Glycoside alcohol metabolism test

Different types of microorganisms have different metabolic pathways and metabolites.
Some biochemical reactions can be used to determine these metabolites.
Biochemical reactions are often used to identify microorganisms that are difficult to distinguish in form and other aspects.
Microbial biochemical identification is an important basis for microbial classification and identification.
1.
At present, most commercial biochemical kits are used to complete biochemical identification tests.
Therefore, this book only focuses on the principles of various biochemical reactions.
If the test method differs from the kit method, the kit method shall prevail, such as For the preparation of reagents, please refer to the relevant national standards
.

Glycoside alcohol metabolism test

1.
Trisaccharide iron (TSI) agar test The ability of different microorganisms to decompose and utilize sugars is very different, or can be used or cannot be used, and those that can be used, either produce gas or not
.

It can be inspected with indicator and fermentation tube

Test method: Pick the bacteria to be tested with an inoculation needle, spread it on the slope and puncture the inoculation, and incubate at (36±1)℃ for 18-24 hours.
The observation results are shown in color chart 1
.

This test can simultaneously observe the fermentation of lactose, glucose and sucrose to produce acid or gas, and hydrogen sulfide (blackening)
.

Glucose is decomposed to produce acid to make the slope yellow first, but due to the small amount, the small amount of acid generated is oxidized by contact with air, and the bacteria use the nitrogenous substances in the culture medium to generate alkaline products, so the slope turns red later.

2.
O-nitrophenol-β-D-galactoside (ONPG) test ONPG is a structural analogue of lactose.
The colorless ONPG is hydrolyzed by β-galactosidase to produce yellow ortho-nitrophenol
.

Bacteria that decompose lactose quickly or slowly, such as Escherichia, Citrobacter, and Klebsiella, are positive

3.
VP test Some bacteria can decompose glucose to produce pyruvic acid in the glucose peptone water medium, which is condensed and decarboxylated to acetyl methyl methanol.
The latter is oxidized to diacetyl by oxygen in the air under a strong alkaline environment .
Diacetyl reacts with the guanidine group in peptone to produce a red compound, which is called the VP(+) reaction
.

experiment method:

(1) O'Meara's method
.

Inoculate the test bacteria in a general culture medium, culture at (36±1)℃ for 48 hours, add 1 mL of O'Meara reagent (40% sodium hydroxide aqueous solution with 0.

(2) Barritt's method
.

Inoculate the test bacteria in a general culture medium and incubate at (36±1)℃ for 4 days.

(3) Fast method
.

Put 2 drops of 0.

This test is generally used to identify the genus Enterobacteriaceae
.

When used in Bacillus, Staphylococcus and other bacteria, the phosphate in the universal medium will hinder the production of acetyl methyl alcohol, so it should be omitted or replaced with sodium chloride

4.
Methyl red test (MR) Each genus of Enterobacteriaceae can ferment glucose and produce pyruvate in the process of decomposing glucose.
During further decomposition, due to different sugar metabolism pathways, lactic acid, succinic acid, acetic acid and formic acid can be produced Various acidic products can reduce the pH of the medium to below 4.
5 and make the methyl red indicator red
.

Test method: Pick a little fresh pure culture, inoculate it in MR-VP medium, culture at (36±1) ℃ or 30 ℃ (30 ℃ is better) for 3 to 5 days, from the next day, take it every day Add 1 to 2 drops of methyl red indicator to 1 mL of culture medium.
The positive is bright red, the weak positive is light red, and the negative is yellow.
See color picture 2
.

If it is found to be positive or still negative until the fifth day, the result can be determined

Methyl red is an acid indicator with a pH range of 4.
4 to 6.
0 and a pK value of 5.
0
.
Therefore, below pH 5.
0, the red color will increase with the increase of acidity, and above pH 5.
0, the yellow will increase with the increase of alkalinity.
When the pH is 5.
0 or close to the upper and lower sides, the discoloration may not be obvious enough.
At this time, the cultivation time should be extended.
Repeat the test
.

5.
Aescin hydrolysis test Some bacteria can hydrolyze esculin to produce esculin, which reacts with the ferrous ions of ferric citrate in the culture medium to produce black compounds and make the culture medium black
.
For example, Klebsiella, Enterobacter and Serratia, Listeria monocytogenes can hydrolyze esculin, and Enterococcus and group D streptococci can also hydrolyze esculin and are resistant to bile
.