Spread Plate Technique: Principle, Procedure, Results

The spread plate technique is a viable counting method employed to plate a liquid sample to isolate or count the bacteria in that sample. A perfect spread plate technique will result in visible and countable colonies of bacteria evenly distributed on the plate.

To get optimum results from the spread plate technique, one 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

Each viable organism grows and divides to yield one colony, known as colony forming units (CFU).

Principle

A liquid sample is diluted in a series of tubes containing sterile water or physiological saline. A fixed volume of sample, usually 0.1 mL, is removed from each tube and placed onto the agar surface. The sample is then spread evenly over the agar surface using a sterile, bent glass rod.

Following incubation, the dispersed cells develop into isolated colonies. The number of colonies present on the plate is counted and multiplied by the dilution factor to calculate the total number of viable organisms initially present in the sample.

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.

Requirements

1. Glasswares
   1. Screw-capped test tubes
   2. Sterile pipettes
   3. Sterile bent glass rods (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.
3. Alcohol (ethanol)

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. Take 1ml of the sample from the first tube and transfer it to the second tube. Label it as 10^-2.
5. Repeat the procedure with all the remaining tubes labeled 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 ethanol. Ethanol is used to sterilize the glass spreader.
3. Flame the glass spreader over a bunsen burner.
4. Spread the sample evenly over the surface of the agar using a cool alcohol-flamed glass rod spreader, carefully rotating the Petri dish underneath at an angle of 45^o 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 and multiply it by the appropriate dilution factor to determine the colony-forming units (CFU) present per ml in the original sample.

CFU/ml = (number of colonies x dilution factor) / volume of culture plated

For example, suppose the plate of the 10^-4 dilution yielded a count of 27 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as follows:

Bacteria/ml = (27) x (10⁴ ) x 10 = 2.7 × 10⁶
(we have multiplied by 10 because we have used 0.1mL while plating the agar plate)

Benefits compared to pour plate technique

1. Only surface colonies develop
2. The organisms are not required to withstand the temperature of liquid agar.

Uses

1. Unlike the streak-plate technique, the spread-plate technique can be performed quantitatively to determine the number of bacteria present in a sample. The spread plate 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.

Reference and further readings

1. Sanders ER. Aseptic laboratory techniques: plating methods. J Vis Exp. 2012;(63):e3064. Published 2012 May 11. doi:10.3791/3064