[Public health microbial sampling. Overview of detection methods] Detection methods of microorganisms in water

First, the detection method of microorganisms in the air-the total number of bacteria

(nutrient agar, 12 1℃ autoclaved for 20min and cultured at 37℃ for 48h)

1.

(1) impact method (two-stage or six-stage microbial sampler)

(2) Natural sedimentation method (exposure for 5min)

Sampling: plum blossom distribution, height:1.2 ~1.5m; Off the wall:1m.

Second, the tea set microbial inspection method

(1) Total number of bacteria

1. Preparation of physiological saline: Dissolve sodium chloride (8.5g) in distilled water (1000mL), subpackage in test tubes, and sterilize each test tube for 20min at 10mL, 12 1℃.

2. Sampling: Wet the cotton swab with normal saline, and apply 50cm2 to the inner and outer edges of the tea set, that is, the contact point between mouth and lips (1~ 1.5cm). Cut off the hand contact point of the cotton swab with sterile scissors, and put the cotton swab into 10mL normal saline for inspection within 4 hours.

3, inspection procedures:

Sample detection → Add two sterile plates (diluted by 10 times in case of serious pollution) to 1mL each → incubate at 37℃ for 48 hours → count colonies → report.

4. Unit: cfu/cm2.

(2) Escherichia coli

1. culture medium: lactose bile salt fermentation broth (double, each tube 10mL,15℃ autoclaved 15min).

2. Detection method: fermentation method (specific content textbook 1 1 page)

3. Sample inspection (the remaining samples are used to determine the total number of bacteria)

4. Gram staining process: (positive bacteria are purple, negative bacteria are red)

(1) Initial dyeing: 1min, washing with water;

(2) Iodine staining: 65438±0min, washed with water;

(3) Decolorization: 30s, water washing;

(4) Staining again: 65438 0 minutes, washing, drying and microscopic examination.

5. Result report: When the lactose fermentation tube finally produces acid and gas, and Gram staining is negative for Bacillus-free, coliforms can be reported to be detected.

Three, towels, bedding microbial detection methods

(1) Total number of bacteria

1. sampling

(1) Towels and pillow towels: after the center of each side is folded by 25cm2;

(2) Bedsheet, Bedsheet: Apply 25cm2 back and forth to the center of the upper and lower parts.

2. The detection procedure is the same as the total number of bacteria in the tea set.

3. Calculation unit: cfu/25cm2.

(2) coliform bacteria (detection procedure is the same as tea set)

1. daubing method: (the collected samples are used to determine the total number of bacteria)

Four, beauty equipment microbial inspection methods

(a) Escherichia coli

1, sampling

Fader: Apply evenly on the front of the fader for three times;

Barber knives, scissors and pedicure tools: paint a sample on both sides of the used knives and scissors; Two knives or two scissors are cut into a sample.

2.5mL samples were poured into a double lactose bile salt fermentation tube and cultured at 37℃ for 24h； hours.

3. Isolation and culture: Inoculating eosin methylene blue plate, and microscopic examination with Gram staining;

4. Confirmation test: Inoculate lactose fermentation tube at 37℃ for 24h, and observe the gas production.

5. Results Report: Gram-negative Bacillus-free bacteria can be reported as positive Escherichia coli.

(2) Staphylococcus aureus

1. medium: 7.5% sodium chloride broth or tryptone soy broth medium.

2. Step:

(1) Pour 5mL of the remaining coliforms into the culture medium and cultivate at 37℃ for 24h； hours;

(2) Inoculated with blood plate, cultured at 37℃ for 24 hours to observe the morphology: round, golden yellow, convex, smooth surface, surrounded by hemolysis circle;

(3) Microscopic examination of selected colonies: Gram-positive (red), arranged like grapes;

(4) Mannitol fermentation test: Inoculate in mannitol fermentation medium, and culture at 37℃ for 24h to observe whether acid is produced;

(5) Plasma coagulase test

Slide method: drop a drop of physiological saline at one end and a drop of plasma at the other end on both sides of a clean slide, inoculate ringed colonies and mix them respectively. Those without coagulation after 5min minutes were negative; If there is a granular clot in plasma, but not in normal saline, it is positive.

Verb (abbreviation of verb) Method for microbiological examination of slippers

Mould and yeast

(Glass beads should be added to normal saline)

1. medium: mold medium, (tiger red) Bengal red medium, potato glucose agar medium (PDA).

2. Sampling: evenly apply a cotton swab to the area of 5cm×5cm where the upper of each slipper contacts the toes, three times, and one pair of slippers is a sample;

3. Shake the test tube 100 times in the palm of your hand with a cotton swab to separate mold spores;

4. Culture: culture in a mold incubator at 25~28℃ for one week.

5. The unit is cfu/50cm2.

Six, bathtub, noodles (feet) basin microbial detection

(1) Total number of bacteria

Sterile saline: bathtub sampling 125mL/ bottle; 50 ml is used for basin sampling. Sampling location: within the basin 1/2 to 1/3 height;

Distribution: Plum blossom distribution in bathtub; Face (foot) basins are distributed on two opposite side walls;

Methods: daubing method (consistent with tea set method)

Unit: cfu/25cm2.

(2) coliform bacteria (same as tea set)

Seven, swimming pool water microbial detection

(1) Total number of bacteria

1. Sampling bottle: acid-free, alkali-free and nontoxic glass container.

2. Before sterilization, add enough 10% sodium thiosulfate solution, and generally add 0. 1mL to 125mL water sample.

Step 3: Sampling

4. Operating steps (same as the total number of bacteria in tea set)

(2) Escherichia coli

1. culture medium: triple concentrated lactose peptone culture medium (distilled water unchanged, other components three times).

