GB/T 18204.3-2025 PDF English
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Examination methods for public places - Part 3: Airborne microorganism indicators
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GB/T 18204.3-2013 | English | 70 |
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Examination methods for public places - Part 3: Airborne microorganism
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Methods of microbiological examination for tea set in public places. Determination of coliform bacteria
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GB/T 18204.3-2025: Examination methods for public places - Part 3: Airborne microorganism indicators ---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GBT18204.3-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 13.060
CCS C 51
Replacing GB/T 18204.3-2013
Examination methods for public places -- Part 3.Airborne
microorganism indicators
Issued on: MAY 30, 2025
Implemented on: DECEMBER 01, 2025
Issued by. State Administration for Market Regulation;
Standardization Administration of the People's Republic of China.
Table of Contents
Foreword... 3
Introduction... 5
1 Scope... 6
2 Normative references... 6
3 Terms and definitions... 6
4 Total bacterial count... 7
5 Total fungal count... 11
6 β-hemolytic streptococcus... 14
7 Legionella pneumophila... 18
Annex A (normative) Requirements for on-site sampling and testing points... 29
Bibliography... 31
Examination methods for public places -- Part 3.Airborne
microorganism indicators
1 Scope
This document describes the on-site sampling and testing methods for the total number
of bacteria, fungi, β-hemolytic streptococcus, and Legionella pneumophila in the air of
public places.
This Part is applicable to the determination of air microbiological indicators in public
places. Refer to this document for other locations.
2 Normative references
The following referenced documents are indispensable for the application of this
document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
GB 4789.28, National food safety standard -- Food Microbiology Testing -- Quality
Requirements for Medium and Reagents
GB/T 6682, Water for analytical laboratory use -- Specification and test methods
GB 19489, Laboratories -- General requirements for biosafety
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1 total bacterial count
The total number of mesophilic aerobic and facultative anaerobic colonies grown and
developed on nutrient agar medium after 48 h of cultivation at 36℃± 1℃.
3.2 total fungal count
The number of colonies formed by culturing on Sabouraud agar medium at 28℃± 1℃
for 3~5 d.
3.3 β-hemolytic streptococcus
Streptococcus pyogenes (or Group A) and Streptococcus agalactiae (or Group B) that
can produce hemolysin and form clearly defined and completely transparent hemolytic
rings (β - type hemolysis) around bacterial colonies on a blood plate.
[Source. GB 4789.11-2014,2.2, modified]
3.4 Legionella pneumophila
A Gram-negative bacterium with blunt ends, flagella, no spores or capsules,
characterized by growth on an activated carbon yeast extract medium containing L-
cysteine and trivalent iron salt buffer. It has been identified as a pathogenic Legionella
bacterium through biochemical and serological tests and is the main pathogen causing
Legionellosis.
[Source. GB/T 40392-2021, 3.2, modified]
3.5 impacting method
A sampling method that uses an impact type air microbial sampler to collect
microorganisms suspended in the air onto the sampling medium through inertial impact.
3.6 natural sinking method
A sampling method that involves exposing the culture medium plate to air and allowing
microorganisms to naturally settle onto the plate by gravity.
4 Total bacterial count
4.1 Impact method
4.1.1 Principle
Use an impact type air microbial sampler. Generate high-speed airflow by passing air
through narrow slits or small holes. Collect microorganisms suspended in the air onto
nutrient agar plates. The bacterial colony count was obtained after 48 h of cultivation
at 36℃± 1℃.
4.1.2 Instruments and consumables
The main instruments and consumables used in this method are as follows.
- Six level sieve impact microbial sampler;
- High pressure steam sterilizer;
- Constant temperature incubator. 36℃± 1℃;
- Aseptic petri dish. diameter of 90 mm;
- pH meter or precision pH test paper.
4.1.3 Nutrient agar medium
4.1.3.1 Composition
Protein peptone. 10.0 g. Beef extract. 3.0 g. Sodium chloride. 5.0 g. Agar. 20.0 g. Pure
water. 1000 mL. Finished culture medium that meets the requirements of GB 4789.28
can also be used. The quality of pure water should meet the requirements of Grade
Three water or above in GB/T 6682.
4.1.3.2 Preparation method
Dissolve peptone, beef extract, and sodium chloride in pure water. Correct the pH to
7.4 ± 0.2.Add agar. Perform high-pressure sterilization at 121℃ for 15 min. When
cooled to 45℃~50℃, pour 15mL of culture into each dish based on sterile operation.
After cooling, make it into a flat plate for later use.
4.1.4 Sampling
4.1.4.1 Sampling points. shall comply with the requirements of A.1 in Annex A.
4.1.4.2 Sampling environmental conditions. Close doors and windows for 15~30 min
during sampling. When sampling in public places where it is difficult to close doors and
windows (such as shopping malls, waiting rooms, etc.), the environmental conditions
at that time should be maintained. Record the operation status of indoor air conditioning
and other equipment, door and window conditions, number of personnel, temperature
and humidity, and weather conditions.
