Definition of microbial growth:-
- In a biological system growth means orderly increase in all chemical components.
- In case of bacteria they should be e in a a balanced growth which means an increase in biomass directly proportional with increase in all type of cell materials like protein, DNA ,RNA, intracellular water etc.
- Increase in biomass is not exactly the growth because the cell may increase their content of storage product such as glycogen or poly- beta-hydroxybutarate.
- There are two common measure of cell growth one is cell count and turbidity.
- The most easy way obvious way to determine microbial number is through direct counting.
1. Direct counting
- petroff hausser counting chamber
- flow cytometer
- membrane filter technique
2.viable cell counting or colony counting
- pour plate technique
- spread plate technique
3. cell mass weighing (dry weight)
4. Indirect counting
- measuring cell mass by turbidometric method.
- Other method
Total cell count:-this method is simple but result is unreliable.
▶petroff hausser counting chamber:-
- Such microscopic counts can be done on either dried sample on a slide or liquid sample.
- Dried sample can be stained to increase the contrast between cells and their background.
- So dried sample do not need this counting chamber so it can be counted directly placing the microbial slide under the microscope.
- For liquid sample this petroff hausser chamber.
- This counting chamber there is a grid with squares of known area is marked on the surface of the glass slide.
- its square on the grid has a volume of loan amount very small but precisely measured.
- The number of cells per unit area grid can be counted under the microscope giving a measure of the number of cell per small chamber volume.
- The number of cells per ml of suspension is calculated by employing a conversion factor based on the volume of the chamber sample.
- The bacteria in several of the central squares are counted under the microscope.
- The average number of bacteria in this square is used to calculate the concentration of cells in the original sample.
- Since there are 25 squares covering an area of 1 mm square the total number of bacteria in 1 mm of the chamber is ( chamber /square) ×( 25 square).
▶ Flow cytometer
- The chamber is 0.02 mm deep.
- Large microorganism such as protest and yeast can be directly counted with electronic counters like culture counterwhich is now known as flow cytometer.
- The microbial suspension is forced through a small hole in the coulter counter.
- An electric current flow through the hole and electrodes placed on both side of the orifice measure its electrical resistance.
- Every time a microbial cell passes through the orifice electrical resistance increase and the cell is counted.
- The Coulter counter gives accurate result with largest cell like red and white blood cell at hospital.
- It is not useful in counting bacteria because of interference by small debris particle the formation of filament and other problem.
▶Membrane filter technique:
- The number of bacteria inaquatic sample is frequently determinant from direct counts after the bacteria have been trapped on special membrane filters.
- In this technique the sample is first filtered through a black polycarbonate membrane filter.
- Then the bacteria are stained with fluorescent dye searches acridine Orange or the DNA strain DAPI and observed microscopically.
- Then strained cell are easily absorbed against the black background of the membrane filter and can be counted when viewed with and fluorescence microscope.
▶Disadvantages of direct counting:-
- Microscopic ccounting is a quick and easy way of estimation of microbial cell number but it has several limitation like:-
- Without special staining technique that cell cannot be distinguished from live cells.
- Small cells are difficult to see under the microscope and some cells are missed.
- Precision is difficult to achieve.
- A phase contrast microscope is required if the sample is not strained.
- Cell suspension of low density have few if any bacteria in the microscope field unless the sample is first concentrate and resuspended in a small volume.
- Motile cell must be immobilized before counting.
- The British in the sample maybe mistaken for microbial cell.
▶viable cell counting:
- A viable cell is one that is able to divide and form off spring.
- Several plaiting methods can be used to determine the number of viable microbes in a sample.
- These are referred as bi I will counting method because the count only those cells that are alive and able to reproduce.
- Two commonly used procedure are spread and pour plate technique.
▶spread plate technique:-
- In this method of volume usually of an appropriately diluted culture is spread over a surface of an agar plate using a sterile glass spreader.
- The plate is then incubated on 10 colonies appear and the number of colonies is counted.
- The surface of the plate must not be to moist so that the liquid soak in and the cell remain stationary.
- Volume greater than 0.1 ML are avoided in this method because the access liquid does not soak in and may cause the colonies to coalesce as the form and making them difficult to count.
▶pour plate technique
- In this method a known volume of culture is pipetted into a sterile petri plate.
