Some standard content:
Daoister Library
Total bacteria count
35.1 Flat blood count method
35.1.1 Scope
Specification for inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the method for determining the total bacteria count in drinking water and its source water. This specification is applicable to the determination of the total bacteria count in drinking water and its source water. 35.1.2 Principle
The total bacteria count refers to the total number of colonies contained in 1mL of water sample obtained after the water sample is cultured under certain conditions (culture medium composition, culture temperature and time: pH, aerobic properties, etc.). The results obtained according to the provisions of this specification only include the total colony count of a group of mesophilic aerobic bacteria that can grow on nutrient agar: 35.1.3 Culture medium and reagents
35.1.3.1 Nutrient agar
35.1.3.1.1 Ingredients:
A Protein A
B Beef extract
C Sodium chloride
D Agar
E Distilled water
10~~20g
1000mL
35.1.3.1.2 Preparation method: Mix the above ingredients, heat to dissolve, adjust the pH to 7.4~~7.6, and divide into glass containers (if domestic agar containing more impurities is used, it should be filtered first), sterilize at 121C for 20min, and store in a cool and dark place for use.
35.1.4 Instruments
35.1.4.1 High-pressure steam sterilizer.
Dry heat sterilizer.
Incubator 3611℃
Electric furnace.
Balance.
Refrigerator.
Magnifying glass or colony counter
pH meter or precision pH test paper
Sterile test tubes, petri dishes (9cm in diameter), graduated pipettes, sampling bottles, etc. 35.1.5 Test steps
35.1.5.1 Drinking water
35.1.5.1.1 Aseptically pipette 1 mL of well-mixed water sample into a sterile plate, pour about 15 mL of nutrient agar medium that has been melted and cooled to about 45°C, and immediately swirl the plate to mix the water sample and the medium thoroughly. A parallel inoculation should be made for each test, and another plate should be poured with only nutrient agar medium as a blank control.
35.1.5.1.2 After cooling and solidification, turn the plate over with the bottom facing up, place it in a 36+1°C incubator for 24 hours, and count the colonies, which is the total number of bacteria in 1 mL of the water sample. 35.1.5.2 Source water
35.1.5.2.1 Aseptically pipette 1 mL of the well-mixed water sample into a test tube containing 9 mL of sterile saline solution, and mix to form a 1:10 dilution
Specifications for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 35.1.5.2.2 Pipet 1 mL of the 1:10 dilution solution into a test tube containing 9 mL of sterile saline solution, and mix to form a 1:100 dilution solution. Dilute to 1:1000, 1:10000 dilution solution, etc. in the same way for use. After each incremental dilution, a 1 mL sterile pipette must be replaced.
35.1.5.2.3 Use a sterile pipette to take 1 mL of 2~3 water samples of appropriate dilutions and inject them into sterile dishes respectively. The following operations are the same as the test steps for drinking water. 35.1.6 Colony count and reporting method
When counting the colonies on the plates, you can observe directly with your eyes, and use a magnifying glass to check if necessary to prevent omissions. After recording the number of colonies on each plate, the average number of colonies at the same dilution should be calculated for use in the next calculation. When calculating the average number at the same dilution, if one of the plates has large flake colonies growing, it should not be used, and the plate without flake colonies should be used as the average number of colonies at the dilution. If the flake colonies are less than half of the plate, and the colony counts in the remaining half are evenly distributed, the half plate can be counted and multiplied by 2 to represent the colony count of the whole blood. Then calculate the average colony count of that dilution.
35.1.7 Selection and reporting of different dilutions 35.1.7.1 First, select the average colony count between 30 and 300 for calculation. If only one dilution has an average colony count within this range, multiply the colony count by the dilution factor and report it (see Example 1). 35.1.7.2 If there are two dilutions and the colony counts they grow are both between 30 and 300, the ratio between the two should be used to determine the dilution. If the ratio is less than 2, the average of the two should be reported (as in Example 2). If it is greater than 2, the total colony count of the smaller dilution should be reported (as in Example 3). If it is equal to 2, the colony count of the smaller dilution shall also be reported (see Example 4). 35.1.7.3 If the average colony count of all dilutions is greater than 300, the average colony count of the highest dilution multiplied by the dilution factor shall be reported (see Example 5).
35.1.7.4 If the average colony count of all dilutions is less than 30, the average colony count of the lowest dilution multiplied by the dilution factor shall be reported (see Example 6).
