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Translation - Chinese Chapter 5
I General principles for microbiology test
1 Scope
This standard gives basic requirements for determination of microbiological in cosmetics.
This standard is applicable to the collection, conservation and preparation of cosmetic samples to be
tested.
2 Instrument & Equipment
2.1 Balance, 0-200g in precision of 0.1g
2.2 Autoclave sterilizer.
2.3 Oscillator.
2.4 Conical flask, 250ml and 150ml.
2.5 Glass beads.
2.6 Glass rod.
2.7 Sterile graduated pipette, 10ml and 1ml.
2.8 Thermostatic water bath.
2.9 Homogenizer or mortar.
2.10 Sterile homogeneous bags.
3 Culture medium and Reagents
3.1 Saline
Compositions: Sodium chloride 8.5g
Distilled water up to 1000ml
Preparation: After dissolution and aliquot for each 90ml conical flask with glass, sterilize in an
autoclave at 121℃ for 20min.
3.2 SCDLP liquid medium
Compositions: Casein peptone 17g
Soybean peptone 3g
Sodium chloride 5g
Dipotassium hydrogen phosphate 2.5g
Glucose 2.5g
Lecithin 1g
Tween 80 7g
Distilled water up to 1000ml
Preparation: Dissolve lecithin in a small amount of distilled water, then mixed with other
Compositions, heat to dissolved, adjust pH to 7.2-7.3, aliquot for 90ml each flask, sterilize in an
autoclave at 121℃ for 20min. Oscillate them to adequately mix sedimentary Tween 80 at the
bottom, standby after cooling down it to around 25℃.
Note: If casein peptone and soybean peptone are unavailable, poly peptone can be used.
3.3 Sterilized liquid paraffin.
Preparation: sterilize 50ml of liquid paraffin in an autoclave at 121℃ for 20min..3.4 Sterilized Tween 80
Preparation: sterilize 50ml Tween 80 in an autoclave at 121℃ for 20min.
4 Dos and Don’ts of Sample Collection
4.1 Collected samples shall be representative, usually the numbers of samples randomly selected is
depending on the size of each batch of packaging unit. For test, shall take totally 10g or 10ml from
at least 2 packing units of samples. When there’re packing units whose weight is less than 20g,
samples shall appropriately be prepared from more packing units.
4.2 Samples to be tested should strictly maintain their original status of packaging. The container
should not be ruptured and should not be opened before test to prevent samples from
contamination.
4.3 Samples should be registered immediately once received, set the serial numbers of test and
arrange the test as soon as possible according to their test procedures. If the test will not be
conducted immediately, the sample should be placed in a cool and dry place at room temperature,
DO NOT refrigerate or freeze.
4.4 If one sample is subjected to multiple test, such as microorganisms, toxicology and chemistry etc.,
it is advised to firstly leave some of the samples for microbial test, and use the remaining samples
for rest test.
4.5 During the test process, from the opening of the packaging to the end of all test activities, it is
necessary to prevent the second-hand contamination and diffusion of microorganisms. All utensils
and materials used should be sterilized in advance and all operation should be carried out in
laboratories that meet the requirements of biosafety.
5 Preparation of samples for test
5.1 Liquid samples
5.1.1 For water-soluble liquid samples, use sterilized pipette to take 10ml samples and mix with
90ml of sterilized saline to prepare 1:10 test sample.
5.1.2 For oily liquid samples, take 10g sample, mix with 5ml of sterilized liquid paraffin, then mix
with 10ml sterilized Tween 80. Oscillate sample for its well mix in water bath at 40℃-44℃ for
10min, then mix with 75ml of sterilized saline (pre-heated in water bath at 40℃-44℃).
Emulsify samples in the water bath at 40℃-44℃ to finally prepare 1:10 suspension.
5.2 Semi-solid samples of cream, frost and emulsion
5.2.1 Hydrophilic samples. Weigh 10g of sample, mix with 90ml of sterilized saline in conical flask
with glass beads, oscillate to evenly mixed, leave for 15min and then collect its supernatant
as 1:10 test sample.
5.2.2 Hydrophobic sample. Weigh 10g of sample, place in a sterilized mortar, add 10ml of sterilized
liquid paraffin, grind it to viscous status, then add 10ml of sterilized Tween 80 and grind it
until fully dissolved, add 70ml of sterilized saline, well mix in water bath at 40-44℃ to prepare
1:10 test sample.
5.3 Solid samples
Weigh 10g of sample, add 90ml of sterilized saline and oscillate them to mix and disperse evenly.
Leave and then take its supernatant as 1:10 test sample.
