GB/T 4789.28-2003 Food hygiene microbiological examination staining methods, culture media and reagents
Some standard content:
ICS 07.100.30
National Standard of the People's Republic of China
GB/T4789.28—2003
Replaces GB/T4789.28—1994
Microbiological examination of food hygiene-Staining methods, culture mediums and reagents reagents2003-08-11 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01 Implementation
CB/T4789.28-2003
This standard is revised from GB/T4789.28-1994 Microbiological Test for Food Hygiene. Compared with GB/T4789.28-1994, this standard is revised as follows: The format of the standard text is revised in accordance with GB/T1.1-2000. The high-salt Czapek medium in 4.78 is deleted. From the date of implementation of this standard, GB/T4789.28-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. Staining Methods, Culture Media and Reagents. Drafting unit of this standard: Nutrition and Food Safety Institute of Chinese Center for Disease Control and Prevention. The main drafters of this standard are Zhou Guilian, Liu Hongdao, Luo Xueyun, Li Fengqin and Fu Ping. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 192
1 Scope
Food Hygiene Microbiological Examination
Staining Methods, Culture Media and Reagents
This standard specifies various staining methods, culture media and reagents. This standard is applicable to the inspection of various microorganisms in food and food poisoning samples. 2 Preparation of staining solution and staining method
2.1 Methylene blue staining method
2.1.1 Lu's alkaline methylene blue staining solution
95% ethanol
0.01% potassium hydroxide solution
Dissolve methylene blue in ethanol and then mix with potassium hydroxide solution. 2.1.2 Staining method
Fix the smear on a flame and wait for it to cool. Add the dye solution, dye for 1min~3tnin, wash with water, wait to dry, and examine under a microscope. 2.1.3 Results
The bacteria are blue.
2.2 Gram staining method
2.2.1 Crystal violet staining solution
Crystal violet
95% ethanol
1% ammonium oxalate aqueous solution
Dissolve crystal violet in ethanol and then mix with ammonium oxalate solution. 2.2.2 Gram iodine solution
Potassium iodide
Distilled water
GB/T4789.28—2003
Mix iodine and potassium iodide first, add a little distilled water, shake thoroughly, and add distilled water to 300mL after complete dissolution. 2.2.3 Safflower counterstain
95% ethanol
Distilled water
Dissolve safflower in ethanol and then dilute with distilled water. 2.2.4 Staining method
2.2.4.1 Fix the smear on a flame, add crystal violet staining solution, stain for 1 minute, and wash with water. Add Gram iodine solution, act for 1 minute, and wash with water. 2.2.4.2
Add 95% ethanol to decolorize, about 305; or drip ethanol all over the smear, immediately pour it off, and then drip ethanol all over the smear, decolorize for 10 seconds.
GB/T4789.28--2003
2.2.4.4 Wash with water, add counterstain solution, and counterstain for 1 minute. Wash with water, wait to dry, and examine under a microscope. 2.2.5 Results
Gram-positive bacteria are purple. Gram-negative bacteria are red. Note: 1110 diluted carbol fuchsin staining solution can also be used as counterstaining solution, and the counterstaining time only needs 10s. 2.3 Acid-fast staining method (Man-Neeler method) 2.3.1 Carbol fuchsin staining solution
Basic fuchsin
95% ethanol
5% phenol aqueous solution
Dissolve fuchsin in ethanol and then mix with phenol solution. 2.3.23% hydrochloric acid-ethanol
Concentrated hydrochloric acid
95% ethanol
2.3.3 Counterstaining solution
Lu's basic methylene blue staining solution.
2.3.4 Staining method
2.3.4.1 Heat the smear over a flame to fix it, add carbol fuchsin staining solution, and slowly heat until steam appears, but do not let it boil. If the staining solution decreases due to evaporation, it should be added at any time. Stain for 5 minutes, pour off the staining solution, and wash with water. 2.3.4.2 Add hydrochloric acid-ethanol to decolorize until no red color falls off (the required time depends on the thickness of the smear, generally 1min~3min), and wash with water.
2.3.4.3 Add Lu's alkaline methylene blue staining solution. Restain for 30s~1min, wash with water, wait to dry, and examine under a microscope. 2.3.5 Result: Acid-resistant bacteria are red, and other bacteria, cells, etc. are blue. 2.4 Koch staining method
2.4.1 Staining solution
2.4.1.10.5% safranin solution.
