Spread Plate Method- Definition, Principle, Procedure, Uses

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The spread plate method is a microbiological laboratory technique for isolating and counting the viable microorganisms present in a liquid sample by spreading a certain volume of the sample over an appropriate solidified culture media . Following the incubation, in a successful spread plate, there will be the formation of evenly distributed discrete colonies all over the surface of the culture media. This technique is used for isolating and counting the total number of viable microorganisms (i.e. calculating the colony-forming units per mL (CFU/mL) in the given sample . It is also for propagating the old culture and mass producing them. It can be used for all of the culturable bacteria and fungi.

The spread plate method is suitable for aerobic and facultative aerobic microorganisms. It is an easy, simple, and economical method; however, it requires the sample to be in liquid or suspension form and needs to be serially diluted . The sample in the spread plate method must be liquid or in suspension. Before plating, the samples are serially diluted . If the objective is to count the CFU/mL then the sample must be diluted to make the microbial load in the sample between 20 – 300 CFU/mL (suitable colony counting range is 20 – 200, some consider it to be 30 – 300, and in average it is taken as 25 – 250) . This can be obtained by pilot test or by using a different range of diluted samples. If the sample is solid or semisolid, it must be first emulsified and then serially diluted to reduce microbial load up to the permitted range.

0.1 mL of the sample (0.1 to 0.2 mL) is pipetted over the center of the solidified agar medium and evenly spread over the surface of the medium using a sterile bent glass rod or swab or glass beads as a spreader . The plates are incubated under the optimum condition following which the numbers of colonies are counted . If the colonies are uncountable or fused or more than 300 CFU/mL or less than 20 CFU/mL, it is recommended to repeat the process for getting the optimum count.

The spread plate method has several advantages over other methods of culturing microorganisms, such as pour plate method and streak plate method. Some of these advantages are:

  • It allows for better observation of colony morphology and enumeration of microorganisms.
  • It avoids overheating and killing of heat-sensitive microorganisms that may occur in pour plate method.
  • It does not require melting and cooling of agar medium as in pour plate method.
  • It does not require multiple inoculations and streaking as in streak plate method.
  • It reduces the risk of contamination by minimizing exposure of agar surface to air.

The spread plate method also has some limitations, such as:

  • It requires extra tools like a spreader and a micropipette.
  • It needs accurate measurement and dilution of sample to obtain countable colonies.
  • It does not support sufficient growth of microaerophiles and anaerobes.
  • It may cause gouging or scratching of agar surface if not done carefully.
  • It may result in uneven distribution of sample if not spread properly.

The spread plate method is widely used in microbiology laboratories for various purposes, such as:

  • To isolate bacteria and fungi from a given sample .
  • To calculate viable microbial load by counting colony formation unit (CFU) per mL .
  • To isolate pure culture of microorganisms from a mixed population .
  • To isolate microorganisms in discrete colonies in order to study their colony characters .
  • To obtain sufficient growth for conducting antimicrobial sensitivity testing and biochemical studies .
  • To study growth curves, metabolic activities, and environmental factors affecting microorganisms.

  • To mass culture stock culture or fresh specimen.

    Objectives of Spread Plate Technique

The spread plate technique is a widely used method for isolating and enumerating microorganisms from liquid samples or suspensions. The main objectives of this technique are:

  • To isolate the microorganisms from the liquid specimen (or suspension) by spreading a known volume of the sample over a solid agar medium and allowing them to grow into discrete colonies .
  • To calculate the viable microbial load by counting the colony forming units (CFU) per mL of the sample. The CFU represents the number of living microorganisms that are able to form visible colonies on the agar medium .
  • To isolate the pure culture of microorganisms from a mixed population by selecting well-separated colonies and subculturing them on fresh agar plates. A pure culture contains only one type of microorganism and is essential for further identification and characterization .
  • To isolate microorganisms in discrete colonies in order to study their colony characteristics, such as shape, size, color, texture, elevation, margin, etc. These features can provide useful clues for the preliminary identification of the microorganisms .

The spread plate technique is suitable for aerobic and facultative anaerobic microorganisms that can grow on solid agar media. It can be used for various types of samples, such as clinical specimens, environmental samples, food samples, industrial samples, etc. It can also be used for different purposes, such as antimicrobial susceptibility testing, screening experiments, growth curve studies, metabolic activity studies, etc .

Principle of Spread Plate Method

The principle of the spread plate method is based on the assumption that each viable microorganism in a diluted liquid sample will grow and form a separate colony on a solid agar medium after incubation. The number of colonies on the plate can be counted and multiplied by the dilution factor to calculate the total number of viable microorganisms in the original sample.

