How do you identify a strain of bacteria?

In general, the identification of an unknown strain isolated and purified from nature or other samples should be done in the following aspects. Individual morphology was observed and Gram stain was performed.

Distinguish between g(+) and g(-) bacteria. and observe its shape, size, presence or absence of spores and their location, etc.; The morphological observation of the strain mainly observes its morphology, size, edge, uplift, transparency, color, odor and other characteristics. Do a motivation test to see if he can move his flagella.

Birth type (Duansheng, Zhousheng.

Physiological and biochemical reactions and serological response experiments. Finally, according to the results of the above experimental items, the unknown bacteria were named by consulting the microbial classification search table.

If you determine the genus and species, you must be a creature of the world.

The morphology, staining, and special structure of bacteria are the earliest and most basic classification criteria; The physiological and biochemical characteristics of bacteria have always been used as the main basis for classification. At present, there are two widely used methods for the classification of bacteria based on physiological and biochemistry, namely traditional classification and numerical classification. For example:

Shapes, i.e.: cocci, bacilli and spirochetes. Lifestyle, divided into two main categories:

Autotrophic and heterotrophic bacteria. The need for oxygen is divided into aerobic and anaerobic bacteria. There are three types of survival temperatures: cold-loving, normal temperature and high temperature.

and some peculiarities in the structure of bacteria. With the traditional classification method, it is classified according to the above characteristics, and the classification at the family, genus and species level mainly depends on biochemical characteristics and antigenic structure.

In addition, it is the genetic classification method, which classifies bacteria according to the degree of homology of nucleic acids, proteins, etc. in the composition.

1 Biochemical identification.

It is the most important one in the identification of bacteria, mainly with the help of the different ability of bacteria to decompose nutrients and the difference of metabolites to identify bacteria, including protein decomposition product test, touch enzyme test, sugar decomposition product test, oxidase test, coagulase test, etc.

2 Serological identification.

It is suitable for bacteria containing more serotypes, and the common method is slide agglutination test, and immunofluorescence method, synergistic agglutination test, convection immunoelectrophoresis, indirect hemagglutination test, enzyme-linked immunosorbent assay and other methods can be used to quickly and sensitively detect the specific antigen of pathogenic bacteria in the sample. Detect unknown antigens (bacteria to be tested) with known antibodies, or detect the corresponding antibacterial antibodies and their titers in the patient's serum with known antigens. Serological identification is simple, rapid, and highly specific, providing a definitive diagnosis for bacterial identification based on biochemical identification.

3 Molecular biology testing.

It is suitable for bacteria that cannot be grown in artificial media, slow growth and high nutritional requirements, and the detection methods include nucleic acid amplification technology, nucleic acid hybridization, biochip and gene sequencing. Common nucleic acid amplification technologies include polymerase chain reaction (PCR), ligase chain reaction, etc., which are mainly used for the detection of pathogenic bacteria such as methicillin-resistant and Mycobacterium tuberculosis. Nucleic acid hybridization includes spot hybridization, in situ hybridization, etc., which are used for the detection of pathogenic bacteria such as Escherichia coli, Salmonella, and Campylobacter jejuni.

Biochips include gene chips and protein chips, which are mainly detection technologies for large-information analysis of genes, proteins, cells and other organisms.

4. Identification by automatic microbial identification system.

It can quickly and accurately identify nearly 1,000 common isolates in clinical practice, and has been widely used in clinical laboratories. Among them, the gene encoding bacterial 16S RNA has become an ideal target sequence for gene isolation, and has gradually become the gold standard for clinical bacterial identification and isolation.

5 Mass spectrometry techniques.

It is a new type of soft ionization biomass spectrometry developed in recent years, which is used to analyze the chemical classification and identification of bacteria, and has the advantages of high sensitivity and high-quality detection range. It is mainly used for the analysis of tandem mass spectrometry of biological macromolecules such as nucleic acids, proteins, and peptides.

Neisseria is a gram-negative coccus, and the most common are Neisseria meningitidis and Neisseria gonorrhoeae.