2.4.1.2 0.5% malachite green solution.
2.4.2 Staining method
2.4.2.1 Fix the smear on a flame, add 0.5% malachite green solution, and heat until bubbles appear, about 2min~3min, and wash with water. 2.4.2.2 Add 0.5% malachite green solution, and re-stain for 40s~50s. Wash with water, wait to dry, and examine under a microscope. 2.4.3 Results
Brucella is red, and other bacteria and cells are green. 2.5 Ort's membrane staining method
2.5.1 Staining solution
Distilled water
Grind and dissolve in a mortar.
2.5.2 Staining method
Fix the smear on a flame, add staining solution, and heat until steam is generated, and continue to stain for 3min. Wash with water, wait to dry, and examine under a microscope. 2.5.3 Results
The anthrax Bacillus cells are reddish brown and the capsule is yellow. 2.6 Wright's staining method
2.6.1 Staining solution
Wright's pigment
Grind and dissolve in a mortar.
2.6.2 Staining method
2.6.2.1 After the smear is naturally dried, add the staining solution and fix for 1 minute. 2.6.2.2 Add an equal amount of distilled water (pH 6.5) and stain for 3 minutes to 5 minutes. 2.6.2.3 Rinse with distilled water, wait until dry, and examine under a microscope. 2.7 Flagella staining method
GB/T4789.28-2003
2.7.1 Preparation of dye solution
2.7.1.1 Solution A: Weigh 5g of tannic acid and 1.5g of ferric cyanide (FeCl), dissolve in 100mL of distilled water, add 1mL of 1% sodium hydroxide solution and 2mL of 15% formaldehyde solution after dissolving. 2.7.1.2 Solution B: Weigh 2g of silver nitrate and dissolve in 100mL of distilled water. Add concentrated ammonium hydroxide solution to 90mL of solution B, until precipitation appears, then add more to make it clear, then carefully add the remaining 10mL of solution B to the clear solution until a slight mist appears (this is a critical operation, so be especially careful). When adding ammonium hydroxide and back-titration with the remaining solution B, shake thoroughly while dripping. The dye solution should be prepared and used on the same day, and it will basically be ineffective after 2d to 3d.
2.7.2 Staining method
Add liquid A to the glass slide in the air, and rinse it gently with distilled water after 4min~6min. Then add liquid B and slowly heat until steam appears, and maintain for about half a minute (be careful not to let the dry surface appear when heating). The part with many bacteria may be dark brown to black. Stop heating, rinse with water, and examine under a microscope after drying. The bacteria and flagella are dark brown to black. 2.8 Alkaline fuchsin staining method
Dissolve 0.5g of alkaline fuchsin dye in 20mL95% ethanol, and then dilute to 100mL with distilled water. If there is insoluble matter, filter it with filter paper, or take the supernatant after standing. Note: This staining solution is used to stain the protein toxin crystals in the thuringia bud cells to distinguish it from Bacillus cereus. 3 Biochemical test culture medium and reagents
3.1 Hugh-Leifson medium (for O/F test) 3.1.1 Ingredients
Protein
Sodium chloride
Dipotassium hydrogen phosphate
Glucose
0.2% bromophenol blue solution
Distilled water
1000ml
3.1.2 Preparation method
Dissolve the protein and salts in water, and adjust the pH to 7.2. Add glucose and agar and boil to dissolve the agar, then add the indicator. After mixing, divide into test tubes, sterilize at 121℃ for 15 minutes, and solidify upright for use. 3.1.3-Test method
Pick a small amount of culture from the slant for puncture inoculation, and inoculate two culture media at the same time. After inoculation, add dissolved 1% agar solution to the surface of one of the culture media, the height is about 1cm, and culture at 36℃±1℃. 3.1.4 Results
The results are shown in Table 1.
GB/T 4789.28—2003
Reaction type
Fermentation type (F)
Oxidation type (O)
Alkali production type (A)
3.2 Sugar fermentation tube
3.2.1 Ingredients
Beef extract
Protein
Sodium chloride
Sodium hydrogen phosphate (NazHPO·12H,O)||tt ||0.2% bromothymol blue solution
distilled water
3.2.2 Preparation method
sealed culture medium
1000mL
open culture medium
3.2.2.1 After the glucose fermentation tube is prepared according to the above ingredients, add glucose at 0.5%, divide it into a small test tube with an inverted small tube, and sterilize it at 121℃ for 15min.
