GB/T 7918.3-1987 English PDF (GBT7918.3-1987)

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GB/T 7918.3-1987: Standard methods of microbiological examination for cosmetics. Fecal coliforms
GB 7918.3-1987
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
UDC 668.58:576:85.07
GB 7918.3-87
Standard methods of microbiological examination for
cosmetics - Fecal coliforms
ISSUED ON: MAY 28, 1987
IMPLEMENTED ON: OCTOBER 01, 1987
Issued by: Ministry of Health of PRC
Table of Contents
1 Method summary ... 3
2 Culture media and reagents ... 3
3 Instruments ... 6
4 Operation steps ... 7
5 Test result report ... 7
Additional information ... 8
NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA
Standard methods of microbiological
examination for cosmetics -
Fecal coliforms
Fecal coliform bacteria originate from the feces of humans and warm-blooded animals.
The detection of fecal coliform bacteria indicates that the cosmetics have been
contaminated by feces and may contain other intestinal pathogens or parasites.
Therefore, fecal coliform bacteria are listed as important health indicator bacteria.
1 Method summary
According to the biological characteristics of fecal coliform bacteria, for example,
Gram-negative non-spore-forming bacteria can ferment lactose to produce acid and gas
when cultured at 44 °C for 24 ~ 48 h, it can produce typical colonies on selective culture
media, decompose tryptophan to produce indigo matrix.
2 Culture media and reagents
2.1 Lactose bile salt culture medium
Ingredients:
Peptone: 20 g
Porcine bile salt: 5 g
Lactose: 5 g
0.4% bromocresol purple aqueous solution: 2.5 ml
Distilled water: 1000 ml
Preparation method: Dissolve peptone, bile salt, lactose in distilled water. Adjust pH to
7.4. Add indicator. Mix well. Dispense into test tubes (add a small inverted tube to each
test tube). Sterilize it at 115 °C (10 lb) for 20 min.
2.2 Double concentration lactose bile salt medium
According to the above lactose bile salt medium composition, the amount of distilled
UDC 668.58:576.85.07
GB 7918.3-87
water remains unchanged, whilst the amount of other ingredients is doubled.
2.3 Eosin methylene blue (EMB) agar
Ingredients:
Peptone: 10 g
Lactose: 10 g
Dipotassium hydrogen phosphate: 2 g
Agar: 20 g
2% eosin aqueous solution: 20 ml
0.5% methylene blue aqueous solution: 13 ml
Distilled water: 1000 ml
Preparation method: First add agar to 900 ml distilled water. Heat to dissolve. Then add
dipotassium hydrogen phosphate peptone. Mix well. Dissolve it. Then make up to 1000
ml with distilled water. Correct the pH value to 7.2 ~ 7.4. Dispense it into flasks.
Sterilize it at 121 °C (15 lb) for 15 min under high pressure for use. Add lactose and
heat to melt agar before use. Cool to about 60 °C. Add sterilized eosin-methylene blue
solution aseptically. Shake well. Pour into a plate for later use.
2.4 Peptone water (for indigo matrix test)
Ingredients:
Peptone (or tryptone): 20 g
Sodium chloride: 5 g
Distilled water: 1000 ml
Preparation method: Heat and melt the above ingredients. Adjust the pH value to 7.0 ~
7.2. Divide into small test tubes. Sterilize at 121 °C (15 lb) for 15 min.
2.5 Indigo matrix reagent
Covank reagent: Dissolve 5 g of p-dimethylaminobenzaldehyde in 75 ml of amyl
alcohol. Then slowly add 25 ml of concentrated hydrochloric acid.
Test method: Inoculate bacteria in peptone water and culture at 44 °C for 24 h. Add 0.3
~ 0.5 ml of Covank reagent along the tube wall. Shake the test tube gently. Positive
ones show deep rose red in the reagent layer.
2.6.2.1 Fix the smear on the flame. Add crystal violet staining solution. Stain for 1 min.
Use water to wash it.
2.6.2.2 Add Gram iodine solution. Let it action for 1 min. Use water to wash it.
2.6.2.3 Add 95% alcohol to decolorize for about 30 seconds. Or add alcohol to the entire
smear. Immediately pour it off. Then add alcohol to the entire smear. Decolorize for 10
seconds. Use water to wash it.
2.6.2.4 Add counterstain solution dropwise. Counterstain for 1 minute. Use water to
wash it. Wait to dry. Examine under a microscope.
2.6.3 Staining results
Gram-positive bacteria are purple, whilst Gram-negative bacteria are red.
Note: If a 1:10 dilute carbolic acid fuchsin stain solution is used as the counterstain solution,
the counterstaining time only takes 10 seconds.
3 Instruments
3.1 Constant temperature water bath or water-proof constant temperature box: 44 °C.
3.2 Thermometer.
3.3 Microscope.
3.4 Glass slide.
3.5 Inoculation loop.
3.6 Electric furnace.
3.7 Conical flask.
3.8 Test tube.
3.9 Small inverted tube.
3.10 pH meter or pH test paper.
3.11 High pressure sterilizer.
3.12 Sterilized pipette.
3.13 Sterilized plate.