Spread Plate Method: Principles, Procedures, and Applications (Comprehensive Guide)

Table of Contents

• What is Spread Plate Method?
• Objectives of Spread Plate Technique
• Principle of Spread Plate Method
• Requirements for Spread Plate Method
• Procedure of Spread Plate Method
• Result Interpretation of Spread Plate Method
• Precautions during Spread Plate Technique
• Applications of Spread Plate Technique
• Advantages of Spread Plate Technique
• Limitations of Spread Plate Technique

What is Spread Plate Method?

The spread plate method is a widely utilized culture technique in microbiology laboratories due to its simplicity and effectiveness. This method is ideal for aerobic and facultative aerobic microorganisms and is recognized for being straightforward, economical, and requiring the sample to be in liquid or suspension form.

In the spread plate method, a specific volume of a liquid sample is spread across a solidified culture medium to isolate and count viable microorganisms. After incubation, a successful spread plate will display evenly distributed, discrete colonies across the media surface.

This technique is used to isolate and count the total number of viable microorganisms, calculating the colony-forming units per mL (CFU/mL) in the sample. Additionally, it is used for propagating and mass-producing cultures. The method is applicable for all culturable bacteria and fungi.

The sample must be in liquid or suspension form and is usually serially diluted before plating. For accurate CFU/mL counting, the microbial load in the sample should be between 20 – 300 CFU/mL, although the suitable counting range is typically considered to be 25 – 250 CFU/mL, with some variations. This range can be determined through a pilot test or by using samples with different dilutions. If the sample is solid or semisolid, it needs to be emulsified and then serially diluted to achieve the desired microbial load.

A volume of 0.1 mL (0.1 to 0.2 mL) of the sample is pipetted onto the center of the solidified agar medium and evenly spread across the surface. The plates are then incubated under optimal conditions, and the number of colonies is counted. If the colonies are too numerous to count, fused, or exceed 300 CFU/mL, or if there are fewer than 20 CFU/mL, it is recommended to repeat the process to achieve an optimal count.

Objectives of Spread Plate Technique

• To isolate microorganisms from a liquid specimen or suspension.
• To calculate the viable microbial load by counting colony-forming units (CFU) per mL.
• To isolate pure cultures of microorganisms from a mixed population.
• To isolate microorganisms in discrete colonies for studying their colony characteristics.
• To obtain sufficient microbial growth for conducting antimicrobial sensitivity testing and biochemical studies.

Principle of Spread Plate Method

When a diluted liquid specimen containing one or more microorganisms, whether of the same or different species, is spread over a suitable solid agar medium, each viable microorganism will multiply and form a separate colony. These colonies can then be counted and expressed in terms of CFU/mL, which is used to calculate the microbial load in the sample.

A certain volume of the diluted sample, typically 0.1 mL (though 0 to 1 mL can be used), is dispensed onto the surface of a pre-sterilized solid medium. A bent glass rod, swab, or glass beads are usually used to spread the sample evenly. After incubating for approximately 24 to 48 hours at 37°C, the viable microorganisms in the sample will grow into discrete visible colonies on the medium's surface. These visible colonies can be counted, and the CFU/mL can be calculated using the appropriate formula.

Requirements for Spread Plate Method

1. Liquid Specimen (or suspension of the solid sample)

The sample must be in liquid form or suspended in a suitable solvent. Solid samples should be dissolved in a solvent that neither inhibits nor promotes the growth of microorganisms and does not react with the culture media components. The sample should be diluted to achieve a well-isolated concentration of 20 – 300 CFU/mL per plate after incubation.

2. Pre-solidified Suitable Solid Culture Media Plates

Use culture media that supports the growth of the desired or probable microorganisms in the specimen. The media should be properly solidified before spreading the diluted sample. Media can be prepared and stored at 4°C for future use, ensuring it is completely solidified if freshly prepared.

3. Test Tubes

Sterile test tubes are necessary for serially diluting the sample.

4. Sterile Distilled Media (or Sterile Broth)

Serially dilute the sample using sterile distilled water or sterile broth, which can also be used to dissolve solid or semi-solid samples.

5. Micropipette

A micropipette with a capacity of 0.1 mL or 1 mL is needed for measuring the sample during serial dilution and inoculation. Alternatively, a sterile graduated pipette can be used.

6. Spreaders

Tools used to spread the inoculum over the agar surface include:

• Bent Glass Rods: Typically bent at one end into an “L” or “J” shape, or forming a closed triangle. Known as “dolly rods,” they are preferred over metallic rods as they cool down quickly and have a smooth surface.
• Glass Beads: Smooth, tiny beads (usually 4 mm in diameter) used in the “Copacabana method.” The sample is dispensed on the agar surface, sterile beads are added, and the plate is shaken to distribute the sample evenly.
• Sterile Cotton Swab: May be used but is not recommended for counting CFU/mL due to potential microorganism absorption.

