1. Observation of morphological structure and cultivation characteristics
1. Observation of the morphological structure of microorganisms is mainly through staining. Observe their shape, size, arrangement, cell structure (including cell wall, cell membrane, nucleus, flagella, spores, etc.) and staining characteristics under a microscope to intuitively understand Morphological and structural characteristics, according to the different morphological structure of different microorganisms to achieve the purpose of distinguishing and identifying microorganisms.
2. The cell group formed by the bacterial cells on the surface of the solid medium is called a colony. Different microorganisms grow and reproduce in a certain medium, and the characteristics of the formed colonies are very different, but the culture characteristics of the same kind of bacteria have certain stability under certain conditions. In this way, different microorganisms can be distinguished and identified. Therefore, the observation of microbial culture characteristics is also an important content in microbial inspection and identification.
1) The culture characteristics of bacteria include the following: On a solid medium, observe the size, morphology, color (whether the pigment is water-soluble or fat-soluble), gloss, transparency, texture, bulge shape, edge characteristics, and migration. Turbidity and precipitation of surface growth (bacterial membrane, ring) in liquid culture. Puncture and inoculate semi-solid medium to observe movement and diffusion.
2) Culture characteristics of mold yeast: Most yeasts do not have filaments, and the colonies formed on the solid medium are very similar to those of bacteria, except that they are larger and thicker than bacterial colonies. Liquid culture is also similar to bacteria, with uniform growth, precipitation or formation of bacterial membranes on the liquid surface. The fungus has branched filaments with thick and long hyphae. In suitable medium, the hyphae extend infinitely along the surface of the medium. Mycelium, aerial mycelium and spore filaments of molds often have different colors, so the color of the colony edge and center, front and back are often different, such as penicillium: green spores, aerial hyphae colorless, base The mycelium is brown. Molds form flocculent, villous and spider web colonies on the solid culture surface.
2. Physiological and biochemical tests
Microbial biochemical reaction refers to the use of chemical reactions to determine the metabolic products of microorganisms. Biochemical reactions are commonly used to identify some microorganisms that are not easily distinguishable in form and other aspects. Therefore, microbial biochemical reaction is one of the important basis for microbial classification and identification.
The biochemical reactions commonly used in microbiological testing are:
1. Glycolysis test
The ability of different microorganisms to decompose and use carbohydrates is very different, and can be used or not, those who can use, or produce gas or not. Available indicators and fermentation tube inspection.
Test method: Aseptic operation, use the inoculation needle or ring to transfer a little pure culture, inoculate the fermentation liquid medium tube, if it is semi-solid medium, use the inoculation needle as a puncture inoculation. After inoculation, incubate at 36 ± 1.0 ° C and observe the results every day to check whether the color of the medium has changed (acid production), whether there are bubbles in the small inverted tube, and tiny bubbles are also positive for gas production. Then check whether there are tiny bubbles along the puncture line and the wall and bottom of the tube. Sometimes it can be seen whether the inoculation bacteria have power. If there is power, the culture can grow in a diffuse manner.
This test is mainly to check the fermentation ability of bacteria on various sugars, alcohols and glycosides, so as to identify various bacteria, so it is often necessary to inoculate multiple tubes at the same time for each test. The commonly used indicators are phenol red, bromocresol purple, bromothymol blue and An-drade indicators.
2. Starch hydrolysis test
Certain bacteria can produce enzymes that break down starch and hydrolyze starch into maltose or glucose. After the starch is hydrolyzed, it will no longer turn blue in case of iodine.
Test method: 18-18 hours of pure culture, spread on starch agar slant or plate (one plate can be inoculated by partition, test several cultures) or directly transplanted in starch broth, at 36 ± 1 ° C Cultivate for 24 ~ 48h, or 5 days at 20 °. Then immerse the iodine reagent directly on the culture surface. If it is a liquid culture, add a few drops of iodine reagent to the test tube. Immediately examine the results, and the positive reaction (the starch is broken down) is a dark blue agar medium, a colorless transparent ring around the colony or culture, or no change in the color of the broth. Negative reaction is no transparent ring or broth is dark blue.
