In this article we will discuss about the techniques used for measuring cell numbers and cell mass of microorganisms.
Measurement (Count) of Cell Numbers:
1. Breed Method:
A known volume of microbial cell suspension (0.01 ml) is spread uniformly over a glass slide covering a specific area (1 sq. cm). The smear is then fixed by heating, stained, examined under oil immersion lens, and the cells are counted.
Customarily, cells in a few microscopic fields are counted because it is not possible to scan the entire area of smear. The counting of total number of cells is determined by calculating the total number of microscopic fields per one square cm. area of the smear.
The total number of cells can be counted with the help of following calculations:
(a) Area of microscopic field = πr2
r (oil immersion lens) = 0.08 mm.
Area of the microscopic field under the oil-immersion lens
= πr2 = 3.14 x (0.08 mm)2 = 0.02 sq. mm.
(b) Area of the smear one sq. cm. = 100sq. mm.
Then, the no. of microscopic fields = 100/0.02=5000
(c) No. of cells 1 sq. cm. (or per 0.01 ml microbial cell suspension)
= Average no. of microbes per microscopic field x 5000
2. Courting Chamber Technique or Direct Microscopic Count (DMC):
The number of cells in a population can be measured by taking direct microscopic count using Petroff-Hausser counting chamber (for prokaryotic microorganisms) or hemocytometers (to larger eukaryotic microorganisms). Prokaryotic microorganisms are more easily counted if they are stained or, if not stained, phase contrast of florescence microscope is employed.
These are specially designed slides that have chambers of known depth with an etched grid on the chamber bottom. Each square on the grid has definite depth and volume (Fig. 19.19). Total number of micro-organisms in a sample can be calculated taking the count of number of bacteria per unit area of grid and multiplying it by a conversion factor (depending on chamber volume and sample dilution used).
More specifically, for convenience, the Petroff-Hausser counting chamber is a specially designed slide accurately ruled out into squares that are 1/400 mm2 in area; a glass coverslip rests 1/50 mm above the slide, so that the volume over a square is 1/20,000 mm3 (i.e., 1/20,000,000 cm3).
A suspension of unstained bacteria can be counted in the chamber employing a phase contrast microscope. If, for example, an average of five bacterial cells occurs in cached ruled square, there is 5 x 20,000,000 or 108 bacterial cells per millimeter.
The direct microscopic method is easy, inexpensive and relatively quick to count bacterial cell number. However, using this method dead cells are not distinguished from living cells and also very small cells are usually missed.
3. Viable Count—Standard Plate Count (SPC) Method:
A bacterial culture need not contain all living cells; there might be some dead cells as well. The culture when grown in proper medium and under standard set or growth conditions, only living cells grow and form colony.
This fact is used to estimate number of living bacterial cells; the estimation of number of living bacterial cells is called viable count. Standard Plate Count (SPC) method is the most commonly used laboratory technique for viable count of bacterial cells in milk, food, water, and many other materials.
Various aspects of SPC are the following:
1. Procedure (Fig. 19.10):
To estimate the number of living bacterial cells in milk, for convenience, the sample of well mixed milk is taken into a pipette. 1 ml of milk dropped and mixed in 99 ml of sterile dilute solution (may be water or nutrient broth or saline solution) taken into a flask.
This results in a dilution of 1: 100 into the flask. Other flaks each containing 99 ml of sterile dilute solution are taken and dilutions of 1: 1000, 1: 10,000, and: 1,000,000 are prepared into them.
Now, 1 ml of each dilution is transferred into separate Petri dishes containing pre-solidified agar medium. The Petri dishes are incubated for 24 hours or more. Each living bacterial cell in dilutions grow in respective Petri dishes reproducing itself until a visible mass of bacterial cells, a colony, develops, i.e., one bacterial cell gives rise to one colony.
The original sample is subsequently diluted till the number of colonies developing on Petri dish fall in the range of 30-300 because the count is almost accurate, and the possibility of interference of one colony with that of another is minimized.
2. Counting of colonies:
Each Petri dish is taken for counting of colonies. Colonies are usually counted by illuminating them from below (dark field illumination) so that they are easily visible, and a large magnifying lens is often used. For this purpose, various instruments such as Quebec colony counter and electronic colony counter are used. Quebec colony counter (Fig. 19.11) is one of the simplest colony counters used in small laboratories.
