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Spread Plate Method for Isolation of Bacteria

In this Spread plate method for isolation of bacteria post we have briefly explained about Spread plate method principle, objectives, requirements, spread plate method procedure, uses and limitations.

Spread Plate Method for Isolation of Bacteria

Individual colonies are isolated using the spread plate method procedure from a diluted sample of the mix population. Different methods for isolating bacteria from a combination sample have been devised, however the spread plate culture method is the most extensively employed in laboratories.

An ideal spread plate should include visible and isolated bacteria colonies that are evenly scattered and countable throughout the plate. The spread plate method procedure main goal is to isolate or count individual bacteria present in a diluted sample including a variety of species.

Principle

A diluted microbial sample containing more than one bacterium is deposited on a solidified agar plate and spread uniformly throughout the surface with an L-shaped glass rod while the media plate is spun on a turntable in the spread plate method procedure.

Cells (CFUs) will be implanted widely enough apart on the agar surface with an accurately diluted sample to grow into independent colonies. The basic idea behind the spread plate method procedure approach is that as the Petri dish rotates, single cells will be deposited on the agar surface with the bent glass rod. These cells will be separated from each other by a sufficient distance to allow the colonies to form independently.

Requirements

Materials

1. Bunsen burner

2. Incubator

3. Marking Pencil

Chemicals

1. 24 hour nutrient broth

2. Nutrient agar plates

3. L-shaped bent glass rod

4. 95 per cent alcohol

Procedure

Spread Plate Method

Spread plate method for isolation of bacteria

1. With a marking pencil, label the nutrition agar plate. Mention the organism’s name, the type of agar used, the date, and the name or initials of the person who created it. To protect the plate from airborne contamination, lift the lid and use it as a shield.

2. Using a clean and sterile Pipette, pipette 0.1 ml of sample from the desired dilution series onto the centre of an agar plate’s surface. Place the cover back on the plate.

3. Because the pipetting equipment used to inoculate the medium is contaminated, properly dispose of it.

4. Dip the L-shaped glass spreader in 90 percent alcohol to sterilise it, then burn the glass spreader. Allow 10–15 seconds for the rod to cool.

5. Lift the cover of the plate after farming the glass rod and use it as a shield against airborne pollution. Then, to chill the inoculum, touch the rod to the agar surface away from it.

6. Hold the plate lid with the base of your thumb and index finger during spreading, and rotate the base with the tip of your thumb and middle finger.

7. Replace the cover after removing the rod from the plate. In order to prepare for the next inoculation, return the rod to the alcohol. There’s no need to re-flame it.

8. Invert the plate and incubate it for the specified amount of time at the specified temperature.

Result and Observation

Observes the colonies on the agar plate after incubation. Some colonies will be independent of one another. Choose any colony on the plate and note its elevation, coloration, and size.

Uses

1. Spread plate method procedure with repeated dilutions is a crucial research tool in microbiology. This method is used to investigate the specimen’s cultural traits.

2. Spread plate method procedure is used to segregate bacteria from a sample containing more than one bacterium into discrete colonies.

3. Spread plate method procedure to investigate a bacteria’s sensitivity and/or resistance to a specific drug or antibiotic.

4. To acquire enough bacterial growth for various assays, this method is used. spread plate method procedure used to figure out how many germs are still alive in a sample.

5. Spread plate method procedure mythos used for transportation or short-term storage of the specimen. Bacteria that are heat-sensitive are unaffected.

Limitations

1. Aerobes prefer this approach, but microaerophilic organisms grow more slowly and The dilutions must be precise.

2. On nutritional agar, inoculum volumes more than 0.1 mL do not absorb well and may cause colonies to consolidate as they form. If the colonies are overcrowded, enumeration difficulties can arise.

Further Readings

Reference