The starch hydrolysis system is a step-by-step process, so the test results have a certain relationship with the strain's ability to produce amylase, the cultivation time, the amount of starch in the medium, and the pH. The pH of the medium must be neutral or slightly acidic, with pH 7.2 being the most suitable. Starch agar plates should not be stored in the refrigerator, so they should be prepared immediately before use.
3. VP test
Certain bacteria can decompose glucose to produce pyruvic acid in the glucose peptone water culture medium. Pyruvate is condensed and decarboxylated to acetylmethyl methanol. The latter is oxidized by air oxygen to diacetyl, diacetyl and peptone in a strong alkaline environment. The guanidine group produces a red compound, called the VP (+) reaction.
experiment method:
1) O'Meara's method: inoculate the test bacteria on a universal medium, and culture at 36 ± 1 ° C for 48h, add O'Meara reagent (add 0.3% creatine Creatine or creatinine Creatinine 40% hydrogen) Sodium oxide aqueous solution) 1ml, shake the test tube for 1 ~ 2min, and leave it at room temperature or a thermostat at 36 ± 1 ° C. If no eosin appears within 4h, it is judged as negative. There are also those who advocate placing the water bath at 48 ~ 50 ° C for 2h to judge the result.
2) Barritt's method: inoculate the test bacteria on a universal medium, incubate at 36 ± 1 ° C for 4 days, and add 2.5ml of α-naphthol (2-na-phthol) pure alcohol solution at a temperature of 36 ± 1 ° C for 4 days. Add 0.2ml of 40% potassium hydroxide aqueous solution, shake for 2 ~ 5min, the positive bacteria often appear red immediately, if there is no red, leave it at room temperature or 36 ± 1 ° C incubator, if it still does not appear red within 2h, it can be judged Is negative.
3) Rapid method: place 2 drops of 0.5% creatine solution in a small test tube, pick an inoculating loop of the ferric sugar agar slant culture of acid production reaction, emulsify and inoculate them, add 3 drops of 5% α-naphthol , 2 drops of 40% sodium hydroxide aqueous solution, and leave it for 5 minutes after shaking to determine the result. Cultures that do not produce acid cannot be used.
This test is generally used for the identification of Enterobacteriaceae. When used in other bacteria such as Bacillus and Staphylococcus, the phosphate in the universal medium can hinder the production of acetylmethyl alcohol, so it should be omitted or replaced with sodium chloride.
4. Methyl Red test
Each genus of Enterobacteriaceae can ferment glucose and produce pyruvic acid during the decomposition of glucose. During further decomposition, due to different sugar metabolism pathways, a large number of acidic products such as lactic acid, succinic acid, acetic acid and formic acid can be produced, which can make the medium The pH value drops below pH 4.5, making the methyl red indicator red.
Test method: Pick a little new pure culture to be tested and inoculate it in a general culture medium, cultivate at 36 ± 1 ° C or 30 ° C (preferably 30 ° C) for 3 to 5 days, from the next day, Take 1ml of culture solution daily and add 1 ~ 2 drops of methyl red indicator. The positive is bright red, the weak positive is light red, and the negative is yellow. The result can be determined until it is found positive or still negative on the 5th day.
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 yellow color increases with acidity, and above pH 5.0, the yellow color increases with alkalinity. When the pH is 5.0 or above, the discoloration may not be obvious. In this case, the culture time should be extended and the test repeated .
5. Indole (Imdole) test
Some bacteria can break down tryptophan in peptone to produce indole. The presence of indole can be demonstrated by the color reaction. Indole combines with p-dimethylaminobenzaldehyde to form rose indole, which is a red compound.