7. Ethanol (70%) 

Used as a chemical sterilizer for sterilizing spreaders (excluding cotton swabs).

8. Bunsen Burner

Used to flame and sterilize glass spreaders and create a sterile working zone.

9. Other Laboratory Facilities 

Standard laboratory equipment and facilities to support the procedure.

Procedure of Spread Plate Method

The general procedure of the spread plate method can be summarized as:

1. Preparation

Arrange all the required materials, put on the PPE, sterilize the work surface, and allow all samples and media to come to room temperature if they were refrigerated.

2. Sample Preparation

• Liquid Samples: Serially dilute to reduce the microbial load to the range of 20 – 300 CFU/mL. (If the sample is assumed to be sterile or the expected microbial load is very low, dilution can be skipped. A prior pilot test can provide an exact value. Serial dilutions up to 10^(-10) can be prepared using different dilutions.)
• Solid or Semisolid Samples: Dissolve in a suitable solvent to prepare a suspension. The suspension should then be serially diluted to reduce the microbial load to the desirable range. (Generally, mix 1 gram of the sample with 9 mL of solvent to achieve a concentration of 10^(-1) g/mL.)
• Spreader Preparation: Sterilize the glass rod by dipping it in a 70% ethanol solution and flaming it over a Bunsen burner. Allow the rod to cool (check if the rod is cool enough by touching a corner of solidified media; if you hear a sizzling sound or if the media melts, cool the rod further).
• Sterilizing Beads: Place beads in a bottle or beaker and autoclave to sterilize them.

3. Labeling Plates

• Label the bottom edge of the plate with the dilution factor, date, name, sample ID, and other required information.

4. Spreading Methods

Spreading with a Bent Glass or Metal Rod:

1. Open the lid of the plate and dispense 0.1 mL of the diluted sample in the center of the Petri plate using a calibrated pipette or micropipette.

2. Using a sterile bent glass rod, uniformly spread the sample all over the plate.

• Using a Turntable: Spin it slowly, hold your spreader gently on the media's surface touching the sample, and gradually spread the sample uniformly. Move the rod back and forth to spread the sample.
• Manual Spreading: Hold the plate in your left hand (or right if left-handed) or keep it still on the bench. Move the spreader in a circular path or back and forth to spread the sample evenly. Finally, move the spreader in a circular motion around the plate's edge to ensure the sample is spread even at the corners.

3. Put on the lid, leave the plate in an upright position, and allow the sample to be absorbed for about 5 minutes. Then incubate the plate in an inverted position.

Spreading with Glass Beads (Copacabana Method):

1. Open a portion of the Petri plate lid with your thumb and index finger and dispense about 10-12 sterile glass beads.

2. Dispense 0.1 mL of the diluted sample in the center of the Petri plate using a calibrated pipette or micropipette.

3. Close the lid and shake the plate horizontally to spread the sample evenly across the agar surface. Repeat the shaking 6-7 times.

4. Rotate the plate clockwise or counterclockwise by 60° and shake the plate horizontally 6-7 times again.

5. Rotate the plate once more in the same direction by 60° and shake it horizontally 6-7 times. (If the sample is not evenly distributed, you can repeat the shaking process.)

6. Leave the plate in an upright position and allow the sample to be absorbed for about 5 minutes.

7. Discard the beads in a container with disinfectant (10% chlorine bleach, ethanol, etc.).

8. Incubate the plate in an inverted position.

By following these steps, the spread plate method effectively isolates and counts viable microorganisms, providing accurate CFU/mL calculations. 

Result Interpretation of Spread Plate Method

After incubation, examine the plates for the development of discrete colonies. Each colony represents one viable microbial cell or colony-forming unit (CFU).

• Homogeneous Colony Morphology:
• If all isolated colonies have the same morphology, it suggests that the specimen contained only one type of microbial genus. However, different genera or species can produce similar colony types. Therefore, perform further tests to confirm the presence of only one type of microorganism.
• Heterogeneous Colony Morphology:
• If isolated colonies have different morphologies, it indicates that the sample contained more than one type of genus or species of microorganisms. These can be purified by sub-culturing each colony onto a separate culture media plate using the streak plate method.
• Counting Colonies and Calculating CFU/mL:
• Count the colonies and calculate the CFU/mL using the appropriate formula to determine the total number of viable microbial cells in the given sample.
• Optimum Colony Count:
• For an optimum count, the number of colonies should be between 20 – 300 CFU/mL. If the count falls outside this range, the procedure must be repeated.
• Less than 20 Colonies: Use a sample of lower dilution.
• More than 300 Colonies: Use a sample of higher dilution for successive repeats.
• Fused Colonies or Overgrown Plate: If colonies are fused or the entire plate is covered with a single colony, report it as "too numerous to count" (TNTC) and repeat the process with a higher dilution sample.

