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

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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:

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.


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.