8×10^5 CFU/mL, which means that the total number of pathogenic Vibrio colonies in the standard curve diluted samples tested by the machine are 2.8×10^4, 5.6×10^3, 2.8×10^3, 5.6×10^2, 2.8×10^2, and 2.8×10^1 CFU/mL, respectively. Based on the conductivity and cultivation time (T, in minutes) obtained from the microbial growth sensor of the standard curve diluted samples, the microbial growth curve can be plotted (Figure 2), and the detection time when the total number of pathogenic Vibrio colonies of different concentrations reached detectable changes can be obtained (Table 3). The relationship between the total number of pathogenic Vibrio colonies and detection time in shrimp culture water can be obtained by plotting the curve based on Table 3 (Figure 3), which is Dt = -121.63 Lg(C) + 795.48, R² = 0.9736. Then, based on the microbial growth curve of the test sample, the detection time T1~T7 when the sample reached detectable changes can be obtained, and the total number of pathogenic Vibrio colonies in the test sample can be obtained by substituting the time into the above relationship.

Table 2 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture（CFU/mL）
The results of plate counting（CFU/mL）
Standard curve samples 2.8×105
F1 2.8×104
F2 5.6×103
F3 2.8×103
F4 5.6×102
F5 2.8×102
F6 2.8×101

Figure 1 Microbial growth curve
Table 3 The total number of pathogenic Vibrio colonies in water samples from shrimp aquaculture, and the detection time T for observable changes in the samples
F1 F2 F3 F4 F5 F6
Total number of pathogenic Vibrio bacteria in aquaculture water (CFU/mL) 2.8×104 5.6×103 2.8×103 5.6×102 2.8×102 2.8×101
The logarithmic (lg) (CFU/mL) value of the total number of pathogenic Vibrio bacteria in aquaculture water 4.447 3.748 3.447 2.748 2.447 1.447
The time required to detect observable changes（min） 238 330 402 456 525 598

Figure 2 The curve depicting the relationship between the total number of pathogenic Vibrio bacteria in aquaculture water and the detection time

Figure 3 The growth curves of the water samples to be tested

Table 4 The quantity of pathogenic Vibrio bacteria present in the water sample under analysis

Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained by the plate counting method Total number of pathogenic Vibrio bacterial colonies (CFU/mL) obtained via the growth curves
Y1 1620 1395
Y2 2310 1853
Y3 1620 1222
Y4 2310 2326
Y5 2310 2156
Y6 1620 1343
Y7 2310 1853

Table 4 demonstrates that the total number of pathogenic Vibrio bacterial colonies detected using microbial growth sensors in various shrimp aquaculture water samples is within the same order of magnitude as the results obtained through plate counting, indicating the method's ability to meet the detection requirements. Furthermore, this approach enables the acquisition of microbial growth curves for deeper research and provides a rapid and efficient means of determining the total number of pathogenic Vibrio bacterial colonies in shrimp aquaculture water samples.

Application Notes

2023-03-14T01:01:12Z

Nick: /* C4D Conductivity Detector */

== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''ER832 ACCS Bacterial Growth Analyser Application Notes'''
* [[Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration]]
* [[Method for Counting the Total Number of Colonies in Ice Cream]]
* [[Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams]]
* [[Rapid Determination Method for Total Number of Pathogenic Vibrio Volonies in Shrimp Aquaculture Water]]

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]

Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams

2023-02-24T00:35:39Z

Nick:

1 Scope

This standard outlines the methodology for determining the total colony count of pathogenic vibrio in clams.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth of pathogenic Vibrio, it metabolizes the large molecules in the medium (i.e., TCBS selective culture medium) with weak electrical conductivity into small molecules and ions with better electrical conductivity, resulting in a change in the conductivity of the liquid medium system. This change can be monitored in real-time using an electronic microbiological growth analyser based on the principle of capacitive-coupled, non-contact conductivity. The corresponding detectable time (Dt) shows a definite inverse relationship with the initial concentration of the cultivable bacteria (C0), which can serve as a quantitative measurement working curve. Therefore, by adopting an electronic microbiological growth analyser to automatically measure the Dt, the number and concentration of pathogenic Vibrio in the clam samples under test can be calculated.

4 Equipment and materials

In addition to the routine sterilization and cultivation equipment found in a microbiology laboratory, the other equipment and materials include:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 A homogenizer

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flasks: 250 mL and 500 mL capacity

4.8 Sterile petri dish, OD 90 mm

4.9 Sterile centrifuge tubes: capacity of 15 mL

5 Medium and reagent

5.1 TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium: Details see A1

5.2 TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:2application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A random sampling technique was employed to collect a 0.2 kg sample of clams. The clam shells were thoroughly washed in tap water and any surface moisture was removed by shaking. The shells were then aseptically opened and both the clam meat and body fluid were collected as the composite sample for analysis.

7.1.2 Using a sterile 10 mL micropipette, 25 mL of the composite sample to be tested was aseptically transferred to a homogenizing cup. Next, 225 mL of TCBS liquid medium was added to the cup. The sample and medium were then homogenized using a rotary-blade homogenizer at a speed of 8000 r/min for 1 minute. The resulting homogenate was diluted 10-fold to prepare a uniform sample suspension.

7.1.3 The 10-fold diluted uniform sample suspension was subjected to four additional 10-fold serial dilutions using TCBS liquid medium, resulting in a total of five sample concentrations. These sample concentrations were labelled F1, F2, F3, F4, and F5 as the standards for subsequent analysis.

7.1.4 Using a sterile 5 mL micropipette, 2 mL of each of the standards F1, F2, F3, F4, and F5 were aseptically transferred to two separate sterile culture dishes. As a negative control, 1 mL of TCBS was added to a separate dish. Approximately 15-20 mL of TCBS agar, cooled to room temperature, was poured into each dish. The dishes were rotated gently to ensure that the TCBS agar, along with the sample standards and the negative control, were well mixed. After the agar had solidified, 4 mL of non-solidified TCBS agar was aseptically added to each plate surface, which was then rotated and incubated at 36°C ± 1°C for 20 hours. Colonies that appeared on plates with a count between 30 and 300 CFU were selected and counted, and the average number of colonies was determined. The bacterial liquid concentrations (C1, C2, C3, C4, and C5 in CFU/mL) of the standards F1, F2, F3, F4, and F5 were then calculated based on the dilution factor.

7.1.5 Using a micro-pipette to extract 6 mL of each of the five standard samples F1, F2, F3, F4, and F5 for testing. The samples were then evenly distributed into three test tubes for each sample (i.e., three parallel tests were performed). Additionally, 6 mL of sterile TCBS liquid culture medium was added to three test tubes and used as a blank control sample (R1, R2, and R3). All 18 test tubes were subsequently inserted into the detection channel of an electronic microbial growth analyser. The software was then opened and the temperature was set to 36°C. The recording cycle for the conductivity (σ) was set to 1 minute. The analyser was then run to obtain the real-time changes in the conductivity of the medium in each test tube (Δσ1, Δσ2, Δσ3, Δσ4, Δσ5, Δσ6, Δσ7, Δσ8, Δσ9, Δσ10, Δσ11, Δσ12, Δσ13, Δσ14, Δσ15, ΔσR1, ΔσR2, ΔσR3) and the corresponding relationship curve between the conductivity and the cultivation time (t) (i.e., Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q12, Q13, Q14, Q15, QR1, QR2, and QR3).

The inflection points (Dt1-Dt15, DtR1, DtR2, and DtR3) on the curves corresponded to the recognizable time of bacterial growth in the test tubes. The mean recognizable times (Dt1m, Dt2m, Dt3m, Dt4m, and Dt5m) of the initial inoculation concentrations (C1-C5) were calculated by averaging the corresponding inflection points. Additionally, the mean recognizable time (Dtrm) for the zero initial inoculation concentration was calculated by averaging the inflection points of the blank control sample.

