GB 27951-2011Hygiene requirements for skin disinfectant (English PDF)

4.3.5 Stability

The validity of the original packaged product is >=12 months.

4.3.6 Requirements for disinfectants in use

The sensory traits, effective ingredient content, pH of the disinfectant in use after opening meet the product quality requirements. The total number of colonies <=50CFU/mL (g), mold and yeast <=10CFU/mL (g). It shall meet the requirements of GB 15982.Pathogenic bacteria (staphylococcus aureus, pseudomonas aeruginosa, hemolytic streptococcus) shall not be detected.

Disinfectant for damaged skin in use shall meet the exit-factory requirements.

5.1 Sensory traits examination

Visually inspect disinfectant color, clarity.

5.2 Determination of physical and chemical indicators

5.2.1 pH determination

Determine according to relevant methods in Disinfection technical specifications.

5.2.2 Active ingredient content

Determine according to relevant provisions in Disinfection technical specifications, GB 6680, GB/T 601.

5.2.3 Determination of lead, mercury and arsenic limits

Determine according to relevant methods in Hygienic specifications for cosmetics.

5.2.4 Stability test

Determine according to relevant methods in Disinfection technical specifications.

5.3 Microorganism killing test

Determine according to relevant methods in Disinfection technical specifications.

5.4 Identification of microbial contamination

See Annex A for inspections of total colony, mold and yeast, pathogenic bacteria and sterility.

5.5 Toxicology test

Determine according to relevant methods in Disinfection technical specifications.

6.1 Disinfection of intact skin

Use disinfectant to wipe or rub to disinfect 2~3 times. Act 1min~5min to achieve disinfection effect.

6.2 Disinfection of damaged skin

Use disinfectant to rub or rinse to disinfect. Act 1min~5min to achieve disinfection effect.

6.3 Disinfection of skin at injection or puncture site

Use disinfectant to wipe to disinfect 2~3 times. Act <=1min to achieve disinfection effect.

A.1 Determination of the total number of colonies

A.1.1 Test equipment

A.1.1.1 Erlenmeyer flask. 250mL.

A.1.1.2 Measuring cylinder. 200mL.

A.1.1.3 Autoclave.

A.1.1.4 Level-100 clean room or level-100 laminar flow ultra-clean workbench.

A.1.1.5 Test tube. 15mm × 150mm.

A.1.1.6 Sterilization plate. 9cm in diameter.

A.1.1.7 Sterilization scale pipette. 10mL, 1mL.

A.1.1.8 Alcohol lamp.

A.1.1.9 Constant temperature incubator. 36°C±1°C.

A.1.1.10 Magnifier.

A.1.1.11 Saline.

A.1.1.12 Common nutrient agar medium.

A.1.1.13 Neutralizer.

A.1.2 Methods

A.1.2.1 Sample processing

Use a sterile pipette to pipette 1.0mL of disinfectant. Add into 9.0mL of sterile saline that contains corresponding neutralizing agent. Shake 20s or beat 80 times. Take 1.10 dilution for testing.

A.1.2.2 Operating steps

Use a sterile pipette to pipette 2mL of 1.10 dilution testing solution. Respectively inject into two sterilization plates, 1mL for each plate. Take another 1mL to inject

A.2.1.12 Saline.

A.2.2 Methods

A.2.2.1 Sample processing. see A.1.2.1.

A.2.2.2 Operating steps. Respectively take 1mL of 1.10, 1.100, 1.1000 test

solutions into sterilization plates. Separately use 2 plates for each dilution. Inject

Sapura agar medium that has been melted and cooled to about 45°C±1°C.

Completely shake well. After solidification, turn the plate. Incubate at 28°C± 1°C for 72h±2h. Count the number of molds and yeasts growing in the plate. If there is mold spread, to avoid affecting the count of other molds and yeasts, the plate shall be taken out in time and counted in 48h±2h. Take another sterilized empty plate without sample. Add about 15mL of Sabaero agar medium. After the agar solidifies, turn the dish. Incubate at 28°C±1°C for 72h±2h, for blank control.

A.2.2.3 Result report. First count the number of mold and yeast colonies

growing on each plate. Calculate the average number of colonies per dilution.

When determining the result, it shall select the plate count of which the number of colonies is within the range of 5~50.Multiplied by the dilution factor shall be the number of mold and yeast contained in the disinfectant per milliliter (or per gram), in CFU/mL (g). If all dilutions are grown aseptically, the reported number shall be < 10CFU/mL (g).

A.3 Test methods for pathogenic bacteria

A.3.1 Test methods for staphylococcus aureus

A.3.1.1 Test apparatuses

A.3.1.1.1 Microscope.

A.3.1.1.2 Constant temperature incubator. 36°C±1°C.

A.3.1.1.3 Centrifuge.

A.3.1.1.4 Sterile pipette. 1mL, 10mL.

A.3.1.1.5 Sterilization test tube. 15mm × 150mm.

A.3.1.1.6 Glass slide.

A.3.1.1.7 Alcohol lamp.

A.3.1.1.8 7.5% sodium chloride broth.

A.3.1.1.9 Blood agar medium.

A.3.1.1.10 Mannitol fermentation medium.