4.1.4.3 Sampling method. Aseptic operation should be performed. Load the nutrient
agar plates step by step into a six-stage sieve impact microbial sampler. Collect data at
a flow rate of 28.3 L/min for 5~15 min. The sampler should be used according to the
instructions.
4.1.4.4 Storage and transportation of samples. Store the collected tablets in an
environment of 4℃. Return to the laboratory for cultivation within 4 h.
4.1.5 Inspection steps
Invert the collected nutrient agar plates and incubate them in a 36℃± 1℃ incubator for
48 h ± 2 h. Perform colony counting.
4.1.6 Inspection results
4.1.6.1 Result calculation
The total bacterial concentration in the air of public places is calculated according to
They shall meet the requirements of 4.1.5.
4.2.6 Inspection results
4.2.6.1 Result calculation
Count the number of bacterial colonies growing on each plate.
4.2.6.2 Result report
The measurement results of the total number of bacteria in the air of a region are
reported as the average of the total number of bacteria measured at all sampling points
in the region. The test results are expressed in colony forming units per plate
(CFU/plate). The calculated total number of bacteria should be rounded to the nearest
integer.
4.2.7 Quality control
It should meet the requirements of 4.1.7.1~4.1.7.5.
5 Total fungal count
5.1 Impact method
5.1.1 Principle
Use an impact type air microbial sampler. Generate high-speed airflow through narrow
slits or small holes to collect microorganisms suspended in the air onto a Sabouraud
agar plate. The fungal colony count was obtained after culturing at 28℃±1℃ for 3~5
d.
5.1.2 Instruments and consumables
The instruments and consumables used in this method are as follows.
- Six level sieve impact microbial sampler;
- High pressure steam sterilizer;
- Fungal incubator. 28℃± 1℃;
- Aseptic petri dish. diameter of 90 mm;
- pH meter or precision pH test paper.
5.1.3 Sabouraud agar medium
5.1.3.1 Composition
Ni - The number of colonies per level of plate, in colony forming units (CFU);
v - Sampling flow rate, in liters per minute (L/min);
t - Sampling time, in minutes (min);
i - Number of tablets.
5.1.6.2 Result report
The determination of total fungal count in the air of a region is reported based on the
maximum value of total fungal count measured at all sampling points in that region.
The test results are expressed in colony forming units per cubic meter (CFU/m3).
5.1.7 Quality control
5.1.7.1 After each batch of culture medium plates is configured, a sterile test should be
conducted. Three tablets can be selected per batch. Cultivate and observe according to
the steps in 5.1.5.The result should be free of bacterial growth.
5.1.7.2 Other quality controls shall comply with the requirements of 4.1.7.2~4.1.7.5.
5.2 Natural sinking method
5.2.1 Principle
Expose the Sabouraud agar plate to air. Microorganisms rely on gravity to naturally
settle onto a flat surface. After laboratory cultivation, the total fungal count shall be
obtained.
5.2.2 Instruments and consumables
The instruments and consumables used in this method are as follows.
- High pressure steam sterilizer;
- Fungal incubator. 28℃± 1℃;
- Sampling bracket;
- Aseptic petri dish. diameter of 90mm;
- pH meter or precision pH test paper.
5.2.3 Sabouraud agar medium
It shall meet the requirements of 5.1.3.
5.2.4 Sampling
5.2.4.1 Sampling points. should meet the requirements of A.2.
5.2.4.2 Sampling environmental conditions. shall comply with the requirements of
4.1.4.2.
5.2.4.3 Sampling method. Aseptic operation should be performed. Place the Sabouraud
agar plate at the sampling point. Open the lid of the dish. Expose for 5 min and cover
the dish with a lid.
5.2.4.4 Sample transportation and storage. shall comply with the requirements of
4.1.4.4.
5.2.5 Inspection steps
They should meet the requirements of 5.1.5.
5.2.6 Inspection results
5.2.6.1 Result calculation
Count the number of fungal colonies growing on each plate.
5.2.6.2 Result report
The measurement results of total fungal count in the air of a region are reported as the
average of total fungal count measured at all sampling points in the region. The test
results are expressed in colony forming units per plate (CFU/plate). The calculated total
fungal count should be rounded to the nearest integer.
5.2.7 Quality control
It should comply with the requirements of 5.1.7.1 and 4.1.7.3~4.1.7.5.
6 β-hemolytic streptococcus
6.1 Principle
Use an impact type air microbial sampler. Generate high-speed airflow through narrow
slits or small holes to collect microorganisms suspended in the air onto a blood agar
plate. After culturing at 36℃± 1℃ for 18~24 h, counting and biochemical identification
are performed to obtain the bacterial count of β-hemolytic streptococcus.