Subsequently, the mean recognizable times (Dt1-C1, Dt2-C2, Dt3-C3, Dt4-C4, and Dt5-C5) were inputted into an Excel spreadsheet to obtain the linear working formula (C (CFU/mL) = -nDt (min) +/- g) and correlation coefficient (r) between the mean recognizable times and the initial pathogenic Vibrio concentration in the clam samples. The constants n and g were dependent on the characteristics of the clam sample source.

Note:

When the correlation coefficient (r) is less than or equal to 0.8, it is necessary to re-determine the working function.

When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Using a micropipette, 2 mL of each sample (sample 1, sample 2, ..., sample n) were individually transferred into separate test tubes. A blank control was prepared using 2 mL of sterile TCBS liquid culture medium. The test tubes were inserted into the detection channels of the electronic microbial growth analyzer in sequence. The instrument software was opened, and the temperature was set to 36°C. The conductivity σ was recorded at intervals of 1 minute. The system was allowed to run for 8 hours, and the corresponding discernible times Dt1, Dt2, ..., Dtn and Dtr were obtained from the curves.

7.2.3 Data acquisition: The obtained values of Dt1, Dt2, ..., Dtn were used to calculate the total number of pathogenic Vibrio colonies in the clam samples. This was done by applying the working function relationship formula C (CFU/mL) = -nDt (min) +/- g to each value, where C represents the total number of pathogenic Vibrio colonies, n is the slope of the working function, Dt represents the detection time, and g is the y-intercept of the working function. The resulting values were multiplied by 10 to obtain the total number of pathogenic Vibrio colonies in the clam samples, reported in CFU/mL.
Note: When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2.4 Results reporting: If the total number of pathogenic Vibrio colonies in the clam sample is less than 100, the result is reported with two significant figures. If the total number is greater than or equal to 100, the third digit is rounded using the "rounding" method and replaced with a zero. Alternatively, the result may be expressed in exponential notation with a base of 10.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 10^1-10^5 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams

2023-02-23T05:21:41Z

Nick:

1 Scope

This standard outlines the methodology for determining the total colony count of pathogenic vibrio in clams.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth of pathogenic Vibrio, it metabolizes the large molecules in the medium (i.e., TCBS selective culture medium) with weak electrical conductivity into small molecules and ions with better electrical conductivity, resulting in a change in the conductivity of the liquid medium system. This change can be monitored in real-time using an electronic microbiological growth analyser based on the principle of capacitive-coupled, non-contact conductivity. The corresponding detectable time (Dt) shows a definite inverse relationship with the initial concentration of the cultivable bacteria (C0), which can serve as a quantitative measurement working curve. Therefore, by adopting an electronic microbiological growth analyser to automatically measure the Dt, the number and concentration of pathogenic Vibrio in the clam samples under test can be calculated.

4 Equipment and materials

In addition to the routine sterilization and cultivation equipment found in a microbiology laboratory, the other equipment and materials include:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 A homogenizer

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flasks: 250 mL and 500 mL capacity

4.8 Sterile petri dish, OD 90 mm

4.9 Sterile centrifuge tubes: capacity of 15 mL

5 Medium and reagent

5.1 TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium: Details see A1

5.2 TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:2application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A random sampling technique was employed to collect a 0.2 kg sample of clams. The clam shells were thoroughly washed in tap water and any surface moisture was removed by shaking. The shells were then aseptically opened and both the clam meat and body fluid were collected as the composite sample for analysis.

7.1.2 Using a sterile 10 mL micropipette, 25 mL of the composite sample to be tested was aseptically transferred to a homogenizing cup. Next, 225 mL of TCBS liquid medium was added to the cup. The sample and medium were then homogenized using a rotary-blade homogenizer at a speed of 8000 r/min for 1 minute. The resulting homogenate was diluted 10-fold to prepare a uniform sample suspension.

7.1.3 The 10-fold diluted uniform sample suspension was subjected to four additional 10-fold serial dilutions using TCBS liquid medium, resulting in a total of five sample concentrations. These sample concentrations were labelled F1, F2, F3, F4, and F5 as the standards for subsequent analysis.

7.1.4 Using a sterile 5 mL micropipette, 2 mL of each of the standards F1, F2, F3, F4, and F5 were aseptically transferred to two separate sterile culture dishes. As a negative control, 1 mL of TCBS was added to a separate dish. Approximately 15-20 mL of TCBS agar, cooled to room temperature, was poured into each dish. The dishes were rotated gently to ensure that the TCBS agar, along with the sample standards and the negative control, were well mixed. After the agar had solidified, 4 mL of non-solidified TCBS agar was aseptically added to each plate surface, which was then rotated and incubated at 36°C ± 1°C for 20 hours. Colonies that appeared on plates with a count between 30 and 300 CFU were selected and counted, and the average number of colonies was determined. The bacterial liquid concentrations (C1, C2, C3, C4, and C5 in CFU/mL) of the standards F1, F2, F3, F4, and F5 were then calculated based on the dilution factor.

7.1.5 Using a micro-pipette to extract 6 mL of each of the five standard samples F1, F2, F3, F4, and F5 for testing. The samples were then evenly distributed into three test tubes for each sample (i.e., three parallel tests were performed). Additionally, 6 mL of sterile TCBS liquid culture medium was added to three test tubes and used as a blank control sample (R1, R2, and R3). All 18 test tubes were subsequently inserted into the detection channel of an electronic microbial growth analyser. The software was then opened and the temperature was set to 36°C. The recording cycle for the conductivity (σ) was set to 1 minute. The analyser was then run to obtain the real-time changes in the conductivity of the medium in each test tube (Δσ1, Δσ2, Δσ3, Δσ4, Δσ5, Δσ6, Δσ7, Δσ8, Δσ9, Δσ10, Δσ11, Δσ12, Δσ13, Δσ14, Δσ15, ΔσR1, ΔσR2, ΔσR3) and the corresponding relationship curve between the conductivity and the cultivation time (t) (i.e., Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q12, Q13, Q14, Q15, QR1, QR2, and QR3).

The inflection points (Dt1-Dt15, DtR1, DtR2, and DtR3) on the curves corresponded to the recognizable time of bacterial growth in the test tubes. The mean recognizable times (Dt1m, Dt2m, Dt3m, Dt4m, and Dt5m) of the initial inoculation concentrations (C1-C5) were calculated by averaging the corresponding inflection points. Additionally, the mean recognizable time (Dtrm) for the zero initial inoculation concentration was calculated by averaging the inflection points of the blank control sample.

Subsequently, the mean recognizable times (Dt1-C1, Dt2-C2, Dt3-C3, Dt4-C4, and Dt5-C5) were inputted into an Excel spreadsheet to obtain the linear working formula (C (CFU/mL) = -nDt (min) +/- g) and correlation coefficient (r) between the mean recognizable times and the initial pathogenic Vibrio concentration in the clam samples. The constants n and g were dependent on the characteristics of the clam sample source.

Note:

When the correlation coefficient (r) is less than or equal to 0.8, it is necessary to re-determine the working function.

When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Using a micropipette, 2 mL of each sample (sample 1, sample 2, ..., sample n) were individually transferred into separate test tubes. A blank control was prepared using 2 mL of sterile TCBS liquid culture medium. The test tubes were inserted into the detection channels of the electronic microbial growth analyzer in sequence. The instrument software was opened, and the temperature was set to 36°C. The conductivity σ was recorded at intervals of 1 minute. The system was allowed to run for 8 hours, and the corresponding discernible times Dt1, Dt2, ..., Dtn and Dtr were obtained from the curves.

