Microbiologists working in the laboratory often have to determine the microbial cell concentration in a given sample and may ‘also compare the amount of microbial growth under various conditions. The enumeration of microorganisms is very important in dairy microbiology, water microbiology, food microbiology as well as in research. In process engineering the knowledge of biomass concentration is also essential.
For any organism, growth will invariably undergo the four phases namely Lag phase, Logarithmic phase (log phase or exponential phase), stationary phase and decline phase (death phase).
The duration of each phase will vary depending on the type of organisms and the knowledge of this growth pattern is of importance to the microbiologists.
Various methods commonly used to enumerate microbial populations and determine growth quantitatively include Turbidimetric method, standard plate count (SPC), measurement of cell mass, Direct microscopic count, chemical estimate (i.e quantitative measurement of proteins, DNA, RNA or other metabolites).
When measuring the growth of a population of bacteria or yeast, one can do it directly by ‘counting the number of cells, or indirectly by measuring some indication of the number of cells, such as the cloudiness of a solution, or production of gas.
It is usual to inoculate a small sample of the microorganism into a sterilised nutrient medium and to place the culture in an incubator at the optimum temperature for growth. Growth can be measured from the time of inoculation.
Two types of cell count are possible, namely viable counts and total counts. The viable count is the total of living cells only. The total count is the total number of cells, living and dead and is often easier to measure.
Viable Counts:
It may be important to know the number of living cells. For example, the effectiveness of pasteurising milk in killing certain bacteria could be measured by making viable counts before and after pasteurization. In an industrial process it is only the living cells which will be contributing to the process, in such cases it is important to know the number of living cells in the culture.
Viable counts are made using spread plates or pour plates which have been earlier explained, though viable yeast cells can be counted using an adaptation or haemocytometry.
Problems Associated with Viable Count
There are a number of problems associated with viable count.
- Some bacteria form chains or groups of cells, for example Streptococci and staphylococci. Each group of cells will give rise to only one colony. The results of viable counts are therefore sometimes expressed as numbers of colony forming units (cfu) rather than number of bacteria.
- If more than one type of bacterium is present, for example in a soil, milk or water sample, conditions will not favour all types equally. Some will therefore grow more rapidly than others, giving variable numbers of visible colonies.
TOTAL COUNTS
Haemocytometer
The number of bacteria or yeast cells in a liquid culture, such as a broth culture, can be counted directly using a microscope. The technique is easier for yeast because the cells are much larger.
With bacteria, an oil immersion lens is required. A special counting slide called a haemocytometer slide is most commonly used. The technique is known as haemocytometry.
To ensure close enough contact between slide and cover – slip, the cover-slip should be pressed down firmly either side of the counting chamber (but don’t break the cover-slip).
It should be moved slightly until coloured lines appear. The liquid sample in the pipette is drawn under the coverslip by capillary action. The slide should be left 2 to 30 minutes to allow cells to settle, making counting easier. The microscope is focused on the grid and the number of cells in the entire grid, or a representative sample, can be counted. At least 600 cells should be counted.
Where cells lie on a boundary, they can be judged as in the square on two sides (e.g upper and right boundaries) and out of the square on the other two sides, rather than trying to count half cells, Each small square is a known area and the depth of liquid below the coverslip is constant (usually either 0.02mm or 0.lmm).
The volume of each square can therefore be calculated, and the number of cells in a given volume can be estimated. The sample should be diluted if there are more than about 30 cells in some of the squares. Remember to allow for any dilution in the calculations.
In the grid, the small squares are each 0.05mm x 0.05mm ~ 0.0025mm2, a total area for the grid of 1mm². If the gap between the cover-slip and the grid is 0.02mm, the total volume above the grid is therefore 1 x 0.02mm = 0.02mm; The number of cells in a given volume of sample can therefore be counted. Remember 1000mm? = lcm? and 1000cm = 1dm (1 litre).
With yeast, methylene blue ‘stain can be used. This stains only dead cells blue. Living cells actively transport the dye back out of the cell. This would enable a viable count to be determined by counting only colourless or very pale blue cells.
Measuring Turbidity
The more cells there are in a suspension, the greater its turbidity or “cloudiness”. The degree of turbidity is measured, by a technique known as turbidimetry.
The simplest way of doing this is to have a set of standard tubes (Brown’s tubes). These can be purchased and contain suspensions of different concentrations of barium sulphate, ranging from transparent (tube 1) to opaque (tube 10). A sample of the suspension of microorganisms under investigation is placed into an identical tube and the turbidity of the suspension is matched with one of the standard tubes. A table is supplied with the tubes how the turbidity of the microorganisms can be converted concentrations of cells for a range of different microorganisms.
Alternatively, turbidity can be measured as the change in percentage transmission of light (or optical density) of the suspension using a colorimeter or a spectrophotometer. The cell mass is directly proportional to the optical density. A red light (red filter) is most useful because it is not interfered with by the yellowish colour of many culture media.
Measuring turbidity is likely to be subject to errors if cells grow in clumps like some bacteria. Also the most accurate estimates are obtained from population density that are not too high or too low. Dilution of the sample may be necessary for an ideal density of cells (10° -10″ cells per cm).
The instruments commonly used for measuring turbidity include spectrophotometer, Nephelometer, colorimeter etc. These are normally measured in absorbance units and estimation of cell mass is normally carried out broth cultures.
