Introduction:
While studying microbiology measurement of microbial growth becomes an important parameter of the study.
Growth of microorganisms can be quantitatively determined by one of the following methods;
Determination of Cell Number:
Total Count or Direct Methods.
Direct Microscopic count / Breed Method.
Counting Chamber / Haemocytometer Method.
Proportional count method.
Electronic Counter Method.
Viable count / indirect Method:
Plate count technique.
Membrane filter count.
Determination of Cell Mass:
Direct Method:
Dry Weight Method.
Measurement of Cell Nitrogen.
Indirect Method.
Turbidimetric Method.
Determination of Cell Activity:
Measurement of biochemical activity (Indirect Method).
Determination of Cell Number:
Total count tells us the number of living and dead cells in a bacterial suspension.
Breed Method:
Also called Direct Microscopic Count.
A known volume of cell suspension (0.01 ml) is spread uniformly over a glass slide within a specific area (1 sq. cm).
The smear is then fixed, stained, examined under the oil immersion lens, and the cells counted.
Since it is impractical to scan the entire area, it is customary to count the cells in a few microscopic fields.
The total cell count is determined by calculating the total number of microscopic fields per 1 sq cm is of cell suspension.
To obtain the total cell count following calculations is used.
(a) Area of the microscopic field = πr2
=3.14x(0.08)2 (Radius of oil immersion lens = 0.08 mm.)
= 0.02 Sq.mm
(b) Area of Smear = 1 sq.cm= 100 sq. mm.
No. of Microscopic fields = 1000.02= 5000.
Total no. of cells/ sq. mm = Avg. no. of cells per field x 5000.
Counting Chamber Method:
Special microscope slides are available with chambers designed to contain a cell suspension above an accurately rules area etched into the glass.
The Petroff-Hausser chamber or haemocytometer is ruled with squares of known area, and is so constructed that a film of known depth can be introduced between the slide and the cover slip.
Consequently, the volume of the liquid overlying each squire is accurately known.
Proportional Count Method:
A standard suspension of particles, for example, plastic- beads, where the number of particles per volume is known, is mixed with an equal amount of cell suspension.
This mixed suspension is spread on the slide, fixed and stained.
The particles and the cells in each microscopic field are then counted.
An average count of the particles and the cell is taken from the number of fields. For example, suppose an average count of 10 particles and 50 cells per field is obtained.
If the number of particles in 1 ml of standard suspension is 25,000 then the number of cells per 1 ml of suspension is:
50/10x25,000 = 1,25,000 cells/ml.
In this method there is no need to measure the amount of the suspension spread on the slide.
Electronic Counter Method:
An electronic instrument called the Coulter counter can also be used for the direct enumeration of cells in a suspension.
The instrument is capable of accurately counting thousands of cells in a few seconds.
The suspending fluid, however, must be free of inanimate particles (e.g. dust), since smaller ones will score as cells and larger ones will plug the aperture through which the cells pass.
Advantages and Disadvantages of Direct Counting Methods:
Direct counting methods are rapid and simple.
The morphology of cells can also be observed when they are counted under the microscope.
The major disadvantage of these methods is that it gives a total cell count which includes both viable and non-viable cells.
Accuracy also declines with very dense and very dilute suspensions because of clumping and statistical errors, respectively.
Very dense suspensions, however, can be counted if they are diluted appropriately.
Determination of the Number of Cells Indirectly by the Plate Count:
The plate count is based upon the assumption that each organism trapped in a nutrient agar medium will multiply and produce a visible colony.
The number of colonies therefore is the same as the number of viable cells inoculated into the medium.
Advantage of the method is that is counts only living organisms
In this procedure an appropriately diluted cell suspension is introduced into a petri dish.
Procedure:
An appropriate melted agar medium is poured into the petri dish and is thoroughly mixed with the inoculum by rotating the plate.
After the solidification of the medium, the plates are inverted and incubated for 18 to 24 hours.
A plate having 30 to 300 colonies is selected for counting the number of organisms.
The plate-count technique is used routinely with satisfactory results for the estimation of bacterial population in milk, water food, and other materials.
The method is highly sensitive, i.e. extremely high or very low populations can be counted.
However, the most obvious advantage of the method is that it counts only living organisms.
Membrane Filters Count:
This method is the same in principle as that of a plate count.
A suspension of microorganisms, such as in water or air, if filtered through a millipore filter membrane.
The organisms are retained on the filter disc.
The disc is then placed in a petri dish containing a suitable medium.
The plates are incubated and the colonies are observed on the membrane surface.
The method has distinct advantages over the plate count.
A large volume of the sample can be analyzed, especially when the number of organisms is very few.
Secondly various types of microorganisms can be detected by using selective media in the plates and under different conditions of growth.
Determination of the cell mass:
Direct Methods:
Measurement of Dry Weight of Cells:
This is the most direct approach for quantitative measurement of a mass of cells.
The sample is centrifuged or filtered and the residue or the pellet is washed a number of times to remove all extraneous matter.
The residue is then dried and weighed.
However, it can be used only with very dense cell suspensions.
This method is tedious and is applicable mainly in research investigations.
It is commonly used for measuring growth of moulds in certain phases of industrial work.
Measurement of Cell Nitrogen:
The major constituent of cell material is protein, and nitrogen is a characteristic constituent of protein.
A bacterial population or cell crop can be measured in terms of cell nitrogen.
The cells are to be harvested as described in the first technique, and then the cell nitrogen is estimated by chemical analysis.
This is also a tedious method, and can be used only with dense cell suspension.
b) Indirect Method:
Turbidity Measurements:
A most widely used technique of measuring cell mass is by observing the light-scattering capacity of the sample.
A suspension of unicellular organisms is placed in a colorimeter or spectrophotometer, and light is passed through it.
The amount of the light absorbed or scattered is proportional to the mass of cells in the path of light.
When cells are growing exponentially, increase in cell mass is directly related to cell number.
This is a rapid and accurate method to estimate dry weight or cell number in unit volume, provided a standard curve is first prepared.
A standard curve can be prepared by measuring bacterial growth simultaneously by two methods, and then establishing a relationship between the values obtained.
For example, an aliquot of samples is removed from the cell suspension, dried, and the weights per milliliter determined.
From the cell suspensions dilutions are prepared, and the organisms are counted by plate-count.
At the same time turbidity measurements of the cell suspension are also determined.
Any two sets of the data can then be plotted (cell weights or cell number against turbidity), as illustrated in Figure 18.33 to obtain a standard curve.
For practical purposes, and within a certain range of concentrations, a linear or straight-line relationship exists.
Thus, by indirectly measuring the turbidity of the suspension, cell weight or cell number can be determined with the help of the standard curve.
This method however, has some limitations.
Turbidity is most effective with suspensions of moderate density.
Suspensions with very high or very low density give erroneous results.
Secondly, it is not possible to measure cultures that are deeply coloured or contain suspended material other than cells.
It must be recognized that turbidity measures both living as well as dead cells.
Determination of Cell Activity:
An indirect method.
Measurement of a specific chemical change produced on a constituent of the medium e.g. acid production from sugar in the nutrient medium.
The amount of acid produced is proportional to the magnitude of the cell suspension.
Alternatively, specific enzymes may be assayed to measure cell growth.