6.2 Instruments and consumables
The instruments and consumables used in this method are as follows.
- Six level sieve impact microbial sampler;
7.1.3.2 BCYE-Cys culture medium
The preparation method is the same as BCYE medium plate, except that L-cysteine
hydrochloride is not added.
7.1.3.3 GVPC medium
NOTE. This culture medium is based on BCYE supplemented with three antibiotics
and glycine.
7.1.3.3.1 GVPC additives
0.3 g/L glycine, polymyxin sulfate B 80000 IU/L, 0.001 g/L vancomycin, 0.08 g/L
actinomycin.
WARNING. Actinobacillone has hepatotoxicity. Gloves and dust masks should be
worn when handling powdered chemical products.
7.1.3.3.2 Preparation of GVPC additives
Dissolve an appropriate amount of polymyxin B sulfate (usually 200 mg) in 100 mL of
pure water to a concentration of 14545 IU/mL. After mixing, perform membrane
filtration for sterilization. Use sterile tubes for subpackaging, 5.5 mL per tube. Store at
-25℃~-15℃. Use at room temperature before reusing.
Dissolve 20 mg of vancomycin hydrochloride in 20 mL of pure water. After mixing,
perform membrane filtration for sterilization. Use sterile tubes for subpackaging. Store
1 mL per tube at -25℃~-15℃. Use at room temperature before reusing.
Dissolve 2 g of actinomycete ketone in 100 mL of pure water. After mixing, perform
membrane filtration for sterilization. Use sterile tubes for subpackaging, 4 mL per tube.
Store at -25℃~-15℃. Use at room temperature before reusing.
The maximum freezing storage time for the above antibiotic additives is 6 months.
7.1.3.3.3 Preparation of GVPC culture medium
Prepare according to the BCYE culture medium preparation method. After adding the
single potassium salt of α-ketoglutarate, add 3 g of glycine. Adjust the pH to 6.8 ± 0.2.
After adding dissolved L-cysteine and iron pyrophosphate, three additional antibiotic
additives are added. Mix well.
7.1.3.4 Gram staining solution
It shall meet the requirements of 6.3.2.
7.1.3.5 Malaurate biochemical reaction tube
pH should be 6.9 ± 0.1.
7.1.3.9 Sampling absorption solution
Weigh 12.0 g of yeast extract and add pure water to 1000 mL. Sterilize under high
pressure at 121℃ for 15 min. Subpack in sterilized centrifuge tubes for later use.
7.1.3.10 Oxidase reagents
Weigh 1.0 g of tetramethyl p-phenylenediamine hydrochloride. Dissolve it in 100 mL
of pure water for later use.
7.1.3.11 Acid buffer solution
7.1.3.11.1 Hydrochloric acid solution [c (HCl)=0.2 mol/L]
Add 17.4 mL of concentrated hydrochloric acid to 1000 mL of pure water.
7.1.3.11.2 Potassium chloride solution [c (KCl)=0.2 mol/L]
Dissolve 14.9 g of potassium chloride in 1000 mL of pure water.
7.1.3.11.3 Preparation of acid buffer solution
Take 3.9 mL of hydrochloric acid solution [c (HCl)=0.2 mol/L] and 25 mL of potassium
chloride solution [c (KCl)=0.2 mol/L]. After mixing, adjust with 1 mol/L potassium
hydroxide. Measure the final pH to 2.2 ± 0.2 using precision pH test strips or pH meters.
Place in a glass bottle with a stopper. The solution should be kept away from light at
room temperature for no more than one month.
7.1.3.12 5% (hydrated) indanone solution
Dissolve 1.75 g of (hydrated) indanone in a mixture of 25 mL of acetone and 25 mL of
butanol. The solution should be refrigerated in the dark for a maximum of 7 days.
7.1.3.13 Diagnosis serum for Legionella pneumophila
Commercialized Legionella pneumophila diagnostic serum can be used.
7.1.4 Sampling
7.1.4.1 Sampling points. shall comply with the requirements of A.1.
7.1.4.2 Sampling environmental conditions. shall comply with the requirements of
4.1.4.2.
7.1.4.3 Sampling method. Pour 20 mL of sampling absorption solution into the aerosol
sampler. Operate according to the sampler manual. The air volume collected for each
aerosol sample is 2 m3.When using the same aerosol sampler to collect air samples
a CO2 concentration (volume fraction) of 5%. Cultivate at 36℃± 1℃ for 2 d. If there
are different types of suspected Legionella colonies, at least 3 suspected colonies of
each type should be selected for culture validation. Legionella colonies grow on BCYE
but not on BCYE-Cys plates. Record the results of each tablet. Biochemical
identification of Legionella colonies.