Warburg Manometeric apparatus, Thumberg tube for dye reduction measurements are other instruments that are used for the estimation of bacterial number. Other forms of microbial growth measurement include Nitrogen content which is an indirect measurement of cell mass in mg nitrogen per ml, Dry weight in mg cells per ml. Others are membrane filter technique and metabolic product in which microbiological assays involves indirect measurement of metabolic activity in amount of product per ml.
Continuous culture system using the chemostat and the turbidometers are also popular methods of measuring the growth of bacterial cells.
NON-COUNTING METHODS
Other methods for measuring the growth of microorganisms can be devised. For example, with fungi, the growth in diameter of the mycelium can be measured over time.
This would be suitable, for example, if the effect of temperature on fungal growth were being investigated, or the effect of an inhibitory substance in the medium. If a fungus is growing in a liquid medium, samples of the culture could be filtered or centrifuged at suitable intervals and the fresh mycelium or dry mass of the mycelium measured.
Advanced methods of estimating growth include microcalorimetry, chemoluminescence, impedance bacteriology, mass spectrometry, gas liquid chromatography etc.
It has been found that if bacteria are stained with acridine orange (AO) and examined under fluorescent microscopy viable or organisms as distinct from dead cells fluoresce with an orange-light. This basic observation been adapted to an ingenious method for determining bacterial content and may be completed within 1 hour.
The method, known as the direct epifluorescent filtration technique (DEFT), consists of filtering the liquid to be tested through a membrane filter, staining the filter with the acridine orange and examining the filter under a fluorescent microscope. The organisms may be counted, thus rendering the technique quantitative.
Flow Cytometry
This technique together with the coulter counting technique depends upon a simple but ingenious device. A potential difference is maintained in a circuit which includes a tube with a small orifice submerged in a conducting liquid.
If a liquid containing particulate matter, blood cells, bacteria or suspensions of inanimate matter is passed down the tube, when a particle passes through the orifice a change in resistance in the circuit occurs and the change may be recorded by the usual detection or print-out devices. Both the number of particles per unit of time and the size may be determined.
There are certain points to be borne in mind, however, with this method:
- In the counting of bacteria, both dead and: living cells will be counted and sized although prestaining with a dye and a sophistication of the instrumentation has been investigated.
- A further possible disadvantage is that the orifice may be blocked during use.
- If the bacterial content of an inanimate suspension, i.e. a medicine such as milk of magnesia, is being examined both bacteria and magnesium hydroxide particles will be detected, although here again methods of distinguishing the two types of particle have been developed.
Microcalorimetry
This method depends on the fact that bacteria like all living organisms produce heat when they metabolize. Because of the small amount of heat produced, especially sensitive calorimetric devices are required hence the name microcalorimetry. The specimen to be evaluated 1s diluted with nutrient medium, and if microorganisms are present they can metabolize, and heat is produced and can be measured. An interesting off shoot of this technique is the fact that differing organisms produce different heat outputs and this may provide a means of identification.
Microcalorimetry may help for organisms to be detected and possibly identified in 2h.
Electrical Conductivity
When organisms grow in liquid media their metabolic products can create a change in the conductivity of the media, a fact noted in 1898. Research has shown that colony numbers of about 10ml are required to produce a measurable conductivity change.
The actual changes in the media may be measured also by changes in impedance (resistance to an alternating current) or the change im its electrical capacity. As with all techniques it has certain limitations. If the level of initial contamination of a product is low, incubation of a sample in a broth will be necessary to increase the organism content nearer to 10ml.
This will add to the time of the test. Another limitation lies in the detection of non-fermentative microorganisms whose metabolic activity produces little change in media conductivity.
Bioluminescence
It has long been known that certain insects (e.g. the _ beetles known as fire flies) and two or three genera of bacteria possess the ability to emit light; this property has been utilized in quality control and research.
1. The use of the fire fly light – emitting system:Light generation depends on the oxidation of a substance known as luciferin. This is a fatty aldehyde such as dodecanal. An enzyme called luciferase, extracted from fire flies, catalyses the oxidation.
The reaction also requires ATP. Thus light emission measures ATP. The detection of bacteria by this method depends on the fact that they, like all living material contain ATP but there arises a potential problem.
Comprehensive kits are available to analysts, bacteriologists and research workers to perform the determination of ATP. They include, or are backed by special light – measuring equipment (luminolmeters) to estimate light emission and follow its estimation.
2. The use of luminous bacteria: With the advent of genetic engineering, it has been possible to insert the light-emitting genes of a natural bioluminiscent organism, the so – called lux cluster, in organisms more relevant to medicine and public health e.g. Escherichia coli ,Salmonella typhimurium, Listeria spp. and Mycobacterium smegmatis amongst others.
The estimation of light in these organisms is used to mark the end-point in an estimation of biocide activity and thermal stress to quote two examples of the application of this method.
Turbidimetric Method
A culture of single-celled microorganism acts as a colloidal suspension which blocks and reflects light passing through the culture. By measuring the degree of light scattering, one can estimate the cell concentration.
A spectrophotometer or colorimeter can be used to measure turbidity. The absorbance (the rate of light scattering) is termed the optical density. The turbidimetric measurement is Often correlated with some other method of cell count for instance standard plate count.
By so doing the turbidity can be “converted to the cell concentration by means of a standard curve.
Limitations of the turbidimetric measurement includes:
- Young and old cells have different refractive indices.
- The medium may change in transmittance during growth.
- A non-linear relationship between turbidity and cell concentration exists at higher cell concentration.