7.2.3 Data acquisition: The obtained values of Dt1, Dt2, ..., Dtn were used to calculate the total number of pathogenic Vibrio colonies in the clam samples. This was done by applying the working function relationship formula C (CFU/mL) = -nDt (min) +/- g to each value, where C represents the total number of pathogenic Vibrio colonies, n is the slope of the working function, Dt represents the detection time, and g is the y-intercept of the working function. The resulting values were multiplied by 10 to obtain the total number of pathogenic Vibrio colonies in the clam samples, reported in CFU/mL.
Note: When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2.4 Results reporting: If the total number of pathogenic Vibrio colonies in the clam sample is less than 100, the result is reported with two significant figures. If the total number is greater than or equal to 100, the third digit is rounded using the "rounding" method and replaced with a zero. Alternatively, the result may be expressed in exponential notation with a base of 10.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 101-105 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams

2023-02-22T23:44:10Z

Nick:

1 Scope

This standard outlines the methodology for determining the total colony count of pathogenic vibrio in clams.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth of pathogenic Vibrio, it metabolizes the large molecules in the medium (i.e., TCBS selective culture medium) with weak electrical conductivity into small molecules and ions with better electrical conductivity, resulting in a change in the conductivity of the liquid medium system. This change can be monitored in real-time using an electronic microbiological growth analyser based on the principle of capacitive-coupled, non-contact conductivity. The corresponding detectable time (Dt) shows a definite inverse relationship with the initial concentration of the cultivable bacteria (C0), which can serve as a quantitative measurement working curve. Therefore, by adopting an electronic microbiological growth analyser to automatically measure the Dt, the number and concentration of pathogenic Vibrio in the clam samples under test can be calculated.

4 Equipment and materials

In addition to the routine sterilization and cultivation equipment found in a microbiology laboratory, the other equipment and materials include:

4.1 16/32 channels CellStatz microbial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 A homogenizer

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flasks: 250 mL and 500 mL capacity

4.8 Sterile petri dish, OD 90 mm

4.9 Sterile centrifuge tubes: capacity of 15 mL

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:2application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A random sampling technique was employed to collect a 0.2 kg sample of clams. The clam shells were thoroughly washed in tap water and any surface moisture was removed by shaking. The shells were then aseptically opened and both the clam meat and body fluid were collected as the composite sample for analysis.

7.1.2 Using a sterile 10 mL micropipette, 25 mL of the composite sample to be tested was aseptically transferred to a homogenizing cup. Next, 225 mL of TCBS liquid medium was added to the cup. The sample and medium were then homogenized using a rotary-blade homogenizer at a speed of 8000 r/min for 1 minute. The resulting homogenate was diluted 10-fold to prepare a uniform sample suspension.

7.1.3 The 10-fold diluted uniform sample suspension was subjected to four additional 10-fold serial dilutions using TCBS liquid medium, resulting in a total of five sample concentrations. These sample concentrations were labelled F1, F2, F3, F4, and F5 as the standards for subsequent analysis.

7.1.4 Using a sterile 5 mL micropipette, 2 mL of each of the standards F1, F2, F3, F4, and F5 were aseptically transferred to two separate sterile culture dishes. As a negative control, 1 mL of TCBS was added to a separate dish. Approximately 15-20 mL of TCBS agar, cooled to room temperature, was poured into each dish. The dishes were rotated gently to ensure that the TCBS agar, along with the sample standards and the negative control, were well mixed. After the agar had solidified, 4 mL of non-solidified TCBS agar was aseptically added to each plate surface, which was then rotated and incubated at 36°C ± 1°C for 20 hours. Colonies that appeared on plates with a count between 30 and 300 CFU were selected and counted, and the average number of colonies was determined. The bacterial liquid concentrations (C1, C2, C3, C4, and C5 in CFU/mL) of the standards F1, F2, F3, F4, and F5 were then calculated based on the dilution factor.

7.1.5 Using a micro-pipette to extract 6 mL of each of the five standard samples F1, F2, F3, F4, and F5 for testing. The samples were then evenly distributed into three test tubes for each sample (i.e., three parallel tests were performed). Additionally, 6 mL of sterile TCBS liquid culture medium was added to three test tubes and used as a blank control sample (R1, R2, and R3). All 18 test tubes were subsequently inserted into the detection channel of an electronic microbial growth analyser. The software was then opened and the temperature was set to 36°C. The recording cycle for the conductivity (σ) was set to 1 minute. The analyser was then run to obtain the real-time changes in the conductivity of the medium in each test tube (Δσ1, Δσ2, Δσ3, Δσ4, Δσ5, Δσ6, Δσ7, Δσ8, Δσ9, Δσ10, Δσ11, Δσ12, Δσ13, Δσ14, Δσ15, ΔσR1, ΔσR2, ΔσR3) and the corresponding relationship curve between the conductivity and the cultivation time (t) (i.e., Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q12, Q13, Q14, Q15, QR1, QR2, and QR3).

The inflection points (Dt1-Dt15, DtR1, DtR2, and DtR3) on the curves corresponded to the recognizable time of bacterial growth in the test tubes. The mean recognizable times (Dt1m, Dt2m, Dt3m, Dt4m, and Dt5m) of the initial inoculation concentrations (C1-C5) were calculated by averaging the corresponding inflection points. Additionally, the mean recognizable time (Dtrm) for the zero initial inoculation concentration was calculated by averaging the inflection points of the blank control sample.

Subsequently, the mean recognizable times (Dt1-C1, Dt2-C2, Dt3-C3, Dt4-C4, and Dt5-C5) were inputted into an Excel spreadsheet to obtain the linear working formula (C (CFU/mL) = -nDt (min) +/- g) and correlation coefficient (r) between the mean recognizable times and the initial pathogenic Vibrio concentration in the clam samples. The constants n and g were dependent on the characteristics of the clam sample source.

Note:

When the correlation coefficient (r) is less than or equal to 0.8, it is necessary to re-determine the working function.

When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Using a micropipette, 2 mL of each sample (sample 1, sample 2, ..., sample n) were individually transferred into separate test tubes. A blank control was prepared using 2 mL of sterile TCBS liquid culture medium. The test tubes were inserted into the detection channels of the electronic microbial growth analyzer in sequence. The instrument software was opened, and the temperature was set to 36°C. The conductivity σ was recorded at intervals of 1 minute. The system was allowed to run for 8 hours, and the corresponding discernible times Dt1, Dt2, ..., Dtn and Dtr were obtained from the curves.

7.2.3 Data acquisition: The obtained values of Dt1, Dt2, ..., Dtn were used to calculate the total number of pathogenic Vibrio colonies in the clam samples. This was done by applying the working function relationship formula C (CFU/mL) = -nDt (min) +/- g to each value, where C represents the total number of pathogenic Vibrio colonies, n is the slope of the working function, Dt represents the detection time, and g is the y-intercept of the working function. The resulting values were multiplied by 10 to obtain the total number of pathogenic Vibrio colonies in the clam samples, reported in CFU/mL.
Note: When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2.4 Results reporting: If the total number of pathogenic Vibrio colonies in the clam sample is less than 100, the result is reported with two significant figures. If the total number is greater than or equal to 100, the third digit is rounded using the "rounding" method and replaced with a zero. Alternatively, the result may be expressed in exponential notation with a base of 10.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 101-105 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration

2023-02-22T23:09:53Z

Nick:

1 Objective

To quantity the Escherichia coli growth dynamics in liquid culture medium (using LB medium as an example): analysis of growth curve, growth Rate, and acceleration to obtain key growth kinetics parameters

2 Introduction

Taking multiple samples of target Escherichia coli and inoculating them into sterile LB liquid culture medium creates the inoculum. Each inoculum is then placed in a disposable test tube, which serves as a sample to be tested. These tubes are inserted into a detection channel of the CellStatz electronic microbial growth analyser, which automatically measures and displays the growth curves of each E. coli sample in real-time, as well as the superimposed growth curves of several samples. After the growth curve determination is complete, the experimenter can use the automatic data processing function of the CellStatz system to obtain the growth rate and acceleration curves, as well as key growth kinetic parameters of the targeted E. coli samples. Generally, positive control uses LB liquid culture medium inoculated with a quality control strain of Escherichia coli at a known concentration, such as 106 CFU/mL, and sterile LB liquid culture medium is used as the negative control.