Use sterilized homogeneous bag, take 10g of the above water-soluble cream, frost and powder
etc., mix with 90ml of sterilized saline. Homogenize for 1min—2min. For hydrophobic cream, frost,
eyebrow pen and lipstick etc., take 10g of sample, mix with 10ml of sterilized liquid paraffin, 10ml
of Tween 80 and 70ml of sterilized saline, homogenize for 3min—5min.II Method for Aerobic Bacterial Count
1 Scope
This standard defines the test method for count of Aerobic Bacterial colonies in cosmetics.
This standard is applicable to the determination of the count of Aerobic Bacterial colonies in cosmetics.
2 Definition
2.1 Aerobic bacterial count
Total number of colonies contained in 1g (1ml) of cosmetic sample which have been treated and
cultured under certain conditions (e.g. medium composition, culture temperature, culture time, pH
Value, aerobic characteristics etc.). The result only contains count of mesophilic aerobic and
facultative anaerobic colonies grow under the conditions defined in this method.
Determination of the total count of colonies is to determine the extent of bacterial contamination of
the sample, which is the comprehensive basis for general evaluation of the hygiene of the sample.
3 Instruments & equipment
3.1 Conical flask, 250ml.
3.2 Cylinder, 200ml
3.3 pH Meter or precise pH Paper.
3.4 Autoclave.
3.5 Tube18mmx150mm.
3.6 Sterilized petri dish, in diameter of 90mm.
3.7 Sterilized graduated pipette, 10ml and 1ml.
3.8 Alcohol burner.
3.9 Thermostatic Incubator,36℃±1℃.
3.10 Magnifier.
3.11 Thermostatic water bath box, 55℃±1℃.
4 Culture medium and reagents
4.1 Saline: see 3.1 of general principles。
4.2 Lecithin, Tween 80 nutrient agar medium
Compositions:
Peptone 20g
Beef extract 3g
Sodium chloride 5g
Agar 15g
Lecithin 1g
Tween 80 7g
Distilled water 1000ml
Preparation: Add lecithin to a small amount of distilled water, heat to dissolved, mix with Tween 80. Mix
rest compositions (except agar) to the rest of the distilled water and dissolved. Add dissolved lecithin,
Tween 80, fully mix and adjust pH to 7.1-7.4, add agar, sterilize in autoclave at 121℃ for 20min, stored
under cool and dark condition.
4.3 0.5% 2,3,5-Triphenyl Tetrazolium Chloride (TTC） Compositions: TTC 0.5g
Distilled water 100ml
Preparation: Filter to remove bacteria after dissolution, or sterilize in an autoclave at 115℃ for 20min,
put in brown reagent bottle and store in refrigerator at 4℃ as standby.
5 Operating procedures
5.1 Use sterilized pipette to draw 2ml of diluted 1:10 sample, drop in two sterilized dishes with 1ml
each. Take another 1ml of sample, inject in 9ml of saline placed in a test tube (DO NOT let
pipette touch the surface of the liquid). Change a new pipette, well mix and 1:100 liquid sample
is then ready. Take 2ml of above 1:100 sample, drop in two sterilized dishes with 1ml each. If
sample contains high concentration of bacteria, keep diluting them to 1:1000, 1:10000…, pipette
of each concentration shall be changed.
5.2 Pour lecithin and Tween 80 nutrient agar medium which are molten and cool to 45℃—50℃ into
petri dish with 15ml each, revolve the petri dish to ensure sample is fully and evenly mixed with
the medium. When Agar is solidified, flip the petri dish and culture in an incubator at 36℃±1 for
48h±2h. Take another sterilized empty dish without a sample, add about 15ml of lecithin Tween
80 nutrient agar medium. After solidification of agar, flip the dish and culture in an incubator at
36℃±1 for 48h±2h as control.
5.3 In order to distinguish between particles and colonies of cosmetics, 1ml of 0.5% TTC solution can
be mixed with 100ml of lecithin Tween 80 agar nutrient medium. If there is bacteria, the colony
shows red after culture, while the color of particle of cosmetics will not change.
6 Method of colony counting
Observe with naked eyes and count colony, then use 5-10 times magnifier to check in case of omission.
After writing down the count of colonies of each dish, the average count of colonies growing in the same
dilution dish then can be calculated. The dish shall not be counted when there are overgrown or
spreading colonies in the dish. If the area of overgrown or spreading colonies is less than half of that
petri dish, and the count of colonies in the remaining half is evenly distributed, the remaining half of the
colony of the dish can be counted and multiplied by 2 as the count of colonies of the whole dish.
7 Method of colony counting and reporting
7.1 Select those petri dishes whose average colony is count between 30 and 300 as the range for
the determination of the total count of colonies. When the average count of colonies of only one
dilution is in line with this range, report the count of the dish colonies multiply with its dilution
multiples as reporting result (see example 1 of table1).
