Micro-08105 3(2-1) MICROBIAL GROWTH AND REQUIREMENTS Dr. Shahzad Ali Assistant Professor

1. Micro-081053(2-1) MICROBIAL GROWTH AND REQUIREMENTS Dr. Shahzad Ali Assistant Professor Department of Wildlife and Ecology UVAS, Ravi Campus, Pattoki

2. MICROBIAL GROWTH The Growth of Bacterial Cultures [page 171(204)] • Bacterial Division • Generation Time • Phases of Growth

3. Bacterial Division • Bacterial growth refers to an increase in bacterial numbers, not an increase in the sizeof the individual cells. • Bacteria normally reproduce by binary fission (Figure 6.12 ). [page 171(204)] • A few bacterial species reproduce by budding; • they form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates. • Some filamentous bacteria (certain actinomycetes) reproduce by producing chains of conidiospores carried externally at the tips of the filaments. • A few filamentous species simply fragment, and the fragments initiate the growth of new cells.

4. Generation Time • The time required for a cell to divide (and its population to double) is called the generation time. • It varies considerably among organisms and with environmental conditions, such as temperature. • Most bacteria have a generation time of I to 3 hours; others require more than 24 hours per generation. • If binary fission continues unchecked, an enormous number of cells will be produced. • If a doubling occurred every 20 minutes- which is the case for E. coli under favorable conditions- after 20 generations a single initial cell would increase to many cells.

5. Phases of Growth • When a few bacteria are inoculated into a liquid growth medium and the population is counted at intervals, • It is possible to plot a bacterial growth curve that shows the growth of cells over time ( Figure 6.15), • There are four basic phases of growth: (173/206) • Lag PHASE • Log PHASE • Stationary PHASE • Death phase

6. Phases of Growth Lag PHASE • For a while, the number of cells changesvery little because the cells do not immediately reproduce in a new medium. • This period of little or no cell division is called the lag phase, and it can last for 1 hour or several days. • During this time, however, the cells are not dormant. • The microbial population is undergoing a period of intense metabolic activity involving, in particular, synthesis of enzymes and various molecules. • (The situation is analogous to a factory being equipped to produce automobiles; there is considerable tooling-up activity but no immediate increase in the automobile population.)

7. Phases of Growth Log PHASE • Eventually, the cells begin to divide and enter a period of growth, called the log phase, • Cellular reproduction is most active during this period, and generation time reaches a constant minimum. • Because the generation time is constant, a logarithmic plot of growth during the log phase is a straight line. • The log phase is the time when cells are most active metabolicallyand is preferred for industrial purposes where, for example, a produce needs to be produced efficiently.

8. Phases of Growth Stationary PHASE • If log growth continues unchecked, large numbers of cells could arise. • For example, a single bacterium (at a weight of9.5 X 10 - 13 g per cell ) dividing every 20 minutes for only 25.5 hours can theoretically produce a population equivalent in weight to that of an 80,000-tonaircraft carrier. • In reality, this does not happen. • Eventually, the growth rate slows, the number of microbial deathsbalances the number of new cells, and the population stabilizes. • This period of equilibrium is called the stationary phase. • What causes exponential growth to stop is not always clear. • The exhaustion of nutrients, accumulation of waste products, and harmful changes in pH may all play a role.

9. Phases of Growth Death phase • The number of deaths eventually exceeds the number of new cells formed, and the population enters the death phase, or logarithmic decline phase. • This phase continues until the population is diminished to a tiny fraction of the number of cells in the previous phase or until the population dies out entirely. • Some species pass through the entire series of phases in only a few days; others retain some surviving cells almost indefinitely

10. Direct Measurement of Microbial Growth • 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. • Population numbers are usually recorded as the number of cells in a milliliter of liquid or in a gram of solid material.

11. Direct Measurement of Microbial Growth Plate Counts • The most frequently used method of measuring bacterial populations is the plate count. • An important advantage of this method is that it measures the number of viable cells. • One disadvantage may be that it takes some time, usually 24 hours or more, for visible colonies to form. • This can be a serious problem in some applications, such as quality control of milk, when it is not possible to hold a particular lot for this length of time. • Plate counts assume that eachlive bacterium growsand divides to produce asinglecolony.

12. Direct Measurement of Microbial Growth Plate Counts • This is not always true, because bacteria frequently grow linked in chains or as dumps • Therefore, a colony often results, not from a single bacterium, but from short segments of a chainor from a bacterial clump. • To reflect this reality, plate counts are often reported as colony-forming units (CFU). • When a plate count is performed, it is important that only a limited number of colonies develop in the plate. • When too many colonies are present, some cells are overcrowded and do not develop; these conditions cause inaccuracies in the count.

13. Direct Measurement of Microbial Growth Plate Counts • The U.S. Food and Drug Administration convention is to count only plates with 25 to 250 colonies, • But many microbiologists preferplates with 30 to 300 colonies. • To ensure that some colony counts will be within this range, the original inoculum is diluted several times in a process called serial dilution (Figure 6.16) .

14. Serial Dilutions • Let's say, for example, that a milk sample has 10,000 bacteria per milliliter. • If 1 ml of this sample were plated out, there would theoretically be 10,000 colonies formed in the Petri plate of medium. Obviously, this would not produce a countable plate. • If 1 ml of this sample were transferred to a tube containing 9 ml of sterile water, each milliliter of fluid in this tube would now contain 1000 bacteria. • If 1ml of this sample were inoculated into a Petri plate, there would still be too many potential colonies to count on a plate. • Therefore, another serial dilution could be made. • One milliliter containing 1000 bacteria would be transferred to a second tube of 9 ml of water. • Each milliliter of this tube would now contain only 100 bacteria, and if • 1 ml of the contents of this tube were plated out, potentially 100 colonies would be formed- an easily countable number. (Figure 6.16-page 175 (208))

15. Pour Plates and Spread Plates A plate count is done by either • Pour plate method • Spread plate method POUR PLATE METHOD • The pour plate method follows the procedure shown in Figure 6.17a. (page 176/209) • Either 1.0 ml or 0.1 ml of dilutions of the bacterial suspension is introduced into a Petri dish. • The nutrient medium, in which the agar is kept liquid by holding it in a water bath at about 50°C, is poured over the sample, which is then mixed into the medium by gentle agitation of the plate.

16. POUR PLATE METHOD • When the agar solidifies, the plate is incubated. • With the pour plate technique, colonies will grow withinthe nutrient agar (from cells suspended in the nutrient medium as the agar solidifies) • as well as on the surface of the agar plate. DRAWBACKS • 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. • Also, when certain differential media are used, the distinctive appearance of the colony on the surface is essential for diagnostic purposes. • Colonies that form beneath the surface of a pour plate are not satisfactory for such tests.

17. Spread plate method • To avoid these problems, the spread plate method is frequently used instead (Figure 6.17b ). • A 0.1 ml inoculum is added to the surface of a pre-poured, solidified agar medium. • The inoculum is then spread uniformly over the surface of the medium with a specially shaped, sterilized glass o r metal rod. • This method positionsall the colonies on the surface and avoids contact between the cells and melted agar.