Introduction
Measuring the growth of microorganisms is an important part of microbiology. The growth of bacteria can be studied by determining the number of cells, the mass of cells or the biochemical activity of the culture. Counting techniques help in estimating the total number of bacteria present in a sample and in assessing only the viable (living) cells capable of forming colonies.
A) Determination of Cell Number
This is one of the most common approaches for measuring microbial growth. It includes two major types of methods:
- Total counting (direct methods): Counts both living and dead cells
- Viable counting (indirect methods): Counts only living cells capable of reproduction
1. Total Count / Direct Methods
These methods count all microorganisms present in a given suspension, including viable and non-viable cells.
a) Direct Microscopic Count (Breed Method)
A known volume of bacterial suspension (0.01 ml) is spread over a 1 sq. cm area on a glass slide. After fixing and staining, cells are counted under oil immersion. The average number of cells per microscopic field is used to calculate total cells per square cm.
b) Counting Chamber Method (Haemocytometer)
In this method, a drop of culture is placed in a special counting chamber (Neubauer chamber) ruled into precise squares. Cells are counted under a phase-contrast microscope. This method is quick and requires minimal equipment.
c) Proportional Count Method
A known number of standard particles (like plastic beads) is mixed with an equal volume of the bacterial suspension. Both particles and cells are counted under the microscope. The ratio of beads to cells helps calculate the total number of bacteria in the sample.
d) Electronic Counter Method (Coulter Counter)
The bacterial suspension passes through a narrow capillary tube that allows only one cell at a time. Each cell interrupts the electrical current and is counted electronically. This method is rapid but counts dust and other particles as well, so the sample must be very clean.
Advantages of Direct Methods
- Rapid and simple
- Useful for observing cell morphology
Disadvantages
- Counts dead and living cells together
- Accuracy decreases in very dense or very dilute samples
2. Viable Count / Indirect Methods
Viable count measures only the living bacteria capable of forming colonies. Each viable cell gives rise to a single colony.
a) Plate Count Technique
This is the most widely used method for viable cell counting. A measured volume of diluted bacterial suspension is pipetted into a sterile Petri plate. Melted agar at 45°C is poured over the inoculum, mixed gently and left to solidify. After incubation at 37°C for 24 hours, colonies are counted. Plates with 30–300 colonies are considered most accurate.
Limitations
- Some microbes may not grow on the selected medium
- Aggregates of cells form a single colony, giving lower counts
b) Membrane Filter Count
A known volume of diluted sample is filtered through a sterile membrane filter. Microorganisms are trapped on the filter surface. The filter disc is placed on a suitable agar medium and incubated. Colonies appear on the filter and are counted.
Advantages
- Large volumes can be tested
- Different microorganisms can be detected by using suitable selective media
B) Determination of Cell Mass
Instead of counting individual cells, the total cell biomass can be measured. This provides an estimate of microbial growth.
1. Direct Methods
a) Dry Weight Measurement
The culture is centrifuged, washed and dried. The dried mass is weighed. This method is suitable for molds and for research studies.
b) Measurement of Cell Nitrogen
Since proteins form a major portion of bacterial cells, nitrogen content can be used to estimate cell mass. After centrifugation and washing, nitrogen is measured chemically. This is useful for dense cultures.
2. Indirect Method
Turbidimetric Method
This is the most widely used method for routine estimation of bacterial mass. The bacterial suspension is placed in a colorimeter or spectrophotometer. Light absorbance (optical density) is measured. Turbidity increases with cell concentration. A calibration curve is used to convert absorbance to cell numbers.
Limitations
- Dead cells contribute to turbidity
- Not suitable for coloured media
- Particles other than bacteria affect reading
C) Determination of Cell Activity
1. Measurement of Biochemical Activity
Growth can also be estimated by measuring metabolic products. As bacteria grow, they produce substances such as lactic acid, carbon dioxide, hydrogen sulphide or enzymes. The quantity of these products correlates with the size of the bacterial population.
Key Points
- Total counting methods include microscopic count, haemocytometer, proportional count and Coulter counter.
- Viable counts include plate count and membrane filtration techniques.
- Cell mass measurement uses dry weight, nitrogen content and turbidity.
- Biochemical activity reflects microbial metabolism and can estimate cell numbers indirectly.
Detailed Notes:
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