HJ 1000-2018Water quality - Determination of total bacteria - Plate count method (English PDF)

6.5 Sodium thiosulfate solution. (Na2S2O3) =0.10 g/ml

Weigh 15.7 g of sodium thiosulfate (6.3), dissolve in an appropriate amount of water, and dilute to 100 ml.

6.6 Ethylenediaminetetraacetic acid disodium solution. (C10H14N2O8Na2·2H2O) =0.15 g/ml

Weigh 15 g of disodium edetate (6.4), dissolve in an appropriate amount of water, and dilute to 100 ml. This solution can be stored for 30 days.

6.7 Glass beads. 3 to 8 mm in diameter.

8.1 Sample Collection

The point layout and sampling frequency are carried out in accordance with the relevant provisions of GB/T 14581, HJ/T 494 and HJ/T 91.

When collecting microbial samples, the sampling bottle (7.1) should not be washed with the sample, and the sample should be collected in a sterile sampling bottle. 3 when collecting surface water samples such as rivers and lake banks, you can directly insert the bottle with the plug into the water by holding the lower part of the bottle, about water 10 to 15 cm, the bottle mouth is in the direction of water flow, pull the stopper, pour the sample into the bottle and close the stopper, and take the sample bottle from the water.

Take out. If there is no water flow, hold the bottle horizontally and push forward. The sampling amount is generally about 80% of the sample bottle capacity.

After the sample is collected, quickly insert the aseptic packaging paper.

When collecting samples from the faucet device, do not use the leaking faucet. Open the faucet to the maximum before collecting water, and drain the water for 3-5 minutes.

Then turn off the faucet, sterilize with a flame for about 3 minutes, or disinfect the faucet with 70% to 75% alcohol.

Open the faucet and let the water drain for 1 min to fully remove the retained impurities in the water pipe. Control the water flow rate during sampling and carefully connect to the bottle.

When collecting surface water, wastewater samples, and samples of a certain depth, it can also be sampled using a sterilized dedicated sampling device.

When stratified sampling is performed at the same sampling point, it should be performed from top to bottom to avoid different levels of disturbance.

If a sample containing active chlorine is collected, add sodium thiosulfate solution (6.5) before sterilization of the sample bottle to remove Inhibition of bacteria by deactivated chlorine (0.1 ml of sodium thiosulfate solution per 125 ml volume); if heavy is collected for samples with higher metal ion content, add disodium edetate solution (6.6) before sterilization of the sample bottle.

To eliminate interference (add 0.3 ml of disodium edetate solution per 125 ml volume).

Note. 15.7 mg sodium thiosulfate (6.3) removes 1.5 mg of active chlorine from the sample. The amount of sodium thiosulfate can be adjusted according to the actual amount of active chlorine in the sample.

8.2 Sample storage

It should be tested within 2 h after sampling. Otherwise, it should be refrigerated below 10 °C but not more than 6 h. After the laboratory is sampled, it cannot for immediate testing, samples should be refrigerated below 4 °C and tested within 2 h.

9.1 Sample dilution

The sample was shaken vigorously 20 to 25 times to disperse the possible bacterial coagulation. Determine dilution based on sample contamination multiple. Pipette 10 ml of the thoroughly mixed sample aseptically and inject a triangular burn containing 90 ml of sterile water (6.2) in the bottle (with appropriate amount of glass beads), mix to a 1.10 dilution sample. Draw a 1.10 dilution of the sample 10 ml into the well Mix 90 ml of sterile water into a conical flask and mix to a 1.100 dilution sample. Dilute to 1.1000, 1.10000 in the same way Release the sample. At least 3 suitable concentrations should be diluted for each sample.

Note. When pipetting different concentrations of diluent, the pipette must be replaced each time.

9.2 Vaccination

Pipette a well-mixed sample or dilute 1 ml of the sample in a sterile operation with a 1 ml sterile pipette.

In a dish, pour 15 to 20 ml of nutrient agar medium (6.1) cooled to 44 to 47 ° C, and immediately shake the plate to make a sample.

Mix the product or diluted sample with the medium. Pour 2 dishes for each sample or diluted sample.

9.3 Cultivation

After the nutrient agar medium in the plate is cooled and solidified, the plate is turned over to make the bottom surface upward (to avoid the uniform growth of bacteria due to surface moisture condensation).

The results were observed in a constant temperature incubator (7.3) for 48 h ± 2 h at 36 °C ± 1 °C.

9.4 Blank test

The laboratory blank is determined by using sterile water. There shall be no colony growth on the plate after cultivation. Otherwise, the sample is determined.

Invalid, should be re-measured after the cause is identified.

10.1 Interpretation of results

When the plate has larger flaky colonies and exceeds half of the plate, the plate does not participate in counting.

When the flaky colonies are less than half of the plate, and the remaining half of the colonies are evenly distributed, the colonies with uniform distribution are counted.

Multiply by 2 to represent the total number of colonies.

Appearance (morphology or color) similar, colonies that are similar but not touching, as long as the distance between them is not less than the minimum the diameter of the colony is counted. Colonies that are in close contact and have different appearances are counted.

10.2 Calculation of results

Multiply the dilution number by the total number or average of each plate colony (the average number of two replicate plates of the same dilution) to calculate the total number of bacteria in a 1 ml sample. The calculation of various situations is as follows.

Preference is given to counting the average number of colonies between 30 and 300, when there is only one dilution factor of the average colony when the number satisfies this range, the average number of colonies multiplied by the dilution factor is the measured value of the total number of bacteria (see Table 1 Example 1).

If there are two dilution factors, the average number of colonies is between 30 and 300, calculate the ratio of the two (multiplely multiplied by their dilution) After a multiple, the ratio of the larger value to the smaller value). If the ratio is less than 2, the average of the two is the measured value of the total number of bacteria;

When the ratio is greater than or equal to 2, the total number of colonies with a small dilution factor is the total number of bacteria (see Table 1, Example 2, Example 3, and Example 4).

If the average number of colonies of all dilution factors is greater than 300, multiply the dilution number by the average number of colonies with the largest dilution factor.

The number is the total number of bacteria measured (see Table 5, Example 5).

If the average number of colonies in all dilutions is less than 30, multiply the dilution number by the average number of colonies with the smallest dilution factor.

The number is the total number of bacteria measured (see Table 1 Example 6).

If the average number of colonies in all dilutions is not between 30 and 300, the average colony is the closest to 300 or 30.

The number is multiplied by the dilution factor to determine the total number of bacteria (see Table 1 Example 7).

Table 1 Dilution multiple selection and total number of colonies

10.3 Results Representation

The measurement result is retained to the whole number, and at most two significant digits are retained. When the measurement result is >=100 CFU/ml, refer to the method of counting; if the undiluted stock solution is aseptically grown on the plate, use the "not detected" or "