The spread plate method involves the following steps:

  • The sample, which must be liquid or in suspension, is serially diluted to reduce the microbial load to a suitable range, usually between 20 and 300 CFU/mL. This range is optimal for obtaining well-isolated and countable colonies on the plate. If the sample is solid or semisolid, it must be first dissolved or emulsified in a sterile solvent before dilution.
  • A fixed volume of the diluted sample, usually 0.1 mL, is pipetted onto the center of a pre-solidified agar plate. The agar medium should be appropriate for supporting the growth of the desired or expected microorganisms in the sample. The plate should be dry and at room temperature to facilitate the absorption of the inoculum by the agar.
  • The inoculum is spread evenly over the surface of the agar using a sterile spreader, such as a bent glass rod, a glass bead, or a cotton swab. The spreader should be cooled before touching the agar to avoid melting or gouging the medium. The spreading should be done gently and uniformly to avoid clumping or overlapping of microorganisms.
  • The plate is left in an upright position for about 5 minutes to allow the inoculum to be absorbed by the agar. Then, the plate is incubated in an inverted position at an optimal temperature and time for the growth of the microorganisms.
  • After incubation, the plate is examined for the presence of discrete colonies on the agar surface. Each colony represents one viable microorganism or one colony-forming unit (CFU) in the inoculum. The colonies can be counted manually or with an automated device and expressed as CFU/mL by multiplying with the dilution factor. The colonies can also be characterized by their morphology, color, size, shape, and other features.

Requirements for Spread Plate Method

The spread plate method requires the following materials and equipment:

  • Liquid Specimen (or suspension of the solid sample): The sample must be either in liquid form or in suspension form. The solid sample must be dissolved in a suitable solvent. The solvent must not show any inhibitory or growth-promoting activity to any of the microorganisms. Also, it must not react with any component of the culture media. The sample must be diluted to an appropriate concentration so that we can get a well-isolated 20 – 300 CFU/mL per plate after the incubation .

  • Pre-solidified Suitable Solid Culture Media Plates: Appropriate culture media that supports the growth of all desired or probable microorganisms in the specimen must be used. The media must be solidified properly before spreading the diluted sample. The media can be prepared and stored at 4°C for future use. If freshly prepared, make sure the media is completely solidified. The thickness of the agar should be roughly 0.3 cm, which can be achieved by pouring 15 to 20 ml of media per 100 x 15 mm plate .

  • Test Tubes: Sterile test tubes are used to serially dilute the sample.

  • Sterile Distilled Water (or Sterile Broth): The sample must be serially diluted using sterile distilled water or sterile broth. They are also used for dissolving the solid or semi-solid sample.

  • Micropipette: A micropipette of 0.1 mL or 1 mL capacity is required for measuring the sample during serial dilution and sample inoculation. A sterile graduated pipette can be used instead of the micropipette.

  • Spreaders: Spreaders are the tools that are used to spread the inoculum over the surface of the agar medium. Bent glass rods, glass beads, and cotton swabs are used as spreaders .

    • Bent glass rod: it is a glass rod that is bent at one end forming either “L” or “J” shaped or forming a closed triangle-like shape at one end. It is often called a “dolly rod”. It is the most commonly used spreader. Metallic rod is not preferred because their surface can be delaminated and they take a longer period to cool down. Glass rods are with smooth surface and can be easily made in a lab and also they cool down quickly.
    • Glass beads: smooth tiny glass beads, usually of 4 mm diameter, are used to spread the inoculum in the “Copacabana method”. The sample is dispensed on the surface of the agar medium. Sterile glass beads are placed over the medium and the whole plate is shaken so that the beads distribute the sample evenly all over the agar surface.
    • Sterile cotton swab: a sterile swab may be used, but it is not recommended when we need to count CFU/mL because some microorganisms may be trapped on the sample that will be absorbed in the swab.

  • Ethanol (70%): 70% ethanol is used as a chemical sterilizer to sterilize the spreaders (except cotton swabs).

  • Bunsen Burner: It is used to flame the glass spreader in order to sterilize them. Besides it can also be used to make a sterile working zone.

  • Other Laboratory Facilities: These include personal protective equipment (PPE), labels, markers, disinfectants, incubator, etc.