Test method: Inoculate a small amount of the pure culture to be tested into the test medium tube. After cultivating at 36 ± 1C for 24h, take about 2ml of the culture solution, add 2 ~ 3 drops of Kovacs reagent, shake the test tube gently, and the red is positive , Or add a small amount of ether or xylene first, shake the test tube to extract and concentrate the indigo matrix, wait for it to float on the surface of the culture solution, then slowly add a few drops of Kovacs' reagent along the wall of the test tube, the contact surface is red, which is positive .
The experiment proves that the indigo base reagent can react with 17 kinds of indigo base compounds to produce a positive reaction. If xylene or ether is used for extraction first, and then the reagent is added, only the indigo base or 5-methyl indigo base will appear in the solvent. Red, so the result is more reliable.
6. Nitrate reduction test
Some bacteria have the ability to reduce nitrate and can reduce nitrate to nitrite, ammonia or nitrogen. The presence of nitrite can be tested with nitric acid reagents.
Test method: Before the test, mix 0.2ml each of the reagent A (sulfanilic acid glacial acetic acid solution) and B (α-naphthylamine ethanol solution) test solutions, take about 0.1ml of the mixed reagent, and add it to the liquid culture or agar The surface of the slant culture is red immediately or within 10 minutes, and the test is positive. If no red appears, it is negative.
When testing with α-naphthylamine, the positive red fades quickly, so the result should be determined immediately after addition. There must be a non-inoculated medium tube as a negative control during the experiment. Alpha-naphthylamine is carcinogenic, so care should be taken when using it.
7. Gelatin liquefaction test
Some bacteria have gelatinases (also known as proteolytic enzymes), which can hydrolyze gelatin into peptides first, and then further hydrolyze them into amino acids, liquefying after losing gel properties.
Test method: Pick the culture of bacteria to be tested from 18 to 24 hours, inoculate a large amount of gelatin in the upper layer of gelatin to a depth of about 2/3 or plant it on a plate medium. Incubate at 20-22 ° C for 7-14 days. The upper layer of gelatin can also be cultivated at 36 ± 1 ℃. Observe the results every day. If the gelatin itself is liquefied due to the high cultivation temperature, it should not be shaken, let it stand in the refrigerator until it solidifies, and then observe whether it is liquefied by bacteria. The observation of the results of the plate test is that the reagent is dropped on the colonies spotted on the medium plate. If it is positive, a clear band should appear around the colony after 10-20 minutes. Otherwise it is negative.
8. Urease test
Some bacteria can produce urease, which decomposes urea to produce two molecules of ammonia, making the medium alkaline and phenol red pink. Urease is not an inducing enzyme because bacteria can synthesize this enzyme regardless of the presence of the substrate urea. The optimum pH of its activity is 7.0.
Test method: Pick 18-24 hours of the culture of the test bacteria to be inoculated in a large amount of liquid culture tube, shake it evenly, and incubate at 36 ± 1 ℃ for 10, 60 and 120 minutes, and observe the results respectively. Or spread and puncture on the agar slope, do not reach the bottom, leave the bottom as a discoloration control. Observe the results after culturing for 2, 4 and 24h. If it is negative, it should continue to be cultured for 4 days for final judgment. It turns pink to be positive.
9. Oxidase test
Oxidase, also known as cytochrome oxidase, is the terminal respiratory enzyme of the cytochrome respiratory enzyme system. Oxidase first oxidizes cytochrome C, and then this oxidized cytochrome C oxidizes p-phenylenediamine to produce a color reaction.
Test method: Add 1 or 2 drops of reagent onto the agar slant culture or blood agar plate colony. The positive one is Kovacs' reagent pink to dark purple, and the Ewing's modified reagent is blue. Negative persons have no color change. The test result should be judged within a few minutes.
10. Hydrogen sulfide (H2S) test
Some bacteria can decompose sulfur-containing amino acids or sulfur-containing compounds in the culture medium, and produce hydrogen sulfide gas. Hydrogen sulfide can produce black precipitates when it meets lead salts or low iron salts.