35.1.7.5 If the average colony count of all dilutions is not between 30 and 300, the average colony count closest to 30 or 300 multiplied by the dilution factor shall be reported: (see Example 7). 35.1.7.6 If no colony grows at all dilutions: then report it as 1 multiplied by the dilution factor. 35.1.7.7 Reporting of colony counts: When the colony count is within 100, it shall be reported as the actual number. When it is greater than 100, two significant figures shall be used. The values after the two significant figures shall be rounded off. In order to shorten the number of zeros after the number, it can also be expressed as an exponent of 10 (see the "Reporting method" column in Table 35-1). Table 35-1 Examples of dilution selection and reporting of total colony counts
Average colony counts at different dilutions
Too many to count
Too many to count
Ratio of colony counts at two dilutions
Total colony count
(CFU/mL)
513000
<1×10
Reporting method
(CFU/mL)
16000 or 1.6×10*
38000 or 3.8×10
27000 or 2.7X10| |tt||1500 or 1.5×10%
510000 or 5.1×10%
270 or 2.7×10
31000 or 3.1×10
<1×10
Daoister Library
36 Total coliform group
Multiple tube fermentation method
36.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation determination method for total coliform group in drinking water and its source water. This specification is applicable to the determination of total coliform group in drinking water and its source water. 36.1.2 Principle
Total coliform bacteria refers to a group of Gram-negative, non-spore-forming bacilli that can ferment lactose, produce acid and gas, and are aerobic and facultatively anaerobic when cultured at 37°C for 24 hours. This group of bacteria mainly comes from human and animal feces and has the general characteristics of indicator bacteria. Therefore, it is used as an indicator of fecal pollution to evaluate the sanitary quality of drinking water. The number of total coliform bacteria in water is expressed as the most probable number (MPN) of total coliform bacteria in 100mL of water sample. 36.1.3 Culture medium and reagents
36.1.3.1 Lactose protein culture medium
36.1.3.1.1 Ingredients
A Protein
B Beef extract
C Lactose
D Sodium chloride
E Bromocresol purple ethanol solution (16 g/L)
F Distilled water
36.1.3.1.2 Preparation method
1000 mL
Dissolve protein, beef extract, lactose and sodium chloride in distilled water, adjust the pH to 7.2-7.4, then add 1 ml of bromocresol purple ethanol solution (16 g/L), mix well, and divide into test tubes with inverted tubes at 115\C Autoclave for 20 min, store in a cool dark place for later use:
36.1.3.2 Double concentrated lactose protein culture medium According to the above lactose protein culture medium (36.1.3.1), except for distilled water, the amount of other ingredients is doubled. 36.1.3.3 Eosin-methylene blue medium
36.1.3.3.1 Ingredients
A Protein
B Lactose
C Potassium dihydrogen phosphate
D Agar
E Distilled water
F Eosin aqueous solution (20 g/L)
G Methylene blue aqueous solution (5 g/L)
20-30 g
1000 mL
36.1.3.3.2 Preparation method: Dissolve protein, phosphate and agar in distilled water, adjust the pH to 7.2, add lactose, mix well and divide into portions, sterilize at 115℃ for 20min, heat to melt agar before use, cool to 50-55, add eosin and methylene blue solution, mix well and pour into a plate. 36.1.3.4 Gram stain solution
36.1.3.4.1 Crystal violet stain solution
A Ingredients a Crystal violet
b Ethanol [β(CH,0H)=95%] 20mL
Daoister Library
c Ammonium oxalate aqueous solution (10g/L)
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 80ml
B Preparation method: Dissolve crystal violet in ethanol [βp(C,H,OH)=95%], and then mix with ammonium oxalate solution Note: Crystal violet cannot be replaced by gentian violet, the former is pure crystal, the latter is not a single component, and false positives are prone to occur. Crystal violet solution will produce precipitation if left for too long and cannot be used again. 36.1.3.4.2 Gram's iodine solution
A Component
Potassium iodide
Distilled water
B Method: Mix iodine and potassium iodide first, add a little distilled water, shake thoroughly: after it is completely dissolved, add distilled water.
36.1.3.4.3 Decolorizing agent: Ethanol [p(C,H.0H)=95%]. 36.1.3.4.4
Safflower counterstain solution
A Component
aSafflower
bEthanol [β(C,HOH)=95%]
cDistilled water
B Method: Dissolve safflower in ethanol, add distilled water after it is completely dissolved Note: If safflower is not available: Phenol fuchsin staining solution (1+10) can be used as counterstaining solution, and the counterstaining time is 10s. 36.1.3.4.5 Staining method
A Smear the cultured material after 18-24h cultivation,
B Fix the smear on flame, add crystal violet stain solution, stain for 1min, wash with water, add Gram iodine solution, act for 1min, wash with water. C
Add decolorizer, shake the slide until no purple color falls off, about 30s, wash with water, D
Add counterstain, counterstain for 1min, wash with water, wait to dry: microscopic examination. 36.1.4 Instruments
36.1.4.1 Incubator: 361℃
Refrigerator: 0~~4℃
Balance.
Microscope.
Plate: 9cm in diameter
Graduated pipette: 1mL, 10mL
Conical flask.
36.1.4.9 Small inverted tube.
36.1.4.10 Glass slide.
36.1.5 Inspection steps
36.1.5.1 Lactose fermentation test
36.1.5.1.1 When inspecting drinking water, take 10mL of water sample and inoculate it into 10mL of lactose protein culture medium, take 1mL of water sample and inoculate it into 10mL of single lactose protein culture medium, take another 1mL of water sample and inject it into 9mL of sterile saline, mix well and take 1mL (i.e. 0.1mL of water sample) and inject it into 10mL of single lactose protein culture medium, and inoculate 5 tubes for each dilution. For treated tap water leaving the factory: if it needs to be inspected frequently or once a day, 5 portions of 10mL of double culture medium can be directly inoculated, and 10mL of water sample can be inoculated in each portion. 36.1.5.1.2 When inspecting source water, if the pollution is serious, the dilution should be increased, and 1, 0.1, 0.01mL or even 0.1, 0.01, 0.001mL can be inoculated: 5 tubes for each dilution, and 15 tubes for each water sample. When inoculating 1mL or lower water samples, it must be diluted 10 times and then 1mL is inoculated. After each incremental dilution, a 1mL sterile graduated pipette is used.