Procedure of Spread Plate Method

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

  • Arrange all the requirements, put on the personal protective equipment (PPE), sterilize the work surface, and allow all the samples and media to come to room temperature if they were refrigerated.
  • Sample preparation
    • Liquid samples must be serially diluted 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, we can skip the dilution. The prior pilot test may give an exact value. You can prepare serial dilution up to 10^-10^ and use different dilutions.)
    • If the sample is solid or semisolid, dissolve it with a suitable solvent to prepare its suspension. The suspension then should be serially diluted to reduce the microbial load at the desirable range. (Generally, 1 gm sample is mixed with 9 ml of solvent to get the concentration of 10^-1^ gm/mL.)
    • Arrange the spreader. The glass rod must be sterilized. For this dip the rod in 70% ethanol solution and flame it over a Bunsen burner. Let the rod cool. (You can check if the rod is cold enough or not by touching a corner of solidified media. If you hear a sizzling sound or if the media melts, then cool the rod further.)
  • Spreading
    • Label the plate at its bottom edge with the dilution factor, date, name, sample ID, and other required information.
    • The spreading can be done by either of the following two methods, viz.:
      • Spreading with a bent glass or metal rod
        • 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 micro-pipette.
        • Using a sterile bent glass rod uniformly spread the sample all over the plate.
          • If you are using a turntable, spin it slowly and then hold your spreader gently on the surface of the media touching the sample, and gradually spread the sample uniformly all over the surface of the media. Moving the rod back and forth will allow you to spread the sample.
          • If you are performing it manually, hold the plate in your left hand (or in your right if you are left-handed) or you can also put it still on the bench. You must move the spreader in either a circular path or in a back and forth motion to spread the sample evenly. At last, move the spreader in a circular motion around the edge of the plate to make sure that the sample is spread even at the corner.
        • Put on the lid, leave the plate in an upright position and allow the sample to be absorbed for around 5 minutes. Then incubate the plate in an inverted position.
      • Spreading with glass beads: “Copacabana Method”
        • Open a portion of the lid of a Petri plate with your thumb and index finger and dispense about 10 -12 sterile glass beads.
        • Dispense 0.1 mL of the diluted sample in the center of the Petri plate using a calibrated pipette or micro-pipette.
        • Close the lid and shake the plate in a horizontal motion so that the beads spread the sample evenly across

Result Interpretation of Spread Plate Method

Following the incubation, the plates are examined for the development of discrete colonies. Each colony represents one viable microbial cell or one colony-forming unit (CFU) that was present in the original sample. The number of colonies on the plate is counted and multiplied by the dilution factor to calculate the total number of viable organisms per milliliter (CFU/mL) in the sample.

To interpret the results of the spread plate method, the following steps are followed:

  • Observe the morphology of the isolated colonies. If all the colonies look similar, it indicates that the sample contained only one type of microorganism. However, different genera or species may produce similar colonies, so further tests are needed to confirm their identity.
  • If the colonies have different morphologies, it indicates that the sample contained more than one type of microorganism. They can be purified by sub-culturing each colony on a separate plate using the streak plate method.
  • Count the colonies on each plate and record the number. Only plates with 20 to 300 colonies are considered suitable for counting, as this range ensures accuracy and precision. Plates with less than 20 colonies are not significant, and plates with more than 300 colonies are too numerous to count (TNTC).
  • Calculate the CFU/mL by using the formula:

$$\text{CFU/mL} = \frac{\text{Number of colonies} \times \text{Dilution factor}}{\text{Volume plated}}$$

  • For example, if a plate with a dilution factor of 10^-5 has 120 colonies and 0.1 mL of sample was plated, then:

$$\text{CFU/mL} = \frac{120 \times 10^{-5}}{0.1} = 1.2 \times 10^7$$

  • This means that there were 12 million viable microorganisms per milliliter in the original sample.
  • Report the result as CFU/mL along with the dilution factor and the volume plated. For example:

120 colonies on a plate with a dilution factor of 10^-5 and a volume plated of 0.1 mL = 1.2 x 10^7 CFU/mL

  • If there are no colonies on any plate, report as no growth or zero CFU/mL.
  • If there are TNTC plates, report as TNTC and repeat the procedure with a higher dilution.