This electronic monitoring-based method is reliable and efficient, making it suitable for investigating the growth kinetics of Escherichia coli and the development of relevant applications.

3 Procedure

3.1 Samples Preparation

1) Sterilize LB liquid medium and divide it into separate containers.

2) Inoculate the activated target E. coli samples (samples A, B, C, D, E, and F) into LB liquid medium to form inoculants A, B, C, D, E, and F.

3) Take 1.8 mL of inoculant A, B, C, D, E, and F, and place them into separate sterile test tubes, labelled as sample tube A, B, C, D, E, and F, respectively.

4) According to the experimental requirements of the researchers, the sample tube can be sealed with a sterile rubber stopper or a sterile 0.22 µm/13 cm disposable filter. In this case, the use of a disposable filter for sealing is illustrated.

5) The positive control sample O (inoculated with 10^6 CFU/mL E. coli quality control strain) and negative control sample N are prepared using the same method and steps as described above, and are pipetted into tubes (tubes O and N) for future use.

Note: During the sample preparation process, it is essential to avoid contamination from surrounding environmental microorganisms. Before loading onto the machine, the sample tubes should be stored at 4 ℃ and for no longer than 1 hour.

3.2 Instrument Preparation

1) Turn on the power to the CellStatz electronic microbial growth analyser and the accompanying computer. The instrument will complete a self-check in approximately 10 seconds.

2) Double-click the shortcut for the CellStatz software on the computer to open the working interface.

3) Set the target temperature for bacterial culture (using 36℃ as an example), and then click "Run". When the ambient temperature is around 25℃, the temperature inside the detection chamber of the analyser will stabilize at 36℃ after approximately 30 minutes. For more details, refer to the instrument manual.

4) In the "Settings" menu of the CellStatz working interface, enter information such as stimulation frequency, stimulation level, working channel, signal acquisition frequency, signal acquisition times, and the file name for saving experimental data. The stimulation frequency, stimulation level, working channel, signal acquisition frequency, and signal acquisition times can also be quickly entered by calling template data. Here, taking commonly used measurement parameters as an example, the stimulation frequency, stimulation level, working channel, signal acquisition frequency, and signal acquisition times are set to 200 MHz, 90%, 1-8 (there are only 8 sample tubes to be inserted into the detection channel of the instrument in this experiment), 60 s, and 1200 times, respectively. The file name for saving the experimental data is "E. coli-101". For more details, refer to the instrument manual.

5) After setting the measurement parameters, click the "OK" button to start recording.

3.3 Measurement of Growth Curves

1) Once the temperature within the detection chamber of the CellStatz electronic microbial growth analyzer stabilizes at 36℃, the prepared sample tubes (A, B, C, D, E, and F) along with the positive control tube (O) and negative control tube (N) are individually inserted into a detection channel of the analyzer. This process is performed sequentially, such that each tube is inserted into channels 1-8 in a consecutive manner.
Note: This operation should be performed as expeditiously as possible, ideally within 1 minute.

2) Upon clicking the "Start" button on the CellStatz work interface, the system initiates the reading of the electrical conductivity, expressed in apparent voltage values, of the solution in each detection tube at intervals of 60 seconds. Subsequently, the system plots these values on a graph with time as the abscissa and the conductivity as the ordinate, generating the primary growth curves 1 through 8. These curves are displayed in real-time in their respective display fields for each working channel. Curves 1 through 6 pertain to the growth curves of sample tubes A through F, respectively, cultivated in LB medium. In contrast, curves 7 and 8 represent the growth curves of the positive control tube O and the negative control tube N, respectively.

3) The experimental operator has the option to select multiple growth curves of interest from within the "Summary" functional area of the work interface. These curves can be viewed concurrently and overlaid upon each other in the "Summary" display field. The user also has the option to visualize the curves (groups) in offset mode by clicking the corresponding option. Additionally, it is possible to establish any of the curves 1 through 8 as a reference curve and view the curves (groups) in correction mode by selecting the "Delta Mode" option.
Note: As a general practice, the growth curve corresponding to the negative sample N, i.e., curve 8, is typically used as the reference curve.
For more details, refer to the instrument manual.

4) Unless manually interrupted, the CellStatz detection system will terminate the measurement procedure automatically upon reaching the specified signal acquisition limit of 1200 times, after which the data will be saved to the designated "E. coli-101" file. This file contains all eight curves, namely, curves 1 through 8. Alternatively, the experimental operator may elect to save the data to the "E. coli-101" file by clicking the "Save" button during the measurement process. Subsequently, the "E. coli-101" file will also comprise all eight curves, curves 1 through 8.
Note: Clicking "Save" will terminate the measurement operation.
For more details, refer to the instrument manual.

3.4 Obtain the growth rate curve, growth acceleration curve, and kinetic parameters

1) By utilizing the "Data Management" function bar located on the CellStatz interface, an individual can selectively choose one or more data sets for subsequent analysis. For example, if one were to choose curves 1 through 4 and curve 8, which correspond to sample A, sample B, sample C, sample D, and negative control N, respectively, these five curves would become activated for further analysis.

2) The experimenter can select one or multiple data sets to be analyzed through the "Data Management" function in the CellStatz user interface. For example, selecting the curves 1, 2, 3, 4, and 8, corresponding to samples A, B, C, D, and negative control N, respectively, activates these five curves.

Using the polynomial algorithm, a degree between 2 and 20 is inputted, and by clicking the "Curve Fitting" button, smooth graphical representations of these five curves are obtained. The experimenter can select the negative control (i.e., curve 8) as a reference from the drop-down menu, in order to obtain the calibrated smooth curves corresponding to samples A, B, C, and D.

3) After selecting the polynomial algorithm and inputting an order between 2 and 20, the user can obtain the smoothed graphs of the five selected curves, including samples A, B, C, D, and negative control N, by clicking on the "Curve Fitting" button. To obtain the corrected smoothed curves of samples A, B, C, and D corresponding to the negative control N, the user can choose the negative control N as the reference in the drop-down menu. The corrected growth rate curves of the four samples can be obtained by clicking the "First Derivative" button. The peak value of the bell-shaped growth rate curve corresponds to the maximum growth rate of E. coli. To facilitate comparison and analysis, the user can select multiple curves to be overlaid and displayed in the same display panel by choosing the growth rate curves 1, 2, and 3, for example. By observing the time (horizontal axis) at which their peak values appear, the corresponding initial inoculum concentrations of the samples can be preliminarily estimated. Generally, the smaller the value on the horizontal axis, the higher the concentration. The peak height (vertical axis) can be used to preliminarily determine the differences in maximum growth. Generally, the larger the value on the vertical axis, the greater the maximum growth. By clicking the "Peak" button, the exact coordinates of the peak values on the curve can be displayed. Combining the horizontal axis values of the working curve (the establishment of the working curve is described in the "Automated Quantitative Determination of E. coli" section) can be used to calculate the initial concentration of E. coli in samples A, B, and C.

4) In order to obtain the growth acceleration curves for the four selected growth rate curves mentioned above, the user can perform second-order derivative processing. For example, if growth rate curves 1, 2, and 3 are selected, the user can click on the "Second Derivative" button to generate three curves, each consisting of a peak and a trough. The abscissa value of the peak corresponds to the moment when the E. coli growth acceleration is maximal, while the abscissa value of the trough corresponds to the moment when the E. coli growth deceleration is maximal. By clicking on the "Peak" and "Trough" buttons, the user can read the specific data of each peak and trough value on the growth acceleration curve.
Note: For more details, refer to the instrument manual.

File:2application block diagram.jpg

2023-02-22T22:55:01Z

Nick:

Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams

2023-02-22T22:54:35Z

Nick: Created page with "1 Scope This standard outlines the methodology for determining the total colony count of pathogenic vibrio in clams. 2 Normative references This standard references certain..."

1 Scope

This standard outlines the methodology for determining the total colony count of pathogenic vibrio in clams.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.