3.2.2.2 After the other various sugar fermentation tubes are prepared according to the above ingredients, divide them into 100mL per bottle, and sterilize them at 121℃ for 15min. Separately prepare 10% solutions of various sugars and sterilize them at the same time. Add 5mL of sugar solution to 100mL culture medium and divide it into small test tubes with aseptic operation.
Note: If the decocted sugar is impure and will hydrolyze itself after heating, it should be sterilized by filtration. 3.2.3 Test method: Pick a small amount of culture from the agar slant and inoculate, culture at 36℃±1℃, and generally observe for 2d~3d. The recovery reaction needs to be observed for 14d~30d.
3.3ONPG culture medium
3.3.1 Ingredients
O-Nitrophenyl-pD-galactopyranoside (ONPG) 0.01mol/L sodium phosphate buffer (pH7.5) 1% protein water (pH7.5)
3.3.2 Preparation method
Dissolve ONPG in buffer, add protein water, sterilize by filtration, and dispense into 10tmm×75mm test tubes, 0.5mL per tube, and plug tightly with rubber stoppers.
3.3.3 Test method
Pick a full loop of culture from the agar slant and inoculate at 36℃±1℃ for 1h~3h and 24h to observe the results. If β-galactosidase is produced, it will turn yellow in 1h~3h, if there is no such enzyme, it will not change color in 24h. 3.4 Buffered glucose protein water (for MR and VP tests) 3.4.1 Ingredients
Dipotassium hydrogen phosphate
Glucose
Distilled water
1000mL
3.4.2 Preparation method
After dissolving, adjust the pH, divide into test tubes, 1mL per tube, and sterilize at 121℃ for 15min. 3.4.3 Methyl red (MR) test
GB/T4789.28—2003
Pick a small amount of culture from the agar slant and inoculate it into this medium, and culture it at 36℃±1℃ for 2d~5d. For Hafnia, it should be cultured at 22℃~25℃. Add one drop of methyl red reagent and observe the result immediately. Bright red is positive, and yellow is negative. Methyl red reagent preparation method: 10mg methyl red is dissolved in 30mL 95% ethanol, and then add 20mL distilled water. 3.4.4V-P testbzxZ.net
Inoculate this medium with agar culture and culture it at 36℃±1℃ for 2d~4d. For Hafnia, it should be cultured at 22℃~25℃, add 0.5mL 6% α-naphthol-ethanol solution and 0.2mL 40% potassium hydroxide solution, shake the test tube thoroughly, and observe the result. Positive reaction will turn red immediately or within a few minutes. If negative, it should be placed at 36℃ ± 1℃ for 4 hours before observation. 3.5 Simon's Citrate Medium
3.5.1 Ingredients
Sodium Nitride
Magnesium Sulfate (MgSO,·7H,O)
Ammonium Dihydrogen Phosphate
Potassium Hydrogen Phosphate
Sodium Citrate
Distilled Water
0.2% Bromothymol Blue Solution
3.5.2 Preparation
1000mL
First dissolve the salts in water, adjust the pH, then add agar and heat to dissolve. Then add the indicator, mix well and dispense into test tubes, sterilize at 121℃ for 15 minutes. Place into a slope. 3.5.3 Test method
Pick a small amount of agar culture for inoculation, and culture at 36℃±1℃ for 4 days. Observe the results every day. In the case of positive results, colonies will grow on the slant, and the culture medium will turn from green to blue.