7.1.5.6 Biochemical identification
7.1.5.6.1 Bacterial staining. Gram staining microscopy is performed on bacterial
colonies. Observe the morphology and color of bacteria. If the bacterial cells are purple,
they are Gram positive bacteria. If the bacterial cells are red, they are Gram negative
bacteria.
7.1.5.6.2 Oxidase test. Use a platinum/iridium inoculation ring or glass rod to pick up
the test colony and place it on filter paper. Add 1 drop of oxidase reagent. If violet or
dark blue appears within 30 s, it is considered positive; If there is no color change within
2 min, it is considered negative.
7.1.5.6.3 Nitrate reduction test. Inoculate the test bacteria onto nitrate culture medium.
Cultivate at 36℃± 1℃ for 1~3 d. Add 1 drop each of solution A and solution B. Observe
the results. If it turns red immediately or within a few minutes, it is considered positive.
If there is no change in the color of the culture medium, it is considered negative.
7.1.5.6.4 Urease test. Inoculate a large amount of the test bacteria into urea agar medium
through puncture. Cultivate at 36℃± 1℃ for 2 and 24 h. Observe the results separately.
If the culture medium turns red, it is considered positive. If the color remains unchanged,
it is negative.
7.1.5.6.5 Gelatin liquefaction test. Inoculate the test bacteria into gelatin culture
medium by puncture. Cultivate at 36℃± 1℃ for 24 h ± 2 h. Remove and place in a
refrigerator at 4℃± 2℃ for 10~30 min before observing the results. When it is still in
a dissolved state or on the surface, it is considered positive in the gelatin liquefaction
test. Those who are insoluble in coagulation are negative.
7.1.5.6.6 Uric acid hydrolysis test. Select the bacterial colony to be tested. Add it to a
test tube containing 0.4 mL of 1% sodium hippurate to prepare a bacterial suspension.
After mixing evenly, incubate in a water bath at 36℃± 1℃ or in a 36℃± 1℃ incubator
for 24 h. Slowly add 0.2 mL of indanone solution along the wall of the test tube. After
incubating in a 36℃± 1℃ water bath or placing in a 36℃± 1℃ incubator for 10 min,
observe the results. If the solution shows a blue purple change within 15 min, it is
considered positive. If there is no color change in the solution or a blue purple change
occurs after 15 min, it is considered negative. In order to avoid false negatives during
the test, standard bacterial strains are used as positive controls.
7.1.5.6.7 Biochemical culture result determination. Gram negative non spore forming
bacteria detected by staining microscopy, with oxidase (-/weak+), nitrate reduction (-),
urease (-), gelatin liquefaction (+), and hydrolyzed hippuric acid (+), can be confirmed
k - Suspected type of Legionella pneumophila (i=1,2,3,..., k).
7.1.6.2 Result report
Qualitative result report. Any sampling point in a region is positive for Legionella
pneumophila, indicating that the measurement result of Legionella pneumophila in the
air of the region is positive. Otherwise, report 'No Legionella pneumophila detected'.
Quantitative result report. The quantitative detection results of Legionella pneumophila
in the air of a region are given based on the maximum value of Legionella pneumophila
measured at all sampling points in the region.
7.1.7 Quality control
7.1.7.1 The culture medium and reagents should meet the quality requirements of GB
4789.28.The purchased finished culture medium should be used within its validity
period.
7.1.7.2 Before sampling begins, ensure that the reagents and materials used are in a
sterile state. Aseptic operation should be carried out during the operation process and
sample transportation. Avoid human pollution.
7.1.7.3 A positive control should be set up (Legionella pneumophila standard strain
ATCC 33152 or other equivalent standard strains).
7.1.7.4 The sampler should be calibrated regularly under load conditions using a flow
meter that has been verified/calibrated to be qualified. The relative deviation should not
exceed 5%, or be calibrated according to the instructions of the sampler. Before
sampling, the airtightness of the sampling system should be checked to ensure that there
is no air leakage.
7.1.7.5 The personal protection of experimental personnel and the disposal of testing
waste should be carried out in accordance with GB 19489.As Legionella pneumophila
is a pathogenic bacterium, experimental operators should have practical experience in
working in a secondary biosafety laboratory.
7.2 Fluorescent polymerase chain (PCR) rapid detection method
7.2.1 Principle
Specific primers and probes are designed for the highly conserved region of the mip
gene in Legionella pneumophila. In the presence of a Legionella pneumophila genome
template in the reaction system, polymerase chain reaction (PCR) is performed and
fluorescent signals are released. The instrument is used to monitor and output the signal
intensity of the corresponding channel during the PCR process in real time, so as to
achieve qualitative analysis of the test results. It is suitable for initial screening of
Legionella pneumophila and rapid identification of suspicious bacterial colonies.
...... Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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