Precautions during Spread Plate Technique

The spread plate technique requires careful attention to detail and proper handling of the samples and media to ensure accurate and reliable results. Some of the precautions that should be followed during this technique are:

  • Follow proper safety protocols. Treat every unknown or clinical specimen as hazardous and follow safety accordingly. Make sure that all the tools and glass wares are sterile. The water or broth used in serial dilution must be sterile.
  • Make sure to sterilize the glass rods by dipping them in 70% alcohol and flaming them before and after their use to spread a specimen. Glass beads can be sterilized by autoclaving. The spreading beads or the 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.
  • The sample must be diluted enough so that the viable microbial load falls between 20 – 300 CFU/mL. Above this range, it will be difficult to count the colony and the colonies may fuse together. Below this range, the result is reported as not significant. The process must be repeated under the same condition if the colony count is below 20 CFU/mL or above 300 CFU/mL.
  • The sample must be 0.1 mL (0 to 1 mL is the permitted range) for spreading. If the sample is 1 mL or more, then there will be fused colonies, and also the sample may not be absorbed by the media leaving it to float on the surface.
  • Media should be properly solidified before use. If it is 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 accurate measurement of sample for inoculation is very important. Hence, always use a micropipette or calibrated pipette.
  • Labeling each Petri plate with an accurate dilution factor is necessary in order to make an accurate calculation of the microbial load following the incubation. Inoculate each plate with the specific specimen whose concentration corresponds to the labeled dilution factor on the plate.
  • Incubate the plate in an inverted position in appropriate condition. Check the plate by 24 hours of incubation, because if delayed over-growth can occur and colonies may fuse making the plate difficult to read. If no growth after 24 hours, incubate it for the next 24 – 48 hours (or more based on probable microorganism) before reporting no growth and discarding.

Applications of Spread Plate Technique

The spread plate technique has various applications in microbiology and related fields. Some of the applications are:

  • Used to isolate bacteria and fungi from a given sample
  • Used in antimicrobial sensitivity testing, and enrichment and screening experiments
  • Used to calculate the number of viable microorganisms (i.e. calculate CFU/mL) in a sample
  • Used in food industries, pharmaceutical industries, soil studies, etc. to isolate and enumerate spoilers or contaminants to check for the quality of the products
  • Used to mass culture the stock culture or fresh specimen
  • Used in clinical laboratories to inoculate the clinical specimens
  • Used to study growth curves, metabolic activities, and biochemical features of microorganisms, and also the effect of environmental factors on them
  • Used in separating pure culture from a mixed culture
  • Used to generate discrete and pure colonies in order to study colony characters, genetic features, and other biochemical features of the isolates

Advantages of Spread Plate Technique

The spread plate technique has several advantages over other methods of culturing microorganisms, such as:

  • It is a simple, easy, and quick method of culturing microorganisms.
  • It can detect a very low microbial load in the sample.
  • It can produce larger and more distinct colonies than the pour plate method, which facilitates the study of colony morphology and identification of microorganisms.
  • It is a qualitative as well as quantitative isolation method that allows both isolation and enumeration (i.e. calculation of CFU/mL) of microorganisms in the sample.
  • It can be used for performing antimicrobial sensitivity testing, enrichment and screening experiments, and growth curve studies.
  • It can use any liquid or suspension sample, as long as it is diluted appropriately.
  • It is suitable for culturing aerobic and facultative anaerobic microorganisms, as well as syntrophic bacteria that can grow in close proximity with distinct colonies.

  • It has a lower risk of contamination if sterile glass beads are used as spreaders, as they do not require opening the lid of the plate.

    Limitations of Spread Plate Technique

Although the spread plate technique is widely used and has many advantages, it also has some limitations that should be considered. Some of the limitations are:

  • It requires extra tools like a spreader or glass beads, which need to be sterilized before and after each use to prevent cross-contamination. Sterilizing the spreader can be time-consuming and may damage the agar surface if not done properly .
  • It needs the sample to be in liquid or suspension form and needs to be serially diluted, which can be complex and error-prone. Solid or semisolid samples must be suspended or dissolved in a suitable solvent, which may not be easy or may affect the viability of some microorganisms .
  • It does not support sufficient growth of microaerophiles and anaerobes, as they may not get enough oxygen or may be exposed to too much oxygen on the surface of the agar. Therefore, this technique is more suitable for aerobic and facultative anaerobic microorganisms .
  • It is unsuitable if the microbial load in the sample is too high or too low, as it may result in too many or too few colonies on the plate. The sample must be diluted enough to get between 20 to 300 CFU/mL, which may require pilot testing or using different dilution ranges. If the colony count is outside this range, the whole procedure must be repeated .
  • It needs specific media pre-solidified before use, which may not be available or suitable for all types of microorganisms. The media must be properly solidified and dried before use, and must support the growth of all desired or probable microorganisms in the sample .
  • It may not preserve the original distribution or proportion of microorganisms in the sample, as some microorganisms may grow faster or slower than others on the agar surface. This may affect the accuracy and representativeness of the results.