Bio-chemical experiments on bacteria
Last Update: 2021-01-20
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bacteria have their own unique enzyme system, so the ability to break down the substrate is different, its metabolites are also different. These
biochemical methods and can be used to distinguish and identify the types of bacteria. Biochemical methods are used to identify different bacteria, called biochemical experiments called bacteria, or biochemical
reactions. Biochemical testing methods are many, here mainly introduces the metabolic test of carbohydrate
1. Sugar (alcohol, glycoside) fermentation test
(1) principle: different types of bacteria contain fermented enzymes of different sugars (alcohols, glycosides), and therefore the metabolic capacity of various sugars (alcohols, gsides) is also different, even if it can break down a certain sugar (alcohol, glycosides), its metabolites can vary from species to species. The ability of bacteria to degrade
acids after degradation of sugars (alcohols, gsides) in
cultures can be used to identify bacterial species.
(2) Method: Add a specific sugar (alcohol, glycoside) of 0.5 to 1.0% (w/v) in the base
(e.g. phenolic red broth base medium pH7.4). There are many types of sugars (alcohols, glycosides) used, depending on the needs of monosaccharides, polysaccharides or polysaccharides, polyols and cyclools, etc., as seen in Table 6-4-1. The pure culture bacteria to be identified were inoculated into the experimental medium and incubated for several hours to two weeks in a incubator at 35 degrees C (depending on the method and strain) to observe the results. If using a micro-fermentation tube, or requires a longer culture time, care should be taken to maintain the humidity around it, so as
(3) Results: Bacteria that break down acids produced by sugars (alcohols, glycosides), indicators in the medium react acidicly (e.g. phenolic red to yellow), gas-producing bacteria can create bubbles in small inverters (Durham tubes), and solid mediums create fissures. There was no change without breaking down the sugar.
(4) Application: sugar (alcohol, glycoside) fermentation test, is the identification of bacteria bio-chemical reaction test in the most important test, different bacteria can ferment different sugars (alcohol, glycosides), such as salmonella can ferment glucose, but can not ferment lactose, E. coli can ferment glucose and lactose. Even if both bacteria can ferment the same sugar, the fermentation results are not the same, such as Shiga bacteria and E. coli can ferment glucose, but the former only produce acid, the latter is acid, gas production, so this test can be used to identify bacteria.
monosaccharose ivy: erythucleose, five carbon sugars: nuclear sugar ucose xycosaccharin, six carbon sugars: glucose fructose semi-lactose sugar
thalose cotton sugar (glucose and fructose and semi-lactose)
polysaccharine (polymolecular fructose) starch
ol side marigoldol wediol glycol sorbitol
2. Glucose metabolic type identification test
(1) principle: bacteria in the process of breaking down glucose, must have molecular oxygen participation, called oxidation type; Fermented bacteria break down glucose in both aerobic or anaerobic environments, while oxidized bacteria can't break down glucose in oxygen-free environments. This test, also known as oxidative fermentation (O/F or Hugh-Leifson, HL) test, can be used to distinguish the metabolic type of bacteria.
(2) Method: Pick a small amount of pure culture (do not pick from the selective plate) inoculated 2 HL culture tubes, in which sterile liquid paraffin with a height of at least 0.5 cm is added to isolate air (as a sealing tube) and the other tube does not (as an open tube). Incubate more than 48h in a 35c incubator.
(3) Results: Neither tube medium produced acid (color unchanged) was negative, both tubes produced acid (yellowing) as fermentation type, liquid paraffin tube did not produce acid, and no liquid paraffin tube produced acid as oxidizing type.
(4): Mainly used in the identification of E. coli and other non-fermented bacteria. E. coli and Vibrio bacteria are fermented and non-fermented bacteria are oxidized or alkali-producing. It can also be used to identify staphylococcus (fermentation type) and microcosm (oxidation type).
3． Methyl red (MR) test
(1) principle: some bacteria in the sugar metabolism process, the breakdown of glucose to produce acetone acid, acetone acid is further broken down into foric acid, acetic acid and amber acid, so that the culture pH dropped below 4.5, the addition of methyl red indicator red. If bacteria break down glucose with little acid production, or the acid produced is further converted into other substances (e.g. alcohols, aldehydes, ketones, gases and water), the culture base pH is above 5.4 and the methyl red indicator is orange.
(2) Method: Inoculate the bacteria to be tested in glucose phosphate protein water, incubate 48h to 96h at 35 degrees C, add 5 to 6 drops of methyl red indicator in 5 ml medium, immediately observe the results.
(3) results were determined: the red is positive, orange is negative, orange is weak positive.
(4) application: often used in the identification of certain genus E. coli, such as E. coli and E. coli, the former positive, the latter negative. E. coli genus and Hafnia genus were negative, while salmonella genus, Syga genus, acid genus and Bacillus deformation genus were positive.