36.1.5.1.3 Place the inoculated tube in a 36±1C incubator and culture for 24±2h. If all lactose protein culture tubes do not produce gas or acid, it can be reported as negative for total coliform bacteria. If there are acid and gas producing ones, follow the following steps. 36.1.5.2 Isolation and culture
The fermentation tubes producing acid and gas shall be transferred to eosin-methylene blue agar plates respectively, and cultured in a 36±1℃ incubator for 18-24h. The colony morphology shall be observed, and the colonies meeting the following characteristics shall be selected: Dark purple-black: colonies with metallic luster, purple-black: colonies without or with slight metallic luster. Light purple-red: colonies with darker centers.
Perform Gram staining, microscopic examination and confirmation test. 36.1.5.3 Confirmation test
If the above staining and microscopic examination show that the colonies are Gram-negative and non-spore-forming bacilli, inoculate lactose protein culture solution at the same time, and culture in a 36±1℃ incubator for 24±2h, the colonies that produce acid and gas are confirmed to have total coliform group. 36.1.5.4 Result report
Based on the number of tubes confirmed to be positive for total coliform group, check the MPN retrieval table and report the MPN value of total coliform group in each 100mL of water sample. If all lactose fermentation tubes are negative, it can be reported that total coliform bacteria are not detected. Table 36-1 MPN for various combinations of positive and negative results when using 5 10mL water samples Number of positive tubes in 5 10mL tubes
Specifications for Drinking Water Inspection (GB5750-85 Revised Edition, Ministry of Health 2001.6) Daoister Library
MPN for different combinations of positive and negative results
When 5 tubes are 0.1mL,
5 tubes are 1mL,
Table 36-2 Using 5 tubes 10mL,
5 tubes each
Of which the number of positive tubes
5 tubes each
tube 1mL
tube 10mL
5 tubes each
5 tubes each
of which Number of positive tubes
5 tubes, each
1mL
5 tubes, each
10mL
Daoister Library
Membrane filter method
36.2.1 Scope
Specification for Inspection of Drinking Water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the membrane filter determination method for coliform group in drinking water and its source water. This specification is applicable to the determination of total coliform group in drinking water and low turbidity source water: 36.2.2 Principle
Filter the water sample with a microporous filter membrane with a pore size of 0.4511m. The bacteria are retained on the filter membrane. Stick the filter membrane on the selective culture medium. After culture, count the number of typical coliform colonies growing on the filter membrane. 36.2.3 Culture medium and reagents
36.2.3.1 Fuchsin sodium sulfite culture medium
36.2.3.1.1 Ingredients
A Protein extract
B Yeast extract
C Beef extract
D Lactose
E Agar
F Dipotassium hydrogen phosphate
G Anhydrous sodium sulfite
H Basic fuchsin ethanol solution (50 g/L)
I Distilled water
36.2.3 .1.2 Preparation of reserve culture medium
15~~20g
1000mL
First add agar to 500mL of distilled water and boil to dissolve. Add potassium hydrogen phosphate, egg white, yeast extract and beef extract to another 500mL of distilled water and heat to dissolve. Pour in the dissolved agar and make up to 1000mL with distilled water. Mix well and adjust the pH to 7.2~~7.4. Add lactose and divide into portions. Autoclave at 115℃ for 20min and store in a cool and dark place for later use.
36.2.3.1.3 Preparation of fuchsin culture medium
Heat and melt the reserve culture medium prepared by the above method, use a sterile pipette to draw a certain amount of alkaline fuchsin ethanol solution (36.2.3.1.1.H) in proportion and place it in a sterile empty test tube, then weigh the required anhydrous sodium sulfite in proportion and place it in another sterile test tube, add a little sterile water to dissolve it, and then boil it in a boiling water bath for 10 minutes to sterilize it, and use a sterile pipette to draw the sterilized sodium sulfite solution: add it dropwise to the alkaline fuchsin ethanol solution until the deep red turns to light pink: add all the sodium sulfite and alkaline fuchsin mixture to the melted reserve culture medium, and mix it thoroughly (to prevent bubbles), and immediately pour 15mL of this culture medium into the sterilized empty plate. After cooling and solidification, put it in the refrigerator for use. This prepared culture medium should not be stored in the refrigerator for more than two weeks. If the culture medium has turned from light pink to deep red, it cannot be used.
36.2.3.2 Lactose protein culture medium, same as 36.1.336.2.4 Instrument
36.2.4.1 Filter
36.2.4.2 Filter membrane, pore size 0.45~-0.6511m diameter According to the filter specifications, the commonly used ones are 3.5cm and 4.7cm.
