Spread Plate Technique: Principle, Procedure, Results

Last updated on June 11th, 2021

Spread plate technique is a viable counting method employed to plate a liquid sample for the purpose of isolating or counting the bacteria present in that sample. A perfect spread plate technique will result in visible and isolated colonies of bacteria that are evenly distributed in the plate and are countable. The technique is most commonly applied for microbial testing of foods or any other samples or to isolate and identify a variety of microbial flora present in the environmental samples e.g. soil.

To get optimum result from spread plate technique students must be careful;

  • To make accurate dilutions using pipettes (master serial dilution technique).
  • To apply a balanced spread technique using a glass spreader to spread the inoculum evenly on the agar surface.
  • To respect the necessary “short” time interval between agar inoculation and spreading.

In this method, the substance to be tested if not in liquid form is ground and dissolved in a suitable liquid medium. The sample is then diluted in 10 fold serial dilutions and plated in an appropriate medium.

Following incubation, the number of colonies present in the plate is counted. Assuming that each viable organism grows and divides to yield one colony, the number of total bacteria present in a sample is calculated.

Requirements

  1. Glasswares: screw-capped test tubes, sterile pipettes, glass rod spreader (bent in the shape of a hockey stick), or commercially available sterile spreaders
  2. Medium: Plate count agar or nutrient agar. The surface of the plate must not be too moist because the added liquid must soak in so the cells remain stationary.

Procedure for Spread Plate Technique

A: Serial dilution 

  1. Prepare a series of at least 6 test tubes containing 9 ml of sterile distilled water.
  2. Using a sterile pipette, add 1ml of sample in the first tube of the set. Label it as 10-1
  3. Mix the contents well by swirling the tube upside down a few times.
  4. From the first tube, take 1ml of the sample and transfer it to the second tube. Label it as 10-2.
  5. Repeat the procedure with all the remaining tubes labeling them until 10-6.

B. Plating 

  1. Pipette out 0.1 ml* from the appropriate desired dilution series onto the center of the surface of an agar plate.
  2. Dip the L-shaped glass spreader (hockey stick) into alcohol.
  3. Flame the glass spreader over a bunsen burner.
  4. Spread the sample evenly over the surface of agar using a cool alcohol-flamed glass rod spreader, carefully rotating the Petri dish underneath at an angle of 45o at the same time.
  5. Incubate the plate at 37°C for 24-48 hours.

*Note: The volume of the liquid should be 0.1 ml or less. Volumes >0.1 ml are avoided because the excess liquid does not soak in and may cause the colonies to coalesce as they form, making them difficult to count.

Calculation of result:

If your spread plate is successful, after incubation you will get isolated countable colonies evenly spread across the surface of the agar. Count the number of colonies using a magnifying device. Once you count the colonies, multiply by the appropriate dilution factor to determine the colony-forming units (CFU) present per ml in the original sample.

CFU/ml = (no. of colonies x dilution factor) / volume of culture plate

For example, suppose the plate of the 10^6 dilution yielded a count of 130 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows:

Bacteria/ml = (130) x (10^6) x 10 = 1.3 × 10^9.
(we have multiplied with 10, because we have used 0.1mL while plating the agar plate)

Uses

Pour plate is one of the recommended assays (others are membrane filtration and spread plate) for a heterotrophic plate count. It produces a quantifiable result for a colony-forming unit per volume tested.

About Nisha Rijal 46 Articles
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

12 Comments

  1. Flaming the glass spreading rod has its hazards and many opt for disposable spreaders. If you are going to use a glass spreading rod then take caution during the flaming process. Make sure that after you dip into the alcohol you put the spreader into the flame pointing down so that the alcohol does not run towards your had. Sometimes the flame is hard to see on the glass rod so DO NOT put the glass spreader back into the alcohol, some do this to be sure things are sterilized, until you are sure the flame has gone out or you will light your alcohol container on fire….and yes I have seen this happen! Make sure to put the alcohol in a container that can be covered in case you need to extinguish an accidental fire.

    • please can you send me the reference name of this method as text book or international testing method for this spread technique
      we can apply this method for other bacteria tests like total coliform , fecal coliforms

      many thanks

    • serial dilution helps to reduce the dense culture of cells to usable or countable cells.In other words, if the product is having too much bacterial count which is nearly uncountable , in that case also serial dilution is useful.
      It is also important to calculate exact cfu/ml. i.e. colony forming unit per ml.

  2. please send me the reference text for spread plate method for testing food
    if you have procedures for testing total coliforms and fecal coliforms in food products , please send me the reference and procedures .

    many thanks .

    • So that there is an evenly distributed growth of bacteria etc. And when we are counting the CFU later, there is little chances of error as bacteria are evenly distributed instead of forming closely spaced clumps of different colonies.

  3. In the sample calculation of CFU/mL that is done, should the answer by 1.3 x 10^9 (1,300,000,000)? I think that the calculation left out the 0.1mL inoculation volume for the plate.

    • same thoughts here. 1.3 x 10^9 is the answer i get considering the volume plated is 0.1mL is instead of 1.0mL.

  4. Hi, based on your example of calculation of results, shouldnt it be described as 130 colonies x 10^6 divided by 0.1 mL since this is spread plate (instead of 1 mL)?

  5. Thank you Nia and all of you for pointing us the mistakes in the calculation part. Now, it has been corrected. We appreciate your time and feedback.

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