Precautions during Spread Plate Technique

• Follow proper safety protocols. Treat every unknown or clinical specimen as hazardous and follow safety accordingly. Ensure all tools and glassware are sterile. The water or broth used in serial dilution must be sterile.
• Sterilize glass rods by dipping them in 70% alcohol and flaming them before and after use. Glass beads can be sterilized by autoclaving. The spreading beads or rod must be at or below 37°C. Don’t use them immediately after flaming; let them cool.
• The solvent used to dissolve the solid sample must be sterile and must not have any growth-supportive or inhibitory effect against any microorganism.
• Dilute the sample enough so that the viable microbial load falls between 20 – 300 CFU/mL. Above this range, it will be difficult to count the colonies, and they may fuse together. Below this range, the result is reported as not significant. Repeat the process under the same conditions if the colony count is below 20 CFU/mL or above 300 CFU/mL.
• The sample volume for spreading should be 0.1 mL (with a permissible range of 0 to 1 mL). If the sample is 1 mL or more, colonies may fuse, and the sample may not be absorbed by the media, causing it to float on the surface.
• Ensure the media is properly solidified before use. If refrigerated, allow it to come to room temperature. Appropriate media selection is necessary for proper and complete isolation.
• Check for any growth or presence of water droplets on the surface of the media before inoculation.
• Accurate measurement of water for serial dilution and sample for inoculation is very important. Always use a micropipette or calibrated pipette.
• Label each Petri plate with an accurate dilution factor to ensure precise calculation of microbial load after incubation. Inoculate each plate with the specific specimen corresponding to the labeled dilution factor on the plate.
• Incubate the plate in an inverted position under appropriate conditions. Check the plate after 24 hours of incubation to prevent overgrowth and colony fusion, which can make the plate difficult to read. If no growth is observed after 24 hours, incubate for an additional 24 – 48 hours (or more based on the probable microorganism) before reporting no growth and discarding.    

Applications of Spread Plate Technique

• The spread plate technique is employed to isolate bacteria and fungi from samples.
• It is used in antimicrobial sensitivity testing, enrichment procedures, and screening experiments.
• This method helps in calculating the number of viable microorganisms (CFU/mL) in a sample.
• It finds applications in industries such as food, pharmaceuticals, and soil studies to isolate and quantify spoilage organisms or contaminants, ensuring product quality.
• The technique is used for mass culturing stock or fresh specimens.
• In clinical laboratories, it is used to inoculate clinical specimens.
• It aids in studying growth curves, metabolic activities, and biochemical features of microorganisms, as well as their responses to environmental factors.
• It is effective in separating pure cultures from mixed cultures.
• The technique generates discrete, pure colonies, allowing for the study of colony characteristics, genetic traits, and other biochemical properties of the isolates.

Advantages of Spread Plate Technique

• The spread plate technique is a straightforward, quick, and easy method for culturing microorganisms.
• It can detect very low levels of microbial load.
• It allows for the study of colony morphology, with colonies typically being larger compared to those produced by the pour plate method.
• This method is both qualitative and quantitative, enabling the isolation and enumeration of microorganisms (i.e., calculation of CFU/mL).
• It is suitable for conducting Kirby-Bauer antimicrobial sensitivity testing.
• The technique can be applied to clinical, industrial, or environmental samples, provided they are in liquid form or can be dissolved to create a suspension.
• It is useful for preparing and maintaining stock cultures.
• It is especially effective for culturing aerobic microorganisms.
• Syntrophic bacteria can be isolated as they can grow in close proximity on the same plate, forming distinct colonies.
• There is minimal risk of contamination when using sterile glass beads as spreaders, as the process is conducted with the culture plate lid closed.

Limitations of Spread Plate Technique

• The spread plate technique requires additional tools, such as a spreader.
• It necessitates that the sample be in liquid or suspension form and requires serial dilution, making the process somewhat complex.
• Solid or semisolid samples must be suspended before inoculation, which can be challenging if the sample is not easily soluble.
• It is not effective for the growth of microaerophiles and anaerobes.
• The technique is unsuitable for samples with very high microbial loads. To achieve the optimal range of 20 – 300 CFU/mL, the sample must be serially diluted, which may require pilot testing.
• There is a risk of gouging the media during spreading, particularly with inexperienced use of a glass rod or if the media is not adequately solidified.
• Sterilization of beads or glass rods after each use is necessary to prevent cross-contamination. Inadequate sterilization can lead to contamination between plates.
• It requires specific media that must be pre-solidified. This means we need prior knowledge of the potential microorganisms in the sample or access to a variety of media.