36.2.4.3 Filtration equipment.
36.2.4.4 Toothless tweezers.
36.2.4.5 Other instruments are the same as 36.1.4.
36.2.5 Inspection steps
36.2.5.1 Preparation
Daoister Library
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 36.2.5.1.1 Sterilization of filter membrane: Put the filter membrane in a beaker, add distilled water, and boil it in a boiling water bath for three times, each time for 15 minutes. After the first two boilings, the water needs to be changed and washed 2-3 times to remove residual solvents. 36.2.5.1.2 Sterilization of filter: Use a lit alcohol cotton ball and flame sterilization. It can also be sterilized at 121℃ for 20 minutes. 36.2.5.2 Filtering water samples
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100 mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath should be higher than the culture medium level in the test tube) and incubate it for 24±2 hours. If all tubes do not produce gas, it can be reported as negative. If there is gas production, transfer it to eosin-methylene blue agar plate and place it at 44.5℃ (incubate it for 18-24 hours. If there are typical colonies on the plate, it is confirmed to be positive for fecal coliform bacteria. 37.1.5.2 When testing unchlorinated water and only want to test fecal coliform bacteria, or when investigating fecal coliform bacteria contamination in source water, direct multiple tube fecal coliform bacteria test can be used. Group method: that is, in the first step of the lactose fermentation test, inoculate according to 36.1.5.1 and culture in a water bath at 44.5 ± 0.5℃. The following steps are the same as 37.1.5.1. 37.1.5.3 Result report
According to the number of positive tubes confirmed as fecal coliform group, check the MPN search table and report the MPN value of fecal coliform group in each 100mL water sample.
Filter membrane method
37.2.1 Scope
Daoister Library
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the filter membrane determination method for fecal coliform group in drinking water and its source water: This specification is applicable to the determination of fecal coliform group in drinking water and its source water. 37.2.2 Principle||tt| |Filter the water sample through a filter membrane with a pore size of 0.4511m. The bacteria are retained on the membrane. Stick the filter membrane on the MFC culture medium. After incubation at 44.5℃, count the typical colonies. 37.2.3 Culture medium and reagents
37.2.3.1 MFC culture medium
37.2.3.1.1 Ingredients
A Pancreatic acid
B Polychlorinated acidbzxZ.net
C Yeast extract
D Sodium chloride
E Lactose
F Bile salt No. 3 or mixed bile saltsG Agar
H Aniline blue
I Distilled water
1000mL
37.2.3.1.2 Preparation method: First add 10g/L of hydroxide in 1000mL of distilled water. Sodium solution [c(Na0H)=0.2mol/L] 10mL: After mixing: take 500mL and add to agar and boil to dissolve. In another 500mL of distilled water, add other reagents except aniline blue, heat to dissolve, pour in the dissolved agar, mix and adjust the pH to 7.4, add aniline blue and boil, quickly remove from the heat source, wait until it cools to about 60'℃, and make a plate. Do not sterilize by high pressure. The prepared culture medium should be stored at 2-10℃ for no more than 96h. This culture medium can also be made into liquid culture medium without adding agar. When using, add 2mL to the sterile absorption pad, and then place the filter membrane on the culture pad for culture.
37.2.3.2EC culture medium (see 37.1.3.1) 37.2.4 Instruments
37.2.4.1 Water-proof constant temperature incubator or constant temperature water bath. 37.2.4.2 Plastic culture blood: 60mm×15mm or 50mm×12mm 37.2.4.3 Other instruments are the same as the total coliform membrane filter method (36.2.4). 37.2.5 Analysis steps
37.2.5.1 Preparation (same as 36.2.5.1 or 36.2.5.1.2) 37.2.5.2 Filtering water samples (same as 36.2.5.2) 37.2.5.3 Culture: After the water sample is filtered, vacuum for about 5 seconds and close the filter valve: remove the filter, use sterile tweezers to grab the edge of the filter membrane, and move it to the M-FC culture medium, with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the flat III upside down and place it in a 44.5℃ water-proof incubator for 22~24 hours. If a constant temperature water bath is used, plastic plates should be used. Cover the plates tightly or seal each plate with waterproof tape. Stack the plates and seal them in a plastic bag: immerse in a constant temperature water bath at 44.5℃ and culture for 24±2 hours. The colonies of fecal coliform bacteria on this culture medium are blue, and the colonies of non-fecal coliform bacteria are gray to cream. 37.2.5.4 Transfer the suspected colonies to EC culture medium and culture at 44.5℃ for 24±2 hours. If gas is produced, it is confirmed to be fecal coliform bacteria. 37.2.5.5 Count the confirmed fecal coliform bacteria and calculate the fecal coliform bacteria density in every 100mL of water. The fecal coliform bacteria count (CFU/100mL) = the counted fecal coliform bacteria count × 100 filtered water sample volume (mL)
..(371)4 Toothless tweezers.
36.2.4.5 Other instruments are the same as 36.1.4.
36.2.5 Inspection steps
36.2.5.1 Preparation
Daoister Library
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 36.2.5.1.1 Sterilization of filter membrane: Put the filter membrane in a beaker, add distilled water, and boil it in a boiling water bath for three times, each time for 15 minutes. After the first two boilings, the water needs to be changed and washed 2-3 times to remove residual solvents. 36.2.5.1.2 Sterilization of filter: Use a lit alcohol cotton ball and flame sterilization. It can also be sterilized by high pressure at 121℃ for 20 minutes. 36.2.5.2 Filtering water samples
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100 mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath should be higher than the culture medium level in the test tube) and incubate it for 24±2 hours. If all tubes do not produce gas, it can be reported as negative. If there is gas production, transfer it to eosin-methylene blue agar plate and place it at 44.5℃ (incubate it for 18-24 hours. If there are typical colonies on the plate, it is confirmed to be positive for fecal coliform bacteria. 37.1.5.2 When testing unchlorinated water and only want to test fecal coliform bacteria, or when investigating fecal coliform bacteria contamination in source water, direct multiple tube fecal coliform bacteria test can be used. Group method: that is, in the first step of the lactose fermentation test, inoculate according to 36.1.5.1 and culture in a water bath at 44.5 ± 0.5℃. The following steps are the same as 37.1.5.1. 37.1.5.3 Result report
According to the number of positive tubes confirmed as fecal coliform group, check the MPN search table and report the MPN value of fecal coliform group in each 100mL water sample.