4． β-semi-lactose glycosidease test (ONPG test)
(1) principle: Lactose fermentation process requires lactose permeable enzyme and β-semi-lactose glycosidease to break down quickly. Some bacteria only have semi-lactose glycosidease, so can only slowly ferment lactose, all lactose rapid fermentation and slow fermentation of bacteria can quickly hydrolysate nitrophenol-β-D-semi-lactose glycoside (O-nitrophenyl-β-gala-gala-ctopyranoside, ONPG) to produce yellow neighbor nitrophenol. For identification of the genus bacillus bacillus, the genus Arizona and the genus salmonella.
(2) method: inoculate the bacteria to be tested in ONPG broth, 35 degrees C water bath or incubator incubation 18 to 24h, observation results.
(3) result: bright yellow is positive and colorless is negative.
(4) application: can be used for the rapid identification of slow fermentation lactose bacteria, this method for the rapid and slow decomposition of lactose bacteria can be tested in a short period of time positive. The genus Eich, genus Bacillus bacillus bacillus, Creeber genus, Hafnia genus, genus Sarepta and genus E. coli were all tested positive, while salmonella genus, genus Bacillus Difficile and Genus Providence were negative.
5． VP test
(1) principle: to determine the ability of bacteria to produce acetyl methyl methanol. Some bacteria, such as E. coli, break down glucose to produce acetone acid, which is further de-de-pyrogenated to form acetyl methyl methanol. Under alkaline conditions, acetyl methyl methanol is oxidized into diacetyl, which in turn binds to arginine-containing substances such as arginine in the culture to form red compounds. That is, V-P test positive.
(2) Method: Inoculate the bacteria to be tested in glucose phosphate protein water, incubate 24 to 48h at 35 degrees C, and add 50g/L alpha-phenol (95% ethanol soluble) liquid) 0.6ml, gently shake
the test tube
, then add 0.2 ml 400g/L KOH, gently shake the test tube 30s to 1min, and then sit still to observe the results.
(3) result: positive for red and negative for yellow or similar copper.
(4) application: mainly used for the identification of E. coli and E. coli. This test is often used in cooperation with the MR test, in general, the former is a positive bacteria, the latter is often negative, and vice versa. However, E. coli bacteria are not always so regular, such as hive hafnia bacteria and strange difficile VP test and MR test are often the same positive.
6． Bile seven-leaf glycoside hydrolytic test
(1) principle: in the presence of 10% to 40% bile, to determine the ability of bacteria hydrolytic seven-leaf glycoside. Helioside is produced by bacterial decomposition of helilutinin, heliolytin and the iron ions in the culture base of ferrochloride to form a black compound.
(2) method: the bacteria will be inoculated in the bile hexaethyl glycoside medium, 35 degrees C incubation 18 to 24h, the observation results.
(3) result: the culture base completely blacked out to positive, unchanged black to negative.
(4) Application: Mainly used to identify the difference between Streptococcus D and other streptococcus, as well as some species of E. coli, some anaerobic bacteria (e.g. fragile Bacillus athrobacteria, etc.) preliminary identification. Group D streptococcus tested positive.
7． Starch hydrolysis test
(1) principle: the bacteria that produce amylase can dissolve starch water into sugar, in the culture base drip iodization, can appear around the transparent area.
(2) method: will be tested bacteria line inoculated in starch
plate or test tube, 35 degrees C incubation 18 to 24h, add a few drops of terradine, immediately observe the results.
(3) results: positive, color-free transparent area around the bacterium, blue elsewhere;
(4) Application: for diphtheria rod-like Bacillus biological type of type, heavy-duty starch hydrolysis test positive, light, medium-negative;
8． Glycelor redness test
(1) principle: glycelor can be broken down by bacteria to produce acetone acid, acetone acid de-de-steroidal base for acetaldehyde, acetaldehyde and colorless redness to produce tantalum compounds, dark fuchsia.
(2) method: take the test bacteria inoculated in the medium of glycero-red broth, incubated at 35 degrees C, observed 2 to 8d. Negative control should be done at the same time.
(3) result: fuchsia was positive and negative with the same color as the control tube.
(4) application: mainly used for the identification of species within the genus salmonella. Typhoid salmonella, type A (C) type of side typhoid salmonella, pig cholera salmonella, Kondolf salmonella and Sendai salmonella this test is negative, type B side typhoid salmonella results are uncertain, other uncommon salmonella mostly positive.
9． Glucose acid oxidation test
(1) principle: some bacteria oxidize potassium gluconate, α-ketone gluconic acid. α-ketone-based glucose is a reductive substance that reacts with Ban's
, resulting in brown or brick-red copper oxide precipitation.
(2) Method: Inoculate the bacteria to be tested in a glucoseate medium (1 ml), incubate 48h at 35 degrees C, add 1 ml of Ban's reagent, boil 10min in a water bath and cool quickly, observed results.
(3) result: yellow-to-brick red precipitation is positive. Unchanged or still blue is negative.
(4) application: mainly used for the identification of false monocytobacteria and E. coli microbiotic sub-groups.
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