1 Scope
This standard specifies the determination method for antibacterial activity of microbe-source enzyme.
This standard is applicable to the determination for antibacterial activity of enzyme produced with microbe.
2 Apparatus and Materials
In addition to the conventional sterilization and cultivation apparatus in microbiological laboratory, other apparatus and materials are as follows:
2.1 Biosafety cabinet.
2.2 Refrigerator: 2~5℃.
2.3 Thermostatic incubator: 36±1℃.
2.4 Thermostatic water bath: 46±1℃.
2.5 Balance: with sensibility of 0.1g.
2.6 Oscillator.
2.7 Aseptic suction tube: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and suction head.
2.8 Aseptic culture dish: with diameter of 90mm.
2.9 Aseptic conical flask : with volume of 250mL and 500mL.
2.10 pH meter or precise pH paper.
2.11 Aseptic paper: see A.8.
2.12 Aseptic forceps.
2.13 Vernier caliper: with scale of 0.1mm.
3 Culture Medium and Reagents
3.1 Tryptone Soy Agar (TSA): see A.1.
3.2 Tryptone Soy Broth (TSB): see A.2.
3.3 Plate Count Agar: see A.3.
3.4 0.1mol/L HCl: see A.4.
3.5 Aseptic normal saline: see A.5.
3.6 50.0μg/mL Ciprofloxacin (CIP) solution: see A.6.
3.7 5.0μg/piece ciprofloxacin paper: see A.7.
4 Test Strains
4.1 Staphylococcus aureus ATCC 6538.
4.2 Escherichia coli ATCC 11229.
4.3 Bacillus cereus ATCC 2.
4.4 Bacillus circulans ATCC 4516.
4.5 Streptococcus pyogenes ATCC 12344.
4.6 Serratia marcescens ATCC 14041.
5 Inspection Procedures
See Figure 1 for the determination and inspection procedures for antibacterial activity of microbe-source enzyme.
Figure 1 Determination and Inspection Procedures for Antibacterial Activity of Microbe-source Enzyme
6 Operation Steps
6.1 Preparation of stock solution of test bacterial suspension
Put the cryopreserved strains of staphylococcus aureus (ATCC 6538), escherichia coli (ATCC 11229), bacillus cereus (ATCC 2), bacillus circulans (ATCC 4516), streptococcus pyogenes (ATCC 12344) and serratia marcescens (ATCC 14041) into test tubes with 5mL TSB respectively for inoculation, culture them at 36±1℃ for 18~24h for the first subculture, respectively pick up 1~2 loops of culture solution for the first passage to subcultivate in 5mL TSB broth, and culture them at 36±1℃ for 18~24h for the secondary subculture and serve as stock solution of test bacterial suspension for standby.
Purity detection for stock solution of test bacterial suspension: respectively pick up one loop of stock solutions of test bacterial suspensions for streak inoculation in TSA plate, culture at 36±1℃ for 20~24h, and observe the purity.
6.2 Concentration determination for stock solution of bacterial suspension
6.2.1 Dilution for stock solution of bacterial suspension
Respectively take out the stock solutions of bacterial suspension prepared in 6.1 for decimal dilution in sequence, bacillus cereus and bacillus circulans are respectively made into 10-4 diluents, while staphylococcus aureus, escherichia coli, streptococcus pyogenes and serratia marcescens are respectively made into 10-5 diluents.
6.2.2 Culture count
Pipet 1mL bacteria diluents prepared in 6.2.1 respectively and put them into aseptic petri dish, carry out two parallel tests for each dilution and adopt aseptic normal saline for blank control. Pour 15~20mL plate count agar, cooling to about 46℃ (may be put into 46±1℃ thermostatic water bath for thermal insulation), into petri dish, rotate the petri dish to mix uniformly. Turn over the plate after agar is solidified, culture it at 36±1℃ for 24h and count, each stock solution of bacterial suspension may be used only when all the bacteria concentrations are greater than 106CFU/mL.
6.3 Preparation of detection plate
Pour 15mL TSA culture medium, melting and cooling to 46±1℃, into aseptic culture dish to make TSA plate for standby after solidification.
Dilute the bacteria cultures after secondary passage in 6.1 with TSA culture medium cooling to 46±1℃ in the proportion of 1:10 (among which, dilute streptococcus pyogenes ATCC 12344 in the proportion of 1:20), after mixing uniformly and sufficiently, take out 10mL to the above-mentioned prepared TSA plate thermal-insulated at 46±1℃, slightly shake the plate to spread out the bacterium solution uniformly, then it is made into detection plate after solidification for standby.
Note: to prevent the culture medium containing bacteria from solidification upon encountering TSA, thus causing unevenness during bacterial suspension spreading, the TSA plate shall be placed at 46±1℃ for thermal insulation and balance for 10min prior to spreading bacterial suspension.
6.4 Preparation of enzyme paper
Accurately weigh (transfer) 1.0g(mL) enzyme and put it into 9mL aseptic normal saline, make into 10% enzyme solution after mixing uniformly and sufficiently. Place all aseptic paper into aseptic petri dish, slowly add 100μL 10% enzyme solution at each paper by drops for complete absorption, prepare 12 pieces of paper for each enzyme sample.
6.5 Paper sticking and culture
Place the paper containing to-be-tested enzyme, positive control paper containing ciprofloxacin (see A.7) and blank negative control paper containing 100μL aseptic normal saline prepared in 6.4 at the detection plates prepared in 6.3 with aseptic forceps respectively, stick 2 pieces scrap at each plate (2 pieces of paper containing to-be-tested enzyme or 2 pieces of paper containing ciprofloxacin or 2 pieces of blank paper), the spacing between the dots of two pieces of paper is greater than 32mm, and each standard strain is provided with one positive control and one negative control plate respectively. Place the plates at 4±0.5℃ refrigerator overnight (12~16h). Transfer to 36±1℃ thermostatic incubator for culture for 24h the next day, then determine the inhibition zone formed by enzyme sample, ciprofloxacin and negative control paper.
7 Antibacterial Activity Result Report
7.1 Result judgment
If the to-be-tested enzyme presents antibacterial activity for 3 or above microbes under test, namely apparent inhibition zone (with diameter ≥16mm for each one) appears around paper, and the inhibition zones of the positive control ciprofloxacin paper formed to staphylococcus aureus, bacillus cereus, serratia marcescens, bacillus circulans and streptococcus pyogenes are greater than 16mm in diameter, it is judged that antibacterial active substance exists in such enzyme sample.
7.2 Result report
Antibacterial activity is detected or not in sample.
1 Scope
2 Apparatus and Materials
3 Culture Medium and Reagents
4 Test Strains
5 Inspection Procedures
Annex A Culture Medium and Reagents