1. Neisseria meningitidis.

1.Biological characteristics: gram-negative diplococcus, the bacteria is kidney-shaped or coffee-bean-like, high nutritional requirements, can decompose glucose and maltose, and produce acid but not gas.

2.Microbiological Testing:

Specimen collection: Most of the specimens are cerebrospinal fluid, the bacteria are easy to autolyze, afraid of cold, heat and high temperature, so it should not be placed in the refrigerator and should be sent for testing immediately.

Direct smear: may be found to be gram-negative diplococci in a kidney-shaped or coffee-bean-like shape.

Isolation culture: Generally, it is cultured in chocolate medium, and the first isolation is placed in 5% 10% CO2, and transparent droplet-like colonies can be seen after 24 hours.

Biochemical identification: decomposition of glucose and maltose, acid production but not gas production, positive oxidase test.

3.Clinical significance: It mainly causes epidemic meningitis (referred to as cerebral encephalitis), which is more common in winter and spring, and has a certain epidemic.

2. Neisseria gonorrhoeae.

1.Biological characteristics: Morphological similar to Neisseria meningitidis and resembles coffee beans.

2.Microbiological Testing:

Specimen collection: purulent discharge, etc., can be autolyzed, and should be sent for testing immediately.

Direct smear: gram-negative diplococcus, kidney-shaped or coffee-bean-like.

Isolation culture: generally cultured in chocolate medium, the initial isolation and placement of 5% 10% CO2, after 24 hours, a round raised, gray-white opaque colony (identified from Neisseria meningitidis) can be seen.

Biochemical identification: decomposition of glucose, no decomposition of maltose, acid production but not gas production (can be distinguished from Neisseria meningitidis).

3.Clinical significance: is the causative agent of gonorrhea (sexually transmitted disease, susceptible to genitourinary infections).

The content comes from the user: Yu Xiulan.

Strain identification. Chapter 1: Strain identification.

China Industrial Microbial Culture Collection and Management Center.

CICC is the only national industrial microbial strain collection and management center in China, with a history of nearly 30 years of strain classification and identification, with advanced biological science instruments and a team of professional personnel engaged in classification and identification. CICC undertakes the entrusted identification of industrial microbial strains in China, and the technical services provided for the identification and evaluation of strains have obtained the "State Administration for Industry and Commerce (SCIC) Business License", providing microbial strain identification services for domestic scientific research institutions, colleges and universities, and production enterprises, involving bacteria, yeast, actinomycetes and filamentous fungi and other microorganisms. The methods of strain identification include morphological observation, physiological and biochemical characteristics, Biolog carbon source automatic analysis and identification, molecular biology identification, API bacterial numerical identification, functional analysis and functional genes, RAPD, SSCP, TLC thin layer chromatography, whole cell fatty acid analysis and identification, (G C)mol determination, DNA DNA homology determination, etc.

Identification of the project. Serial number: Service item, culture category.

1. Identification of pure strains (including morphology, physicochemical, and molecular) bacteria, yeast, actinomycetes, filamentous fungi, 2. Identification of functional genes (PHES, GRYA, ATPD, etc.) Lactobacillus, Bacillus, Bifidobacterium 3

4. Identification of contaminated strains of products

5. Microbiota analysis and isolation of pure breeds

6. Analysis of cell wall chemical components (amino acids) bacteria, actinomycetes 7. Analysis of cell wall chemical components (sugar) bacteria, actinomycetes.

8 DNA (g+C)mol content: bacteria, actinomycetes, 9 DNA, DNA, hybridized bacteria, yeast, actinomycetes, filamentous fungi, 10 fatty acids, bacteria, actinomycetes, cicccicc

First of all, you need to make sure that the strain to be identified must be pure. If the culture is not pure, the observed phenomenon is a mixed phenomenon, which naturally does not give the correct result. Therefore, prior to identification, the strain must first be purified.

There are generally two purification methods: plate scribing single colony isolation method and single cell isolation method. The former is easy to operate, has good effect and has a wide range of applications.

