MZ/T 140-2019Technical specifications for examination of pathogenic bacteria in funeral home (English PDF)

4.1 Detection method

The total number of bacteria in the air at the funeral site was determined by impact method or natural sedimentation method sampling, nutrient agar medium culture count.

4.2 Impact method

4.2.1 Instruments and equipment

Six-stage sieve impact air microbiological sampler, high-pressure steam sterilizer, constant temperature incubator, refrigerator, flat dish (90 mm diameter).

4.2.2 Medium

4.2.2.1 Ingredients of nutrient agar medium.

Peptone 10.0 g

Beef paste 3.0 g

Sodium chloride 5.0 g Agar 15.0 g 1000 mL of distilled water

4.2.2.2 Preparation method. after mixing the above ingredients, heat and dissolve, adjust the pH value to 7.2 ~ 7.6, set the volume to 1000 mL, and pressurize at 121 °C Sterilize for 20 min. Pour the sterilized nutritional agar into a petri dish with a diameter of 90 mm in 15 mL, and set it at 4 °C for use.

4.2.3 Sampling

4.2.3.1 Sampling points

Set the sampling point according to the size and location of the room. The sampling layout method is. when the indoor area is <=30 m2, set 3 points of the inner, middle and outer diagonals, the inner and outer points are located 1 m away from the wall; when the indoor area is > 30 m2, 4 corners and 5 points in the center are set, and the 4 corners are located 1 m away from the wall.

The sampling point is 1.2 m to 1.5 m above the ground.

4.2.3.2 Sampling method

When sampling, place the six-stage sieve impact microbiological sampler at each sampling point, the flowmeter is set to 28.3 L/min, and the sampling time is 5 min ~ 15 minutes, cover the dish immediately after sampling.

4.2.4 Inspection steps

The nutrient agar plate after collecting bacteria was placed in a 36 ° C ± 1 ° C incubator, and the colonies were counted after 48 h of culture.

After the test sampling is completed, the same batch of unused medium should be cultured at the same time as the above test sample or cultured after inoculation, as negative Contrast. If there is bacterial growth in the negative control group, it means that the culture medium used is contaminated and the test is invalid.

Retest.

4.2.5 Results Report

4.2.5.1 Calculation of the total number of bacteria at the sampling point. count the colonies, record the results and calculate the total number of bacteria in the air according to the following formula.

In the formula.

Q-the total number of bacteria in the air, in units of colony forming units per cubic meter (cfu/m3);

N-the total number of colonies on the plate, the unit is colony forming unit (cfu);

L-gas production per minute, the unit is liter per minute (L/min);

T-Sampling time in minutes (min).

4.2.5.2 Measurement results of total bacteria in a region. The measurement results of total bacteria in the air in a region are determined according to the bacteria in all sampling points in the region.

The maximum of the total measured values is given.

4.3 Natural settlement method

4.3.1 Instruments and equipment

Autoclave, thermostatic incubator, plate (90 mm diameter), sampling rack.

4.3.2 Medium

See 4.2.2.

4.3.3 Sampling

4.3.3.1 Sampling point. see 4.2.3.1.

4.2.3.1 Sampling method. Place the nutrient agar plate at the sampling point, open the lid of the dish, and expose for 5 min.

4.3.4 Inspection steps

See 4.2.4.

4.3.5 Results Report

Count the number of colonies growing on each plate, find the average number of colonies at all sampling points, and check the results as the number of colonies per plate (cfu /Dish) given.

5.1 Detection methods

Sampling method and blood agar culture medium were used to determine beta-hemolytic streptococci in funeral sites.

5.2 Instruments and equipment

See 4.2.1.

5.3 Medium

5.3.1 Blood agar plate composition.

Peptone 10.0 g

Beef paste 3.0 g

Sodium chloride 5.0 g Agar 20.0 g Defibrillated sheep blood 5 mL ~ 10 mL 1000 mL of distilled water

5.3.2 Preparation method. Peptone, sodium chloride, and meat paste are heated and dissolved in distilled water, the adjusted pH value is 7.4 ~ 7.6, agar is added, and 121 °C Autoclave for 20 min. After cooling to 50 ° C, add defibrinated sheep blood aseptically, and shake well.

5.4 Sampling

See 4.2.3.

5.5 Inspection steps

5.5.1 Culture method. The blood agar plate after sampling is cultured at 36 °C ± 1 °C for 24 h to 48 h.

5.5.2 Observation of results. After culture, off-white colonies with small diameters ranging from 0.5 mm to 0.7 mm were formed on blood agar plates.

The colonies are transparent or translucent, and the surface is smooth and opalescent; microscopic examination is Gram-positive Bacillus aureus, round or oval, arranged in a chain, Influenced by culture and operating conditions, the length of the chain is between 4-8 cells or dozens of cells; there are obvious 2 mm-4 mm around the colony A well-defined, completely transparent, colorless hemolytic ring. Those that meet the above characteristics are beta-hemolytic streptococci.

5.6 Results Report

5.6.1 Calculation of hemolytic streptococcus results at the sampling point. colony count, record the results and calculate beta-hemolytic streptococci in the air according to the following formula Total number of colonies.

In the formula.

A-Total number of hemolytic streptococcus colonies in the air, in units of colony forming units per cubic meter (cfu/m3);

N-the total number of colonies on the plate, the unit is colony forming unit (cfu);

L-gas production per minute, the unit is liter per minute (L/min);

T-Sampling time in minutes (min).

5.6.2 Measurement results of hemolytic streptococcus in a region. The measurement results of hemolytic streptococcus in a region are based on all sampling points in the region the maximum of the beta-hemolytic streptococcus measurements is given.

