Spread Plate Method

 

Spread plate method is used in microbiological laboratories to isolate and count viable microorganisms within a liquid sample. The method, preferred for its simplicity and cost-effectiveness, provides evenly distributed colonies on solid culture media. It is especially useful for the study of aerobic and facultative aerobic microorganisms.

Spread plate method

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Spread plate method is based on spreading a specific volume of diluted liquid specimen over the surface of a solid agar medium. The principle of the method is to grow distinct colonies after incubation. This allows the researchers to count colony-forming unit (CFU) per millilitre of the sample. Only 0.1 ml of the sample is usually used. The spreading can be done either by a bent glass rod, glass beads or cotton swabs. By adhering to the protocol, researchers can achieve reliable means for isolating and quantifying viable microorganisms.

Objectives

Isolation of microorganisms from liquid specimens or suspensions

Calculation of viable microbial load (CFU/ml)

Isolation of pure cultures from mixed populations

Study of colony morphology and growth characteristics

Propagation of old cultures and mass production for antimicrobial sensitivity testing and biochemical studies

Materials and methods

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Materials

• Liquid sample or suspension of solid samples
• Pre-solidified solid culture media plates (Petri dish)
• Sterile test tubes and distilled media
• Micropipette
• Spreaders (bent glass rod, glass beads, or cotton swabs)
• Ethanol (70%) and Bunsen burner for sterilization
• Personal protective equipment (PPE)
• Other laboratory facilities, such as laminar flow hood

Step-by-step method

Spread plate method

1. Preparation: Prepare all the required materials, sterilize the workspace, put on the required PPE, and bring samples and media to room temperature.
2. Sample preparation: Use serial dilution for the liquid sample to reduce microbial load. The ideal measurement is between 20-300 CFU/ml. If you are using a solid sample, dissolve it in a sterile solvent and then dilute.
3. Labelling: Write down the name of the sample along with the dilution factor and any other necessary information in the bottom edge of the Petri dish.
4. Spreading the sample: Dispense 0.1 ml of the sample using a micropipette in the centre of the agar medium. Spread the sample evenly using either the sterilized glass rod or the "Copacabana method" with glass beads.
5. Incubation: Allow the sample to be absorbed by agar medium and incubate the plate in an inverted position under optimal conditions.

Spreading the sample with sterilized glass rod

1. Dispense 0.1 ml of the diluted sample into the centre using a micropipette.
2. Gently spread the sample using bent glass rod.
3. Allow the sample to be absorbed for about 5 minutes and incubate.

Spreading the sample using the "Copacabana method"

1. Carefully add 10-12 sterile glass beads on the agar medium.
2. Dispense 0.1 ml of the sample using a micropipette.
3. Gently shake the plate in a horizontal motion. This allows the glass beads to evenly distribute the sample on the surface.
4. Rotate the plate by 60 degrees and shake again.
5. Repeat the shaking in the same direction by another 60 degrees.
6. Allow the sample to absorb by keeping the plate upright for 5 minutes.
7. Discard the glass beads.
8. Incubate the plate.

Best practice

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It is important to avoid touching the edge of the plate with the loop when streaking or inoculating. This can introduce contaminants to the medium and cause inaccurate results. Before inoculating, ensure that the plates are free of any condensation to ensure optimal conditions. If any moisture is present, leave the plates at room temperature over night or place them in a laminar flow hood to dry. Keep the inoculum volume low to avoid prolonged soaking times and to prevent colony clumping. Lastly, do not use agar directly from the fridge. To ensure the medium has equilibrated properly, bring the agar to room temperature before use.

Results interpretation

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After the incubation, examine the plates. Each colony represents one viable microbial cell or one CFU. If the colony morphologies are identical, it suggests that the sample contained one type of microbial species. However, as some species produce similar colonies, it is recommended to run further tests such as streak plate method. On the other hand, if the colony morphologies vary, it is likely that the sample contained a mix of microbial species. Count the colonies and use the CFU/ml formula to determine the number of viable microbial cells.

If the count falls outside of the ideal range (20-300 CFU/ml), repeat the whole procedure with lower or higher dilution. If the colonies fuse together or the whole plate is covered in one colony, record the experiment as "too numerous to count" and repeat the process with adjusted dilution.

Spread plate method applications

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Spread plate method can be used for both isolation and study of characteristics of bacteria and fungi. It is useful for antimicrobial sensitivity testing as well as enrichment and screening experiments. Thanks to its ability to display distinct colonies, it is used to calculate the number of viable microbes. Thus having applications in food industries, pharmaceutical testing, and soil studies. Additionally, spread plate method can be used for mass culture stock cultures and fresh specimens. Lastly, the method is instrumental in separating pure cultures and growing pure colonies.

Advantages

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• Simple, quick and cost-effective
• Suitable for culturing aerobic microorganisms
• Allows both qualitative and quantitative analysis
• Low risk of contamination when following the proper technique

Limitations

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• Requires liquid or suspension samples
• Not suitable for anaerobic or microaerophilic organisms
• May require serial dilution and specific tools like spreaders
• High microbial loads may complicate colony counting

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