A.3.2.2.3 Separate culture. Pick cultures from the culture film. Conduct crossed inoculation on the cetyltrimethylammonium bromide agar plate. Incubate at

36°C±1°C for 18h~24h. For pseudomonas aeruginosa on this medium, the colony is flat and amorphous, spreading to the periphery or spreading slightly.

The surface is wet. The colony is grayish white. The water-soluble green pigment is often diffused in the culture medium around the colony.

A.3.2.2.4 Staining microscopy. Pick suspicious colonies. Smear. Conduct Gram stain. Those who are Gram-negative under microscope shall be tested for oxidase.

A.3.2.2.5 Oxidase test. Take a small piece of clean white filter paper and put it in a sterilization dish. Use a sterile glass rod to pick up the suspicious colonies of pseudomonas aeruginosa and apply it to the filter paper. Then add a drop of freshly-prepared 1% dimethyl-p-phenylenediamine test solution. Within

15s~30s, when pink or purple appears, it is positive for oxidase test. If the culture does not change color, the oxidase test is negative.

A.3.2.2.6 Pyocyanin test. Take 2~3 suspicious colonies. Respectively inoculate on pseudomonas aeruginosa determination medium. Incubate at 36°C±1°C for

24h±2h. Add 3mL~5mL of chloroform. Shake well to dissolve the pyocyanin in the chloroform solution. When the chloroform extract is blue, pipette chloroform into another test tube and add about 1mL of 1mol/L hydrochloric acid. After oscillation, let stand for a while. If there is pink to purple in the upper hydrochloric acid, it is positive, which means that pyocyanin exists in the test object.

A.3.2.2.7 Nitrate reduction gas production test. Pick suspicious pure cultures of pseudomonas aeruginosa. Inoculate in nitrate peptone water medium. Incubate at 36°C±1°C for 24h±2h. Observe the results. Where there is gas in the small inverted tube in nitrate peptone water culture medium, it is positive, which means that the bacteria can reduce nitrate and decompose nitrite to produce nitrogen.

A.3.2.2.8 Gelatin liquefaction test. Take pure culture of suspicious colonies of pseudomonas aeruginosa. Puncture and inoculate in gelatin medium. Incubate at 36°C±1°C for 24h±2h. Take out and put in the refrigerator for 10min~30min.

If it is still dissolved or the surface is dissolved, the gelatin liquefaction test shall be positive. If it is coagulation insoluble, it shall be negative.

A.3.2.2.9 42°C growth test. Pick suspicious pure cultures of pseudomonas aeruginosa. Inoculate on the plain agar slant medium. Place in a 42°C±1°C incubator and cultivate for 24h~48h. If pseudomonas aeruginosa can grow, it shall be positive while pseudomonas fluorescens cannot grow.

A.3.2.3 Result report

A.3.3.2.3 Separate culture. Pick cultures from the culture film. Conduct crossed inoculation on the blood plate. Incubate at 36°C±1°C for 18h~24h. The type of hemolytic streptococcus on the blood plate is grayish white, translucent or opaque, with needle-shaped protrusions, smooth surface, neat edges, and beta hemolytic circle around.

A.3.3.2.4 Staining microscopy. Pick suspicious colonies. Smear. Conduct Gram stain. It is Gram-positive, chain-like cocci under microscope.

A.3.3.2.5 Catalase test. Use the inoculating ring to pick up the central culture of single colony that has been incubated for 18h~24h and place it on a clean glass slide. Use a dropper to add 30% H2O2 to the bacteria on the slide (the operation sequence cannot be reversed, otherwise it is easy to have false positives). Immediately observe for bubbling. Record the results. When there are bubbles, it shall be positive. Hemolytic streptococcus B is negative.

A.3.2.2.6 Streptokinase test. Pipette 0.2mL of potassium oxalate plasma (0.02g of potassium oxalate plus 5mL of human plasma and mix well. After centrifugal precipitation, absorb supernatant). Add 0.8mL sterile saline and mix well. Then add 0.5mL of 24h broth culture to be tested and 0.25mLof 0.25% calcium chloride. Mix well. Put in 36°C±1°C water bath. Observe every 2min (usually coagulable within 10min). Continue to observe and record the time of dissolution after plasma coagulation. If it does not melt within 2h, move it to the incubator and observe the result for 24h. If all melt, it is positive. When it still does not melt within 24h, it shall be negative.

A.3.2.2.7 Bacitracin sensitivity test. Apply the concentrated bacteria solution of the tested bacteria to the blood plate. Use sterile forceps to take 0.04 unit of bacitracin paper and place it on the surface of the plate. At the same time, a known positive strain is used as a control. Incubate at 36°C±1°C for 18h~24h.

Those with bacteriostatic zone are positive.

A.3.3.3 Result report. After the tested disinfectant is separated and cultured by enriching bacteria, when cocci is proved as Gram-positive, chain-like; catalase is negative, streptokinase test is positive, and sensitive to bacitracin, it may report that streptococcus hemolyticus is detected.

A.3.4 Sterility test

A.3.4.1 Test equipment

A.3.4.1.1 Aerobic-anaerobic medium.

A.3.4.1.2 Fungal medium for sterility test (hereinafter referred to as fungal medium).

A.3.4.1.3 Neutralizer.

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