3.6 Klein Citrate Medium
3.6.1 Ingredients
Sodium citrate
Glucose
Yeast extract
Cysteine monohydrochloride
Potassium dihydrogen phosphate
0.2% phenol red solution
Distilled water
3.6.2 Preparation method
1000ml
Heat and dissolve, divide into test tubes, sterilize by high pressure at 121℃ for 15min, and place into a slant. 3.6.3 Test method
Inoculate the entire slant with agar culture, and culture at 36℃±1℃ for 7 days. Observe the results every day. The culture medium turns red for positive results. 197
GB/T4789.28—2003
3.7 Sodium malonate culture medium
3.7.1 Ingredients
Yeast extract
Ammonium sulfate
Dipotassium hydrogen phosphate
Potassium dihydrogen phosphate
Sodium chloride
Sodium malonate
0.2% bromothymol blue solution
Distilled water
3.7.2 Preparation method
1000mL
First dissolve yeast extract and salts in water, add indicator after pH correction, divide into test tubes, and sterilize at 121℃ for 15min. 3.7.3 Test method
Inoculate with fresh agar culture, culture at 36℃±1℃ for 48h, and observe the results. Positives change from green to blue. 3.8 Ammonium Glucose Medium
3.8.1 Ingredients
Sodium chloride
Magnesium sulfate (MgSO.: 7H.O)
Diammonium dihydrogen phosphate
Dipotassium hydrogen phosphate
Glucose
Distilled water
0.2% bromovanillin blue solution
3.8.2 Preparation
1000mL
First dissolve the salts and sugar in water, adjust the pH, then add agar, heat to dissolve, then add the indicator, mix well and dispense into test tubes, sterilize at 121℃ for 15min, and place into a slope. 3.8.3 Test method
Use the inoculation needle to gently touch the surface of the culture, and make a very dilute suspension in the saline tube. No turbidity can be observed by naked eye. The number of bacteria in each inoculation loop should be between 20 and 100. After sterilizing the inoculation loop, pick the bacterial solution for inoculation, and inoculate a common slant in the same way as a control. Incubate at 36℃±1℃ for 24h. The positive ones have normal-sized colonies growing on the glucose ammonium slant; the negative ones do not grow, but grow well on the control medium. If very small colonies grow on the glucose ammonium slant, it can be regarded as a negative result. Note: The container should be soaked in cleaning solution before use. Rinse it with clean water and distilled water, and make a cotton plug with new cotton, and use it after dry heat sterilization. If you are not careful during operation and there is contamination by impurities, it is easy to cause false positive results. Sodium hippurate culture medium
3.9.1 Ingredients
Sodium hippurate
Meat extract
3.9.2 Preparation
Dissolve sodium hippurate in meat extract, dispense into small test tubes, and draw a horizontal line on the wall of the tube. To mark the height of the liquid level in the tube, sterilize at high pressure at 121℃ for 20min.
3.9.3 Reagents
Dissolve 12g of ferric nitride (FeCl·6H2O) in 100mL of 2% hydrochloric acid solution. 3.9.4 Test method
GB/T4789.28-—2003
Inoculate with pure culture, culture at 42℃ for 48h, and observe whether the culture medium reaches the mark on the wall of the test tube. If it is insufficient, add distilled water to the original amount. After centrifugation, take 0.8 mL of the supernatant, add 0.2 mL of ferric chloride reagent, mix well immediately, and observe the results after 10 min~15 min.
3.9.5 Results
The appearance of permanent precipitation is positive.
3.10 Nutritional gelatin
3.10.1 Ingredients
Protein
Beef extract
Distilled water
pH6.8~7.0
3.10.2 Preparation method
1000mL
Heat to dissolve, adjust to pH7.4~7.6, dispense into small tubes, sterilize at 121℃ for 10min, take out and cool quickly to solidify. The final pH should be 6.8~7.0 after rechecking.
3.10.3 Test method
Inoculate with agar culture, culture at 22℃～25℃, observe the results every day, and record the liquefaction time. Or culture at 36℃±1℃, take out every day, put in the refrigerator for 30min and then observe the results. 3.11 Phenylalanine culture medium
3.11.1 Ingredients
Yeast extract
DI-phenylalanine (or L-phenylalanine 1g) Disodium hydrogen phosphate
