Bacterial Biochemical Reaction Test
Bacterial Biochemical Reaction Test
Some bacteria belong to different species or types, but their morphology and colony characteristics are basically the same, relying only on the observation of morphology and colony, can not be distinguished. However, these different species and types of bacteria, their material metabolism and products are often different, which can be used to identify the bacteria, that is, the biochemical reaction of bacteria, biochemical reaction test must use pure species of bacteria. The purpose of this experiment is to understand the preparation method of culture medium commonly used for bacterial biochemical reactions and to learn several biochemical reactions commonly used for the identification of Enterobacteriaceae.
Operation method
Bacterial biochemical reaction tests
Materials and Instruments
Bacteria Move I. Preparation of common culture media for bacterial biochemical reactions For more product details, please visit Aladdin Scientific website.
Peptones Sodium chloride Distilled water Peptone water Glucose Lactose Mannitol Sucrose Maltose Bromocresol violet Bromomuscumolan Phenol red
Test tubes Sugar fermentation tubes
1. Peptone water
Ingredients: peptone 1.0 g, sodium chloride 0.5 g, distilled water 100 ml.
Dissolve the above ingredients by heating and correct pH 7.6, divide into test tubes, autoclave at 15 lbs 15 min and set aside. Commonly used in the preparation of sugar fermentation medium or for checking bacteria in indigo substrate production test, etc..
2. Sugar fermentation tubes
Ingredients: peptone water, sugars such as glucose, lactose, mannitol, sucrose or maltose, indicators such as bromocresol violet (B.C.P), bromomuscovanillan (B.T.B) or phenol red (P.R).
Add some kind of sugar (0.5-1%) and indicator to peptone water, dispense in small test tubes with an inverted tube (Figure 2-5), and autoclave for 15 lb 20 min for use. Commonly used in sugar fermentation tests.
3. Glucose peptone water
Ingredients: peptone 0.5 gglucose 0.5 gDipotassium hydrogen phosphate 0.5 gDistilled water 100 ml
The above ingredients were mixed and dissolved to correct the pH to 7.0, divided into test tubes and autoclaved at 10 lb for 20 min. Used for V-P test and Methyl Red test.
4. Citrate medium
Ingredients: sodium citrate 0.2gMagnesium sulfate 0.02gAmmonium dihydrogen phosphate 0.1 gDipotassium hydrogen phosphate 0.1 gSodium chloride 0.5 gAgar (rinsed continuously and thoroughly under running water) 2.0 gDistilled water 100 mlB.T.B alcohol solution (0.5%) 1.6 ml
After heating to dissolve the various components to correct the pH to 6.8, then add 0.5% B.T.B ethanol solution to mix, divided into test tubes, 8 pounds 15 min autoclave sterilization, remove and set into the slant while hot. Used for citrate utilization test.
5. Lead acetate medium
Ingredients: Plain agar medium 100 mlSodium thiosulfate 0.25 gLead acetate solution (10%) 1 ml
First dissolve ordinary agar medium, then add sodium thiosulfate, correct pH to 7.2, boil and filter, autoclave for 10 lbs 20 min, remove and aseptically add sterilized 10% lead acetate solution 1 ml, mix well and mount in small test tubes, solidify upright and prepare for use. Used to detect the production of hydrogen sulfide.
6. Urea medium
Ingredients: peptone 0.1 gsodium chloride 0.5 gPotassium dihydrogen phosphate 0.2 gAgar 2.0 gDistilled water 100 mlPhenol red solution (0.6%) 0.2 mlGlucose solution(10%) 0.1 mlUrea (20%) 1 ml
Dissolve peptone, sodium chloride, potassium dihydrogen phosphate and agar by heating in distilled water, correct pH7.4, filter and add 0.6% phenol red solution to mix, autoclave at 8 lbs for 15 min, remove and leave to cool to 60°C, add sterilized 10% glucose solution and 20% urea, divide into small test tubes and condense into a slant for standby. For urea decomposition test.
II. Bacterial biochemical reaction tests
1. Fermentation of sugars
Inoculate the bacteria in sugar fermentation tubes and incubate at 37°C for 18-24 h to observe the results. If necessary, incubate for a longer period of time before determining the results.