Test method: In the medium containing sodium thiosulfate and other indicators, inoculate along the wall of the tube, incubate at 36 ± 1 ℃ for 24 ~ 28h, and the medium is black as positive. Negative should continue to be cultured until 6 days. The lead acetate paper strip method can also be used: inoculate the test bacteria in general nutrient broth, and then hang the lead acetate paper strip over the culture medium, so as not to be splashed as appropriate; use a pipe plug to press and place it at 36 ± 1 ℃ for culture 1 ~ 6 days. The note turned black to be positive.
11. Trisaccharide iron (TSI) agar test
Test method: Use the inoculation needle to pick the suspicious colony or pure culture of the bacteria to be tested, puncture the inoculation and spread on the inclined surface, incubate at 36 ± 1 ℃ for 18 ~ 24h, and observe the results.
In this test, it can be observed that lactose and sucrose are fermented to produce acid or gas (yellow); hydrogen sulfide (black). Glucose is decomposed to produce acid, which can cause the slope to turn yellow first, but due to the small amount, the generated small amount of acid is oxidized by contact with air, and the bacteria use the nitrogen-containing substance in the culture medium to generate alkaline products, so the slope becomes red later Because the bottom is in an anaerobic state, the acids are not oxidized, so they remain yellow.
12. Hydrogen sulfide-indigo matrix-dynamic (SIM) agar test
Test method: pick colonies or inoculate needles with pure needles to inoculate about 1/2 depth, incubate at 36 ± 1 ℃ for 18 ~ 24h, and observe the results. The culture showed black as hydrogen sulfide positive, turbid or growing outward along the puncture line as motive force, then add a few drops of Kovacs' reagent to the culture surface and let stand for 10 minutes, if the reagent is red, it is positive for indigo matrix. The uninoculated lower part of the medium can be used as a control.
This test is used for the preliminary biochemical screening of Enterobacteriaceae bacteria. The combined use with trisaccharide iron agar can significantly improve the screening efficiency.
3. Serological test
Serological reaction means that the corresponding antigen and antibody act under certain conditions in vitro, and the precipitation and agglutination phenomenon can be seen with the naked eye. In food microbiological testing, serological reactions are often used to identify the isolated bacteria to finally confirm the test results.
General characteristics of serological response:
1) The binding of antigenic bodies is specific. When there is a common antigenic body, cross-reaction will occur.
2) The binding of the antigen body is the binding of the molecular surface. Although this binding is quite stable, it is reversible.
3) The combination of antigen bodies is carried out in a certain proportion, and only when the ratio is appropriate, can a visible reaction occur.
4) The serological reaction is roughly divided into two stages, but there is no strict boundary between them. The first stage is the antigen-body specific binding stage. The reaction speed is very fast, and the reaction can be completed in a few seconds to a few minutes, but no visible phenomenon occurs. The second stage is the visible stage of the antigen body reaction, which manifests as agglutination, precipitation, complement binding reaction and so on. The reaction speed is slow, it takes a few minutes, a few tenths or even longer. Moreover, in the second-stage reaction, changes in environmental factors such as electrolyte, PH, and temperature directly affect the results of the serological reaction.
It is customary to classify classic serological reactions into three categories: agglutination reactions, precipitation reactions, and complement binding reactions.
1. Agglutination reaction
Particulate antigens (bacteria, erythrocytes, etc.) bind to the corresponding antibodies, and the phenomenon of agglutination visible with the participation of electrolytes is called agglutination reaction. The antigen is called lectin and the antibody is called lectin. In this reaction, because the amount of antibody per unit volume is large, the antibody should be diluted when performing quantitative experiments.