7.2 If there are two dilutions multiples whose average colony count is between 30 and 300, then the
ratio of the total count of two colonies should be determined. If this ratio is less than or equal to
2, the average count of colonies shall be reported. If the ratio is greater than 2, the number of
colonies in a petri dish with a lower dilution should be reported as reporting result (see example
2 and 3 of the table1).
7.3 If the average colony count of all dilutions is greater than 300, the average count of colonies with
the highest dilution should be multiplied by the dilution multiples as reporting result (see example
4 of the table1).
7.4 If the average colony count of all dilutions multiples are less than 30, the average count of
colonies with the lowest dilution should be multiplied by the dilution multiples as reporting result
(see example 5 of table1).
7.5 If the average count of colonies of all dilutions are beyond of 30 to 300, one of the dilution
multiple is greater than 300, while another neighboring dilution multiple is less than 30, report
the average count of colonies multiplied by the dilution multiples of the colonies closest to 30 or
300 as reporting result (see example 6 of table1).
7.6 If all dilution multiples are sterile, the count of reports is less than10 CFU per gram or per ml.7.7 Report the actual bacterial count when the count of colonies is within 10, if the count of colonies
is greater than100, then use two-digit valid number. The value of the number behind the twodigit valid number shall be calculated by using rounding method. To shorten the number of 0
behind the number, exponent of 10 should be used to express the result (see report mode
column of table1). For the count of colonies is reported as “Not counted”, the dilution multiple of
the sample shall be indicated.
Table 1 Bacterial counting results and reporting methods
No. Average count of colonies with
different dilution multiples
10-1
10-2
10-3
Ratio of bacteria of two
dilution multiples
Total count of colonies
（CFU/ml or CFU/g）
Report method
（CFU/ml或
CFU/g）
1 1365 164 20 -- 16400 16000 or 1.6x104
2 2760 295 46 1.6 38000 38000 or 3.8x104
3 2890 271 60 2.2 27100 27000 or 2.7x104
4 Not counted 4650 513 -- 513000 510000 or 5.1x105
5 27 11 5 -- 270 270 or 2.7x102
6 Not counted 305 12 -- 30500 31000 or 3.1x104
7 0 0 0 -- ＜1x10 ＜10
CFU：Colony-Forming Unit.
7.8 Samples sampled by weight shall report by using CFU/g as unit, while samples sampled by
volume should report by using CFU/ml as unit. III Test Method for Thermotolerant Coliform Bacteria
1 Range
This standard defines test method for thermotolerant coliform bacteria in cosmetics.
This standard is applicable to the determination of thermotolerant coliform bacteria in cosmetics.
2 Definitions
2.1 Thermotolerant coliform Bacteria
A group of aerobic and facultative anaerobic gram-negative Bacillus spp, can ferment lactose to
produce acid and gas when culture at 44.5℃ for 24h-48h.
The bacterium mainly comes from faeces of human and warm-blooded animals, can be used as a
faecal contamination indicator when evaluating the hygienic quality of cosmetics to infer the
possibility of contamination of intestinal pathogens in cosmetics.
3 Instrument
3.1 Thermostatic water bath or water-jacket thermostat:44.5℃±0.5℃.
3.2 Thermometer.
3.3 Microscope.
3.4 Slide glass.
3.5 Inoculating loop.
3.6 Electromagnetic hotplate.
3.7 Conical flask, 250ml
3.8 Test tube: 18mmx150mm.
3.9 Small inverted tube.
3.10 pH meter or pH paper.
3.11 Autoclave sterilizer.
3.12 Sterilized graduated pipette, 10ml and 1ml.
3.13 Sterilized petri dish: in diameter of 90mm.
4 Culture medium and reagents
4.1 Double lactose bile salt (containing neutralizer) medium
Compositions: Peptone 40g
Pig Bile salt 10g
Lactose 10g
0.4% Bromine Cresol Purple Aqueous solution 5ml
Lecithin 2g
Tween 80 14g
Distilled water 1000ml
Preparation: Dissolve lecithin and Tween 80 into a small amount of distilled water. Dissolve
peptone, bile salts and lactose into the rest of the distilled water, mix them up and adjust pH to
7.4. Then well mix up with 0.4% bromine cresol purple aqueous solution, aliquot with test tubes
with 10ml each with a small inverted tube inside. Sterilized in an autoclave at 115℃ for 20min.
4.2 Eosin Methylene Blue (EMB) agar
Compositions: Peptone 10g
Lactose 10g
Dipotassium hydrogen phosphate 2g Agar 20g
2% Eosin aqueous solution 20ml
0.5% Methylene blue aqueous solution 13ml
Distilled water 1000ml
Preparation: Add agar to 900ml of distilled water, heat to dissolved, then add dipotassium
hydrogen phosphate peptone, mix to ensure their evenly dissolving. Add distilled water to 1000ml
totally. Adjust pH to 7.2-7.4, make aliquot for three conical flasks, sterilize in an autoclave at
121℃ for 15min as standby. When use it in test, add lactose and heat the agar until molten. Cool
down to 60℃ and add sterilized 2% Eosin aqueous solution under aseptic condition, shake to
evenly dissolved. Pour in a petri dish as standby.