Methods for strain identification generally include conventional identification and molecular biology methods

1) Routine identification: colony morphology observation, Gram staining, fimbrial staining, carbohydrate decomposition experiment, indole experiment, starch hydrolysis experiment, V-P experiment, methyl red experiment, citrate utilization experiment, nitrate reduction experiment, contact enzyme experiment, etc.

2) Molecular biology methods: bacterial 16srdna species identification: DNA extraction, PCR amplification of specific primers, PCR product purification and sequencing, phylogenetic tree analysis; The same is true for the identification of fungal 18S rdDNA ITS.

Traditional methods of bacteria do bai

Gram staining, motility test, various metabolisms, and then Chaberger's bacterium handbook; If the huigo is used, the total DNA is answered, the full-length fragment of 16 srdna is amplified by PCR, compared with the NCBI database, and the phylogenetic tree identification is constructed by selecting the closely related sequence.

In the case of fungi, they are mainly identified by morphology, especially sexual reproductive morphology; In the case of molecules, the ITS region is generally amplified, compared, and identified.

Determine the species first.

1.Selective medium to make a general judgment (such as SS - Shiga Salmon, ECCA--- large intestine, HE --- many bacteria can be, alkali peptone water --- cholera. According to the morphological size and growth of the colony, in fact, this step is also to purify the bacteria, but the direction of the bacteria is probably determined, and it is also helpful for the selection of the next biochemical reaction;

2.Colonies on the plate can be reacted to life for further verification, e.g., KIA disaccharide, oxidase reaction, V-P motility test.

3.If the above is consistent with the biochemical characteristics of the bacterium, you can do serum identification, and you can identify the genus.

Determine the genus 4At this time, the serotype can be identified by serum, that is, the genus.

This general microbiology laboratory is conditional.

The above steps can also be done with biochemical slats, such as api20, which will be a little more expensive, but it will be much faster.

If there are conditions, there is a biological microplate reader, a new product of the Academy of Military Sciences, which can directly make the species of bacteria, which is a bit expensive.

Of course, PCR is still the fastest method, but because it is an unknown bacteria, there is no range, and it is also very troublesome, so the primers have to be prepared more.

The smear I said upstairs is now used less, and the trouble is dead, unless you have more experience and can see it at once, otherwise you can only see that it is cocci, vibrio, bacilli, and it doesn't make much sense.

But if you detect the target, you can check the national standard, which is almost a few steps.

First of all, it is necessary to determine whether it is a pure colony, macroscopic view of the colony morphology, whether there are notches on the edges, whether they are convex or flat, the color of the front and back, and then staining microscopy. The fungus looks at the hyphae to see if there are septas, spore stems, false roots, spore spikes, and so on. Bacterial staining is usually done with a Gram stain to see if it is a bacillus, a cocci or a vibrio.

After these tasks are done, physiological and biochemical tests are carried out, and in this way, the genus can basically be identified. To identify the species, you must take it to sequencing, bacteria test 16 srna, fungi test 18 srna, conditional laboratories can test by themselves, no conditions can be sent to special institutions to test, generally not expensive, more than ten yuan a base.

Hope it helps.

I agree with the opinion of the netizen "third person", my method is more traditional and classic. Your method is faster and more efficient.

The question you asked is indeed too big to describe in detail, I can only briefly say it:

First of all, you need to determine what conditions your strain needs to grow, anaerobic or aerobic, etc., and then choose the appropriate medium for isolation and culture (if the bacterial content is low, then you also need to increase bacteria). Then select the suspicious colonies on the plate for biochemical identification, at this time, you need to judge them according to the colony morphology, Gram staining, etc., and then judge what kind of bacteria you picked the colonies according to the results of the biochemical reaction.

Obviously, the most difficult step is the last step, now it is generally used to identify what bacteria are in the bacterial identification slats (manual or instrumental), a slats have many biochemical items, and a set of codes is derived from the results of all biochemical items, which are queried by the codes.

Do it according to the biochemistry of bacteria.