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GB/T 18204.4-2013 English PDF (GBT18204.4-2013)

GB/T 18204.4-2013 English PDF (GBT18204.4-2013)

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GB/T 18204.4-2013: Examination methods for public places -- Part 4: Microorganism on a surface of public articles

This Part of GB/T 18204 specifies the sampling and test methods for the total bacterial count, total fungi count, coliforms, staphylococcus aureus and streptococcus hemolyticus of public articles in public places. This Part is applicable to the test of the total number of bacteria, total bacterial number, total fungi count, coliforms, staphylococcus aureus and streptococcus hemolyticus of public articles in public places, and other places can be implemented by reference.
GB/T 18204.4-2013
GB
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 13.060
C 51
Replacing GB/T 18204.2~18204.8-2000, GB/T 18204.11~18204.12-2000
Partially replacing GB/T 17220-1998
Examination Methods for Public Places ?€?
Part 4: Microorganism on a Surface of Public Articles
ISSUED ON: DECEMBER 31, 2013
IMPLEMENTED ON: DECEMBER 01, 2014
Issued by: General Administration of Quality Supervision, Inspection and Quarantine;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 5
2 Terms and Definitions ... 5
3 Plate Counting Method for Total Bacterial Count ... 6
4 Coliform Multi-Tube Fermentation Method ... 9
5 Plate Identification Method for Staphylococcus Aureus ... 13
6 Plate Counting Method for Total Fungi Count ... 16
7 Culture Method of Streptococcus Hemolyticus ... 18
Appendix A (Normative) Microbiological Sampling Method from Public Articles in Public Places ... 22
Examination Methods for Public Places ?€?
Part 4: Microorganism on a Surface of Public Articles
1 Scope
This Part of GB/T 18204 specifies the sampling and test methods for the total bacterial count, total fungi count, coliforms, staphylococcus aureus and streptococcus hemolyticus of public articles in public places.
This Part is applicable to the test of the total number of bacteria, total bacterial number, total fungi count, coliforms, staphylococcus aureus and streptococcus hemolyticus of public articles in public places, and other places can be implemented by reference. 2 Terms and Definitions
For the purposes of this Document, the following terms and definitions apply. 2.1 Total bacterial count
The total number of mesophilic aerobic and facultative anaerobic colonies grown and developed on the nutrient agar medium at 35??C ~ 37??C for 48h after the sampling and processing of the public articles.
2.2 Coliforms
Cultivate gram-negative non-spore bearing bacilli that can ferment lactose, produce acid, produce gas, produce aerobic and facultative anaerobic at 37??C for 24h. 2.3 Staphylococcus aureus
Plasma coagulase-positive Gram-positive Staphylococcus that grows well on Baird Parker medium or blood plate medium, and decomposes mannitol to produce acid. 2.4 Total fungi count
The total number of colonies formed on Rose Bengal or Sabouraud agar medium at 25??C ~28??C for 3d~7d.
2.5 Streptococcus hemolyticus
It belongs to the genus streptococcus and is the gram-positive bacteria that Pork bile salt (or beef and sheep bile salt): 5g
Lactose: 10g
Bromocresol purple aqueous solution (mass concentration=0.04%): 25mL
Distilled water: 1000mL
Preparation method: Dissolve peptone, bile salt and lactose in distilled water; adjust the pH to 7.4; add bromocresol purple solution; and mix well. Then dispense into test tubes with inverted tubes, 10 mL per tube. After autoclaving at 68.96kPa (115??C, 10 lb) for 20min.
NOTE: The other compositions in the double-material lactose bile salt fermentation tube has doubled except distilled water.
4.12 Eosin-methylene blue agar
Compositions:
Peptone: 10g
Lactose: 10g
Dipotassium hydrogen phosphate: 2g
Agar: 17g
Eosin aqueous solution (mass concentration = 2%): 20mL
Methylene blue aqueous solution (mass concentration=0.5%): 10mL
Distilled water: 1000mL
Preparation method: Dissolve peptone, phosphate and agar in distilled water; adjust the pH to 7.2; and dispense into flasks. After autoclaving at 68.96kPa (115??C, 10 lb) for 20min, add lactose and heat to melt the agar before use; cool to 50??C ~55??C; add eosin and methylene blue solution; shake well; and pour into the plate. 4.1.3 Lactose fermentation tube
Compositions:
Peptone: 20g
Lactose: 10g
Bromocresol purple aqueous solution (mass concentration=0.04%): 25mL
5.3.4 Pick typical colonies for smear staining microscopy, which are gram-positive and arranged in grapes.
5.3.5 Plasma coagulase test: Pipette 0.5mL of 1:4 fresh plasma into a sterile cuvette; and then add 0.5mL of 24h broth culture of the bacteria to be tested. Mix well, put it in a 36??C??1??C incubator or water bath; and observe once every 30min. If there is a clot within 24h, it is positive. At the same time, 0.5 mL of broth culture and broth medium of known plasma coagulase positive and negative strains are respectively added to a sterile cuvette and mixed with 0.5 mL of 1:4 plasma as a control.
5.4 Result report
Where there is suspicious colony growth on the above-mentioned selected plate, the staining microscopic examination proves that it is Gram-positive staphylococcus, and the plasma coagulase test is positive, the the detection of staphylococcus aureus can be reported.
6 Plate Counting Method for Total Fungi Count
6.1 Medium and reagents
6.1.1 Normal saline
See 3.1.1.
6.1.2 Rose Bengal medium
Compositions:
Peptone: 5g
Glucose: 10g
Potassium dihydrogen phosphate: 1g
Magnesium sulfate (MgSO4 ?€? 7H2O): 0.5g
Agar: 20g
Chloramphenicol: 0.1g
Distilled water: 1000mL
Rose Bengal aqueous solution (mass concentration=1???3000): 100mL
Preparation method: Dissolve peptone, glucose, potassium dihydrogen phosphate, magnesium sulfate and agar in distilled water; then add Rose Bengal solution; Minced beef: 500g
Sodium chloride: 5g
Peptone: 10g
Dipotassium hydrogen phosphate: 2g
Distilled water: 1000mL
Preparation method: Add 500g of minced fascia-free fat-free beef to 1000mL of distilled water; mix them and put in the refrigerator overnight. Remove the slick oil on the liquid surface; and boil it in water for 30min to completely condense the meat residue into a block; filter it with a flannel; and squeeze it and collect all the filtrate under pressure; and add water to make up the original amount. Add peptone, sodium chloride and phosphate; adjust the pH to 7.4~7.6 after dissolving; boil and filter; add 1% glucose; autoclave at 121??C for 15min.
7.1.2 Pick's Broth
Compositions:
Tryptone's beef heart infusion (mass concentration = 1%): 200mL
Crystal violet saline solution (mass concentration=0.04%): 10mL
Sodium trinitride solution (mass concentration = 0.125%): 10mL
Defibrinated rabbit blood (or sheep blood): 10mL
Preparation method: The above-mentioned sterilized ingredients are mixed in sequence by aseptic operation and stored in the refrigerator for later-use. 7.1.3 Blood agar: see 5.1.4.
7.1.4 Potassium oxalate human plasma: Put 0.01g of potassium oxalate into a sterile cuvette; then add 5 mL of human blood; mix well. Centrifuge for precipitation, and pipette the supernatant to obtain potassium oxalate human plasma.
7.1.5 Calcium chloride (mass concentration = 0.25%).
7.1.6 Sterilized normal saline (mass concentration = 0.85%).
7.1.7 Gram staining solution: see 4.1.4.
7.2 Apparatus
7.2.1 Autoclave steriliser.
7.2.2 Constant temperature water bath: 36??C??1??C.
7.2.3 Microscope.
7.2.4 Electric stove or microwave oven.
7.2.5 Sterile test tubes, plates (diameter of 9cm), graduated pipettes, etc. 7.2.6 Glass slides.
7.2.7 Homogenizer.
7.2.8 Centrifuge.
7.2.9 Balance.
7.3 Operation procedures
7.3.1 See Appendix A for sampling method.
7.3.2 Take 1mL of liquid test sample and add 9mL of glucose meat infusion broth; or directly streak inoculation it on the blood plate. If the test sample is contaminated severely, the pick?€?s broth shall be inoculated with the same amount; cultivate for 24h at 36??C??1??C; inoculate the blood plate; place at 36??C??1??C and cultivate for 24h. Pick up small colonies with haemolytic circles; separate them on the blood plate; and observe the haemolysis and Gram staining.