3 Principle of testing

During the growth of pathogenic Vibrio, it metabolizes the large molecules in the medium (i.e., TCBS selective culture medium) with weak electrical conductivity into small molecules and ions with better electrical conductivity, resulting in a change in the conductivity of the liquid medium system. This change can be monitored in real-time using an electronic microbiological growth analyser based on the principle of capacitive-coupled, non-contact conductivity. The corresponding detectable time (Dt) shows a definite inverse relationship with the initial concentration of the cultivable bacteria (C0), which can serve as a quantitative measurement working curve. Therefore, by adopting an electronic microbiological growth analyser to automatically measure the Dt, the number and concentration of pathogenic Vibrio in the clam samples under test can be calculated.

4 Equipment and materials

In addition to the routine sterilization and cultivation equipment found in a microbiology laboratory, the other equipment and materials include:

4.1 16/32 channels bacterial growth analyser

4.2 Sterile surgical scalpels and forceps, disposable glass test tubes (referred to as "test tubes" for short)

4.3 A homogenizer

4.4 Micropipettes and tips: 100 μL (1 μL graduation), 1 mL (0.01 mL graduation), 5 mL (0.1 mL graduation), 10 mL (0.1 mL graduation)

4.5 Analytical balance: sensitivity of 0.001 g

4.6 Thermostatic incubator: 36°C ± 1°C

4.7 Sterile conical flasks: 250 mL and 500 mL capacity

4.8 Sterile petri dish, OD 90 mm

4.9 Sterile centrifuge tubes: capacity of 15 mL

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See the block diagram below:

[[File:2application block diagram.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 A random sampling technique was employed to collect a 0.2 kg sample of clams. The clam shells were thoroughly washed in tap water and any surface moisture was removed by shaking. The shells were then aseptically opened and both the clam meat and body fluid were collected as the composite sample for analysis.

7.1.2 Using a sterile 10 mL micropipette, 25 mL of the composite sample to be tested was aseptically transferred to a homogenizing cup. Next, 225 mL of TCBS liquid medium was added to the cup. The sample and medium were then homogenized using a rotary-blade homogenizer at a speed of 8000 r/min for 1 minute. The resulting homogenate was diluted 10-fold to prepare a uniform sample suspension.

7.1.3 The 10-fold diluted uniform sample suspension was subjected to four additional 10-fold serial dilutions using TCBS liquid medium, resulting in a total of five sample concentrations. These sample concentrations were labelled F1, F2, F3, F4, and F5 as the standards for subsequent analysis.

7.1.4 Using a sterile 5 mL micropipette, 2 mL of each of the standards F1, F2, F3, F4, and F5 were aseptically transferred to two separate sterile culture dishes. As a negative control, 1 mL of TCBS was added to a separate dish. Approximately 15-20 mL of TCBS agar, cooled to room temperature, was poured into each dish. The dishes were rotated gently to ensure that the TCBS agar, along with the sample standards and the negative control, were well mixed. After the agar had solidified, 4 mL of non-solidified TCBS agar was aseptically added to each plate surface, which was then rotated and incubated at 36°C ± 1°C for 20 hours. Colonies that appeared on plates with a count between 30 and 300 CFU were selected and counted, and the average number of colonies was determined. The bacterial liquid concentrations (C1, C2, C3, C4, and C5 in CFU/mL) of the standards F1, F2, F3, F4, and F5 were then calculated based on the dilution factor.

7.1.5 Using a micro-pipette to extract 6 mL of each of the five standard samples F1, F2, F3, F4, and F5 for testing. The samples were then evenly distributed into three test tubes for each sample (i.e., three parallel tests were performed). Additionally, 6 mL of sterile TCBS liquid culture medium was added to three test tubes and used as a blank control sample (R1, R2, and R3). All 18 test tubes were subsequently inserted into the detection channel of an electronic microbial growth analyser. The software was then opened and the temperature was set to 36°C. The recording cycle for the conductivity (σ) was set to 1 minute. The analyser was then run to obtain the real-time changes in the conductivity of the medium in each test tube (Δσ1, Δσ2, Δσ3, Δσ4, Δσ5, Δσ6, Δσ7, Δσ8, Δσ9, Δσ10, Δσ11, Δσ12, Δσ13, Δσ14, Δσ15, ΔσR1, ΔσR2, ΔσR3) and the corresponding relationship curve between the conductivity and the cultivation time (t) (i.e., Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, Q9, Q10, Q11, Q12, Q13, Q14, Q15, QR1, QR2, and QR3).

The inflection points (Dt1-Dt15, DtR1, DtR2, and DtR3) on the curves corresponded to the recognizable time of bacterial growth in the test tubes. The mean recognizable times (Dt1m, Dt2m, Dt3m, Dt4m, and Dt5m) of the initial inoculation concentrations (C1-C5) were calculated by averaging the corresponding inflection points. Additionally, the mean recognizable time (Dtrm) for the zero initial inoculation concentration was calculated by averaging the inflection points of the blank control sample.

Subsequently, the mean recognizable times (Dt1-C1, Dt2-C2, Dt3-C3, Dt4-C4, and Dt5-C5) were inputted into an Excel spreadsheet to obtain the linear working formula (C (CFU/mL) = -nDt (min) +/- g) and correlation coefficient (r) between the mean recognizable times and the initial pathogenic Vibrio concentration in the clam samples. The constants n and g were dependent on the characteristics of the clam sample source.

Note:

When the correlation coefficient (r) is less than or equal to 0.8, it is necessary to re-determine the working function.

When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2 Examine samples

7.2.1 Sample preparation: The same method as described in section 7.1.1 was used to prepare the samples for analysis.

7.2.2 On-machine testing: Using a micropipette, 2 mL of each sample (sample 1, sample 2, ..., sample n) were individually transferred into separate test tubes. A blank control was prepared using 2 mL of sterile TCBS liquid culture medium. The test tubes were inserted into the detection channels of the electronic microbial growth analyzer in sequence. The instrument software was opened, and the temperature was set to 36°C. The conductivity σ was recorded at intervals of 1 minute. The system was allowed to run for 8 hours, and the corresponding discernible times Dt1, Dt2, ..., Dtn and Dtr were obtained from the curves.

7.2.3 Data acquisition: The obtained values of Dt1, Dt2, ..., Dtn were used to calculate the total number of pathogenic Vibrio colonies in the clam samples. This was done by applying the working function relationship formula C (CFU/mL) = -nDt (min) +/- g to each value, where C represents the total number of pathogenic Vibrio colonies, n is the slope of the working function, Dt represents the detection time, and g is the y-intercept of the working function. The resulting values were multiplied by 10 to obtain the total number of pathogenic Vibrio colonies in the clam samples, reported in CFU/mL.
Note: When Dtrm > 480 min, it indicates that the system has no false positive results.

7.2.4 Results reporting: If the total number of pathogenic Vibrio colonies in the clam sample is less than 100, the result is reported with two significant figures. If the total number is greater than or equal to 100, the third digit is rounded using the "rounding" method and replaced with a zero. Alternatively, the result may be expressed in exponential notation with a base of 10.

7.3 Test report

The experimental report should include the following essential details:

- Complete identification of the sample, including all necessary information.
- Description of the sampling method used (if applicable).
- All operational details and potential factors that may have affected the test results, which are not specified in this standard.
- Presentation of the obtained results, along with an indication of the representation method used.
- Final results, referencing the results obtained from any reproducibility checks.

8 Additional information:

- The systematic error of this method is ≤20%.
- The linear range of this method is 101-105 CFU/mL.
- Separate working functions should be established for clam samples from different locations.
- The validity of the working function needs to be verified every six months using the plate counting method.
- Personnel and laboratory comparisons have been completed.