Filter membrane method
37.2.1 Scope
Daoister Library
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the filter membrane determination method for fecal coliform group in drinking water and its source water: This specification is applicable to the determination of fecal coliform group in drinking water and its source water. 37.2.2 Principle||tt| |Filter the water sample through a filter membrane with a pore size of 0.4511m. The bacteria are retained on the membrane. Stick the filter membrane on the MFC culture medium. After incubation at 44.5℃, count the typical colonies. 37.2.3 Culture medium and reagents
37.2.3.1 MFC culture medium
37.2.3.1.1 Ingredients
A Pancreatic acid
B Polychlorinated acid
C Yeast extract
D Sodium chloride
E Lactose
F Bile salt No. 3 or mixed bile saltsG Agar
H Aniline blue
I Distilled water
1000mL
37.2.3.1.2 Preparation method: First add 10g/L of hydroxide in 1000mL of distilled water. Sodium solution [c(Na0H)=0.2mol/L] 10mL: After mixing: take 500mL and add to agar and boil to dissolve. In another 500mL of distilled water, add other reagents except aniline blue, heat to dissolve, pour in the dissolved agar, mix and adjust the pH to 7.4, add aniline blue and boil, quickly remove from the heat source, wait until it cools to about 60'℃, and make a plate. Do not sterilize by high pressure. The prepared culture medium should be stored at 2-10℃ for no more than 96h. This culture medium can also be made into liquid culture medium without adding agar. When using, add 2mL to the sterile absorption pad, and then place the filter membrane on the culture pad for culture.
37.2.3.2EC culture medium (see 37.1.3.1) 37.2.4 Instruments
37.2.4.1 Water-proof constant temperature incubator or constant temperature water bath. 37.2.4.2 Plastic culture blood: 60mm×15mm or 50mm×12mm 37.2.4.3 Other instruments are the same as the total coliform membrane filter method (36.2.4). 37.2.5 Analysis steps
37.2.5.1 Preparation (same as 36.2.5.1 or 36.2.5.1.2) 37.2.5.2 Filtering water samples (same as 36.2.5.2) 37.2.5.3 Culture: After the water sample is filtered, vacuum for about 5 seconds and close the filter valve: remove the filter, use sterile tweezers to grab the edge of the filter membrane, and move it to the M-FC culture medium, with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the flat III upside down and place it in a 44.5℃ water-proof incubator for 22~24 hours. If a constant temperature water bath is used, plastic plates should be used. Cover the plates tightly or seal each plate with waterproof tape. Stack the plates and seal them in a plastic bag: immerse in a constant temperature water bath at 44.5℃ and culture for 24±2 hours. The colonies of fecal coliform bacteria on this culture medium are blue, and the colonies of non-fecal coliform bacteria are gray to cream. 37.2.5.4 Transfer the suspected colonies to EC culture medium and culture at 44.5℃ for 24±2 hours. If gas is produced, it is confirmed to be fecal coliform bacteria. 37.2.5.5 Count the confirmed fecal coliform bacteria and calculate the fecal coliform bacteria density in every 100mL of water. The fecal coliform bacteria count (CFU/100mL) = the counted fecal coliform bacteria count × 100 filtered water sample volume (mL)
..(371)4 Toothless tweezers.
36.2.4.5 Other instruments are the same as 36.1.4.
36.2.5 Inspection steps
36.2.5.1 Preparation
Daoister Library
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 36.2.5.1.1 Sterilization of filter membrane: Put the filter membrane in a beaker, add distilled water, and boil it in a boiling water bath for three times, each time for 15 minutes. After the first two boilings, the water needs to be changed and washed 2-3 times to remove residual solvents. 36.2.5.1.2 Sterilization of filter: Use a lit alcohol cotton ball and flame sterilization. It can also be sterilized by high pressure at 121℃ for 20 minutes. 36.2.5.2 Filtering water samples
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100 mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath should be higher than the culture medium level in the test tube) and incubate it for 24±2 hours. If all tubes do not produce gas, it can be reported as negative. If there is gas production, transfer it to eosin-methylene blue agar plate and place it at 44.5℃ (incubate it for 18-24 hours. If there are typical colonies on the plate, it is confirmed to be positive for fecal coliform bacteria. 37.1.5.2 When testing unchlorinated water and only want to test fecal coliform bacteria, or when investigating fecal coliform bacteria contamination in source water, direct multiple tube fecal coliform bacteria test can be used. Group method: that is, in the first step of the lactose fermentation test, inoculate according to 36.1.5.1 and culture in a water bath at 44.5 ± 0.5℃. The following steps are the same as 37.1.5.1. 37.1.5.3 Result report
According to the number of positive tubes confirmed as fecal coliform group, check the MPN search table and report the MPN value of fecal coliform group in each 100mL water sample.
