The term growth in microbiology indicates the magnitude of total population.
Microbial growth measurement is essential for:
- Determining the growth and reproduction rate,
- Determining the suitability of water, milk, and food for human consumption,
- Counting microflora of soil,
- Monitoring the fermentation process,
- Checking the suitability of raw material for preparing pharmaceutical products, and
- Checking the microbial titre in commercial products (e.g., bio-fertiliser, vaccine, probiotic tablets, etc.).
Quantitative measurement of bacterial growth can be carried out using the following techniques:
Total Count/Direct Methods
- Direct microscopic count/Breed method,
- Counting chamber method/haemocytometer method,
- Electronic cell counter/coulter method, and
- Proportional count method.
Viable Count/Indirect Method
- Plate count method, and
- Membrane filter count method.
For counting the microorganisms they should be detected first. The oldest counting technique involves using a microscope to magnify individual cells so that they become visible to naked eye. Direct counting techniques do not rely on cell population growth. Different direct counting methods are discussed below:
Direct Microscopic Count/Breed Method : In this method, 0.01ml of bacterial cell suspension is uniformly smeared over a glass-slide within 1sq.cm specific area. It is then stained and examined under ‘oil immersion objective’. The entire 1sq.cm area cannot be examined at the same time, thus few microscopic areas are observed.
The total number of microscopic fields per 1sq.cm area is the total number of cells.
The total number of bacterial cells can be obtained by the following calculations:
Area of microscopic field = π r²
Where, r (radius of oil immersion objects) =0.08 mm
Area of microscopic field = 3.14×(0.08)²
Area of microscopic field = 0.02sq. mm
Area of the smear =1 sq. cm=100 sq. mm
Number of microscopic fields =100/0.02=5,000
Total number of cells /1 sq. cm= Average number of cells per field ×5,000, i.e., Number of cells/ 0.01ml of suspension. On multiplying this number with 100 , number of cells /ml of suspension is obtained.
This method quickly analyses milk in dairy to determine the contamination level before pasteurisation.
This method also determines relative effectiveness of antibiotics against pathogenic bacteria.
This method does not determine the number of viable cells.
Counting Chamber Method : In this method, special microscope slides having grid or lines etched on its surface are used for easy and accurate counting.
In Petroff-Hauser chamber or haemocytometer (named so, as originally it was used for counting blood cells), a film of known depth is introduced between the slide and cover slip. When counting, the number of cells per ml of original sample is determined by multiplying the sample’s dilution factor.
Haemocytometer creates a volumetric grid divided into cubes of different sizes for accurate counting of the number of particles in a cube and calculating the sample concentration.
- This method is fast.
- This method needs minimum equipment and other requirements.
- This method can study bacterial morphology.
- This method cannot calculate the number of viable cells.
- This method may involve statistical errors.
Electronic Cell Counter\Coulter Method : In this method, electronic cell counter is used to count the number of cells. The bacterial cell suspension is passed through a capillary tube with microscopic diameter through which only one cell can pass at a time.
A light beam is directed to a photodetector instrument through a capillary tube. The disturbances in light beam due to passage of bacterial cell allow reading of a single unit, which is then displayed by a digital reader.
- This method accurately counts thousands of cells in a few seconds.
- This method checks large amount of suspension in a very less duration.
- Large non-living particles may clog the pore.
- This method does not provide a viable count as dead cells are also counted.
Proportional Count Method : In this method, a standard suspension of particles (e.g., plastic bead) and an equal amount of bacterial cell suspension are mixed. The resultant suspension is spread over the slide and stained. The number of fields gives the average count of the particles and the cell.
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