SN/T 4656.7-2020Import and export textiles biosafety inspection method - Part 7: Pseudomonas aeruginosa (English PDF)

6.1 Weighing method

Open the submitted sample aseptically, cut the sample evenly with sterile scissors (4.1), and accurately weigh the cut out on the electronic balance (4.2) 25 g of the sample, cut into pieces, add it to a sterile homogenization bag (4.3) containing 225 mL of sterile diluent, and beat with a flapping homogenizer (4.4) for 1 min to 2 min, mix thoroughly to obtain a 1.10 sample homogenate.

6.2 Multi-point sampling elution method

Open the sample to be tested in a sterile method, arrange 5 sampling points evenly around and in the middle of the sample, and use a sterile specification plate (4.5) according to each the sampling points are cut according to the area of 4 cm×5 cm (20 cm2), and the sampling area of every 20 cm2 is 1 test sample, and a total of 5 samples are collected for each sample.

For sample inspection, the sampling area is 100 cm2.Put the 5 samples collected above into a sterile homogenization bag (4.3) containing.200 mL of sterile diluent, Use a flapping homogenizer (4.4) to beat for 1 min to 2 min, mix thoroughly, and use the sample homogenate as the stock solution.

6.3 Cotton swab smearing method

Aseptically open the sample to be tested, moisten a sterile dry cotton swab (4.6) with sterile diluent, and distribute the control evenly around and in the middle of the sample 5 sampling points, use a sterile specification plate (4.5) to smear evenly in the area of 20 cm2 for each sampling point, and use 1 for each sampling point Sterile dry cotton swab (4.6), spread evenly in the area of 4 cm×5 cm (20 cm2), immediately cut off the cotton with sterile scissors (4.1) Swab the hand contact part, put the smeared part into a sterile homogenization bag (4.3) containing 50 mL of sterile diluent and mix well to make 1.10 Sample homogenate.

6.4 Selection of sample preparation method

6.4.1 The sample preparation is generally based on method 6.1 as the reference method.

6.4.2 When the sample is larger or thicker or porous, method 6.2 should be used for sample preparation. When using method 6.2, if the sample is tested If the area is too large or too small, the sampling points can be increased or decreased proportionally.

6.4.3 When the sample has a dense texture and is not easy to cut and prepare; or the sample is expensive and the customer requires non-destructive testing, the sample preparation should be used in 6.3 method.

6.4.4 When using methods 6.1 and 6.2, if the test sample absorbs a large amount of water and cannot absorb enough sample for conversion, the diluent may be suitable.

When increased until enough to suck out the conversion.

7.1 Principle

Pseudomonas aeruginosa has acetamidase, which can use acetamide as a carbon source and decompose acetamide to produce alkali. Sample inoculated with acetamide After the base, the target bacteria can make the acetamide medium red; the combined Pseudomonas aeruginosa is oxidase-positive, can reduce nitrate to produce gas and in the characteristics of growth at 42 °C can accurately determine Pseudomonas aeruginosa.

7.2 Inspection procedure

7.3 Operation steps

7.3.1 Dilution of samples

Use a micropipette (4.7) to pipette 1 mL of the prepared sample homogenate, and slowly inject it into the sample containing 9 mL of sterile in the small triangular flask (4.8) (note that the tip of the tip does not touch the liquid surface), shake the small triangular flask or use a sterile tip to repeatedly pipette the mixture is evenly mixed to prepare a homogeneous sample solution of the next dilution. According to the above operating procedures, prepare a 10-fold serial dilution sample homogenate. Every pass Increase and dilute once, and replace with a sterile tip.

7.3.2 Sample inoculation

According to the actual contamination of the sample, select 2 to 3 suitable dilution sample homogenates (including the original solution), and each dilution is divided into Do not pipet 1 000 µL of sample homogenate to inoculate two dry acetamide agar (5.3) plates with 500 µL and 500 µL inoculum respectively.

Then use a sterile L-shaped glass holder (4.10) to evenly coat the entire plate, taking care not to touch the edge of the plate. If the sample liquid is not easy to absorb, you can the plate is placed in a constant temperature incubator (4.11) at 36 °C ± 1 °C for 1 h. After the sample is absorbed, the plate is turned over and placed upside down in the incubator.

Raise for 24 h ± 2 h.

7.3.3 Count of typical or suspicious colonies

Colony morphology of Pseudomonas aeruginosa on acetamide agar (5.3) plate. milky white flat colonies, moist, neat edges, colonies the surrounding medium turns red. Select the typical or suspicious Pseudomonas aeruginosa colonies, and the two plates with the same dilution are all typical or suspicious for the plates with the total number of suspected colonies in the range of 20 CFU to.200 CFU, count the number of typical or suspicious colonies.

7.3.4 Confirmation test

7.3.4.1 Overview

Pick at least 5 (all less than 5) typical or suspicious colonies from the counted acetamide agar (5.3) plate, and do the following confirmatory test.

7.3.4.2 Staining microscopic examination

Pick a single colony on the acetamide agar (5.3) plate, smear it, stain with Gram stain (5.4), and under the microscope (4.13) Observe its colony morphology. Pseudomonas aeruginosa is a gram-negative bacterium under the microscope. It is slender and varies in length, sometimes in the shape of a ball or thread.

Arranged in pairs or short chains.

7.3.4.3 Oxidase test

Take a piece of clean filter paper and place it in a sterile glass plate (4.9), use a micropipette (4.7) to suck up the oxidase reagent (5.5) and add it dropwise Put one drop on the filter paper, only make the filter paper wet, not too wet, use the inoculation loop (4.12) to pick a single colony on the acetamide agar (5.3) plate Spread it on the filter paper, at 10...