Filter membrane method
37.2.1 Scope
Daoister Library
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the filter membrane determination method for fecal coliform group in drinking water and its source water: This specification is applicable to the determination of fecal coliform group in drinking water and its source water. 37.2.2 Principle||tt| |Filter the water sample through a filter membrane with a pore size of 0.4511m. The bacteria are retained on the membrane. Stick the filter membrane on the MFC culture medium. After incubation at 44.5℃, count the typical colonies. 37.2.3 Culture medium and reagents
37.2.3.1 MFC culture medium
37.2.3.1.1 Ingredients
A Pancreatic acid
B Polychlorinated acid
C Yeast extract
D Sodium chloride
E Lactose
F Bile salt No. 3 or mixed bile saltsG Agar
H Aniline blue
I Distilled water
1000mL
37.2.3.1.2 Preparation method: First add 10g/L of hydroxide in 1000mL of distilled water. Sodium solution [c(Na0H)=0.2mol/L] 10mL: After mixing: take 500mL and add to agar and boil to dissolve. In another 500mL of distilled water, add other reagents except aniline blue, heat to dissolve, pour in the dissolved agar, mix and adjust the pH to 7.4, add aniline blue and boil, quickly remove from the heat source, wait until it cools to about 60'℃, and make a plate. Do not sterilize by high pressure. The prepared culture medium should be stored at 2-10℃ for no more than 96h. This culture medium can also be made into liquid culture medium without adding agar. When using, add 2mL to the sterile absorption pad, and then place the filter membrane on the culture pad for culture.
37.2.3.2EC culture medium (see 37.1.3.1) 37.2.4 Instruments
37.2.4.1 Water-proof constant temperature incubator or constant temperature water bath. 37.2.4.2 Plastic culture blood: 60mm×15mm or 50mm×12mm 37.2.4.3 Other instruments are the same as the total coliform membrane filter method (36.2.4). 37.2.5 Analysis steps
37.2.5.1 Preparation (same as 36.2.5.1 or 36.2.5.1.2) 37.2.5.2 Filtering water samples (same as 36.2.5.2) 37.2.5.3 Culture: After the water sample is filtered, vacuum for about 5 seconds and close the filter valve: remove the filter, use sterile tweezers to grab the edge of the filter membrane, and move it to the M-FC culture medium, with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the flat III upside down and place it in a 44.5℃ water-proof incubator for 22~24 hours. If a constant temperature water bath is used, plastic plates should be used. Cover the plates tightly or seal each plate with waterproof tape. Stack the plates and seal them in a plastic bag: immerse in a constant temperature water bath at 44.5℃ and culture for 24±2 hours. The colonies of fecal coliform bacteria on this culture medium are blue, and the colonies of non-fecal coliform bacteria are gray to cream. 37.2.5.4 Transfer the suspected colonies to EC culture medium and culture at 44.5℃ for 24±2 hours. If gas is produced, it is confirmed to be fecal coliform bacteria. 37.2.5.5 Count the confirmed fecal coliform bacteria and calculate the fecal coliform bacteria density in every 100mL of water. The fecal coliform bacteria count (CFU/100mL) = the counted fecal coliform bacteria count × 100 filtered water sample volume (mL)
..(371)1 Preparation
Daoister Library
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 36.2.5.1.1 Sterilization of filter membrane: Put the filter membrane in a beaker, add distilled water, and boil it in a boiling water bath for three times, 15 minutes each time. After the first two boilings, the water needs to be changed and washed 2-3 times to remove residual solvents. 36.2.5.1.2 Sterilization of filter: Use a lit alcohol cotton ball and flame sterilization. It can also be sterilized by high pressure at 121℃ for 20 minutes. 36.2.5.2 Filtering water samples
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100 mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath should be higher than the culture medium level in the test tube) and incubate it for 24±2 hours. If all tubes do not produce gas, it can be reported as negative. If there is gas production, transfer it to eosin-methylene blue agar plate and place it at 44.5℃ (incubate it for 18-24 hours. If there are typical colonies on the plate, it is confirmed to be positive for fecal coliform bacteria. 37.1.5.2 When testing unchlorinated water and only want to test fecal coliform bacteria, or when investigating fecal coliform bacteria contamination in source water, direct multiple tube fecal coliform bacteria test can be used. Group method: that is, in the first step of the lactose fermentation test, inoculate according to 36.1.5.1 and culture in a water bath at 44.5 ± 0.5℃. The following steps are the same as 37.1.5.1. 37.1.5.3 Result report
According to the number of positive tubes confirmed as fecal coliform group, check the MPN search table and report the MPN value of fecal coliform group in each 100mL water sample.
