Quantitative measurement of bacterial growth(total and viable count).pptx

Detailed Knowledge and methods for the Quantitative measurement of bacterial growth(total and viable count).

1. UNIT I 1.3 Quantitative measurement of bacterial growth(total and viable count) Presented by: Mohammad Abuzar( M. Pharm ) Assistant Professor School of Pharmacy AIKTC, New Panvel .

2. CONTENTS 2

3. 3 INTRODUCTION When a few  bacteria  a re inoculated into a liquid growth medium or any solid culture media and the population is counted at intervals. The growth of microbial populations can be measured in a number of ways. Some methods measure cell numbers; other methods measure the population’s total mass, which is often directly proportional to cell numbers. It is orderly increase in a cellular constituent. When microorganisms reproduce by binary fission or budding then it also leads to increase in the number of cells.

4. 4 MEASUREMENTS OF MICROBIAL GROWTH There are various ways to measures microbial growth for the determination of growth rates and generation times. For the measurement of growth either mass or population number is followed because growth leads to increase in both. Growth can be measured by one of the following types of measurements: Cell count  this method involves the measurement of growth either by microscopy or by using an electronic particle counter or indirectly by a colony count. Cell mass  in this growth can be measured directly by weighing or by a measurement of nitrogen concentration in cells or indirectly by the determination of turbidity using spectrophotometer. Cell activity  in this growth can be measured indirectly by analysis of the degree of biochemical activity to the size of population.

5. 5 SOME SPECIFIC PROCEDURE WILL ILLUSTRATE THE APPLICATION OF EACH TYPE OF MEASUREMENT Direct microscopic count Electronic enumeration of cell numbers The plate count method Turbidity estimation of bacterial numbers Determination of nitrogen content Determination of dry weight of cells Filtration method Most Probable Number (MPN) Method

6. Measurement of Microbial Growth Total count Direct Method Measurement of cell numbers by direct counting using a counting chamber Easy, inexpensive, and relatively quick Gives information about the size and morphology of microorganisms Petroff- Hausser counting chamber – counting procaryotes Hemocytometers can be used for both procaryotes and eucaryotes Procaryotes are more easily counted in these chambers if they are stained, or when a phase-contrast or a fluorescence microscope Disadvantages of counting chamber The microbial population must be fairly large for accuracy because such a small volume is sampled It is also difficult to distinguish between living and dead cells in counting chambers without special techniques.

7. 7 These specially designed slides have chambers of known depth with an etched grid on the chamber bottom. The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and any sample dilutions required

8. 8 Coulter Counter Larger microorganisms such as protozoa, algae, and non-filamentous yeasts The microbial suspension is forced through a small hole or orifice. 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. Coulter Counter – Advantages and disadvantages The Coulter Counter gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

9. 9 Membrane filtration The sample is 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

10. 10 Viable count Colony counting using streak plate or spread plate technique This method allows the determination of the number of cells that will multiply under certain defined conditions. Plate count method can be done in two ways either by spread plate method or by pour plate method. This method of bacterial counting is most commonly used with satisfactory results for the estimation of bacterial populations in  milk , water, foods and many other materials. This technique has some drawbacks because some relatively heat-sensitive microorganisms may be damaged by the melted agar and will therefore be unable to form colonies

11. 11 Membrane filtration technique Counts of colonies growing on special membrane filters having pores small enough to trap bacteria Sample is drawn through a special membrane filter The filter is then placed on an agar medium or on a pad soaked with liquid media Incubated until each cell forms a separate colony colony count gives the number of microorganisms in the filtered sample

12. 12 Measurement of cell mass Increases in the total cell mass, as well as in cell numbers, accompany population growth Therefore techniques for measuring changes in cell mass can be used in following growth Determination of microbial dry weight Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an advantageous technique for measuring the growth of fungi. Time-consuming, however, and not very sensitive Turbidity and Microbial Mass Measurement 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.

13. 13

14. 14 Determination of Nitrogen Content Cell material is mostly composed of proteins, and nitrogen is a characteristic component of proteins. Our ability to describe bacterial populations or cell crops can be determined by measuring bacterial nitrogen. Firstly, cells are harvested and washed free of medium, after which nitrogen is determined by a quantitative chemical analysis.

15. 15 Most Probable Number (MPN) Method MPN (most probable number) is another method of determining bacteria numbers in a sample. Statistical estimating techniques rely on the principle that the greater the number of bacteria contained in a sample, the more dilution is needed in order to reduce that number to the point that no bacteria remain in the tubes after dilution. If the microbes on which the count is being performed are not suitable for solid media (such as chemoautotrophic nitrifying bacteria), then the MPN method is useful. It is also helpful for identifying microbes in liquid differential media using the growth of bacteria (such as lactose-fermenting coliform bacteria, for example). A MPN is just a statement that there’s a 95% chance that bacterial populations fall within a certain range and that it represents the most likely scenario

16. 16

17. 17 Factors to Consider 1. Viability: Viable count methods provide information about living, reproducing cells. Distinguishes between living and dead cells, providing a more accurate representation of the growth potential. 2. Selective Media: Using selective media helps in counting specific bacterial groups or species. Allows for a more targeted assessment of microbial populations. 3. Sampling Technique: Proper sampling is crucial to obtaining representative counts. Aseptic techniques must be followed to avoid contamination. 4. Incubation Conditions: Incubation conditions (temperature, atmosphere) should mimic the natural environment of the bacteria being studied. 5. Equipment Calibration: Equipment such as pipettes and dilution devices must be calibrated for accuracy.

18. 18 Total count : counting the living and dead cells Viable count : counting only the living cells that are capable of multiplication Total count is determined by counting chambers, coulter counter, cell mass determination, turbidity measurement, membrane filtration Viable count can be determined by plate count and membrane filtration Summary

19. 19 W.B. Hugo and A.D. Russel: Pharmaceutical Microbiology, Blackwell Scientific publications, Oxford London. Prescott and Dunn., Industrial Microbiology, 4th edition, CBS Publishers & Distributors, Delhi. Pelczar , Chan Kreig , Microbiology, Tata McGraw Hill edn . Malcolm Harris, Balliere Tindall and Cox: Pharmaceutical Microbiology. Rose: Industrial Microbiology. Probisher , Hinsdill et al: Fundamentals of Microbiology, 9th ed. Japan Cooper and Gunn’s: Tutorial Pharmacy, CBS Publisher and Distribution. Peppler : Microbial Technology. I.P., B.P., U.S.P.- latest editions. Ananthnarayan : Text Book of Microbiology, Orient-Longman, Chennai Edward: Fundamentals of Microbiology. 12. N.K.Jain : Pharmaceutical Microbiology, Vallabh Prakashan , Delhi REFERENCES

20. 20