1 Scope
This standard specifies the method for examination of staphylococcus aureus in foods.
In this standard, Method I is applicable to qualitative examination of staphylococcus aureus in foods, Method II to enumeration of staphylococcus aureus in foods containing more staphylococcus aureus and Method III to enumeration of staphylococcus aureus in foods containing fewer staphylococcus aureus.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Constant temperature incubator: 36℃±1℃.
2.2 Refrigerator: 2℃~5℃.
2.3 Thermostatic water bath: 36℃~56℃.
2.4 Balance: with sensibility of 0.1g.
2.5 Homogenizer.
2.6 Oscillator.
2.7 Aseptic pipette: 1mL (with scale division of 0.01mL), 10mL (with scale division of 0.1mL) or micropipettor and pipette tips.
2.8 Aseptic conical flask: 100mL and 500mL.
2.9 Aseptic culture dish: 90mm in diameter.
2.10 Coating rod.
2.11 pH meter or pH colorimetric tube or precise pH paper.
3 Culture Media and Reagents
3.1 7.5% sodium chloride broth: see A.1.
3.2 Blood agar plate: see A.2.
3.3 Baird-Parker agar plate: see A.3.
3.4 Brain heart infusion broth (BHI): see A.4.
3.5 Rabbit plasma: see A.5.
3.6 Diluent: phosphate buffer solution: see A.6.
3.7 Nutrient agar small slant: see A.7.
3.8 Gram stain solution: see A.8.
3.9 Aseptic normal saline: see A.9.
Method I Qualitative Examination of Staphylococcus Aureus
4 Examination Procedures
See Figure 1 for the qualitative examination procedures of staphylococcus aureus.
Figure 1 Examination Procedures of Staphylococcus Aureus
5 Operation Steps
5.1 Treatment of sample
Weigh 25g of sample, put into an aseptic homogenizing cup containing 225mL of 7.5% sodium chloride broth, homogenize at 8 000r/min~10 000r/min for 1min~2min, or put into an aseptic homogenizing bag containing 225mL of 7.5% sodium chloride broth, flap with a slap type homogenizer for 1min~2min. For liquid sample, pipet 25mL of sample into an aseptic conical flask containing 225mL of 7.5% sodium chloride broth (proper amount of aseptic glass beads may be put into the flask in advance), shake and mix well.
5.2 Enrichment
Cultivate the above sample homogeneous solution at 36℃±1℃ for 18h~24h. The staphylococcus aureus shows turbid growth in 7.5% sodium chloride broth.
5.3 Isolation
Subject the enriched cultures to streak inoculation on Baird-Parker agar plate and blood agar plate respectively, cultivate at 36℃±1℃ for 18h~24h for blood agar plate and 24h~48h for Baird-Parker agar plate.
5.4 Preliminary identification
The staphylococcus aureus on Baird-Parker agar plate are round, with smooth, salient, and humid surface, 2mm~3mm in diameter, gray-black to black in color and lustrous with light-colored (non-white) border and surrounded by turbid ring (precipitation) accompanied with a distinct zone outside. When contacting the colony with inoculating needle, it is viscous like butter. Non-fat soluble strains may be observed sometimes, which are almost the same in appearance except turbid ring and distinct zone. Compared with typical colony, the colony isolated from long-kept frozen or dehydrated foods are generally light black with more rough appearance and drier texture. The colonies forming on the blood agar plate are relatively large, round, smooth, salient, humid and golden yellow (sometimes white), surrounded by visible and completely transparent hemolytic circle. The above-mentioned suspicious colonies are selected for Gram stain microscopic examination and plasma coagulase test.
5.5 Confirmatory identification
5.5.1 Staining microscopic examination: staphylococcus aureus are gram-positive coccus, arranged in grape shape, without spores and capsules and with diameter of 0.5μm~1μm.
5.5.2 Plasma coagulase test: select five suspicious colonies at least (select all if less than five) from Baird-Parker agar plate or blood agar plate, inoculate into 5mL BHI and nutrient agar small slant respectively, cultivate at 36℃±1℃ for 18h~24h.
Take 0.5mL of fresh-prepared rabbit plasma into a small test tube, add 0.2mL~0.3mL of BHI culture, oscillate and shake well, put the tube into incubator or water bath at 36℃±1℃, observe every half an hour within 6h. If coagulation occurs (clots when the tube is tilted or inverted) or coagulation volume is greater than 50% of the original volume, it is judged as positive. Meanwhile, broth culture for plasma coagulase test containing both positive and negative staphylococcus strains are used as control. Commercial reagent may also be used for plasma coagulase test according to the instructions.
If the result is suspicious, select colonies from nutrient agar small slant into 5mL of BHI, cultivate at 36℃±1℃ for 18h~48h and repeat the test.
5.6 Examination of staphylococcal enterotoxin (optional)
To identify suspicious food poisoning sample or staphylococcus aureus strains generating staphylococcal enterotoxin, the staphylococcal enterotoxin shall be detected according to Appendix B.
6 Result and Report
6.1 Result judgment: it is judged as staphylococcus aureus if complying with 5.4 and 5.5.
6.2 Result report: whether staphylococcus aureus is detected in 25g(mL) sample or not.
Method II Plate Counting Method of Staphylococcus Aureus
7 Examination Procedures
See Figure 2 for the examination procedures of staphylococcus aureus by plate counting method.
Figure 2 Examination Procedures of Staphylococcus Aureus by Plate Counting Method
8 Operation Steps
8.1 Dilution of sample
8.1.1 Solid and semi-solid sample: weight 25g of sample, put in an aseptic homogenizing cup containing 225mL of phosphate buffer solution or normal saline, homogenize at 8 000r/min~10 000r/min for 1min~2min, or put in an aseptic homogenizing bag containing 225mL of diluent, slap with a slap type homogenizer for 1min~2min, to prepare the 1:10 homogeneous sample solution.
8.1.2 Liquid sample: use aseptic pipette to pipet 25mL of sample into an aseptic conical flask containing 225mL of phosphate buffer solution or normal saline (proper amount of aseptic glass beads are put into the flask in advance), mix well to prepare the 1:10 homogeneous sample solution.
8.1.3 Use 1mL aseptic pipette or micropipettor to pipet 1mL of 1:10 homogeneous sample solution, slowly inject it into an aseptic test tube containing 9mL of phosphate buffer solution or normal saline along the tube wall (the pipette or pipette tip shall not touch the diluent), shake the tube or repeatedly insufflate and flap with a 1mL aseptic pipette, mix well to prepare the 1:100 homogeneous sample solution.
8.1.4 Prepare decimal serial dilutions of homogeneous sample solution according to the operation procedures of 8.1.3, change a new 1mL aseptic pipette or pipette tip once per serial dilution.
Contents
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Media and Reagents
Method I Qualitative Examination of Staphylococcus Aureus
4 Examination Procedures
5 Operation Steps
6 Result and Report
Method II Plate Counting Method of Staphylococcus Aureus
7 Examination Procedures
8 Operation Steps
9 Calculation of Result
10 Report
Method III MPN Enumeration of Staphylococcus Aureus
11 Examination Procedures
12 Operation Steps
13 Result and Report
Appendix A Culture Media and Reagents
Appendix B Examination of Staphylococcal Enterotoxin
Appendix C MPN Retrieval Table of Staphylococcus Aureus