4.3 Peptone water (for Indole test)
Compositions: Peptone (or bacto-tryptone) 20g
Sodium chloride 5g
Distilled water 1000ml
Preparation: Heat the above composition molten, adjust pH to 7.0-7.2, make aliquot in small test
tubes, sterilize in an autoclave at 121℃ for 15min。
4.4 Kovacs reagent
Kovacs reagent: Dissolve 5g of 4-Dimethylaminobenzaldehyde in 75ml of amyl alcohol, then
slowly add 25ml of concentrated hydrochloric acid.
Test method: Inoculate bacteria in peptone water, culture at 44.5℃±0.5℃ for 24h±2h. Stream
0.3ml-0.5ml Kovacs reagent along pipe walls, lightly shake the test tube. The positive reaction will
be showing deep rose red at the reagent layer.
Note: Peptone should be rich in tryptophan, each batch of peptone purchased shall be verified
with known strains prior to its using.
4.5 Gram staining solution:
4.5.1 Preparation of staining liquid
4.5.1.1 crystal violet staining solution:
Crystalline violet 1g
95% Ethanol 20ml
1% Ammonium oxalate aqueous solution 80ml
Dissolve the crystalline violet in ethanol and then mixed with ammonium oxalate solution.
4.5.1.2 Gram Iodine solution:
Iodine 1g
Potassium iodide 2g
Distilled water 300ml
Mix iodine with potassium iodide, dissolve with a little amount of distilled water, shake until
fully dissolved, and add distilled water to 300ml.
4.5.1.3 Destaining solution: 95% ethanol.
4.5.1.4 Counterstain solution:
（1) Safranine solution:
Safranine 0.25g
95% ethanol 10ml Distilled water 90ml
Dissolve the safranine in ethanol and dilute with distilled water.
（2) Diluted carbolfuchsin staining: take 10g of alkaline fuchsin, grind it into powder, add 100ml of 95%
ethanol, leave overnight, filter by filtration paper. Take 10ml of the liquid, mix with 90ml of 5% carbonate
aqueous solution, that is carbolfuchsin staining. Take 10ml of this liquid and mix with 90ml of water to
prepare diluted carbolfuchsin staining.
4.5.2 Staining method
4.5.2.1 Fixed smear on the flame, drip crystalline violet staining liquid, stain for 1min, wash
with water.
4.5.2.2 Drop Gram-iodide liquid, leave for 1min, wash with water.
4.5.2.3 Drip 95% ethanol destaining solution for approx. 30s, or fill the ethanol on full
smear, pour it out immediately, again drip the entire smear with ethanol, destain for
10s, wash away with water.
4.5.2.4 Drip counterstain solution, leave for 1min, wash with water, leave until dry, and then
perform microscopy.
4.5.3 Staining results
Gram-positive bacteria show purple while Gram-negative bacteria show red.
Attention: If 1:10 diluted carbolfuchsin liquid is used for counterstaining, then 10s is sufficient for the afterstaining.
5 Operating Procedures
5.1 Take 10ml of 1:10 diluted sample, add to 10ml of double lactose bile salt (containing neutralizer)
medium, culture in an incubator at 44.5℃±0.5 for 24h, if produced neither acid nor gas, continue to
culture to 48h, if it yet produces neither acid nor gas, negative result of thermotolerant coliform
bacteria shall be reported.
5.2 If acid and gas are produced, streak and inoculate to EMB agar dish, culture at 36℃±1℃ for 18h24h. Meanwhile take 1 to 2 drops of culture liquid and inoculate into peptone water culture at
44.5℃±0.5℃ for 24h±2h.
After culture, observe if there’re typical colonies grow on the dish. The typical colony of
thermotolerant coliform bacteria on the EMB agar medium show deep purple and black with round
shape, have regular edge with smooth and moist surface, usually with metallic luster. Some of
them are in deep purple and black, without or with slightly metallic luster, or pink purple, the deep
colony at the center of the dish is also usually thermotolerant coliform bacteria. Attention should
be paid on their careful selection.
5.3 Pick the above suspected colony and smear it for Gram staining microscopy test.
5.4 In the Peptone water culture medium, add about 0.5ml of Kovacs reagent, observe Indigo
reaction. Positive reaction shows rose red whilst negative reaction keeps its original reagent color.
6 Test Results Report
According to fermented lactose produces acid and gas, typical colony on the plate proved to be Gramnegative short bacillus, and positive to indigo test, then the thermotolerant coliform can be reported as
detected in the tested sample. IV Test method for pseudomonas aeruginosa
1 Scope
This standard defines the test method of pseudomonas aeruginosa in cosmetics.
