Microbiology Examination Methods for the isolation of bacterial colonies

Plate marking method and dilution coating method, plate method has already been said, so let's not talk about it, let's talk about the coating method. The so-called dilution coating is to dilute the bacterial solution 100 times (with a pipette.

After mixing well, take a few drops of the mixed bacterial solution with a pipette, add it dropwise to the pre-prepared plate, and evenly coat the bacterial solution with a ring glass rod to cover the medium.

Cover the lid and put it in a 30-degree incubator for 24 hours. However, I personally think that the scribing plate method is good, and it is easy to select and take bacteria.

Plate scribing method:

1. Pour the sterile plate, prepare sterile water, prepare the medium, sterilize, and pour the plate.

2. The sample of the bacteria to be isolated is dissolved in water to make a suspension or bacterial suspension.

3. Aseptic operation, dip an inoculation loop liquid, and separate it in a plate.

4. The plate is incubated in the incubator at a suitable temperature.

After hours or 48 hours, an observation plate is performed and a single colony transfer bevel is picked.

6. After the culture slope is overgrown with fungus, microscopic examination, if it is not pure, repeat the above experimental steps.

Dilution coating method.

1. Prepare sterile water, sterile plate medium, sterile pipette and coater.

2. Prepare bacterial suspension with sterile water.

3. Dilute the concentration of the bacterial suspension, according to the concentration of the bacterial suspension (such as 10 of 9 times per bacterial ml), dilute to the minus 7 times of 10.

4. Take the liquid in 10 negative negative 5 power sterile water, inoculate it on a plate, and coat it.

5. Spread the plate, mark it, and put it in the incubator for culture.

6. If a single colony grows, pick a single colony to transfer to the bevel.

7. After the culture slope is overgrown with fungus, microscopic examination, if it is not pure, repeat the above experimental steps.

This method can also be found in the microbiology experiment book.

One additional sentence: There are other isolation methods, such as difficult to isolate bacteria, which can be used by gradient centrifugation, anaerobic tube rolling method, etc.

There are mainly 1. Dilution.

BAI inverted plate method.

First, the DU microbial suspension was diluted in a series of DAO (e.g., :

10000), then take a small amount of different dilutions, mix with the agar medium that has been melted and cooled to about 50, shake well, pour into the sterilized petri dish, and make an agar plate that may contain bacteria after the agar solidifies, and the colonies can appear after a certain period of incubation. If properly diluted, a single scattered colony can appear on the surface of the plate or in agar medium, which may have been formed by the propagation of a single bacterial cell.

The individual colonies are then picked or repeated several times to obtain a pure culture.

2., dilution coating plate method.

The melted medium is poured into a sterile plate to make a sterile plate, after cooling and solidifying, a certain amount of microbial suspension droplets are added to the surface of the plate, and then the bacterial solution is evenly dispersed to the entire plate surface with a sterile glass coating stick, and a single colony is picked after culture.

3. Plate marking method.

Microbial samples were separated by multiple "point-to-line" dilutions on the surface of the solid medium.

4. Dilution shake tube method.

First, a series of test tubes containing sterile agar medium are heated to melt the agar and then cool and keep it at about 50, the material to be separated is diluted gradient with these test tubes, the test tube is quickly shaken evenly, and after condensation, a mixture of sterilized liquid paraffin and solid paraffin is poured on the surface of the agar column to separate the medium from the air.

Summary. There may be several reasons for the low number of colonies obtained:1

Microorganisms have strict requirements for culture conditions, such as culture medium, temperature, pH value, oxygen content, etc., which will affect the growth of microorganisms, and if the conditions are not suitable, it will lead to slow growth or death of microorganisms; 3.Improper technical operation: non-standard operation, poor sealing of petri dishes, contamination, etc. will also affect the results of the experiment.

For example, labware should be sterilized before isolation to avoid contamination by exogenous bacteria. 4.Improper storage conditions:

Isolated bacteria can lead to a decrease in the number of colonies if they are not stored properly, such as too high or too low temperature, too long a storage time, etc.

What are the reasons for the low number of colonies obtained in bacterial isolation culture and purification experiments?

It should include personal reasons for operation and cultivation conditions.

In isolation, culture and purification experiments, if the sample itself contains a variety of microorganisms, it will affect the growth and isolation of specific microorganisms; 2.Unsuitable culture conditions: microorganisms have strict requirements for culture conditions, such as culture medium, temperature, pH value, oxygen content, etc., which will affect the growth of microorganisms, and if the conditions are not suitable, it will lead to slow growth or death of microorganisms; 3.

Improper technical operation: non-standard operation, poor sealing of petri dishes, contamination, etc. will also affect the results of the experiment. For example, labware should be sterilized before isolation to avoid contamination by exogenous bacteria.

4.Improper storage conditions: Isolated bacteria can lead to a decrease in the number of colonies if they are not stored properly, such as too high or too low temperature, too long storage time, etc.

There are many ways to isolate and purify microorganisms. For example, the culture medium method.

There are mainly 1Scribing method 2Inverted plate method 3

Coating method Select different media according to the isolated strains, dilution and mixing inverted plate method, dilution coating plate method, plate marking separation method, dilution shake tube method, liquid medium separation method, single cell separation method, selective culture separation method, etc. The first three of these methods.

Spore separation is generally used. It can be used for all strains.

Strip or coat the plate.

Pure cultures of pathogenic bacteria are often required in various experiments to study the morphology, physiology, ecology and pathogenicity of pathogenic bacteria to host plants. Isolation and culture is one of the most basic techniques for plant disease experiments. The method of isolation of pathogenic bacteria varies depending on the material.

The most common methods for fungal diseases are Laxian tissue isolation, direct fungal extraction and fungal surface disinfection.

1) The main steps of the tissue separation method are: taking the diseased and healthy junction diseased tissues; Soak in 70% ethanol for a few seconds; sterilize in 10% calcium hypochlorite solution for 3 5min; Wash 3 times in sterile water for 3min each time; It is then transferred directly to PSA plate medium (or other suitable medium) and incubated inverted in an incubator to allow colonies to grow before purification.

2) The main steps of the direct fungus extraction method are: taking the diseased tissue with fungus; Under a low-power microscope, use a glass needle or other thin needle-like material to directly obtain the conidia or mycelium of the fungus; Or take the conidia with a glass ball, smear it on the agar plate with antibiotics that inhibit bacteria, invert the plate and culture it in the incubator, after 6 hours, wait for the spores to germinate, and use a thin inoculation needle to directly pick the conidia or the bud tube of the spores germinated by the fungus under a low-power microscope; Transfer the mycelium, conidia or sprout tubes obtained above into PSA inclined medium or plate medium (or other suitable medium); Place the medium in an incubator and incubate.

3) The main steps of the direct surface disinfection method of bacteria are: taking the hypnozoite (such as sclerotia or mycelona); Soak in 70% ethanol for 3 8min; or soak the state chain in 70% B wheel track alcohol for a few seconds, and disinfect in 10% calcium hypochlorite solution for 5 8min; Wash 3 times in sterile water for 3min each time; It is then transferred directly to PSA plate medium (or other suitable medium) and incubated inverted in an incubator to allow colonies to grow before purification.