Spread Plate Technique: Principle, Procedure, Results

By Nisha Rijal •  Updated: 05/03/22 •  4 min read

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 results from the spread plate technique students must be careful;

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 on 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 to 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 by 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.

Nisha Rijal

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.

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