This standard is applicable to the determination of pseudomonas aeruginosa in cosmetics.
2 Definition
2.1 Pseudomonas aeruginosa
Belongs to the genus of Pseudomonas, Gram-negative bacillus, positive to oxidase, can produce
colonies of pseudomonas aeruginosa. In addition, it can liquefy gelatin, reduce nitrate to nitrite and can
grow under conditions at 42℃±1℃.
3 Instrument
3.1 Thermostatic Incubator: 36℃±1℃ and 42℃±1℃.
3.2 Conical flask, 250ml.
3.3 Test tube: 18mm x 150mm.
3.4 Sterilized Petri dish: in diameter of 90mm.
3.5 Sterilized graduated pipette, 10ml and 1ml.
3.6 Microscope.
3.7 Slide.
3.8 Inoculating needles, inoculating rings.
3.9 Induction.
3.10 Autoclave sterilizer.
3.11 Thermostatic water bath.
4 Culture medium and reagents
4.1 SCDLP liquid medium
See 3.2 of the general principles.
4.2 Hexadecyl trimethyl ammonium bromide (CTMAB) medium
Compositions: Beef extract 3g
Peptone 10g
Sodium chloride 5g
CTMAB 0.3g
Agar 20g
Distilled water 1000ml
Preparation: except agar, mix above compositions and heat to dissolved, adjust pH to 7.4-7.6, add agar,
sterilized in an autoclave at 115℃ for 2omin, and then make it as plate as standby.
4.3 Acetamide medium
Compositions: Acetamide 10.0g
Sodium chloride 5.0g DiPotassium hydrogen phosphate anhydrous 1.39g
Anhydrous potassium dihydrogen phosphate 0.73g
Magnesium sulfate (Mgso4.7H2O) 0.5g
Phenol red 0.012g
Agar 20g
Distilled water 1000ml
Preparation: Add all Compositions (except agar and phenol infrared,) to distilled water, heat to
dissolved, adjust pH to 7.2, add agar and phenol red, sterilize in an autoclave at 121℃ for 20min, then
prepare it in dish as standby.
4.4 Medium for determination of pseudomonas aeruginosa
Compositions: Peptone 20g
Magnesium chloride 1.4g
Potassium sulfate 10g
Agar 18g
Glycerol (chemical pure) 10g
Distilled water 1000ml
Preparation: Add Peptone, magnesium chloride and potassium sulfate to distilled water, warm them to
dissolve, adjust pH to 7.4, add agar and glycerol, heat to dissolve, make aliquot in test tube, sterilize in an
autoclave at 115℃ for 20min as standby in form of dish.
4.5 Gelatin medium
Compositions: Beef extract 3g
Peptone 5g
Gelatin 120g
Distilled water 1000ml
Preparation: Add all composition to the distilled water to soak for 20min, timely stirring and heating at to
dissolved, adjust pH to 7.4, load into the test tube, sterilized in an autoclave at 115℃ for 20min and then
erect the tube to form a high-rise layer as standby.
4.6 Nitrate peptone water medium
Compositions: Peptone 10g
Yeast immersion cream 3g
Potassium nitrate 2g
Sodium nitrite 0.5g
Distilled water 1000ml
Preparation: Add peptone and yeast extract to distilled water, heat to dissolved, adjust pH to 7.2, boil
and filter then replenish sufficient liquid, add potassium nitrate and sodium nitrite, dissolve and mix, divide
and make aliquot test tubes with a small inverted tube, sterilize in an autoclave at 115℃ for 20min as
standby.
4.7 Ordinary agar slant medium
Ingredients: Peptone 10g
Beef extract 3g
Sodium chloride 5g
Agar 15g
Distilled water 1000ml
Preparation: Dissolve all rest of the compositions (except agar) in distilled water and adjust pH to 7.2-
7.4, add agar, heat to dissolve, make aliquot test tubes, sterilize at 121℃ for 20min then make a slant
medium as standby.
5 Operating Procedures5.1 Enrichment culture: Take 10ml of 1:10 diluted sample solution and mix with 90ml of SCDLP liquid
medium, culture in an incubator at 36℃±1℃ for 8h-24h. If there’re pseudomonas aeruginosa
grow, an extra layer of thin bacteria will show on the surface of the culture liquid, and the culture
solution is often yellow to greenish or blue-green.
5.2 Isolated culture: Pick the culture from the thin film of the culture liquid, streak and inoculate on the
plate of CTMAB agar medium. Culture in an incubator at 36℃±1℃ for 18h—24h. When
pseudomonas aeruginosa is on this medium, its colony is flat and unshaped, diffused to the
periphery or spread slightly, the surface is moist with grayish white in color, and the medium
around the colony is often diffused with water-soluble pigment.
