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In Microbiology, isolation and enumeration of bacteria, fungi, actinobacteria, virus, algae, protozoa are commonly performed for various ecosystems, to assess the microbial richness. There are several methods being adopted for it. Isolation refers the separation of a strain from a natural, mixed population of living microbes, as present in the environment. Enumeration refers counting the total microbial cells present in it. Most of the investigations, enumeration is always coupled with isolation, if any specific microbial strain is needed for the further analyses. The two common methods of enumeration are a) microscopic count; b) viable count.
Microscopic count
A total count of microbial numbers can be achieved using a microscope to observe and enumerate the cells present in a culture or natural sample. The method is simple, but the results can be unreliable.
The most common total count method is the microscopic cell count. Microscopic counts can be done on either samples dried on slides or on samples in liquid. Dried samples can be stained to increase contrast between cells and their background. With liquid samples, specially designed counting chambers are used. In such a counting chamber, a grid with squares of known area is marked on the surface of a glass slide. When the coverslip is placed on the chamber, each square on the grid has a precisely measured volume. The number of cells per unit area of grid can be counted under the microscope, giving a measure of the number of cells per small chamber volume. The number of cells per milliliter of suspension is calculated by employing a conversion factor based on the volume of the chamber sample. A second method of enumerating cells in liquid samples is with a flow cytometer. This is a machine that employs a laser beam and complex electronics to count individual cells.
Microscopic counting is a quick and easy way of estimating microbial cell number. However, several limitations are there:
- Without special staining techniques, dead cells cannot be distinguished from live cells.
- Small cells are difficult to see under the microscope, and some cells are inevitably missed.
- Precision is difficult to achieve.
- A phase-contrast microscope is required if the sample is not stained.
- Cell suspensions of low density (less than about 10^6 cells/milliliter) have few if any bacteria in the microscope field unless a sample is first concentrated and resuspended in a small volume.
- Motile cells must be immobilized before counting.
- Debris in the sample may be mistaken for microbial cells.
Viable Counts
Viable cell is referred as one that is able to divide and form offspring, and in most cell-counting situations, these are the cells we are most interested in. For these purposes, we can use a viable counting method. To do this, we typically determine the number of cells in a sample capable of forming colonies on a suitable agar medium. For this reason, the viable count is also called a plate count. The assumption made in the viable counting procedure is that each viable cell can grow and divide to yield one colony. Thus, colony numbers are a reflection of cell numbers. There are at least two ways of performing a plate count: the spread-plate method and the pour-plate method. In the spread-plate method, a volume (usually 0.1 ml or less) of an appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader. The plate is then incubated until colonies appear, and the number of colonies is counted. In the pour-plate method, a known volume (usually 0.1–1.0 ml) of culture is pipetted into a sterile Petri plate. Melted agar medium, tempered to just about gelling temperature, is then added and mixed well by gently swirling the plate on the benchtop. Because the sample is mixed with the molten agar medium, a larger volume can be used than with the spread plate.
With both the spread-plate and pour-plate methods, it is important that the number of colonies developing on or in the medium not be too many or too few. On crowded plates some cells may not form colonies, nd some colonies may fuse, leading to erroneous measurements. If the number of colonies is too small, the statistical significance of the calculated count will be low. To obtain the appropriate colony number, the sample to be counted must almost always be diluted. Because one may not know the approximate viable count ahead of time, it is usually necessary to make more than one dilution. Several 10-fold dilutions of the sample are commonly used for this. Hence this method is also known as “Serial-dilution and plate-count” method.
The number of colonies obtained in a viable count experiment depends not only on the inoculum size and the viability of the culture, but also on the culture medium and the incubation conditions. The colony number can also change with the length of incubation. Furthermore, the size of colonies may vary. If some tiny colonies develop, they may be missed during the counting. Viable counts can be subject to rather large errors for several reasons. These include plating inconsistencies, such as inaccurate pipetting of a liquid sample, a nonuniform sample (for example,a sample containing cell clumps), insufficient mixing, and other factors. Data are often expressed as the number of colony-forming units (cfu) obtained rather than the actual number of viable cells, because a colony-forming unit may contain one or more cells.
Despite the difficulties associated with viable counting, the procedure gives the best estimate of the number of viable cells in a sample and so is widely used in many areas of microbiology. For example, in food, dairy, medical, and aquatic microbiology, viable counts are employed routinely. The method has the virtue of high sensitivity, because as few as one viable cell per sample plated can be detected. This feature allows for the sensitive detection of microbial contamination of foods or other materials.
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