Second step

(1) speculative test

Add 100mL water sample into two large test tubes (with small test tubes) filled with 50mL triple concentrated lactose peptone culture solution; Add 10 ml water sample into 10 test tube (with small test tube) containing 5mL triple concentrated lactose peptone culture medium. Shake well and cultivate.

(2) Plate separation

Inoculated with eosin methylene blue, cultured and observed; Typical colony morphology: deep purple or reddish purple with metallic luster.

(3) Re-fermentation experiment

Gram staining, lactose fermentation tube inoculation, culture, observation.

(4) Look-up table: MPN value of coliforms in 1000mL water sample.

Eight, drinking water microbial inspection methods

(1) sampling

1. The sampling container shall be made of clean, colorless and nontoxic polyethylene plastic and hard glass (also called borosilicate glass). Sample bottles must be specially designed for special use, and bottles filled with concentrated solution in the laboratory shall not be used as sample bottles. Usually we use 500ml autoclaved flask for sampling.

2 containers, bottle stoppers and bottle caps should be able to withstand sterilization temperature.

All items used in the inspection must be completely sterilized. Before sterilization, it should be thoroughly cleaned, autoclaved at 12 1℃ for 20min, or dried at 160℃ for 2h.

4. Refrigerate and test within 4 hours. If there is free residual chlorine, add 0.65438±0ml sodium thiosulfate solution every 65438±025ml before disinfection of the sampling bottle.

(2) Total number of bacteria (the operation steps are the same as those of the tea set, remember to make a blank control)

(III) Total coliforms

1. Lactose fermentation test

(1) Add 10mL water sample into 10mL double lactose fermentation tube; Add 1 ml water sample into 10 ml single lactose fermentation tube; Add 0. 1 ml water sample into 10 ml single lactose fermentation tube; Inoculate 5 test tubes at each dilution.

(2) Culture at 37℃ for 24 hours, and observe the phenomenon of acid production and gas production.

2. Isolation and culture (inoculation with eosin methylene blue, culture)

3. Confirmation experiment (Gram staining, microscopic examination, lactose inoculation fermentation tube)

4. Registration of results: look-up table.

(4) Thermostable Escherichia coli

1. medium: EC medium.

2. Operation steps: Escherichia coli multi-tube fermentation.

3. Culture temperature: 44.5℃ for 24h.

(5) Escherichia coli

1. medium: EC-MUG medium (after the medium is heated and dissolved, it is necessary to check whether there is fluorescence under 366nm violet light).

2. Culture temperature: 44.5℃ for 24h.

3. Observation: whether there is blue fluorescence when irradiated with 366nm ultraviolet lamp in the dark;

4. Calculate the number of positive test tubes and look up the table to get the most possible number. The result was reported as MPN/ 100 ml.

Nine, public places central air conditioning system

(1) Legionella pneumophila in condensed water and cooling water

(1) sampling

1. Sampling container: glass bottles, polyethylene bottles and wide-mouth bottles can be selected and disinfected before use.

2. Sampling quantity: According to aseptic operation, take about 500ml water sample (or sediment, sludge and other samples) from each sampling point.

3. Neutralization: For the samples disinfected by chlorine or ozone, before sterilization, add sodium thiosulfate solution into the sampling container for neutralization.

4. Transport and storage of samples: It is best to send the samples to the laboratory within 2 days, and do not freeze them, but keep them away from light and heat, and keep them at room temperature for no more than 15 days.

(2) Heat treatment: take 1 ml eluted sample and heat it in a 50℃ water bath for 30 minutes.

Acid treatment: Take 5ml of eluted sample, adjust the pH to 2.2, gently shake and leave it for 5min.

(3) The inoculation plate is placed in a CO2 incubator with a concentration of 2.5% at 35~37℃. When the culture was observed, the plate was turned over, incubated for 65438±00d days, and kept moist.

(4) observation

Legionella grows slowly and is easily covered by other bacteria, so it needs to be observed under a stereoscopic microscope every day. Legionella colonies have various colors, usually white, gray, blue or purple, but also dark brown, gray green and deep red; The colony is neat and the surface is smooth, which is a typical ground glass. Under ultraviolet light, some have fluorescence.

(2) Total number of bacteria in air supply

In aseptic operation, a six-stage screen air impact sampler was used, the air velocity was 28.3L/min, and the sampling time was 5~ 15min. After the bacteria were collected, they were cultured on nutrient agar plates at 35~37℃ for 48 h, and the number of colonies was counted.

(3) Total number of fungi in air supply

The sampling method is as above. The collected fungi were cultured in Shack agar medium at 28℃ for 5 ~ 7 days, and the results were observed every day and recorded on the 7th day. If there are too many fungi, the results can be counted on the fifth day, and the culture time can be recorded and converted into cfu/m3.

(4) β -hemolytic streptococcus in gas source

On the blood agar plate, tiny colonies with gray and convex surface and diameter of 0.5mm~0.7mm are formed, which are transparent or translucent, smooth and milky, and there are hemolytic rings around the colonies. Microscopically, it is Gram-positive Bacillus, round or oval, arranged in a chain.

(5) Microbial inspection on the inner surface of air duct

Sampling area: The sampling area of each point should be 50cm2.

Sampling methods: scraping method and wiping method.

The culture and counting methods of bacteria and fungi are the same as above.