Spread Plate Enumeration - Introduction, Principle, Formula, Procedure, Enumeration, Advantage, Limitation

Introduction to Spread Plate Enumeration

Spread plate enumeration is a type of plate count method or standard count method of enumeration of microorganisms that only counts viable cells. In addition to enumeration, it isolates organisms from a mixed culture.

To determine the number of bacteria present in any sample, a series of dilutions are done so that each dilution will only contain a sparse concentration of microorganisms.

Principle of Spread Plate Enumeration

Principle of Spread Plate Enumeration is based on the principle that a single viable microbial cell that has been separated by dispersion from one another in a confined space of an agar medium will give rise to a macroscopically visible separate colony.

In spread plate enumeration, the dilution is usually done in a series of test tubes containing sterile distilled water or physiological saline which does not contain any nutrients. A measured amount of suspension is taken from diluted samples and poured on the surface of the agar medium which is then evenly spread with the help of a sterile bent glass rod.

Formula of Spread plate enumeration

The number of organisms can be determined by using the formula for Spread plate enumeration:

No. of bacteria = no. of colonies x Dilution factor

Where Dilution factor = 1/dilution

If the inoculum size is 0.1ml

No. of bacteria = no. of colonies x Dilution factor x 10

Procedure of Spread plate enumeration

The procedure of Spread plate enumeration is as follows:

1. Take sterile dilution blanks with each containing 9ml and mark it as 10^-1, 10^-2,......10^-n

2. Take 1ml or 1 gram from the sample with the help of a sterile pipette and mix it with the first dilution blank measured 10^-1, thus preparing an initial dilution

3. Shake gently to mix the content to obtain uniform distribution of cells

4. From the initial dilution i.e. 10^-1, take 1 ml of suspension by using a sterile pipette and mix it with 10^-2

5. Repeat the procedure until the desired dilution (10^-n) is obtained

6. From selected dilutions, 0.1ml of the suspension is transferred onto the agar plate

7. Using sterile spreaders such as curved glass rods, immediately spread the inoculum uniformly

8. Incubate the inoculated agar plates at 35-37°C for 18-24 hours

Enumeration of Spread plate enumeration

Enumeration of Spread plate enumeration is done by using the formula:

No. of bacteria = no. of colonies x Dilution factor

If the inoculum size is 0.1ml, then

No. of bacteria = no. of colonies x Dilution factor x 10

For Eg:

Let's suppose the plate of the 10-³ dilution yielded a count of 311 colonies. Then, the number of bacteria in 1 ml of the original sample can be calculated as:

Bacteria/ml = (311) x (10³) x 10 = 3.11 × 10⁶

Advantages of Spread plate enumeration

The disadvantage of Spread plate enumeration is as follows:

• Unlike the inpour plate method, the organisms do not need to withstand the high temperature of the liquid agar thus psychrophiles are able to grow

• All microbial cells can grow on the agar surface while in the pour plate method, aerobic cells may be trapped inside the agar resulting in less colony forming unit/ml count

• Although strict aerobes are favored in this technique, microaerophilic microbes can also grow slowly

Disadvantage of Spread plate enumeration

The disadvantage of Spread plate enumeration is as follows:

• If the colonies are too crowded or mixed, enumeration may be difficult. Thus, volumes more than 0.1ml is rarely used during inoculation onto the medium.

• If a large size of inoculum is used (more than 0.1ml), the agar may not be able to soak up the excess liquid, and colonies may have coalesced

• Some bacteria such as Proteus are highly motile and can swarm the entire plate resulting in mixed colonies