Where in the absence of CTMAB agar, Acetamide medium can also be used for isolation. Streak
and inoculate bacterial liquid on the plate, culture at 36℃±1℃ for 24h±2h, pseudomonas
aeruginosa grows well on this medium with flat colony shape and irregular edges, the medium
around the colony shows red, and there’re no other bacteria grow.
5.3 Staining microscopy: pick suspicious colonies, coat on the smear, perform Gram staining, the
result of Gram-negative microscopy should subject to oxidase test.
5.4 Oxidase Test: Lay a small piece of clean white filter paper on a sterilized petri dish, use a sterile
glass bar to pick a suspicious colony of pseudomonas aeruginosa and coat on a filter sheet, drip
a drop of the newly formulated 1% dimethyl-p-phenylenediamine test fluid, when pink or purplish
red appears in15s-30s, it is positive to oxidase test. If the color of the culture does not change, it
is negative to oxidase test.
5.5 Test of pyocyanine: pick 2-3 suspicious colonies, respectively inoculate on the pyocyanine
determination medium, culture at 36℃±1℃ for 24h±2h, add 3ml-5ml of chloroform, fully oscillate
the culture to dissolve in the chloroform solution. When the chloroform extract is blue, transfer
chloroform to another test tube and add 1ml of 1mol/L hydrochloric acid, oscillate and leave for a
little while. Report positive if color shows pink to purplish red at the upper layer of hydrochloric
acid solution which indicates there is pyocyanine in the sample.
5.6 Nitrate reduction and gas production test: Pick suspected pure culture of colonies of
pseudomonas aeruginosa, inoculate in nitrate peptone water medium, culture at 36℃±1℃ for
24h±2h, observe the results. When there’s gas in the small inverted tube in the nitrate peptone
water medium that is positive reaction, which indicates the bacteria can reduce nitrate and
decompose nitrite to nitrogen.
5.7 Gelatin liquefaction test: Take the pure culture of suspicious colonies of pseudomonas
aeruginosa, stab inoculate in gelatin medium, culture at 36℃±1℃ for 24h±2h, transfer to a
refrigerator at 4℃±2℃ for 10min-30min, positive result of liquefaction test shall be reported if
gelatin is still in the form of dissolution or only the surface is dissolved. While negative result of
liquefaction test shall be reported if it’s still in the form of solid.
5.8 42℃ growth test: pick suspected pure culture of pseudomonas aeruginosa, inoculate on the
ordinary agar slant medium, culture at 42℃±1℃ for 24h-48h, positive result should be reported if
pseudomonas aeruginosa can grow, while similar fluorescent pseudomonas cannot grow.
6 Test Results Report
After the sample was isolated and cultured and proved as gram-negative bacillus, positive to oxidase and
pseudomonas aeruginosa test, then can report the pseudomonas aeruginosa are detected in the tested
sample. If the test of pyocyanine is negative while test of gelatin liquefaction, test of nitrate reduction gas
production and test of 42℃ growth are all positive, pseudomonas aeruginosa could still be reported as
detected in the tested sample. V Test method for staphylococcus aureus
1 Scope
This standard gives method of detection and identification of staphylococcus aureus in cosmetics.
This standard is applicable to the detection and identification of staphylococcus aureus in cosmetics.
2 Definitions
2.1 Staphylococcus aureus
Gram-positive cocci, grape-like clusters, no spores, no capsule, can decompose mannitol, positive reaction
to plasma coagulase.
3 Instruments & equipment
3.1 Microscope.
3.2 Thermostatic Incubator: 36℃±1℃.
3.3 Centrifuge.
3.4 Sterilization graduated pipette, 10ml and 1ml.
3.5 Test tube: 18mmx150mm.
3.6 Slide.
3.7 Alcohol burner.
3.8 Conical flask, 250ml.
3.9 Autoclave sterilizer.
3.10 Thermostatic water bath.
4 Medium and reagents
4.1 SCDLP liquid medium
See 3.2 of the general principles.
4.2 Nutrient broth
Compositions: Peptone 10g
Beef extract 3g
Sodium chloride 5g
Distilled water up to 1000ml
Preparation: Heat the above compositions to dissolved, adjust pH to 7.4, make aliquot and sterilize in
autoclave at 121℃ for 15min.
4.3 7.5% of sodium chloride broth
Compositions: Peptone 10g
Beef extract 3g
Sodium chloride 75g
Distilled water up to 1000ml
Preparation: Heat the above compositions to dissolved, adjust pH 7.4, make aliquot and sterilize in an
autoclave at 121℃ for 15min.
4.4 Baird Parker plate Compositions: Bacto-tryptone 10g
Beef extract 5g
Yeast extract 1g
Sodium pyruvate 10g
Glycine 12g
Lithium chloride (LiCl·6H2O) 5g
Agar 20g
Distilled water 950mL
pH7.0±0.2
Preparation of enrichment: mix 50ml of vitelline saline with 10ml of bacterial-filtered 1% potassium
tellurite solution and store in the fridge.