1) Direct agglutination reaction
The reaction that occurs when the particulate antigen directly binds to the corresponding antibody is called a direct agglution reaction.
a. Slide agglutination method. It is a conventional qualitative test method. The principle is to use known antibodies to detect unknown antigens. Commonly used to identify strains and blood types. If the serum containing dysentery bacillus antibody and the bacterial solution to be tested are mixed one drop at a time, after a few minutes of seeding, if a visible agglomerate is visible to the naked eye, that is, a positive reaction, the bacterium is dysentery bacillus. This method is fast and simple, but it cannot be quantitatively determined.
b. Test tube agglutination method. It is a quantitative test method. Mostly use known antigens to detect the presence or absence of corresponding antibodies in serum. Commonly used to help diagnose certain infectious diseases and conduct epidemiological investigations. For example, Fei's reaction is a test tube agglutination test to diagnose and treat typhoid fever. Because the content of antibody is to be determined, the serum to be examined is diluted with isotonic saline to different concentrations, and then the same amount of antigen is added, 37 ℃ or 56 ℃, observed for 2 ~ 4 hours, the highest dilution of serum still has obvious agglutination The phenomenon is the agglutination titer of the antiserum.
2) Indirect agglutination reaction
The soluble antigen (antibody) is first adsorbed on the surface of a granular microsphere that is not related to immunity, and then interacts with the corresponding antibody (antigen). In the presence of electrolyte, agglutination can occur, called indirect agglutination reaction ( Indirect agglutination). The carrier increases the reaction area of ​​soluble antigen. When a small amount of antigen is bound to the antibody on the carrier. There is a visible reaction with high sensitivity.
2. Precipitation reaction
The soluble antigen binds to the corresponding antibody, and in the presence of an appropriate amount of electrolyte, after a certain period of time, a visible precipitate is formed, which is called a precipitation reaction (Precipitation). The antigen in the reaction is called precipitin, and the antibody is precipitin. Due to the large amount of antigen per unit volume, in order not to make the antigen excessive, the antigen should be diluted, and the dilution of the antigen is used as the titer of the precipitation reaction.
1) Ring precipitation reaction: It is a qualitative test method that can detect unknown antigens with known antibodies. Inject a known antibody into a special test tube, and then slowly add an equal amount of antigen along the wall of the tube. If the antigen corresponds to the antibody, a white precipitation ring will appear at the interface of the two liquids.
2) Flocculent precipitation reaction: Mix the known antigen and antibody in a test tube (such as a concave glass slide), if the antigen and antibody correspond, and the ratio between the two is appropriate, a visual flocculent precipitate will appear, which is positive reaction.
3) Agar diffusion test: using soluble antigen and antibody to diffuse in semi-solid agar, if the antigen and antibody correspond, and the ratio between the two is appropriate, a white precipitation line will appear in a part of its diffusion. Each pair of antigen and antibody can form a precipitation line. With several pairs of antigens and antibodies, several precipitation lines can be formed separately. Agar diffusion can be divided into two types: one-way diffusion and two-way diffusion. Unidirectional diffusion is a quantitative experiment. It can be used for the determination of immune protein content. The two-way diffusion is mostly used for qualitative experiments. Because the method is simple and easy to use, it is often used for the analysis and identification of complex antigen components.
3. Complement binding reaction
Complement fixation reaction (Complement fixation reaction) is an antigen-antibody reaction using sheep red blood cells and hemolysin as an indicator system with the participation of complement. Complement is non-specific and can bind to any group of antigen-antibody complexes to cause a reaction. If the complement binds to the complex of sheep red blood cells and hemolysin, hemolysis will occur, and if it binds to bacteria and the corresponding antibody complex, bacteriolysis will occur. Therefore, the entire test requires five components, complement, system to be tested (known antibody or antigen, unknown antigen or antibody) and indicator system (sheep cells and hemolysin). The test principle is that complement does not bind antigen or antibody alone. If bacteriolysis occurs, it is the result of the binding of complement to the system to be tested, indicating that the antigen and antibody are corresponding. This reaction is complicated in operation, high in sensitivity and strong in specificity, and can detect a small amount of antigens and antibodies, so it has a wide range of applications.
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