7.3.3 Morphology and staining: The bacterium is spherical or oval, with a diameter of 0.5??m~1??m; and is arranged in a chain. The chain length varies, the shorter one is composed of 4~8 cells, while the longer one is composed of 20~30 cells. The length of the bacterium is often related to the type of bacteria and the growth environment; long chains are easy to appear in liquid culture; short chains are often present in solid medium, without spore formation or flagella, and inability to move.
7.3.4 Cultivation characteristics: The bacteria have high nutritional requirements; and they do not grow well on ordinary culture medium. They grow well in medium supplemented with blood and serum. When streptococcus hemolyticus grows in serum broth, the bottom of the tube is flocculent or granular. The colonies on the blood plate are white-grey, translucent or opaque, smooth surface, opalescent, and the diameter is about 0.5mm~0.75mm. It is a small round protruding colony. There is a haemolytic circle around the streptococcus hemolyticus.
7.4 Result report
It is a grey-white colony on the blood plate; the colony is transparent or translucent, the surface is smooth with opalescence; and the microscopic examination is gram- positive non-spore cocci, round or oval, in a chain-like arrangement; and there is a Appendix A
(Normative)
Microbiological Sampling Method from Public Articles in Public
Places
A.1 Scope
This appendix specifies the basic requirements for the microbiological sampling method from public articles in the public places.
A.2 General requirements
Randomly select the public articles to be used after cleaning and disinfection; aseptically operate; use sterile dry cotton swabs to infiltrate in 10mL of sterile normal saline (absorb about 1mL of solution). And apply evenly back and forth on the appropriate parts of the articles for the sample collection; and then cut off the part of the cotton swab touched by the hand with sterilized scissors; put the cotton swab into the remaining 9 mL of normal saline; and sent it for inspection within 4h. A.3 Collection location and sampling area
A.3.1 Cup
Sampling in a circle at the contact point between the inner and outer edges of the tea set and the mouth, that is, at a height of 1cm~5cm. The total sampling area is 50cm2. A.3.2 Cotton fabrics
A.3.2.1 Evenly smear for 5 times on the central 5cm??5cm (25cm2) area of the two sides of the towel, pillow towel, and bath towel after folding in half. The sampling area of every 25cm2 is 1 sample; and a total of 2 samples are collected for each article. A.3.2.2 Evenly smear for 5 times on the upper and lower parts of the bed sheet and sheet, that is, the area of 5cm??5cm (25cm2) in contact with the neck and feet; and the sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.2.3 Randomly select two areas of 5cm??5cm (25cm2) on pyjamas and pyjama trousers to respectively and evenly smear for 5 times. The sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.3 Sanitary ware
A.3.3.1 Bathtub: Respectively smear and sample within a half of the height of the side wall of the basin and 5cm??5cm (25cm2) in the bottom centre of the basin; and the sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.3.2 Face (feet) basin: Respectively smear and sample within a half of the height of the basin with respect to the two side walls within the range of 5cm??5cm (25cm2); and the sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.3.3 Toilet pan: Respectively smear and sample two 5cm??5cm (25cm2) areas on the front bend of the toilet circle; and the sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.3.4 Massage bed (chair): Respectively smear and sample two 5cm??5cm (25cm2) areas in the middle of the bed (chair) surface; and the sampling area of 25cm2 is 1 sample; and a total of 2 samples are collected for each article.
A.3.4 Footwear
Evenly smear for 5 times in the area of 5cm??5cm where the toe of each shoe is in contact with the toe. One pair of shoes is one sample; and the total sampling area is 50cm2.
A.3.5 Shopping cart (basket)
Respectively and evenly smear for 5 times in the two 5cm??5cm areas on the handle of the car (basket). One article is one sample, and the total sampling area is 50cm2. A.3.6 Beauty salon and nail products
A.3.6.1 Hair-cut clipper: Respectively and evenly smear for 3 times on the front part of the clipper up and down; when the sampling area reaches 25cm2, it shall be regarded as 1 sample.
A.3.6.2 Hair-cut knife and scissors: Respectively smear for 1 time on both sides of the knife and scissors; when the sampling area reaches 25cm2, it shall be regarded as 1 sample.
A.3.6.3 Beauty and manicure products: Smear and sample at the contact point with the human body; when the sampling area reaches 25cm2, it shall be regarded as 1 sample.
A.3.6.4 Pedicure tool: Smear the sample at the contact point between the pedicure tool and the human body, when the sampling area reaches 50cm2, it shall be regarded as 1 sample.
A.3.7 Other articles

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