Sodium chloride
Distilled water
3.11.2 Preparation
1000mL
After heating and dissolving, divide into test tubes, sterilize at 121℃ for 15min, and make a slope. 3.11.3 Test method
Pick a large amount of culture from the agar slope, transfer to phenylalanine agar, and culture at 36℃±1℃ for 4h or 18h~24h. Add 2~3 drops of 10% ferric chloride solution and let it flow down from the slant culture. Those positive for phenylalanine denitrase will be dark green. 3.12 Amino Acid Decarboxylase Test Medium
3.12.1 Ingredients
Protein
Alcohol yeast extract
Glucose
Distilled water
1.6% bromocresol purple-ethanol solution
L-amino acid or DL-amino acid
1000mL
0.5 or 1g/100mL
GB/T4789.28—2003
3.12.2 Preparation
After heating and dissolving the ingredients other than amino acids, dispense into 100mL bottles and add various amino acids: lysine, arginine and ornithine. Add 1-amino acid at 0.5% and DL-amino acid at 1%. Adjust the pH to 6.8. No amino acids are added to the control medium. Dispense into sterile small test tubes, 0.5 mL per tube, add a layer of liquid paraffin on top, and sterilize at 115℃ for 10 minutes. 3.12.3 Test method
Pick the culture from the agar slant and inoculate, culture at 36℃±1℃ for 18h~24h, and observe the results. For amino acid decarboxylase positive, the culture medium should be purple due to alkali production. For negative, there is no alkaline product, but the culture medium turns yellow due to acid production by glucose. The control tube should be yellow. 3.13 Protein water (for indigo matrix test) 3.13.1 Ingredients
Protein (or trypsin)
Sodium chloride
Distilled water
3.13.2 Preparation method
1000ml
Prepare according to the above ingredients, dispense into small test tubes, and sterilize at 121℃ for 15 minutes. 3.13.3 Indigo matrix reagent
3.13.3.1 Kovank reagent: Dissolve 5g of p-dimethylaminoformaldehyde in 75mL of amyl alcohol, then slowly add 25mL of concentrated hydrochloric acid. 3.13.3.2 Ou-Bo reagent: Dissolve 1g of p-dimethylaminobenzaldehyde in 95mL of 95% ethanol, then slowly add 20mL of concentrated hydrochloric acid. 3.13.4 Test method
Pick a small amount of culture for inoculation, and culture at 36℃±1℃ for 1d~~2d, and 4d~5d if necessary. Add about 0.5mL of Kovank reagent and shake the test tube gently. The positive ones will show deep red in the reagent layer; or add about 0.5mL of Ou-Bo reagent, flow down along the tube wall and cover the surface of the culture solution. The positive ones will show rose red in the contact with the liquid surface. Note: The protein should contain rich tryptophan. After each batch of protein is purchased, it should be identified with known bacteria before use. 3.14 Ferrous Sulfate Agar (for Hydrogen Sulfide Test) 3.14.1 Ingredients
Beef Extract
Yeast Extract
Protein Extract
Ferrous Sulfate
Sodium Thiosulfate
Sodium Nitride
Distilled Water
3.14.2 Preparation
1000mL
Heat to dissolve, calibrate pH, dispense into test tubes, sterilize at 115℃ for 15min, take out and stand upright to solidify. 3.14.3 Test Method
Pick agar culture, puncture along the tube wall, culture at 36℃±1℃ for 1d~2d, and observe the results. Hydrogen sulfide producers turn the culture medium black.
Note: To determine the production of hydrogen sulfide by Enterobacteriaceae, trisaccharide iron agar or this culture medium should be used. 3.15 Urea agar
3.15.1 Ingredients
Protein
Glucose
Potassium dihydrogen phosphate
0.4% phenol red solution
Distilled water
20% urea solution
pH7.2±0.1
3.15.2 Preparation
1000mL
GB/T4789.28—2003
Mix the ingredients except urea and agar, and adjust the pH, add agar, heat to dissolve and dispense into flasks. Autoclave at 121℃ for 15min. Cool to 50℃～55℃, and add urea solution that has been sterilized and filtered. The final concentration of urea is 2%, and the final pH should be 7.2±0.1. Dispense into sterile test tubes and place them on a slant for later use. 3.15.3 Test method
Pick agar culture for inoculation, culture at 36℃±1℃ for 24 h, and observe the results. Urease-positive culture medium turns red due to alkali production.