- Melted agar medium is then added and mixed well by gently swirling.
- Because the sample is mixed with the molten agar medium a larger volume can be used then spread plate.
- With this method the organism to be counted must be able to with stand brief exposure to the temperature of molted agar.
- The colonies form throughout the plate not just on the surface.
- So all the colonies may be counted.
- Pour plate method is used to enumerate cells from natural sample but at the same time any debris in the sample must be distinguishable from actual bacterial colonies on the count will be erroneous.
▶Diluting cell and counting
- Both in spread and pour plate method it is important that the number of colonies developing on the plate not be too many or too few.
- On crowd place some cells may not form colonies and some colonies main fuse leading to erroneous measurement.
- If the number of colonies is too small the statistical significance of the calculate count will be low.
- The useful practice which is statistical more valued is the colony on the place that have between 30 and 300 colonies.
- Now to obtain the appropriate colony number the sample to be counted most almost alway 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 dilution of the sample are commonly used to make 10 fold dilution. then makes 1.0 ml of sample 29 ml of solution. If 104 dilution needed then we can make 1.0 ml of sample 299 ml salient to make 10-2 and it can be diluted for other in this manner as for the required dilutions.
▶Cell mass weighing:-
- Increase in the total cell mass as well as in cell numbers a company population growth.
- Show the technique for measuring changes in cell mass can be used in following methods.
- The most direct approach is the determination of microbial dry weight.
- Cell growing in liquid medium are collected by centrifugation was dried in an oven at 90 degree Celsius and 110 degree Celsius and then weighed.
- This technique is useful to microbial growth of filamentous fungi.
- It is the time consuming and not very sensitive method.
- Because bacteria weigh very little it is necessary to centrifuge several hundred millimetre of culture to collect a sufficient quantity.
Measuring cell mass by turbidometric method:-
- Spectrophotometry can also be used to measure cell mass.
- This method are more rapid and sensitive.
- The depend on the fact that microbial cells scatter light that strikes them.
- Because microbial cells in a population are of roughly contest size the amount of scattering is directly proportional to the biomass of cell present and indirectly related to the cell number.
- When sample is more turbid it indicates more cell present there.
- Hands more light scattered which is measured by spectrophotometer.
- When the concentration of bacteria reaches about 10 to the power 7 cells per ml the medium become cloudy or turbid.
- Further increase in concentration result in greater turbidity and less light is transmitted through the medium.
- The extent of light scattering can be measured by spectrophotometer and is almost linearly related to the cell concentration at low absorbents levels.
- So population growth can be easily measured as long as the population is high enough to give the table turbidity.
- This is the indirect method because measurement of cell mass is proportional to the cell number.
- Turbidity is measured by spectrophotometer.
- In which light passes through cell suspension dected the and scattered light.
- Mostly 480 NM ,540 NM and 600 NM are used for bacterial turbidity.
- Measurement of the dense cell suspensions are more accurate at longer wavelength.
- It is decrease in scattered light caused by turbidity that is the measured quantity.
▶Optical density versus cell number
- At high cell concentration light scattered away from the spectrometer photocell by one cell can be scattered back by another.
- Two other photocell this makes it appear as if light had never been scattered in the first place.
- At high densities the correlation between cell number and turbidity divides from linearity and the od measurement made our than less accurate.
- Turbidity measurement is quick and easy to perform without destroying or significantly disturbing the sample.
- The same sample can be checked repeatedly and the measurement plotted on a semi logarithm plot versus time.
- Many microbes grow in suspension in liquid medium and many others do not.
- Some bacteria from small to large slums and in such instance OD measurement may be quiet inaccurate as a measure of total microbial mass.
- So for od to accurately reflect cell mass in a liquid culture clumping and biofilms have to minimised.
- This can be done by string ,shaking or by keeping the cell wall mixed during the growth process to prevent the formation of cell aggregates and biofilms.
- Dead cell also interfere during measurement so this method is accurate only at early log phase.
- If the amount of a substance in each cell is constant the total quantity of that cell constitute directly related to the total microbial cell mass.
- Sample of washed cell collected from a known volume of a medium can be analysed for total protein or nitrogen.
- An increase in the microbial population will be reflected in higher total protein levels.
- Similarly chlorophyll determination is used to measure algal and cyanobacteria population.