Preparation: Add all compositions to the distilled water, boil to completely dissolved, cool to 25℃±1℃
and correct pH. Make aliquot bottles with 95ml each, sterilize in autoclave at 121℃ for 15min. When put into
use, heat to agar molten, take each 95ml to add 5ml of enrichment of vitelline potassium tellurite which was
preheated to 50℃, shake to dispersed evenly and then pour it into the plate. The medium should be
compact and opaque. Store in refrigerator and must not exceed 48h±2h prior to its use.
4.5 Blood agar medium
Components: Nutrient agar 100ml
Defibrous sheep blood (or rabbit blood) 10ml
Preparation: Heat to melt the nutrient agar and leave until cool to around 50℃, add defibrous sheep
blood under aseptic condition, shake to dissolved evenly, prepare in the form of plate, put in refrigerator as
standby.
4.6 Mannitol fermentation medium
Compositions: Peptone 10g
Sodium chloride 5g
Mannitol 10g
Beef extract 5g
0.2% Thymol blue solution 12ml
Distilled water 1000ml
Preparation: Add peptone, sodium chloride and beef extract to distilled water, heat to molten,
adjust pH to 7.4, add mannitol and indicator, full mix, make aliquot test tube, sterilize at
68.95kPa(115℃ 10 lb) for 20min as standby.
4.7 Liquid Paraffin
See 3.3 of the general principles.
4.8 Plasma preparation of rabbits (human)
Take 3.8% sodium citrate solution, sterilize in autoclave at 121℃ for 30min, each one of them
add 4 times of rabbit (human) whole blood, well mix and leave for a while. Centrifuge it at
2000rpm-3000rpm for 3min—5min. The blood cells sink and take the upper plasma.
5 Operating Procedures
5.1 Enrichment: Take 10ml of 1:10 diluted sample, inoculate to 90ml of SCDLP liquid medium and
culture at 36℃±1℃ for 24h±2h.
Note: In case of this medium is not available, 7.5% sodium chloride broth can be used. 5.2 Isolation: Take 1-2 inoculating rings of above enrichment culture medium, streak to inoculate on
Baird Parker plate medium. If Baird Parker medium is unavailable, use blood agar plate, culture at
36℃±1℃ for 48h. The colony on the blood agar plate is golden, round, opaque, smooth on the
surface and there’s a hemolysis ring around it, while on Baird Parker plate medium the colony is
round, smooth, bulging, moist, the color is gray to black and the edge is pale, surrounded by a
turbid belt, and there’s transparent band at its periphery. It feels soft like cream resin when touch
the colony with inoculation needle. Occasionally there will be similar non-liposoluble colonies but
without turbid belt or transparent band. Pick a single colony, isolate and purify on a blood agar plate,
culture at 36℃±1℃ for 24h±2h.
5.3 Staining microscopy: Pick isolated and purified colony, coat on smear and perform Gram staining,
conduct microscopy. Staphylococcus aureus is Gram-positive bacteria, aggregated in grape-like
clusters, no spores or capsule. The thalli of pathogenic staphylococcus is smaller and it’s about
0.5µm-1µm in diameter.
5.4 Fermentation test of mannitol: Take above isolated colony and inoculate on mannitol fermentation
medium, add 2mm-3mm height of sterilized liquid paraffin on the liquid surface of medium, culture
at 36℃±1℃ for 24h±2h, staphylococcus aureus should be able to ferment mannitol to produce
acid.
5.5 Test of plasma coagulase: Draw 0.5ml of 1:4 fresh plasma, place in a small sterilized test tube and
add 0.5ml the bacteria sample to be examined which has been cultured for 24h±2h with broth
medium. Mix evenly, place in thermostat or thermostatic water bath at 36℃±1℃, observe every
half hour. It’d be positive if the there’re clots present within 6h. Simultaneously respectively take
0.5ml of broth culture and broth medium of known strains positive and negative to plasma
coagulase, add 0.5ml of aseptic 1:4 plasma respectively and mix evenly as a control.
6 Report test result
When those suspicious colonies grow on the above selective plate are proved to be Gram-positive
staphylococcus by using of staining microscopy, are able to ferment mannitol to produce acid and are
positive to plasma coagulase test, then can report staphylococcus aureus are detected in the tested
sample.VI Test methods for mold and yeast
1 Scope
This standard defines the test methods for counting mold and yeast in cosmetics.
This standard is applicable to the determination of mold and yeast in cosmetics.
2 Definitions
2.1 Determination of mold and yeast count
After culture of samples under certain conditions, the total count of live mold and yeast contained
in 1g or 1ml of cosmetics, indicates the contamination extent and general hygiene status of
cosmetics.