Filter membrane method
37.2.1 Scope
Daoister Library
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the filter membrane determination method for fecal coliform group in drinking water and its source water: This specification is applicable to the determination of fecal coliform group in drinking water and its source water. 37.2.2 Principle||tt| |Filter the water sample through a filter membrane with a pore size of 0.4511m. The bacteria are retained on the membrane. Stick the filter membrane on the MFC culture medium. After incubation at 44.5℃, count the typical colonies. 37.2.3 Culture medium and reagents
37.2.3.1 MFC culture medium
37.2.3.1.1 Ingredients
A Pancreatic acid
B Polychlorinated acid
C Yeast extract
D Sodium chloride
E Lactose
F Bile salt No. 3 or mixed bile saltsG Agar
H Aniline blue
I Distilled water
1000mL
37.2.3.1.2 Preparation method: First add 10g/L of hydroxide in 1000mL of distilled water. Sodium solution [c(Na0H)=0.2mol/L] 10mL: After mixing: take 500mL and add to agar and boil to dissolve. In another 500mL of distilled water, add other reagents except aniline blue, heat to dissolve, pour in the dissolved agar, mix and adjust the pH to 7.4, add aniline blue and boil, quickly remove from the heat source, wait until it cools to about 60'℃, and make a plate. Do not sterilize by high pressure. The prepared culture medium should be stored at 2-10℃ for no more than 96h. This culture medium can also be made into liquid culture medium without adding agar. When using, add 2mL to the sterile absorption pad, and then place the filter membrane on the culture pad for culture.
37.2.3.2EC culture medium (see 37.1.3.1) 37.2.4 Instruments
37.2.4.1 Water-proof constant temperature incubator or constant temperature water bath. 37.2.4.2 Plastic culture blood: 60mm×15mm or 50mm×12mm 37.2.4.3 Other instruments are the same as the total coliform membrane filter method (36.2.4). 37.2.5 Analysis steps
37.2.5.1 Preparation (same as 36.2.5.1 or 36.2.5.1.2) 37.2.5.2 Filtering water samples (same as 36.2.5.2) 37.2.5.3 Culture: After the water sample is filtered, vacuum for about 5 seconds and close the filter valve: remove the filter, use sterile tweezers to grab the edge of the filter membrane, and move it to the M-FC culture medium, with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the flat III upside down and place it in a 44.5℃ water-proof incubator for 22~24 hours. If a constant temperature water bath is used, plastic plates should be used. Cover the plates tightly or seal each plate with waterproof tape. Stack the plates and seal them in a plastic bag: immerse in a constant temperature water bath at 44.5℃ and culture for 24±2 hours. The colonies of fecal coliform bacteria on this culture medium are blue, and the colonies of non-fecal coliform bacteria are gray to cream. 37.2.5.4 Transfer the suspected colonies to EC culture medium and culture at 44.5℃ for 24±2 hours. If gas is produced, it is confirmed to be fecal coliform bacteria. 37.2.5.5 Count the confirmed fecal coliform bacteria and calculate the fecal coliform bacteria density in every 100mL of water. The fecal coliform bacteria count (CFU/100mL) = the counted fecal coliform bacteria count × 100 filtered water sample volume (mL)
..(371)1 Preparation
Daoister Library
Standards for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) 36.2.5.1.1 Sterilization of filter membrane: Put the filter membrane in a beaker, add distilled water, and boil it in a boiling water bath for three times, 15 minutes each time. After the first two boilings, the water needs to be changed and washed 2-3 times to remove residual solvents. 36.2.5.1.2 Sterilization of filter: Use a lit alcohol cotton ball and flame sterilization. It can also be sterilized by high pressure at 121℃ for 20 minutes. 36.2.5.2 Filtering water samples
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100 mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath should be higher than the culture medium level in the test tube) and incubate it for 24±2 hours. If all tubes do not produce gas, it can be reported as negative. If there is gas production, transfer it to eosin-methylene blue agar plate and place it at 44.5℃ (incubate it for 18-24 hours. If there are typical colonies on the plate, it is confirmed to be positive for fecal coliform bacteria. 37.1.5.2 When testing unchlorinated water and only want to test fecal coliform bacteria, or when investigating fecal coliform bacteria contamination in source water, direct multiple tube fecal coliform bacteria test can be used. Group method: that is, in the first step of the lactose fermentation test, inoculate according to 36.1.5.1 and culture in a water bath at 44.5 ± 0.5℃. The following steps are the same as 37.1.5.1. 37.1.5.3 Result report
According to the number of positive tubes confirmed as fecal coliform group, check the MPN search table and report the MPN value of fecal coliform group in each 100mL water sample.
Filter membrane method
37.2.1 Scope
Daoister Library
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the filter membrane determination method for fecal coliform group in drinking water and its source water: This specification is applicable to the determination of fecal coliform group in drinking water and its source water. 37.2.2 Principle||tt| |Filter the water sample through a filter membrane with a pore size of 0.4511m. The bacteria are retained on the membrane. Stick the filter membrane on the MFC culture medium. After incubation at 44.5℃, count the typical colonies. 37.2.3 Culture medium and reagents
37.2.3.1 MFC culture medium
37.2.3.1.1 Ingredients
A Pancreatic acid
B Polychlorinated acid
C Yeast extract
D Sodium chloride
E Lactose
F Bile salt No. 3 or mixed bile saltsG Agar
H Aniline blue
I Distilled water
1000mL
37.2.3.1.2 Preparation method: First add 10g/L of hydroxide in 1000mL of distilled water. Sodium solution [c(Na0H)=0.2mol/L] 10mL: After mixing: take 500mL and add to agar and boil to dissolve. In another 500mL of distilled water, add other reagents except aniline blue, heat to dissolve, pour in the dissolved agar, mix and adjust the pH to 7.4, add aniline blue and boil, quickly remove from the heat source, wait until it cools to about 60'℃, and make a plate. Do not sterilize by high pressure. The prepared culture medium should be stored at 2-10℃ for no more than 96h. This culture medium can also be made into liquid culture medium without adding agar. When using, add 2mL to the sterile absorption pad, and then place the filter membrane on the culture pad for culture.
