Quantitative Techniques in Microbiology

1. Quantitative Techniques in Microbiology Dr Paul D. Brown paul.brown@uwimona.edu.jm BC10M: Introductory Biochemistry

2. Quantitative Techniques • Measurement of cell numbers/cell mass • Spread and pour plate methods for viable cell counts

3. Bacterial Growth • Orderly increase in the quantity of cellular constituents • Depends on cell’s ability to form new protoplasm from nutrients available in the environment • In most bacteria, • involves increase in cell mass and number of ribosomes • duplication of the bacterial chromosome • synthesis of new cell wall and plasma membrane • partitioning of the two chromosomes • septum formation, and cell division. • This asexual process of reproduction is called binary fission.

4. Bacterial Growth • Two aspects to growth • Population growth • Individual cell growth • Cells have a growth pattern which leads to a doubling in their number at division, and this determines the pattern of culture growth. But the two growth aspects are not necessarily the same.

5. Exponential growth results from binary fission

6. Measuring cell growth • Cell mass • Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation • Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content • Indirect measurement of chemical activity such as rate of O2 (or CO2) production or consumption, etc. • Turbidity (optical density) measurement (directly related to cell mass or number; easy but not sensitive to small numbers)

7. Examples of growth curves measured by light scattering and the problem with the method at high cell densities

8. Turbidity measurement

9. Turbidity

10. Counting Chamber

11. Measuring cell growth • Cell number • Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones. Need dense (>107 cells per ml) suspensions • Electronic counting chambers count numbers and measure size distribution of cells. • Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface. Gives the number of viable bacteria (CFU) in the sample.

12. Method Application Comments Direct microscopic count Enumeration of bacteria in milk or cellular vaccines Cannot distinguish living from nonliving cells Viable cell count (colony counts) Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc. Very sensitive if plating conditions are optimal Turbidity measurement Estimations of large numbers of bacteria in clear liquid media and broths Fast and nondestructive, but cannot detect cell densities less than 107 cells per ml Measurement of total N or protein Measurement of total cell yield from very dense cultures  Only practical application is in the research laboratory Measurement of biochemical activity e.g. O2 uptake, CO2 production, ATP production, etc. Microbiological assays Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers Measurement of dry weight or wet weight of cells or volume of cells after centrifugation Measurement of total cell yield in cultures  Probably more sensitive than total N or total protein measurements Some methods used to measure bacterial growth

13. Cell counting • Plate counts • Measures viable cells; each viable cell will produce a single countable colony • May have to dilute cells to get good number on plate (range: 30-300) • 100-1000 colonies are possible on a plate • Serial dilution and plating • Colony forms in local region on agar plate • Agar not metabolized • Agar melts at 90˚C but does not resolidify until below 45˚C.

14. Serial dilution and plating

15. Nutritional Factors • Required elements: • Major elements (autotrophs vs. heterotrophs) • Trace elements • Growth factor requirements vary greatly (from versatile to fastidious organisms • Energy sources: phototrophs vs. chemotrophs • Great nutritional diversity allows prokaryotes to live just about anywhere • Photautotrophs • Chemolithoautotrophs • Photoheterotrophs • chemoorganoheterotrops