Bacteria such as decomposition of sugar, the production of acid, so that the indicator color (general record symbol is "ten"); some bacteria in the decomposition of sugar at the same time also produces gas, the inverted tube bubbles appear (record symbol for the "⊕"); such as the bacteria do not decompose the sugar, the indicator does not change color, do not see the culture solution cloudy (record symbol for the "-").
2. IMViC consists of the following four tests.
(1) Production of indole substrate (Production of indole)
Inoculate the bacteria in peptone water and incubate at 37℃ for 18～24 h. Some bacteria have tryptase, which can decompose tryptophan in peptone and produce indigo substrate, which is colorless and can't be checked directly, then add several drops of indigo substrate reagent (Kovacs reagent) on the liquid surface of the culture medium, and those whose contact surfaces show rose red are positive, and those who are still yellow are negative, and if the color is not obvious, then add 4～5 drops of ether to make the ether dispersed in the liquid, and if there are indigo substrate in culture liquid, it can be extracted into the ether layer with more obvious color reaction. ～If the color is not obvious, add 4-5 drops of ether and shake the test tube to make the ether dispersed in the liquid. If there is indigo matrix in the culture medium, it can be extracted into the ether layer and the color reaction is more obvious.
(Note) Kofacs reagent: take 4g of Paradimethylaminobenzaldehyde (Paradimethylaminobenzaldehyde), add 380 ml of 95% alcohol and 80 ml of concentrated hydrochloric acid.
(2) Methyl red test
Inoculate the bacteria in glucose peptone water and incubate at 37℃ for 48 h, then add 3 drops of methyl red reagent to it. Bacteria such as E. coli decompose glucose to produce pyruvic acid, but pyruvic acid is not condensed to acetyl methyl methanol, more acid in the medium, low pH, so it is red and positive. While Aerobacter aerogenes condenses pyruvate to acetylmethylmethanol, there is less acid in the medium and the pH is high, so it is yellow and negative.
Note: Methyl red reagent: take 0.04 g of methyl red and dissolve in 100 ml of 60% alcohol. The reagent is red when acidic and yellow when alkaline.
(3) V-P test (Voges-Proskauer test)
Bacteria inoculated in glucose peptone water, 37 ℃ culture 48h, and then add an equal amount of 40% potassium hydroxide solution (containing 0.3% creatine) to the culture medium. Bacillus aerogenes decompose glucose to generate pyruvate, pyruvate is condensed to acetyl methyl methanol, the latter is oxidized by oxygen in the air when encountered with alkali, generating diacetyl, diacetyl and guanidine compounds in the medium to react, generating red compounds, the medium becomes red, it is positive for the V-P test, and other bacteria can not generate acetyl methyl methyl methanol, the medium does not become colorful, it is negative.
(4) Utilization of citrate (CITRUS)
Bacteria were inoculated on citrate medium and incubated at 37℃ for 48 h. Bacteria such as Aerobacter aerogenes could utilize citrate as a carbon source, decompose citrate to produce carbonate, which made the medium alkaline, and the indicator in the medium changed from light green to dark blue, which was positive for citrate utilization test. E. coli can not decompose citrate, and the color of the medium remains unchanged, which is negative.
3. Hydrogen sulfide production test (Production of H2S)
Inoculate the bacterial puncture in lead acetate medium and observe the result after 24 h incubation at 37℃. Some bacteria can decompose the sulfur-containing amino acids in the medium to produce hydrogen sulfide, and the puncture site is dark brown, which is a positive reaction. If there is no change in the color of the medium, the reaction is negative.
4. Splitting of urea (SUR) test
Inoculate the bacteria in urea medium and incubate at 37℃ for 18～24 h. Aspergillus can decompose urea rapidly (2～4 h) to produce a large amount of ammonia, which makes the medium become alkaline and purplish-red, and it is a positive reaction.
Nowadays, many kinds of micro-volume, rapid, semi-automatic or fully automatic bacterial biochemical reaction kits and testing instruments have been widely used in clinics. Such as SCEPTOR bacterial identifier, is an automated identification system, including microbial identification and drug sensitivity test two parts, through the 24 biochemical reaction and 59 kinds of antibiotic sensitivity test, the results of the automatic entry into the computer, the information by the computer to make a quick judgment, the results are reliable, easy to operate.