Biochemical reactions commonly used in microbial identification

Summary

The biochemical reactions of microorganisms have simple and convenient applications for the identification of common microorganisms, thus demonstrating the diversity of their physiological characteristics. For example, (1) Determine whether a microorganism is a gram-positive or negative cell. (2) To determine initially whether a microorganism is anaerobic.

Operation method

Biochemical reactions commonly used in microbial identification

Principle

1. Some bacteria have the ability to synthesize amylase and can secrete extracellular amylase. Amylase can hydrolyze starch to maltose and glucose, and starch hydrolyzed no longer turns blue when exposed to iodine. 2. Lipase produced by bacteria can break down fats in the culture medium to produce glycerol and fatty acids. Fatty acids can cause the pH of the medium to drop, which can be tested by adding neutral red as an indicator to the lipid medium. The neutral red indicator range is pH 6.8 (red) - 8.0 (yellow). When bacteria break down fats to produce fatty acids, red spots appear in the medium around the colony. 3. Some bacteria secrete proteases to break down gelatin and produce small molecules. If the bacteria have the ability to decompose gelatin, the medium can be changed from a solid state to a liquid state. 4. Buttermilk contains mainly lactose, casein and other components. The utilization of buttermilk by bacteria mainly refers to the decomposition and utilization of lactose and casein. Litmus is often added to buttermilk as an acid-base indicator and redox indicator. Litmus is mauve when neutral, red when acidic, blue when basic, and partially or completely decolorized when reduced. Bacterial utilization of buttermilk can be divided into three cases: (1) acid coagulation: bacteria ferment lactose, produce many acids, so that litmus buttermilk red, when the acidity is very high, can make the buttermilk coagulation, which is called acid coagulation. (2) rennet coagulation: some bacteria can secrete rennet, so that the casein in the milk coagulation, this coagulation occurs in a neutral environment. Usually this bacteria also have the ability to hydrolyze proteins, thus producing alkaline substances such as ammonia, making litmus blue. (3) Peptonization: Casein is hydrolyzed, turning buttermilk into a clear, transparent liquid. Peptonization can be carried out under acidic or alkaline conditions, and generally litmus pigment is reduced and discolored.

Materials and Instruments

Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas mucilaginous, alkali-producing bacillus, Proteus vulgaris
Grease medium Starch medium Gelatinized medium Litmus buttermilk medium
Petri dish Inoculation ring Alcohol lamp Test tube Inoculation needle

Move

I. Starch hydrolysis test

1. Prepare starch medium plate: pour the starch medium which is melted and cooled down to about 50℃ into the sterile petri dish, and make the plate after solidification.
2. Inoculation: mark the bottom of the plate with a marker pen to divide it into two parts, and write the name of the bacterium on each part respectively, take a small amount of bacteria to be tested by the inoculation ring, and spot the center of the opposite part on the surface of the medium, and one of the strains should be Bacillus subtilis do Control bacteria
3. Cultivation: Place the inoculated petri dish in a constant temperature box at 28℃ for 24h.
4. Detection: Remove the plate, open the lid of the petri dish, drop a small amount of iodine solution on the plate, and gently rotate it so that the iodine solution spreads evenly over the whole plate. If a colorless transparent circle appears around the colony, it means that the starch has been hydrolyzed, indicating that the bacterium has the ability to decompose starch. The size of the transparent circle can be used to illustrate the strength of the ability of the test strain to hydrolyze starch.
Second, grease hydrolysis test
1. Put the triangular bottle containing grease medium into boiling water bath to melt, take out and fully oscillate so that the grease is uniformly distributed, then pour it into the sterile petri dish and wait for solidification to form a flat plate.
2. Inoculation: inoculate on both sides of the same petri dish, and one of them is Staphylococcus aureus as a control bacterium. Put in 37℃ constant temperature box culture 24h, take out and observe the color of the plate bacterial moss, if there is a red spot, that is to say that the fat is hydrolyzed, this reaction is a positive reaction. The size of the red spot indicates the strength of the ability of the tested strain to hydrolyze fat.
Third, gelatin liquefaction test
1. Inoculation: Use puncture inoculation method to inoculate E. coli or Enterobacter aerogenes in gelatin medium.
2. Cultivation: Put in 20 ℃ thermostat incubator to cultivate for 48h. If the bacteria do not grow in 20 ℃, then it should be put in the optimal temperature for cultivation.
3. Observation of the results: Observe the medium to have or not to have liquefaction and the shape of the liquefaction. Because gelatin solidifies when it is lower than 20℃ and liquefies by itself when it is higher than 25℃, if the bacteria are cultivated at higher than 20℃, the observation should be put in the ice bath, if the gelatin is liquefied by the bacteria, the gelatin won't be solidified again even at low temperature.
IV. litmus buttermilk test
1. Inoculation: Inoculate the sticky milk alkali-producing bacillus and Pseudomonas aeruginosa into litmus buttermilk medium.
2. Cultivation: Cultivate the inoculated test tubes at a constant temperature of 37℃ for 7d, and additionally keep one litmus buttermilk medium without inoculation as a control.
3. Observation of the results: Take out the cultures, and take the test tubes without inoculation of any bacterium as a control, and observe the changes of the growth of the inoculation of different bacterial growths.

V. Methyl red test (M.R. test)
Inoculate E. coli and Aerobacter aerogenes into glucose peptone water medium respectively, incubate at 32℃ for 24h, add a few drops of methyl red indicator and observe the results, those presenting red color will be considered as positive and those presenting yellow color will be considered as negative.

Caveat

1. in the starch hydrolysis experiment, observe the growth of various bacteria, and gently rotate the iodine solution when adding it so that it spreads evenly over the whole plate.

2. Each experiment needs to set up negative and positive control groups to ensure the experimental results.

3. Cell growth is fast and needs to be observed and recorded in time.

4. Mark the cells with a marker before inoculation and inoculate them with the right number.

Common Problems

1. how to explain that amylase is an extracellular enzyme rather than an intracellular enzyme: starch is a large molecule, it is difficult to enter into the cell, and even requires high energy-consuming cytotoxicity, thus through the action of extracellular enzymes, starch is hydrolyzed into smaller molecules of glucose and then transported into the cell, which doesn't require high energy-consuming cytotoxicity.

2. The role of iodine: Since starch will generate glucose after hydrolysis, glucose is a polyhydroxy aldehyde, so you can use the characteristic properties of the aldehyde group to verify, such as silver mirror reaction, Ferrin's reagent, etc., a positive indicates that the hydrolysis of starch produces glucose. Negative indicates that starch has not been hydrolyzed.

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