Breed Count Method - Introduction, Principle, Wilson's chart, Procedure, Formula, Limitation, Advantage
Introduction to Breed Count Method
The number of microorganisms, which can be determined by the breed count method, at any given time can be determined by following the course of the growth cycle. As exponential growth is a balanced phase, the growth rate is determined by the properties of cell mass and cell number.
The breed count method detects the number of both viable and non-viable bacteria, algae, or yeast cells present in samples such as broth culture, beer, fruit juices, milk, and water. It is a direct cell count method and is done by direct microscopic examination.
American bacteriologist Robert Breed designed this technique of enumeration.
Principle of Breed Count Method
In the breed count method, a known volume of sample (0.1ml) is directly spread over a 1cm2 area of the slide in a uniform way which is then dried and stained. Then the principle of the method involves counting the average number of organisms per microscopic field.
Formula for Breed Count Method
The enumeration formula of microorganisms for the breed count method in the sample is calculated by using the formula.
N (T.C.) = n x F
where,
N (T.C.) = total number of microorganisms/ml
n = average number of microorganisms in one microscope field
F = (Area of smear / Area of microscopic field (A) ) x 100
A = π r2 (r = radius of microscopic field)
As this method can't distinguish between living and dead microbial cells, the breed count method always gives a high number of organisms in comparison to the indirect methods such as a plate count method.
Wilson's chart for Breed Count Method
The number of microscopic fields in Wilson's chart to be counted depends upon the number of microorganisms observed under one microscopic field.
Average no. of microorganism | No. of fields to be counted |
|---|---|
0-3 | 64 |
4-6 | 32 |
7-12 | 16 |
13-25 | 8 |
26-50 | 4 |
51-100 | 2 |
>100 | 1 |
Procedure for Breed Count Method
The procedure for breed count method involves following steps:
Determination of area of 1 microscopic field
Place the stage micrometer on the microscope's stage and observe it under oil immersion with a sharp focus
Count the number of divisions in the stage micrometer visible under one microscopic field
Determine the radius and area of 1 microscopic field using the formula given above
For Eg:
* If the diameter of 1 microscopic field = 13 divs of the stage micrometer
* Each division of stage micrometer = 0.01mm
Diameter of microscopic field = 13 x 0.01 mm = 0.13 mm
Radius of microscopic field = diameter/2 = 0.13/2 = 0.06mm
Hence,
Area of one microscopic field = π r2 = 3.14 x 0.062 = 0.01 mm2
Enumeration of microbes
Take a clean, sterile 1cm2 engraved glass slide
Using a sterile calibrated loop or pipette, make a uniform smear within the marked area
Air dry and heat fix the test microbial smear
Use simple staining using methylene blue and observe the slide under the microscope
Get concurrent readings on Wilson's chart
Calculate the number of microorganisms per ml of sample
For Eg:
* Area of smear = 1cm2 = 100mm2
* If the area of one microscopic field = 0.01 mm2
No. of fields under lens (F) = (Area of smear/Area of microscopic field (A) ) x 100 = (100/0.01) x 100 = 1,000,000 fields
If n = average number of microorganisms per microscopic field = 0.2
Then,
Total no. of organism per ml (Total Count) = n x F = 0.2 x 1,000,000 = 2x106/ml
Advantages of Breed Count Method
The advantages of the breed count method are as follows:
It is a faster method of microbial enumeration in comparison to Viable Plate Count or standard plate count (SPC)
Even if the organisms require specific growth requirements such as in the cases of thermophiles, and psychrophiles, it can be counted
Detection of Streptococcus in milk samples will indicate that the animal is suffering from mastitis
Limitation of Breed Count Method
The disadvantages/limitation of the breed count method are as follows:
Both viable and non-viable cells are counted as there is no effective way to differentiate between the two
A suspension may contain a very high number of cells which makes this technique difficult
As high magnification is required to visualize the bacteria, it limits the volume of liquid that can be examined
Some bacterial cells are very small and thus are missed/overlooked
The precision of this depends highly on the experience of the microbiologist as some cells can be missed while manually counting
This method is tedious and not suitable in cases of suspensions with low density of microbial cells