Microbial Cell Enumeration - Introduction, Direct Cell Count, Indirect Cell Count, Turbidimetric Enumeration
Introduction to Microbial Cell Enumeration
The number of microorganisms 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.
Enumeration of microbial cells can be categorized into direct cell count and indirect cell count.
Types of Microbial Cell Enumeration
There are two types of Microbial Cell Enumeration- direct cell count method and indirect cell count method.
Direct cell count method
The number of bacterial cells per milliliter in the suspension can be significantly counted by a direct method. It is done by microscopically counting the individual cells in a determined small volume with any degree of accuracy. This type of counting is done with the help of a special type of microscopic slide called counting chambers. Such a direct count is known as total cell count and includes both viable and non-viable organisms.
Direct microbial cell enumeration methods include:
Breed smear enumeration for dried sample
Neubauer or Petroff Hausser counting chamber for liquid samples
Electronic cell count for liquid samples - (no. of cells/ml)
Microscopic count (no. of cells/ml)
Advantages of direct count
It is a faster method of microbial enumeration in comparison to SPC (standard plate count)
Even if the organisms require specific growth requirements such as in the cases of thermophiles, psychrophiles, and non-viable microbes it can be counted
Detection of Streptococcus in milk samples will indicate that the animal is suffering from mastitis
Limitation of direct count
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 a low density of microbial cells
Indirect cell count
The indirect microbial cell enumeration methods include:
Determining cell mass (dry weight of cells/ml)
Determination of particular cell components ie. DNA, RNA, protein, and ATP
Oxygen utilization
Carbon-dioxide production
Nitrogen determination (mg N2/ml)
Measurement of biochemical activity (milli equivalents of acid/ml or per culture)
Plate count method by serial dilution - only counts viable cells (cfu/ml)
Member filter enumeration (only counts viable cells)
Turbidimetric enumeration by photometer, spectrophotometer, colorimeter, nephelometer (optical density/absorbance)
* cfu = colony forming unit
Plate count method
The enumeration of microbial cells can also be done by the plate count method. It 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.
The result in this method is obtained after preparing appropriate dilutions and culturing them on an appropriate medium. This determines the viable cells in the initial population of the microorganism by counting the number of colonies that grow on the inoculated medium after inoculation and multiplying that number by the dilution factor which is called viable count. This method is more sensitive than total cell count as even a single viable cell in the suspension can be enumerated.
The combination of total cell count and viable plate count can be used to effectively determine the viable cells in the population.
Determination of cell components
Sometimes because of the complexity of media/nutrient requirements or microbial growth patterns such as filaments or clump growths, direct enumeration methods is not appropriate. In such cases, growth can be enumerated by indirect cell counts such as measuring the number of particular cell components ie. DNA, RNA, protein, and ATP are present in the cultured medium.
Turbidimetric enumeration
Since physically measuring the dry mass of bacterial cells is time-consuming and insensitive, the methods commonly used for measuring the cell mass of the microorganisms are optical methods. In other words, the cell mass is determined by the amount of light scattered by a suspension of cells. The principle lies in the fact that small particles scatter light proportionally and depend on the concentration of bacterial cells present. Such measurements are done by photometer or spectrophotometer or nephelometer.