In this, the Petri dish containing bacterial colonies is mounted on a platform. When the Petri dish is illuminated from beneath, the visible colonies can be counted with the help of its lens that provides X1.5 magnification.
Electronic colony counter is highly improved device. The Petri dish is placed on its illuminated stage, the count bar is depressed, and the precise number of colonies is instantly displayed on a digital readout.
3. Calculation of count:
The probable number of bacteria per ml in original sample can be estimated by multiplying bacterial colony count by the reciprocal of the dilution and of the volume used.
For example, if bacterial colony count is 50 for 1 : 10,000 dilution when volume used is 1 ml, then The number of colony forming bacterial cells = 50 x 10,000 x 1 = 5 x 105
4. Limitations of SPC:
(i) Only bacteria that will be counted are those which can grow on the medium used and under the conditions of incubation provided.
(ii) Each viable bacterial cell that is capable of growing under the culture conditions provided may not necessarily result in one colony. The development of one colony from one bacterial cell can only take place when the bacterial suspension is homogenous and no aggregates of cells are present in it.
However, if the bacterial cells possess the tendency to aggregate, e.g., cocci in clusters (staphylococci), chains (streptococci), or pairs (diplococci), the resulting counts will be lower than the number of actual bacterial cells. For this reason the counts are often reported as colony forming units (CFU) per millilitre rather than number of bacterial cells per millilitre.
5. Advantages of SPC:
SPC is easy to perform and can be used to measure bacterial populations of any magnitude. It is very sensitive technique and even very small number of bacterial cells can be counted using it. Theoretically, if 1 ml sample contains as few as one bacterial cell, the latter develops one colony upon transferring the sample into medium containing Petri dish.
4. Coulter Counter:
Coulter counter (Fig. 19.12) is an electronic device used to count number of bacteria and other micro-organisms such as protozoa, microalgae and yeasts. This device is provided with a tiny orifice 10-30 pm in diameter. This orifice connects the two compartments of the counter which contain an electrically conductive solution (electrodes).
In this method, the sample of bacterial cells is forced through the small orifice (small hole). On the both sides of the orifice, electrodes are present to measure the electric resistance or conductivity when electric current is passed through the orifice.
Every time a bacterial cell passes through the orifice, electrical resistance between the two compartments (electrodes) increases momentarily or the conductivity drops. The generates an electrical signal which is automatically counted.
Each electrical signal represents the counting of one bacterial cell. The Coulter counter gives accurate results with larger cells. The precaution to be taken in this method is that the suspension of samples should be free of any cell debris or other extraneous matter.
5. Membrane-Filter Technique:
Microbial cell numbers are frequently determined using special membrane filters possessing millipores small enough to trap bacteria. In this technique, a water sample containing microbial cells is passed through the filter (Fig. 19.13). The filter is then placed on solid agar medium or on a pad soaked with nutrient broth (liquid medium) and incubated until each cell develops into a separate colony.
Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with the medium and the microorganism. A colony count gives the number of microorganisms in the filtered sample, and specific media can be used to select for specific microorganisms. This technique is especially useful in analysing aquatic samples.
Measurement of Cell Mass:
1. Dry Weight Technique:
The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the microorganisms are removed from the medium by filtration and the microorganisms on filters are washed to remove all extraneous matter, and dried in desiccator by putting in weighing bottle (previously weighed).
The dried microbial content is then weighed accurately. This technique is especially useful for measuring the growth of micro fungi. It is time consuming and not very sensitive. Since bacteria weigh so little, it becomes necessary to centrifuge several hundred millions of culture to find out a sufficient quantity to weigh.
2. Measurement of Nitrogen Content:
As the microbes (bacteria) grow, there is an increase in the protein concentration (i.e. nitrogen concentration) in the cell. Thus, cell mass can be subjected to quantitative chemical analysis methods to determine total nitrogen that can be correlated with growth. This method is useful in determining the effect of nutrients or antimetabolites upon the protein synthesis of growing culture.
3. Turbidometric Estimation (Turbidometry):
Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number.
One visible characteristic of growing bacterial culture is the increase in cloudiness of the medium (turbidity). When the concentration of bacteria reaches about 10 million cells (107) per ml, the medium appears slightly cloudy or turbid.
Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured.
Greater the turbidity, lesser would be the transmission of light through medium. Thus, light will be transmitted in inverse proportion to the number of bacteria. Turbidity can be measured using instruments like spectrophotometer and nephelometer (Fig. 19.14).