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#Microbialgrowth #RohitMane #ScientistRacademy 1 Introduction A number of techniques are available in order to measure the growth of microbial populations. There are different techniques used for the measurement of microbial growth those are useful and efficient in the growth of microorganisms. The techniques are efficient in the way of environmental conditions provided to the microorganisms. All methods are used for the isolation, identification, characterization, purification of microorganisms. All methods are performed in the lab for the isolation and preservation of microorganisms but these all techniques were not useful for all type of microorganisms because of specific conditions used for each microbe such as nutrients, pH, temperature and many other conditions. Measurement of microbial growth is carried out by different methods depicted as follows: Indirect methods Dry Weight Turbidity Direct methods Direct Microscopic Count Membrane Filtration Serial Dilution Plate count: Spread plate count and Pour plate count 1. Indirect methods Dry weight: Used for filamentous bacteria and fungi. The microorganisms grown in liquid media, filtered, dried & weighted. The method does not include any amount of water content in it. Biomass: (weight of paper + mycelia) - (weight of paper) It is time consuming and hence not very sensitive. Turbidity: As bacteria multiply in a liquid media, the media becomes turbid. Spectrophotometer/photometer/calorimeter is used in order to measure the turbidity at 540 nm or 600 nm or 660 nm. A beam of light is transmitted through a bacterial suspension to a light-sensitive detector. The fact that microbial cells scatter light striking them, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. The extent of light scattering can be measured and is almost linearly related to bacterial concentration at low absorbance levels. Turbidity affects on pH and temperature of the microbial culture. The turbidity is shown in figure 4.2. 2. Direct measurement of cell numbers: Microorganisms can be measured directly on the plate or slide therefore known as direct measurement of cell number. Bacteria counted on plate counts are referred to as colony forming units as a single cell or a clump of bacterial cells can lead to a colony which contains many cells. The colonies when they are counted in plate count method are to be present sparsely for accurate counting as overcrowding can lead to incorrect counting. To solve this, one has to adapt the serial dilution method in order to get an accurate count. Advantage: It measures the number of viable cells. Disadvantage: Time consuming and expensive 1. Serial dilution: A serial dilution is a series of sequential dilutions used to decrease the microbial load. Supposing one has to accurately count the number of cells given in a solution, and then serial dilution needs to be performed. Each dilution will decrease the concentration of bacteria by specific amount. By calculating the total dilutions, it is possible to count that how many bacteria we started with. 2.Membrane Filtration: Used to study if the quantity of the bacteria is very small as in liquid samples like lakes, streams etc. Membranes with different pore sizes are used to trap different microorganisms. The sample is drawn through these special membrane filters and placed on an agar medium or on a pad soaked with liquid media. After incubation, the number of colonies can be counted and the number determined in the original sample. Selective media or differential media can be used for specific microorganisms. This is mostly used for analyzing aquatic samples. Mostly sand filter and diatomaceous earth filters are used. 2. Direct microscopic count: Petroff-Hausser counting chamber It also gives information about the size and morphology of the microorganisms. In the direct microscopic count, a counting chamber consisting of a ruled slide and a coverslip is employed. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. The chamber for example, has small etched squares 1/20 of a millimeter (mm) by 1/20 of an mm and is 1/50 of an mm deep. The volume of one small square therefore is 1/20,000 of a cubic mm or 1/20,000,000 of a cubic centimeter. There are 16 small squares in the large double-lined squares that are actually counted, making the volume of a large double-lined square 1/1,250,000 cc. The normal procedure is to count the number of bacteria in five large double-lined squares and divide by five to get the average number of bacteria per large square. This number is then multiplied by 1,250,000 since the square holds a volume of 1/1,250,000 cc, to find the total number of organisms per cc in the original sample.