MEASURMENTS OF BACTERIAL GROWTH

GROWTH OF BACTERIA CANNOT BE MEASURED DIRECTLY BY SEEING THEM AS THEY ARE MICROSCOPIC STRUCTURES THEREFORE WE HAVE TO USE SEVERAL METHODS WHICH ARE DESCRIBED IN THIS PRESENTATION

1. MEASURMENTS OF BACTERIAL GROWTH

2. INTRODUCTION Growth in a biological system is an orderly increase in the quantity of cellular constituents and which depends upon the ability of the cell to new protoplasm from nutrients available. But, in bacteria growth involves increase in cell mass and no. of ribosomes ,duplication of bacterial chromosomes,septum formation and cell divisions. Method of growth measurements involve both direct and indirect techniques:- [A] Direct method [B] Indirect methods

3. DIRECT METHODS Cell count Haemocytometer It is a simplest, most convenient and cheapest method of determining the no. of cell in a sample is to use a haemocytometer and microscope. It is a special slide that has counting chambers with a known volume of liquid. The engraved grid on the surface of counting chambers ensures the no. of particles in a defined volume of liquid. The haemocytometer is placed on the microscope stage and cell suspension is counted.

4. Viable plate count Viable plate count is used to estimate the no. of viable cells that are present in a sample. This method relies on bacteria growing a colony on a nutrient medium. The colony become visible to the naked eye and the no. of colonies on a plate can be counted. To be effective , on average between 30 and 300 colonies of the target bacterium are grown.

5. Coulter count Coulter counter is an apparatus for counting and sizing particles and cells. A typical coulter counter has one or more microchannels that separate two chambers containing electrolyte solutions. When a particle flows through one of the microchannels it results in the electrical resistance change of the liquid filled microchannels. This resistance change can be recorded as electric current.

6. Membrane filter count Using the membrane filter technique, sample is passed through the membrane using a filter funnel and vaccum system. Any organism in sample is concentrated on the surface of the membrane. The membrane is then placed in a special plate with appropriate medium. The passage of nutrients through the filter facilitates the growth of microorganisms in the form of colonies on the upper surface of membrane. Now, the discrete colonies can easily be counted.

7. Direct microscopic count In the direct microscopic count, a counting chamber consists of ruled slide and coverslip. Number of bacteria in small known volume is directly counted microscopically and the no. of bacteria in original sample is determined by extrapolation. Petroff-Hausser counting chamber is commonly used in dairy industry.

8. 2. Cell mass Wet weight A known volume of culture sample is centrifuged, then , the wet weight of pellet is measured by using pre weighed filter paper . Dry weight A known volume of culture sample is centrifuged , pellet is dried by removing water and then dry weight of pellet is measured by using pre weighed filter paper.

9. Total nitrogen content Major constituent of cell material is protein and since nitrogen is a characteristic part of protein , bacterial population can be measured in terms of bacterial nitrogen. Bacteria average approx. 14% N2 on a day weight basis . To measure growth by this technique, first harvest the cells and wash them free of medium and perform a quantitative chemical analysis of nitrogen.

10. Turbidity When bacteria growing in a liquid medium are mixed , the culture appears turbid. This is because a bacterial culture act as a colloidal suspension that block and reflects light passing through culture. Light absorbed by the bacterial suspension will be directly proportional to the concentration of the cells in the culture. By measuring the amount of light absorbed one can estimate the number of bacteria present. The instrument used to measure turbidity is spectrophotometer.

11. INDIRECT METHODS The bacterial growth can be indirectly estimated by detecting specific changes caused in growth medium as a result of activity and multiplication of bacterial cells. It includes detecting activity cell products such as acid and gas production.

12. OXYGEN CONSUMPTION The dye reduction tests such as methylene blue and resazurin reduction tests is based on the fact that the color imparted to milk by the addition of a dye such as methylene blue will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism cause the color to disappear. The agencies responsible for the oxygen consumption are the bacteria. Thus, the time of reduction is taken as a measure of the number of organisms in milk although actually it is likely that it is more truly a measure of the total metabolic reactions proceeding at the cell surface of the bacteria.

13. CO2 PRODUCTION Gas production by bacteria is another major activity which can be taken up as an index of bacterial growth. Detection of gas production using Durham tube and change in color of the growth medium due to reduction of pH sensitive ingredients present in medium are commonly used for detection of acid and gas producing coliforms and yeasts. 

14. REFERENCES 1. http://ecoursesonline.iasri.res.in/mod/page/view.php?id=5205 2. http://loretocollegebiology.weebly.com/measuring-bacterial-growth.html 3. https://nptel.ac.in/courses/102103015/12

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