6.1 Detection method

The method for measuring the coliform on the appliance is a multi-tube fermentation method.

6.2 Media and reagents

6.2.1 Normal saline

Sodium chloride 9.0 g 1000 mL of distilled water Method. Weigh 9.0 g of sodium chloride in 1000 mL of distilled water and aliquot it into test tubes. Each tube is 10 mL and autoclaved at 121 ° C for 20 min.

6.2.2 Nutrition Agar

See 4.2.2.1.

6.2.3 Lactose bile salt culture solution

Peptone 20.0 g

Pig bile salt 5.0 g

Lactose 5.0 g

0.4% bromocresol purple aqueous solution 2.5 mL 1000 mL of distilled water Method. Dissolve peptone, pig bile salt and lactose in 1000 mL of distilled water, adjust pH to 7.2 ~ 7.5, add indicator, fully Mix well and aliquot into test tubes containing small tubes (inverted tubes). Autoclave at 115 °C for 30 min and store in a cold and dark place for later use.

6.2.4 Eosin blue medium (EMB medium)

Peptone 10.0 g

Lactose 10.0 g

Dipotassium hydrogen phosphate 2.0 g Agar 17.0 g 20% eosin aqueous solution 20 mL 13% 0.5% US blue solution in water 1000 mL of distilled water Method. Add agar to 900 mL of distilled water, heat to dissolve, then add dipotassium hydrogen phosphate and peptone, mix well to dissolve, and then Add distilled water to make up to 1000 mL, and adjust the pH to 7.2 ~ 7.5. Filter with absorbent cotton and gauze while hot, add lactose, mix well, and set Measure and place in a flask, autoclave at 115 °C for 30 minutes, and store in a cold and dark place for later use.

Just before use, heat and thaw the stock culture medium, and then cool it down to about 60 °C.

2% eosin aqueous solution and 0.5% methylene blue aqueous solution, shake well (to prevent the generation of air bubbles), pour into the dish for later use.

6.2.5 Lactose fermentation tube

Peptone 20.0 g

Lactose 10.0 g

0.4% bromocresol purple aqueous solution 2.5 mL 1000 mL of distilled water Method. Dissolve the peptone and lactose in distilled water, adjust the pH to 7.2 ~ 7.5, add the indicator, and aliquot it into a small tube (inverted tube) 3 mL of each test tube, autoclaved at 115 °C for 30 min, stored in a cold and dark place for later use.

6.2.6 Gram stain

Purchase the configured Gram staining solution and store it in a cool place at room temperature.

6.3 Instruments and equipment

Autoclave, dry heat sterilizer, constant temperature incubator, refrigerator, electric stove, balance, vortex mixer, sterile test tube, plate, scale Pipette, sterile cotton swab, pH meter.

6.4 Sample collection

Randomly extract the public supplies to be used after cleaning and disinfection, aseptic operation, use sterile dry cotton swabs, and sterilize them in 10 mL After infiltrating in saline, smear evenly on the appropriate parts of the supplies and collect samples, and then use sterilizing scissors to cut off the parts where the cotton swabs touch the hands Place the cotton swab into the remaining 9 mL of normal saline and submit it for inspection within 4 hours.

Sampling requirements. smear the samples in contact with the human body, and the sampling area must be above 25 cm2 per sample

6.5 Detection and analysis steps

6.5.1 Sample dilution

Shake the test tube containing the cotton swab after sampling to make a 1.10 diluted sample. The whole process needs to be performed in a sterile operation table.

6.5.2 Lactose and bile salt fermentation test

Pour the test sample into the double-lactose bile salt fermentation culture medium, and place it in a 36 °C ± 1 °C incubator for 24 hours to observe whether it produces acid or gas.

If no acid or gas is produced, it is negative for coliform. If yellowing and gas generation occur, follow the steps below.

6.5.3 Isolation culture

Take an inoculating loop culture solution from the acid-producing and gas-producing fermentation tube, transfer it to eosin blue agar plate, and place it in a 36 °C ± 1 °C incubator Breed for 18 h to 24 h. Then take out, observe the colony morphology, and do Gram staining and confirmation test.

Dark purple, metallic luster colonies;

Purple-black colonies with no or slightly metallic luster;

Fuchsia red, darker colonies.

6.5.4 Confirmatory test

On the above plate, pick one or two suspected coliform colonies for staining and microscopic examination; inoculate lactose fermentation tubes at the same time, set at 36 °C Incubate for 24 h in an incubator at ± 1 ° C and make a record.

6.6 Calculations and results

If the lactose fermentation tube finally produces acid and gas, and Gram-staining-free Bacillus aureus, the coliform group can be reported.

7.1 Testing method

The method for measuring Staphylococcus aureus on utensils is the plate identification method.

7.2 Medium and reagents

7.2.1 Trypticase broth broth

Trypanase (or tryptone) 17 g Plant Peptone (or Soy Peptone) 3 g Sodium chloride 100 g Dipotassium hydrogen phosphate 2.5 g Glucose 2.5 g 1000 mL of distilled water Preparation method. after mixing and dissolving the above ingredients, adjust the pH value to 7.2 ~ 7.3, aliquot, and autoclave at 121 °C for 20 min.

7.2.2 Sodium chloride broth

Peptone 10.0 g

Beef Paste 3.0 g

Sodium chloride 75.0 g 1000 mL of distilled water Preparation method. heat and dissolve the above ingredients, adjust the pH value to 7.4, aliquot, and autoclave at 121 °C for 20 min.

7.2.3 Baired Parker Tablet

Tryptone 10 g

Beef paste 5 g

Yeast extract 1 g