Measurement of microbial growth

Topic includes various techniques of microbial growth Measurements

1. Measurement of Microbial Growth Dr. Kalavati Prajapati

2. Measurement of Microbial Growth Measurement of Cell count Measurement of Viable cell Measurement of Cell Mass Measurement of Cell constituents

3. A. Measurement of Cell count: To determine microbial numbers is through direct counting. Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. Petroff-Hausser counting chambers can be used for counting procaryotes Hemocytometers can be used for both procaryotes and eucaryotes .

4. 1. The Petroff-Hausser Counting Chamber.(Direct microscopic count) 400 to500 magnification. The average number of bacteria in these squares is used to calculate the concentration of cells in the original sample. Since there are 25 squares covering an area of 1 mm 2 , the total number of bacteria in 1 mm 2 of the chamber is (number/square) (25 squares). The chamber is 0.02 mm deep and therefore, bacteria/mm 3 = (bacteria/square) (25squares) (50).

5. 2.Electronic counters(Coulter Counter) Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted An electrical current flows through the hole, and electrodes placed on both sides of the orifice measure its electrical resistance. Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted. Limitation : counts of all cells, whether alive or dead. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

6. Culture sample Capillary tube Light beam Photodetector Digital Readout Electronoic Cell Counter

7. 3.Dry slide/ direct microscopic count Preapare a smear of bacterial culture to study Stained with suitable dye Used to quick checkof milk as it enters the dairy to determine the level of contamination before pasteurization.

8. 1 . The Membrane Filtration Procedure. Usually the counts obtained with this approach are much higher than those from culture techniques because some of the bacteria are dead. Sample is first filtered through a black polycarbonate membrane filter to provide a good background for observing fluorescent objects. The bacteria then are stained with a fluorescent dye such as acridine orange or DAPI and observed microscopically. Acridine orange–stained microorganisms glow orange or green and are easily counted with an epifluorescence microscope B.Measurement of Viable cell

9. Filtration Direct Measurements of Microbial Growth Figure 6.17a, b

10. 2.Spread-plate and pour-plate techniques(Viable cell count ) Plating techniques are simple, sensitive, and widely used for viable counts of bacteria and other microorganisms in samples of food, water, and soil. it is not possible to be absolutely certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU) rather than the number of microorganisms. Limitations: Suitability of medium Suitability of incubations conditions. Hot agar used in the pour-plate technique may injure or kill sensitive cells.

11. Plate Counts: Perform serial dilutions of a sample Figure 6.15, top portion

12. Inoculate Petri plates from serial dilutions

13. After incubation, count colonies on plates that have 25-250 colonies (CFUs) Plate Count

14. Multiple tube MPN test Count positive tubes and compare to statistical MPN table . 3. Direct Measurements of Microbial Growth (MPN)

15. B. Measurement of Cell Mass 1. Dry Weight Estimation (Gravimetric method): The most direct approach is the determination of microbial dry weight. Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity (mg dry wt /ml).

16. 2.Turbidometric and spectrophotometric method method: Determination of microbial mass by measurement of light absorption. As the population and turbidity increase, more light is scattered and the absorbance reading given by the spectrophotometer increases. Advantage: More rapid, sensitive techniques depend on the fact that microbial cells scatter light striking them. Because 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

17. C. Measurement of cell constituents: 1.Determination of N-contents. Major constituents is protein in cell. One can also measure a bacterial population or cell crop in terms of bacterial nitrogen. From total cell dry weight 14% is N. For the N measurement first harvest the cells then wash them with free of medium and perform a quantitative chemical analysis of N.(mg N/ ml). It is only applicable for concentrated population.

18. 2.Measurement of a specific chemical change produced on a constituent of the medium ( Biochemical activity). Glucose fermentation in medium. The cell concentration is measured from acid produced by culture.( miliequivalent acid produced/ ml culture). Other biochemical reactions also used for measurement of growth at particular phase.

19. 3. ATP bioluminometry ATP is rapidly lost from dead cells. It is useful to determine viable cell. This techniques utilize enzyme-substrate complex. Luciferase enzyme obtained from firefly Photinus pyralis converts Luciferin to oxyluciferin by bioluminometer . ATP+O 2 +luciferin Luciferase Oxyluciferin+AMP+CO 2 +PPi+ Photon of light (562 nm) Very rapid and sensitive under optimum conditions as little as 10 -15 mol of ATP can be detected.

20. 4. Online estimation It provide real time estimation of biomass, and minimizes the requirements for repeated sampling and off-line estimation. O.D. based or capacitance based probe, Carbon dioxide evolution estimation, Oxygen utilization estimation

21. 1 . Which of the following is used to grow bacterial cultures continuously? Chemostat Coulter Counter Hemostat Petroff-Hausser chamber 2. Which of the following techniques would be best to use to determine the number of living cells in a culture? Viable plate count Coulter counter Microscopic count Most Probable Number Multiple Choice Questions

22. 3.Which instrument is used for counting procaryotes Chemostat Coulter Counter Hemostat Petroff-Hausser chamber 4. Which enzyme is used in ATP bioluminometry method Luciferase Peroxidase Oxy luciferase None of these

23. 5.Electronic counter method is not highly used because… Counts all cells Count dead cell debris Counts alive or dead. All of these 6.Which techniquie ids used for viable count of bacterial cells Membrane filtration Plate method a and b None of these

24. 7. All of the following are drawbacks to the use of spectrophotometry in estimating microbial growth EXCEPT:    It cannot distinguish between live and dead cells.   It can not be used if there are less than one million cells per milliliter.    It will underestimate the true number of cells if they are not uniformly in suspension.    It kills the organisms as they are being counted. 8. Full form of CFUs Colony formal unit Colony forming unit Counting forming Unit None of these

25. 9 . Turbididmetric measurements of bacterial population size are usually made with_________ Peter hoff hauser chamber Spectrophotometer Centrifuge Dry slide technique 10. ___________are usedf to maintain culture in exponential phase in industrial ferementation . Chemostat e Turbidostate Diauxic growth a and b

26. References Prescott, L.M., Harley, J.P., Klein, D.A. (2002). Microbiology . Fifth Edition. Wm. C. Brown Pub. Dubuque, Iowa. pp. 112-125. Pelczar , M.J., Chan, E.C.S., Krieg, N.R.(1993) Introduction to Microbiology . Fifth Edition. Tata Mc- Graw – Hill Edition.pp-119-132 Ingraham , J.L. and Ingraham C. A(2008) Introduction to Microbiology. Third Edition Modi H.A. (2014) A handbook of Elementary Microbiology . Shanti Prakashan . pp-203-216