B1 ? Product collection and sample processing
The same batch number of two transport packaging at least 12 assorted sales package samples, 1/4 sample for testing, 1/4 sample for retention, the other 1/2 sample (can be sealed) for re-examination, if necessary. Sampling of the assorted sales package should not be cracked, not to be opened before the test.
Open at least 3 packages for testing under class 100 cleanup aseptically, take a sample from each package, and accurately weigh 10g ± 1g of sample. Cut and add to 200ml of sterilized saline, mix thoroughly to get a saline sample. Liquid products use the original liquid directly as a sample solution.
If the sample contains a lot of water-absorbent resin material and can not be sucked out enough sample solution, the amount of dilution can be increased by 50ml each time, until it can be sucked out enough sample solution for testing. Adjust the dilution accordingly when calculating the total number of bacterial colonies and fungal colonies.
B2 ? Bacterial colony count and initial contaminants
This method is applicable to the detection of initial contaminants and bacterial colony count (hereinafter collectively referred to as bacterial colony count).
B2.1 Operation Procedure
After the above saline sample solution settles naturally, take the supernatant for colony counting. *** Inoculate 5 dishes, add 1ml of sample solution to each dish, and then pour 15-20ml of melted nutrient agar medium cooled to about 45℃ into each dish and mix well. After the agar solidified, turn the petri dish over and incubate at 35℃±2℃ for 48h, then calculate the number of colonies on the plate.
B2.2 Reporting results
The plate with flaky growth of colonies is not suitable for use; count the colonies on the plate that meets the requirements, according to the formula (B1) to calculate the results:
X1 = A × (K ÷ 5)
In the formula, X1 - the total number of bacterial colonies cfu/g or cfu/mL;
The total number of bacterial colonies cfu/g or cfu/mL. cfu/mL;
A - total number of bacterial colonies on 5 plates of nutrient agar medium;
K - dilution.
When the number of colonies is less than 100, it is reported as the actual number, and when it is more than 100, two significant digits are used.
If the total number of colonies of the sample exceeds the provisions of this standard, according to B2.3 for retesting and reporting of results.
B2.3 retest method
The retention of the retest samples in accordance with the previous method of retesting 2 times, the average of the 2 results are up to the provisions of this standard, it is determined that the samples examined are qualified; of which there are any 1 results of the average exceeds the provisions of this standard, the samples examined are determined to be unqualified.
B3 ? Coliform detection method
B3.1 procedure
Take a sample of 5ml inoculation 50ml, lactose bile salt fermentation tube, placed in 35 ℃ ± 2 ℃ incubation for 24h, such as no acid production and gas production, it is reported as coliform negative.
If acid production and gas production, then the line inoculated with erythromelane agar plate, set 35 ℃ ± 2 ℃ culture 18 ~ 24h, observe the morphology of colonies on the plate. Typical coliform colonies are black or reddish purple, rounded, with neat edges, smooth and moist surface, often with a metallic luster, but also purple-black, without or with a slight metallic luster, or pink, the center of the deeper colonies.
Take the suspected colonies 1-2 for Gram staining microscopy, while inoculated with lactose intoxication tubes, placed in 35 ℃ ± 2 ℃ culture for 24h, observe the gas production.
B3.2 Results report
Where the lactose bile salt fermentation bone acid production and gas production, lactose fermentation tube acid production and gas production, in the Erythromycin blue plate has a typical coliform colonies, Gram staining for the negative non-germinative bacilli, can be reported to the sample examined detected E. coli.
B4 ? Pseudomonas aeruginosa detection method
B4.1 operating procedures
Take 5ml of sample solution, add to 50ml SCDLP culture medium, mix well, set 35 ℃ ± 2 ℃ culture 18 ~ 24h. If Pseudomonas aeruginosa grows, the culture of the agrofacial surface of the culture medium presents a thin film of bacteria, the culture medium is often yellow-green or blue-green. Pick the culture from the thin film of the culture fluid, line inoculation hexadecane trimethylamine bromide agar plate, set at 35 ℃ ± 2 ℃ culture 18 ~ 24h, observe the characteristics of the colony. Pseudomonas aeruginosa grows well on this medium, the colony is flat, the edge is not neat, the medium around the colony is slightly pink, other bacteria do not grow.
Take the suspect colony smear for gram staining, microscopic examination of gram-negative bacteria should be the following tests:
Oxidase test: take a small piece of clean white filter paper in a sterilized dish, with a sterile glass rod to pick the suspect colony coated in the filter paper, and then add a drop of newly formulated 1% dimethyl p-phenylenediamine test solution on its top, within 30s there is a pink or purple red, as a positive oxidase test. oxidase test is positive, and those that do not change color are negative.
Pyrethrin test: take 2-3 suspected colonies, respectively inoculated in the pyrethrin determination medium slant, 35 ℃ ± 2 ℃ culture 24h, add ?3 ~ 5 ml, sufficiently oscillate to make the culture of pyrethrin may be present in the dissolution of the pyrethrin, to wait for the trichlorethylene-methane was blue, with a pipette to another test tube and add 1 mol / L hydrochloric acid 1mL, oscillation, and then leave it for a few moments. Shake and let stand for a few moments. If the upper layer appears pink or purple-red is positive, indicating the presence of chlorpyrifos.