Appendix A

A.1: TCBS (Thiosulfate Citrate Bile Sucrose) Liquid Medium

A.1.1 Composition

Peptone: 10.0 g

Yeast extract: 5.0 g

Sodium citrate: 10.0 g

Sodium thiosulfate: 10.0 g

Sodium chloride: 10.0 g

Bile salt: 5.0 g

Sodium taurocholate: 3.0 g

Ferric citrate: 1.0 g

Sucrose: 20.0 g

Bromothymol blue: 0.04 g

Thymol blue: 0.04 g

Distilled water: 1000.0 mL

A.1.2 Preparation

The components listed in A.1.1 were dissolved in distilled water and the pH was adjusted to 8.6±0.2. The solution was heated to boiling until all the components were completely dissolved. The mixture was then cooled and kept for further use.

A.2: TCBS (Thiosulfate Citrate Bile Sucrose) Agar Medium

A.2.1 Composition

Peptone 10.0 g

Yeast extract 5.0 g

Sodium citrate 10.0 g

Sodium thiosulfate 10.0 g

Sodium chloride 10.0 g

Bile powder 5.0 g

Sodium taurocholate 3.0 g

Ferric citrate 1.0 g

Sucrose 20.0 g

Bromothymol blue 0.04 g

Thymol blue 0.04 g

Agar 15.0 g

Distilled water 1,000.0 mL

A2.2 Method
The components in A.2.1 are dissolved in distilled water, and the pH is adjusted to 8.6±0.2. The mixture is then heated and boiled until fully dissolved, and allowed to cool to about 50°C before use.

Application Notes

2023-02-22T22:49:18Z

Nick: /* C4D Conductivity Detector */

== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''ER832 ACCS Bacterial Growth Analyser Application Notes'''
* [[Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration]]
* [[Method for Counting the Total Number of Colonies in Ice Cream]]
* [[Rapid Determination Method for Total Colony Count of Pathogenic Vibrio in Clams]]

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]

Method for Counting the Total Number of Colonies in Ice Cream

2023-02-22T22:46:46Z

Nick: Created page with "1 Scope This standard outlines the methodology for determining the total count of colonies of both aerobic and facultative anaerobic bacteria in ice cream. 2 Normative refer..."

1 Scope

This standard outlines the methodology for determining the total count of colonies of both aerobic and facultative anaerobic bacteria in ice cream.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.GB 4789.2-2016 Food Microbiological Analysis - Total Colonies Count

3 Principle of testing

During microbial growth, large molecules with low electrical conductivity present in the medium undergo metabolic processes that transform them into small molecules and ions with higher electrical conductivity, such as amino acids. As a result, the conductivity of the medium changes. The detectable time for this change is inversely proportional to the logarithm of the initial microbial concentration. This allows for the determination of the concentration and quantity of culturable bacteria in the sample to be tested through the use of a working curve.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, other equipment and materials are as follows:

4.1 16/32 channels CellStatz electronic microbial growth analyzer

4.2 Sterile scalpel

4.3 Micropipette and suction head: the measuring ranges are 100 μL, 1 mL and 5 mL, respectively

4.4 Scale: accuracy is 0.0001 g

4.5 Constant Temperature Incubator: 36 ℃±1 ℃, 30 ℃±1 ℃

4.6 Table Concentrator: ±1 ℃

4.7 Fridge: 2 ℃~5 ℃，-18℃~-20℃

4.8 Sterile petri dish, OD 90 mm

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See block diagram shown below:

[[File:Example.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 To begin the experiment, a 100 μL aliquot of a specific mixed bacterial flora solution stored at -20℃ should be taken and inoculated into 4 mL of LB liquid medium. The culture should then be incubated overnight at 36℃ with shaking at 180 r/min.

7.1.2 The concentration of the activated bacterial solution will be determined by employing the plate counting method described in GB 4789.2-2016.

7.1.3 Following quantification of the bacterial solution, different concentration gradients will be established for the contamination of 1 mL of ice cream solution. Specifically, the bacterial concentration of the contaminated ice cream solution will be set at 1.0x10^7, 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL. Subsequently, 1 mL of each contaminated ice cream solution will be mixed uniformly with 9 mL of LB liquid medium to generate standard growth curves with known bacterial concentrations of 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL.

7.2 Procedure for samples to be examined

7.2.1 To begin the analysis, an appropriate amount of the ice cream sample will be obtained using a random sampling method. The representative samples will be collected using aseptic techniques and placed in sterilized containers to allow for natural melting. In cases where the ice cream is packaged, the opening of the packaging will be swabbed with 75% ethanol before sampling to ensure sterility.

7.2.2 A 1 mL aliquot of the ice cream solution will be transferred using a pipette and added to 9 mL of LB liquid medium. The solution will then be thoroughly mixed to prepare a bacterial solution containing 10% LB medium from the ice cream sample for analysis using the designated equipment.

7.3 Negative control sample
A 1 mL aliquot of sterile ice cream solution will be transferred and added to 9 mL of LB liquid medium. The mixture will be thoroughly mixed to prepare a negative control sample for analysis.

7.4 Culture the colonies

The prepared standard curve samples and the ice cream samples to be tested will be loaded into separate 2 mL test tubes. Each concentration level will have three reference samples. These test tubes will be placed into the designated equipment and incubated at 36°C for 12 hours. Conductivity measurements will be taken at 60-second intervals to obtain the necessary data.

7.5 Data processing

The data obtained from the microbial growth sensor, including electrical conductivity and culture time (T, in minutes), will be used to plot the microbial growth curve. By analyzing the relationship between the concentration of the standard bacterial solution and its corresponding detection time, a quantitative working function C bacteria (CFU/mL) = mT (min) + n will be obtained. The detection time (T) of the ice cream sample will be substituted into this function to obtain the total number of bacterial colonies present in the sample. The resulting number will be multiplied by 10 to determine the total number of bacterial colonies in the ice cream sample.

7.6 Results

7.6.1 If the total number of bacterial colonies in the samples obtained during session 7.5 is less than 100, the data shall be reported with two significant figures.

7.6.2 When reporting the total number of colonies of the samples obtained from session 7.5, the appropriate significant figures should be used based on the magnitude of the number. If the total number of colonies is less than 100, a two-digit significant number should be used. However, if the total number of colonies is greater than 100, the third digit should be rounded off and replaced by a zero. Alternatively, the number can be expressed in exponential form as a power of 10.

7.7 Test report

The test report should indicate:

- All the information necessary for the complete identification of the sample;

- Sampling method adopted, if any;

- All operational details not specified in this standard and some factors that may have affected the test results;

- The obtained result, indicating the method of representation adopted;

- Final results cited if repeated checks have been made;

- Quantitative work functions should be established separately for each type and specification of ice cream products;

- The working function needs to be validated by plate counting every six months.

8 Standard specification

This standard was completed on 6th September 2021.

Appendix A

A1 LB liquid medium

A1.1 Components

Yeast Extract 5g

Tryptone 10 g

Sodium chloride 10 g

Distilled Water 1000 mL

A1.2 Preparation

To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.

A2 Plate count agar

A2.1 Components

Tryptone 5.0g

Yeast Extract 2.5g

Glucose 1.0g

June Fat 15.0g

Distilled Water 1000 mL

A2.2 Preparation

To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.

Method for counting the total number of colonies in ice cream

2023-02-21T03:46:35Z

Nick:

Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration

2023-02-21T03:37:39Z

Nick: Created page with "1 Objective To quantity the Escherichia coli growth dynamics in liquid culture medium (using LB medium as an example): analysis of growth curve, growth Rate, and acceleration..."

Application Notes

2023-02-21T03:34:36Z

Nick: /* C4D Conductivity Detector */

== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''ER832 ACCS Bacterial Growth Analyser Application Notes'''
* [[Quantification of Escherichia coli Growth Dynamics including Growth Kinetics, Growth Rate, and Acceleration]]
* [[Method for Counting the Total Number of Colonies in Ice Cream]]

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]

Method for counting the total number of colonies in ice cream

2023-02-17T05:28:37Z

Nick:

1 Scope

This standard outlines the methodology for determining the total count of colonies of both aerobic and facultative anaerobic bacteria in ice cream.