3.16 Potassium cyanide (KCN) culture medium:
3.16.1 Ingredients
Protein tannin
Sodium chloride
Potassium dihydrogen phosphate
Sodium hydrogen phosphate
Distilled water
0.5% potassium cyanide solution
3.16.2 Preparation method
1000mL
Prepare the ingredients except potassium cyanide and dispense them into flasks. Autoclave at 121℃ for 15 min. Place in a refrigerator to cool down fully. Add 2.0mL of 0.5% potassium cyanide solution to every 100mL of culture medium (final concentration is 1:10000), and dispense into 12mm×100mm sterile test tubes, about 4mL per tube. Immediately plug with sterile rubber stopper and place in a 4℃ refrigerator. It can be stored for at least two months. At the same time, use the culture medium without potassium cyanide as the control culture medium and dispense into test tubes for standby use. 3.16.3 Test method
Inoculate agar culture into protein stale water to form diluted bacterial solution, pick 1 loop and inoculate into potassium cyanide (KCN) culture medium. Pick another loop and inoculate into control culture medium. Culture at 36℃±1℃ for 1d~2d and observe the results. If there is bacterial growth, it is positive (no inhibition), and if there is no bacterial growth after 2d, it is negative (inhibition). Note: Potassium cyanide is a highly toxic drug. Be careful when using it and do not infect it alive to avoid poisoning. In summer, dispensing of culture medium should be done in a refrigerator. The main reason for the failure of the test is that the seal is not tight, potassium cyanide gradually decomposes, and hydrorhinic acid gas escapes, resulting in a decrease in drug concentration and bacterial growth, thus causing a false positive reaction. Special attention should be paid to every link during the test. 3.17 Nitrate culture medium
3.17.1 Ingredients
Potassium nitrate
Protein Chen
Distilled water
3.17.2 Preparation method
1000mL
Dissolve, adjust pH, and divide into test tubes, about 5mL per tube, and sterilize at 121℃ for 15min. 201
GB/T4789.28-—2003
3.17.3 Nitrate reduction reagent
3.17.3.1 Solution A: Dissolve 0.8g of p-aminobenzenesulfonic acid in 100mL of 2.5mol/L acetic acid solution. 3.17.3.2 Solution B: Dissolve 0.5g of methylamine in 100mL of 2.5mol/L acetic acid solution. 3.17.4 Test method
After inoculation, culture at 36℃±1℃ for 1d~4d, add one drop of solution A and one drop of solution B, and observe the results. When nitrate is reduced to nitrite, it will turn red immediately or within a few minutes.
Note: There are three reasons for the negative result of this test: bacteria cannot reduce nitrate; nitrite continues to decompose to produce ammonia and nitrogen; the culture medium is not suitable for the growth of bacteria. If you want to check whether the nitrate in the culture medium has not been decomposed, you can add a little zinc powder to reduce nitrate to nitrite and turn red. 3.18 Oxidase test
3.18.1 Reagents
3.18.1.11% dimethyl-p-phenylenediamine hydrochloride solution: prepare a small amount freshly and store in a refrigerator away from light. 3.18.1.21% α-naphthol-ethanol solution. 3.18.2 Test method
3.18.2.1 Take a clean white filter paper and dip it into the colony. Add one drop of dimethyl p-phenylenediamine hydrochloride solution. The positive ones will show pink and gradually deepen. Add another drop of α-naphthol solution. The positive ones will show bright blue within half a minute. Negative ones will not change color within two minutes. 3.18.2.2 Take the reagent with a capillary pipette and drop it directly on the colony. The color reaction is the same as above. 3.19 Cytochrome oxidase test
3.19.1 Reagents
3.19.1.11% dimethyl p-phenylenediamine hydrochloride solution. 3.19.1.21% α-teaphenol-ethanol solution. 3.19.2 Test method
Take a slant culture cultured at 37℃ (or below 37℃) for 20 hours, drop 2 to 3 drops of each reagent from the upper end of the slant, and tilt the slant slightly to allow the reagent mixture to flow through the culture on the slant. If it is a plate culture, the reagent mixture can be dropped on the colony.
3.19.3 Result
Those that show blue color within 2 minutes are positive. Most positive cultures show strong positive reaction within half a minute, and weak or suspicious reaction after 2 minutes are regarded as negative results. 3.20 Catalase test
3.20.1 Reagents
3% hydrogen peroxide solution: Prepare before use. 3.20.2 Test method
Pick an inoculation loop of the colony on the solid culture medium, place it in a clean test tube, add 2mL of 3% hydrogen peroxide solution, and observe the results. 3.20,3 Result
Those that bubble within half a minute are positive, and those that do not bubble are negative. 3.21 Peroxidase test
3.21.1 Reagents
3.21.1.12% catechol solution.
3.21.1.23% hydrogen peroxide solution.
3.21.2 Test method
Pick an inoculation loop of the colony on the solid culture medium, place it in a clean test tube, and drop 1mL of 2% catechol solution and 1mL of 3% hydrogen peroxide solution. Let it stand at room temperature (20℃) for 30min~60min, and observe the results. 3.21.3 Results
For a positive reaction, the bacteria will turn dark brown, and for a negative reaction, the bacteria will not change color. 202
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