Identification of indicator bacteria for sterilization

- Apr 09, 2020-

Method principle


The sterilized waste water is filtered through a filter with a pore size of 0.45 μm, bacteria and spores are trapped on the filter, and the filter is placed on a tyrosine-selective medium and cultured at 37 ° C for 72 h to 96 h The tyrosinase produced by the black variant of Bacillus subtilis can decompose and use tyrosine to form melanin, and produce black characteristic colonies. The characteristic colonies were further identified by staining microscopy and biochemical tests. The black variant of Bacillus subtilis was Gram-positive Bacillus, vegetative cells were rod-shaped, arranged individually or in short chains, the spores were oval, mesozoic, and did not swell. Can use glycerin, mannose, reduce nitrate, hydrolyze starch, can not use amygdalin, mannitol. A black variant of Bacillus subtilis was detected or not detected according to the results report.

Instruments and equipment


Sampling bottle: 500 ml wide mouth glass bottle with screw cap or ground stopper.


Constant temperature incubator: Allowable temperature deviation 36 ℃ ± 1 ℃.


High-pressure steam sterilizer: 121 ° C adjustable.


pH meter: accurate to 0.1 pH units, or more accurate. You can also use a pH cuvette or pH precision test paper.


Microscope: objective lens 10 ×, 20 ×, 40 ×, oil lens 100 ×, eyepiece 10 × or 15 ×.


Analytical balance: Sensitivity 0.0001 g. Petri dish: 90 mm in diameter.


Filtering device: equipped with sand core filter and vacuum pump, the filtration pressure should not exceed -50kPa.


Inoculation loop and forceps.


Pipette: 1 ml ± 0.01 ml, 10 ml ± 0.1 ml. You can also use a metered adjustable pipette.


Measuring cylinder: 100 ml. Erlenmeyer flask: 100 ml. Alcohol lamp.


Aseptic operation equipment: sterile room or ultra-clean workbench, biological safety cabinet.


Note: Pipettes, sampling bottles, petri dishes, triangle bottles and other glassware should be bandaged according to the requirements of aseptic operation before the test. The high-pressure steam sterilizer (7.3) is sterilized at 121 ° C for 20 minutes, dried, and set aside.


Sample Collection


The point layout and sampling frequency shall be implemented in accordance with the relevant regulations of HJ / T 494 and HJ / T 91.


When collecting microbial samples, the sampling bottle (7.1) shall not be washed with samples, and the samples shall be collected in sterilized sampling bottles.


If a sample containing active chlorine is collected, add sodium thiosulfate solution (6.13) before sterilizing the sampling bottle to remove the bacteria's inhibitory effect of active chlorine (add 0.1 ml of sodium thiosulfate solution per 125 ml volume) ); If a sample with a high content of heavy metal ions is collected, add a solution of disodium ethylenediaminetetraacetate (6.14) before sterilizing the sampling bottle to eliminate interference (0.3 ml of ethylenediaminetetrade per 125 ml volume) Disodium acetate solution).


Note: 15.7 mg of sodium thiosulfate (6.11) can remove 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.


to cultivate


Remove the filter with sterilized tweezers and place it on the tyrosine agar medium (6.1). The filter should retain the bacteria facing up. The filter should be completely close to the medium. There should be no air bubbles between the two. Then use paraffin. Seal the petri dish with a quality parafilm (6.17), invert the petri dish, place it in a constant temperature incubator (7.2), and incubate at 36 ° C ± 1 ° C for 72 h to 96 h. After 96 h of culture, if no colonies grow on the medium, the test is terminated; if colonies grow on the medium, pick black characteristic colonies on a nutrient agar medium (6.2), and culture at 36 ° C ± 1 ° C for 24 hours. h, to be identified. At the same time, the tyrosine agar medium Petri dishes with picked colonies are stored at 2 ° C to 5 ° C for recheck if necessary.


Note: When the color of the colony cannot be judged, it can be streaked again and inoculated into tyrosine agar medium (6.1) and observed after cultivation; if the colony is gray or gray-black, it can be observed after prolonging the cultivation time appropriately, and the cultivation time should not exceed 7 days

Staining microscopy


Pick the suspicious colonies (9.3) and stain them with Gram staining solution (6.9) and spore staining solution (6.10) respectively. For detailed operation and observation of results, see Appendix B. If you use commercially available reagents, follow the instructions. Then microscopic examination. Left-Gram stain; right-spore stain. Figure 2 Microscopic examination of Bacillus subtilis black variant staining


Biochemical test


Pick characteristic colonies (9.3) and inoculate them into starch medium (6.3), nitrate medium (6.4), glycerol red broth medium (6.5), mannose biochemical medium (6.6), and mannitol biochemical culture (6.7) and amygdalin biochemical culture medium (6.8). For detailed operations and results observation, please refer to Appendix B. If a commercially available culture medium is used, follow the instructions.

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