2 Normative references

This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.GB 4789.2-2016 Food Microbiological Analysis - Total Colonies Count

3 Principle of testing

During microbial growth, large molecules with low electrical conductivity present in the medium undergo metabolic processes that transform them into small molecules and ions with higher electrical conductivity, such as amino acids. As a result, the conductivity of the medium changes. The detectable time for this change is inversely proportional to the logarithm of the initial microbial concentration. This allows for the determination of the concentration and quantity of culturable bacteria in the sample to be tested through the use of a working curve.

4 Equipment and materials

In addition to the routine sterilization and culture equipment in the microbiology laboratory, other equipment and materials are as follows:

4.1 16/32 channels bacterial growth analyser

4.2 Sterile scalpel

4.3 Micropipette and suction head: the measuring ranges are 100 μL, 1 mL and 5 mL, respectively

4.4 Scale: accuracy is 0.0001 g

4.5 Constant Temperature Incubator: 36 ℃±1 ℃, 30 ℃±1 ℃

4.6 Table Concentrator: ±1 ℃

4.7 Fridge: 2 ℃~5 ℃，-18℃~-20℃

4.8 Sterile petri dish, OD 90 mm

5 Medium and reagent

5.1 LB liquid medium: Details see A1

5.2 Plate counting AGAR medium: Details see A2

6 Test procedure

See block diagram shown below:

[[File:Example.jpg]]

7 Detailed test procedure

7.1 Develop the standard growth curves for the total number of colonies

7.1.1 To begin the experiment, a 100 μL aliquot of a specific mixed bacterial flora solution stored at -20℃ should be taken and inoculated into 4 mL of LB liquid medium. The culture should then be incubated overnight at 36℃ with shaking at 180 r/min.

7.1.2 The concentration of the activated bacterial solution will be determined by employing the plate counting method described in GB 4789.2-2016.

7.1.3 Following quantification of the bacterial solution, different concentration gradients will be established for the contamination of 1 mL of ice cream solution. Specifically, the bacterial concentration of the contaminated ice cream solution will be set at 1.0x10^7, 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL. Subsequently, 1 mL of each contaminated ice cream solution will be mixed uniformly with 9 mL of LB liquid medium to generate standard growth curves with known bacterial concentrations of 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL.

7.2 Procedure for samples to be examined

7.2.1 To begin the analysis, an appropriate amount of the ice cream sample will be obtained using a random sampling method. The representative samples will be collected using aseptic techniques and placed in sterilized containers to allow for natural melting. In cases where the ice cream is packaged, the opening of the packaging will be swabbed with 75% ethanol before sampling to ensure sterility.

7.2.2 A 1 mL aliquot of the ice cream solution will be transferred using a pipette and added to 9 mL of LB liquid medium. The solution will then be thoroughly mixed to prepare a bacterial solution containing 10% LB medium from the ice cream sample for analysis using the designated equipment.

7.3 Negative control sample
A 1 mL aliquot of sterile ice cream solution will be transferred and added to 9 mL of LB liquid medium. The mixture will be thoroughly mixed to prepare a negative control sample for analysis.

7.4 Culture the colonies

The prepared standard curve samples and the ice cream samples to be tested will be loaded into separate 2 mL test tubes. Each concentration level will have three reference samples. These test tubes will be placed into the designated equipment and incubated at 36°C for 12 hours. Conductivity measurements will be taken at 60-second intervals to obtain the necessary data.

7.5 Data processing

The data obtained from the microbial growth sensor, including electrical conductivity and culture time (T, in minutes), will be used to plot the microbial growth curve. By analyzing the relationship between the concentration of the standard bacterial solution and its corresponding detection time, a quantitative working function C bacteria (CFU/mL) = mT (min) + n will be obtained. The detection time (T) of the ice cream sample will be substituted into this function to obtain the total number of bacterial colonies present in the sample. The resulting number will be multiplied by 10 to determine the total number of bacterial colonies in the ice cream sample.

7.6 Results

7.6.1 If the total number of bacterial colonies in the samples obtained during session 7.5 is less than 100, the data shall be reported with two significant figures.

7.6.2 When reporting the total number of colonies of the samples obtained from session 7.5, the appropriate significant figures should be used based on the magnitude of the number. If the total number of colonies is less than 100, a two-digit significant number should be used. However, if the total number of colonies is greater than 100, the third digit should be rounded off and replaced by a zero. Alternatively, the number can be expressed in exponential form as a power of 10.

7.7 Test report

The test report should indicate:

- All the information necessary for the complete identification of the sample;

- Sampling method adopted, if any;

- All operational details not specified in this standard and some factors that may have affected the test results;

- The obtained result, indicating the method of representation adopted;

- Final results cited if repeated checks have been made;

- Quantitative work functions should be established separately for each type and specification of ice cream products;

- The working function needs to be validated by plate counting every six months.

8 Standard specification

This standard was completed on 6th September 2021.

Appendix A

A1 LB liquid medium

A1.1 Components

Yeast Extract 5g

Tryptone 10 g

Sodium chloride 10 g

Distilled Water 1000 mL

A1.2 Preparation

To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.

A2 Plate count agar

A2.1 Components

Tryptone 5.0g

Yeast Extract 2.5g

Glucose 1.0g

June Fat 15.0g

Distilled Water 1000 mL

A2.2 Preparation

To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.

File:Example.jpg

2023-02-17T05:20:43Z

Nick: Nick uploaded a new version of File:Example.jpg

File:Example.jpg

2023-02-17T05:18:54Z

Nick:

Method for counting the total number of colonies in ice cream

2023-02-17T05:15:19Z

1 Scope
This standard outlines the methodology for determining the total count of colonies of both aerobic and facultative anaerobic bacteria in ice cream.
2 Normative references
This standard references certain provisions contained in the reference standard. For dated citation standards, subsequent changes or revisions will not apply. However, parties who agree to follow this standard are encouraged to consider using the latest version of the reference standard described below.GB 4789.2-2016 Food Microbiological Analysis - Total Colonies Count
3 Principle of testing
During microbial growth, large molecules with low electrical conductivity present in the medium undergo metabolic processes that transform them into small molecules and ions with higher electrical conductivity, such as amino acids. As a result, the conductivity of the medium changes. The detectable time for this change is inversely proportional to the logarithm of the initial microbial concentration. This allows for the determination of the concentration and quantity of culturable bacteria in the sample to be tested through the use of a working curve.
4 Equipment and materials
In addition to the routine sterilization and culture equipment in the microbiology laboratory, other equipment and materials are as follows:
4.1 16/32 channels bacterial growth analyser
4.2 Sterile scalpel
4.3 Micropipette and suction head: the measuring ranges are 100 μL, 1 mL and 5 mL, respectively
4.4 Scale: accuracy is 0.0001 g
4.5 Constant Temperature Incubator: 36 ℃±1 ℃, 30 ℃±1 ℃
4.6 Table Concentrator: ±1 ℃
4.7 Fridge: 2 ℃~5 ℃，-18℃~-20℃
4.8 Sterile petri dish, OD 90 mm