37.2.3.2EC culture medium (see 37.1.3.1) 37.2.4 Instruments
37.2.4.1 Water-proof constant temperature incubator or constant temperature water bath. 37.2.4.2 Plastic culture blood: 60mm×15mm or 50mm×12mm 37.2.4.3 Other instruments are the same as the total coliform membrane filter method (36.2.4). 37.2.5 Analysis steps
37.2.5.1 Preparation (same as 36.2.5.1 or 36.2.5.1.2) 37.2.5.2 Filtering water samples (same as 36.2.5.2) 37.2.5.3 Culture: After the water sample is filtered, vacuum for about 5 seconds and close the filter valve: remove the filter, use sterile tweezers to grab the edge of the filter membrane, and move it to the M-FC culture medium, with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the flat III upside down and place it in a 44.5℃ water-proof incubator for 22~24 hours. If a constant temperature water bath is used, plastic plates should be used. Cover the plates tightly or seal each plate with waterproof tape. Stack the plates and seal them in a plastic bag: immerse in a constant temperature water bath at 44.5℃ and culture for 24±2 hours. The colonies of fecal coliform bacteria on this culture medium are blue, and the colonies of non-fecal coliform bacteria are gray to cream. 37.2.5.4 Transfer the suspected colonies to EC culture medium and culture at 44.5℃ for 24±2 hours. If gas is produced, it is confirmed to be fecal coliform bacteria. 37.2.5.5 Count the confirmed fecal coliform bacteria and calculate the fecal coliform bacteria density in every 100mL of water. The fecal coliform bacteria count (CFU/100mL) = the counted fecal coliform bacteria count × 100 filtered water sample volume (mL)
..(371)2 Filter the water sample
Use sterile tweezers to pick up the edge of the sterilized filter membrane, place it with the rough surface facing up on the sterilized filter bed, fix the filter, inject 100mL of water sample (if the water sample contains a large number of bacteria, reduce the amount of filtered water sample: or dilute the water sample) into the filter, open the filter valve, and filter at -0.5 atmospheres. 36.2.5.3 Culture
After the water sample is filtered, vacuum for about 5 seconds: close the filter valve. Remove the filter, use sterile tweezers to pick up the edge of the filter membrane, and transfer it to the fuchsin sodium sulfite culture medium (36.2.3.1), with the filter membrane retaining bacteria facing up. The filter membrane should be completely close to the culture medium, and no bubbles should be left between the two. Then turn the plate upside down and place it in a 37℃ constant temperature box for culture for 22~-24h.
36.2.6 Observation results
36.2.6.1 Select the colonies that meet the following characteristics for Gram staining and microscopic examination: purple-red colonies with metallic luster, dark red colonies without or with slight metallic luster, and light red colonies with darker center color.
36.2.6.1.1 For Gram-negative non-spore-forming bacilli, inoculate with lactose protein culture medium and culture at 37℃ for 24h. If there is acid and gas production, it is judged as positive for total coliform bacteria: 36.2.6.1.2 Calculate the total coliform bacteria growing on the filter membrane and report it as the total coliform bacteria in 100mL of water sample (CFU/100mL)
Total coliform bacteria colony count (CFU/100mL) = total coliform bacteria colony count × 100 filtered water sample volume (mL)
.......(36 -1)
Daoister Library
Fecal coliform bacteria
Multiple tube fermentation method
37.1.1 Scope
Specification for the inspection of drinking water (GB5750-85 revised edition, Ministry of Health 2001.6) This specification specifies the multiple tube fermentation method for the determination of fecal coliform bacteria in drinking water and its source water. This specification is applicable to the determination of fecal coliform bacteria in drinking water and its source water. 37.1.2 Principle
The coliform bacteria in the natural environment are distinguished from the coliform bacteria in feces by increasing the culture temperature. The coliform bacteria that can still grow at 44.5℃ are called fecal coliform bacteria. 37.1.3 Culture medium and reagents
37.1.3.1 EC culture medium
37.1.3.1.1 Ingredients
A Trypsin Chen
B Lactose
C Bile salt No. 3 or mixed bile salts
D Dipotassium hydrogen phosphate
E Potassium dihydrogen phosphate
F Sodium chloride
G Distilled water
1000ml
37.1.3.1.2 Preparation method: Dissolve the above ingredients in distilled water, divide into test tubes with inverted tubes, autoclave at 115℃ for 20 minutes, and the final pH is 6.9+0.2
37.1.3.2 Eosin-methylene blue agar (same as 36.1.3.3). 37.1.4 Apparatus
37.1.4.1 Constant temperature water bath: 44.5~~0.2℃ or water-insulated constant temperature incubator. 37.1.4.2 Others are the same as the multiple tube fermentation method for total coliform bacteria (36.1.4.1 to 36.1.4.9). 37.1.5 Test steps
37.1.5.1 Take 1 drop from the positive tube (producing acid and gas) in the total coliform lactose fermentation test and transfer it to EC culture medium. Place it in a 44.5℃ water bath (the water level in the water bath sh
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.