According to the unique morphology and culture characteristics of mold and yeast, this method
culture sample on the Rose Bengal medium at 28℃±2℃ for 5 days, then count mold and yeast
grown.
3 Instrument & Equipment
3.1 Thermostatic Incubator: 28℃± 2℃.
3.2 Oscillator.
3.3 Conical flask, 250ml.
3.4 Tube: 18mmx150mm.
3.5 Sterilized petri dish: 90mm in diameter.
3.6 Sterilization graduated pipette, 10ml and 1ml.
3.7 Cylinder, 200ml.
3.8 Alcohol burner.
3.9 Autoclave sterilizer.
3.10 Thermostatic water bath.
4 Medium and reagents
4.1 Saline
See section 3.1 of general principles.
4.2 Rose Bengal medium
Composition: Peptone 5g
Glucose 10g
Potassium hydrogen phosphate 1g
Magnesium sulfate (7H2O） 0.5g
Agar 20g
1/3000 Rose Bengal Solution 100ml
(Tetrachloro-tetraiodofluorescein)
Distilled water 1000ml
Chloramphenicol 100mg
Preparation: Dissolve the above components (except Rose Bengal) in the distilled water and mix
with Rose Bengal solution. Make aliquot and sterilized in autoclave at 121℃ for 20min. Dissolve
chloramphenicol in another small amount of ethanol, add to the medium after filtration. If
chloramphenicol is unavailable, use 30mg streptomycin per 1000ml.5 Operation Procedures
5.1 Dilution of sample
See 5.1 of the total count of Aerobic bacterial colonies.
5.2 Inject 1ml of 1:10, 1:10 and 1:1000 diluted sample into sterilized petri dishes separately, 2 petri
dishes for each dilution. Introduce the Rose Bengal medium which is molten and then cooled to
45℃±1℃, well shaken. When agar solidify, flip the dish and culture at 28℃±2℃ for 5 days,
observe and record. Take another sterilized empty dish without sample, add about 15ml of Rose
Bengal medium, after Agar is solidified, flip the petri dish and culture at 28℃±2℃ for 5 days as
control.
5.3 Calculation: Count the number of mold and yeast colonies grown on each dish, calculate the
average number of colonies of different dilutions. When determining the result, the dishes which
contain 5-50 colonies should be selected, multiplied by dilution multiples, then obtain the number
of mold and yeast contained per 1g (or 1ml). The count of colonies beyond this range should be
reported according to the reporting methods of the total count of colonies.
5.4 Use CFU/g (ml) as unit to report the number of mold and yeast contained in cosmetics per gram
(or per ml)
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Bio
A social observer, Chinese culture
ambassador, and civilizations facilitator. Has a profound understanding of Chinese culture, excellent command of written Chinese, and insightful perception
of different cultures with more than 20 years of mid-senior management at large SOE (State Owned Enterprise) and various
MNCs.
Simon was born and raised in a family
of traditional Chinese culture in northwest China, completed his college life in southeast of China and is now living in Shanghai. He has very
broad interests in Chinese, social sciences, languages, literature, Chinese calligraphy,
martial arts, history, geography, art, natural science, and technology. He commands
excellent Chinese language including Mandarin and many Chinese dialects and can not
only well understand Classical Chinese, appreciate ancient Chinese literary
works, poetry, music, arts and paintings, but also write in Classical and
Modern Chinese articles, poems and Chinese Couplets (Duilian), have good
Chinese handwriting, name new companies, and write copywriting for various
organizations.
Simon’s background is in Agriculture, Food,
Nutrition, Chemicals, Biology, Cosmetics, Life Science, and Natural sciences
with broad services, industrials, and engineering know-how. Thanks to his broad industry engagement at third-party MNCs of Testing, Inspection, and Certification, Simon engaged in many state-level
translations and standardizations of International Standards and Regulations, localized, trained, implemented, and managed the Cultures, Compliance, Risks, Safety, Security, Regulatory, and Sustainability issues in the Greater China region.
During his middle school and university
study, Simon served as Rep. of Chinese subject, Minister of Propaganda
Department of faculty, and journalist of University Newspaper.
Simon is also very active
in a lot of social groups of Classical Chinese Culture.
Examples of past works:
1. Naming: Named the No. 4
Chang’e Lunar Rover of China as Walker (Xing Zhe, 行者). Voted as No. 1 nationally.
2. Naming: Named Chinese
company name for an MNC company in Chemical Industry. Rewarded as No. 2.
3. Copywriting: Vision/Mission
of the hospital of the University ofChinese Academy of Science (Shenzhen).
Rewarded No. 3.
4. Chinese Couplets: Wrote
Couplets for one MNC in a company-wide contest. Rewarded as No. 1.
Expertise