5 Medium and reagent
5.1 LB liquid medium: Details see A1
5.2 Plate counting AGAR medium: Details see A2

6 Test procedure
See block diagram shown below:
[[File:Example.jpg]]

7 Detailed test procedure
7.1 Develop the standard growth curves for the total number of colonies
7.1.1 To begin the experiment, a 100 μL aliquot of a specific mixed bacterial flora solution stored at -20℃ should be taken and inoculated into 4 mL of LB liquid medium. The culture should then be incubated overnight at 36℃ with shaking at 180 r/min.
7.1.2 The concentration of the activated bacterial solution will be determined by employing the plate counting method described in GB 4789.2-2016.
7.1.3 Following quantification of the bacterial solution, different concentration gradients will be established for the contamination of 1 mL of ice cream solution. Specifically, the bacterial concentration of the contaminated ice cream solution will be set at 1.0x10^7, 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL. Subsequently, 1 mL of each contaminated ice cream solution will be mixed uniformly with 9 mL of LB liquid medium to generate standard growth curves with known bacterial concentrations of 1.0×10^6, 1.0×10^5, 1.0×10^4, 1.0×10^3, 1.0×10^2, 1.0×10^1, and 0 CFU/mL.
7.2 Procedure for samples to be examined
7.2.1 To begin the analysis, an appropriate amount of the ice cream sample will be obtained using a random sampling method. The representative samples will be collected using aseptic techniques and placed in sterilized containers to allow for natural melting. In cases where the ice cream is packaged, the opening of the packaging will be swabbed with 75% ethanol before sampling to ensure sterility.
7.2.2 A 1 mL aliquot of the ice cream solution will be transferred using a pipette and added to 9 mL of LB liquid medium. The solution will then be thoroughly mixed to prepare a bacterial solution containing 10% LB medium from the ice cream sample for analysis using the designated equipment.
7.3 Negative control sample
A 1 mL aliquot of sterile ice cream solution will be transferred and added to 9 mL of LB liquid medium. The mixture will be thoroughly mixed to prepare a negative control sample for analysis.
7.4 Culture the colonies
The prepared standard curve samples and the ice cream samples to be tested will be loaded into separate 2 mL test tubes. Each concentration level will have three reference samples. These test tubes will be placed into the designated equipment and incubated at 36°C for 12 hours. Conductivity measurements will be taken at 60-second intervals to obtain the necessary data.
7.5 Data processing
The data obtained from the microbial growth sensor, including electrical conductivity and culture time (T, in minutes), will be used to plot the microbial growth curve. By analyzing the relationship between the concentration of the standard bacterial solution and its corresponding detection time, a quantitative working function C bacteria (CFU/mL) = mT (min) + n will be obtained. The detection time (T) of the ice cream sample will be substituted into this function to obtain the total number of bacterial colonies present in the sample. The resulting number will be multiplied by 10 to determine the total number of bacterial colonies in the ice cream sample.
7.6 Results
7.6.1 If the total number of bacterial colonies in the samples obtained during session 7.5 is less than 100, the data shall be reported with two significant figures.
7.6.2 When reporting the total number of colonies of the samples obtained from session 7.5, the appropriate significant figures should be used based on the magnitude of the number. If the total number of colonies is less than 100, a two-digit significant number should be used. However, if the total number of colonies is greater than 100, the third digit should be rounded off and replaced by a zero. Alternatively, the number can be expressed in exponential form as a power of 10.
7.7 Test report
The test report should indicate:
- All the information necessary for the complete identification of the sample;
- Sampling method adopted, if any;
- All operational details not specified in this standard and some factors that may have affected the test results;
- The obtained result, indicating the method of representation adopted;
- Final results cited if repeated checks have been made;
- Quantitative work functions should be established separately for each type and specification of ice cream products;
- The working function needs to be validated by plate counting every six months.
8 Standard specification
This standard was completed on 6th September 2021.
Appendix A
A1 LB liquid medium
A1.1 Components
Yeast Extract 5g
Tryptone 10 g
Sodium chloride 10 g
Distilled Water 1000 mL
A1.2 Preparation
To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.
A2 Plate count agar
A2.1 Components
Tryptone 5.0g
Yeast Extract 2.5g
Glucose 1.0g
June Fat 15.0g
Distilled Water 1000 mL
A2.2 Preparation
To prepare the solution, the specified ingredients should be added to distilled water, and then the mixture should be boiled and stirred until fully dissolved. The pH of the solution should be adjusted to 7.0±0.2 using an appropriate buffer solution. Next, the solution should be divided into either test tubes or conical bottles, and sterilized via autoclaving at 121 ℃ for a duration of 15 minutes.

Application Notes

2023-02-17T05:13:10Z

Nick: /* C4D Conductivity Detector */

== Electrochemistry ==

'''Application Notes'''

* [[How to Choose a Potentiostat]]
* [[Cleaning and Polishing Voltammetric Electrodes]]
* [[Reference Electrode Potentials]]
* [[Plotting the IV Curve of a Solar Cell in EChem Software]]
* [[Analysis of Antioxidants using Cyclic Voltammetry]]
* [[Connecting 3rd Party Potentiostats to the e-corder]]
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Specifications of Ion Selective Electrodes]]
* [[Troubleshooting Issues with the Potentiostat]]

'''Electrochemistry Techniques'''

* [[Software for Electrochemistry Techniques]]
* [[Cyclic Voltammetry: Hints and Tips]]
* [[Anodic Stripping Voltammetry: Hints and Tips]]
* [[Potentiometric Stripping Analysis (PSA)]]

== Chromatography PowerChrom ==

* [[Shimadzu GCs and HPLCs with PowerChrom]]
* [[HP5890 GC with PowerChrom]]
* [[Waters HPLC with PowerChrom]]
* [[Ion Chromatographs with PowerChrom]]
* [[Running Multiple Recording Units with PowerChrom]]

== Data Recording in Chart ==

* [[Basics of Data Acquisition]]
* [[Exporting Chart Data]]
* [[Problems with Noise]]
* [[Multiple e-corders with Chart]]
* [[File:EAN004_Chart_–_A_Tutorial.pdf]] (1.4 MB PDF)
* [[Using the SRS QCM200 Quartz Crystal Microbalance with an e-corder]]
* [[Recording from Figaro Gas Sensors]]
* [[Jenway Spectrophotometer with the e-corder]]
* [[Recorder Comparative Specification – Comparing the Specifications of Various eDAQ Data Recording Hardware Units]]

== C4D Conductivity Detector ==

'''ER832 ACCS Bacterial Growth Analyser Application Notes'''
* [[Method for counting the total number of colonies in ice cream]]

'''Connecting to third-party capillary electrophoresis instruments'''

* [[Connecting the C4D to the Agilent 1600 CE]]
* [[Connecting the C4D to the Agilent 7100 CE]]
* [[Connecting the C4D to the Beckman Coulter CE]]
* [[Connecting the C4D to the PrinCE]]
* [[Connecting the C4D to the WynSep Wyn-CE]]

'''Technical Notes'''

* [[The eDAQ 150 micron C4D headstage]]
* [[C4D for Ion Chromatography with the ET125 Headstage]]
* [[Appropriate Frequency to use with Different Background Electrolytes and Capillary Inner Diameters]]
* [[The Three Classes of C4D Products: Detectors, Monitors and Meters]]

'''Procedures'''

* [[Procedure for Microchip Electrophoresis with C4D with the ET225 and ER455]]
* [[Procedure for Microchip Electrophoresis with C4D with the ET121]]
* [[Procedure for Capillary Electrophoresis with C4D]]
* [[Optimizing the C4D Settings using the C4D Profiler V2 Software]]
* obsolete: [[C4D Profiler in PowerChrom Software]]

'''Application Notes'''

* [[C4D Analysis of Acetylchorine Chloride on a Prince CE System]]
* [[Analysis of Inorganic Cations by CE-C4D]]
* [[Anions by MCE-C4D]]
* [[Electro-osmotic Flow Measurement in a Microfluidic Chip]]
* [[Simultaneous Analysis of Anions and Cations in Drinking Water by CE-C4D]]
* [[Measuring Total Dissolved Inorganic Carbon of Seawater using Flow Injection Analysis and C4D]]
* [[Measuring the Conductivity of Melt Water from Arctic Ice Cores]]
* [[Measuring the Conductivity of a Liquid in a Pipette Tip]]
* [[Monitoring Bacterial Growth using a Capacitively-Coupled Contactless Conductivity Detector (C4D)]]

== SDx Tethered Membranes ==

* [[Tethered Membranes]]
* [[Ionophores in Society]]
* [[SDx Protocol 1]]

== Technical Notes ==

* [[Signal Sources and Amplifiers]]
* [[Using an e-corder over the Internet]]
* [[Troubleshooting Communication between Hardware and Computer, Hardware Unavailable, Cannot Find a Hardware Unit]]
* [[Troubleshooting Connecting Front-End Amplifiers to the e-corder]]