Characteristics and study of prokaryotic growth How do we grow bacteria in the laboratory? What is required for growth?

1. Characteristics and study of prokaryotic growth How do we grow bacteria in the laboratory? What is required for growth? How do we measure bacterial growth? What do bacteria do while they are growing? How do bacteria grow in the environment?

2. What is a pure culture? All of the organisms are descended from a single cell (and therefore are identical) Bacteria do not grow that way naturally Most bacteria cannot be cultured that way So why do it?

3. Historically (19th century): Koch’s postulates –applied to prove that a particular organism caused disease -isolate organism from diseased animal -establish a pure culture of the organism (and prove it) - infect another animal with cultured bacteria does this animal get sick? are the same microbes isolated from the second animal? Koch proved that B.anthracis causes anthrax and that M. tuberculosis causes tuberculosis this way Koch also developed new methods for bacterial growth

4. What is required for pure culture? Sterile apparatus Aseptic technique Appropriate media solid media: separable colonies

5. Bacteria “grow” (i.e, divide) by binary fission With each generation the population doubles “generation time,” or doubling time, varies with bacterium and growing conditions Can be as little as 20 minutes Thus bacterial contamination can be rapidly established

7. What sorts of factors affect bacterial growth? temperature

8. How can they grow at such a wide range of temperatures? Implications substances that function at extreme temperatures for prevention of contamination for pathogenesis (why do we develop fevers?)

9. Oxygen requirements Availability varies in the environment and on/in our bodies

10. pH Bacteria maintain an internal neutral pH, but may survive in a wide range of acidic or alkaline environments How? H. pylori- produce urease; generates ammonia and raises pH of immediate environment Other bacteria use proton pumps acidophiles- out alkalophiles- in

11. Salt tolerance Synthesize or pump solutes inside the cell (and draw water) Osmotolerant organism- can grow in dry environments (like skin) Halophiles- require high salt conditions High solutes concentrations are historically used to preserve foods

12. What do bacteria require to grow? Nutrients Energy source Some organisms require growth factors (fastidious) Which is easiest to grow in the laboratory?

14. Some media are richer than others Some are used to look at mixed cultures of microorganisms Selective- allow some organisms to grow but not others Differential- bacteria have different characteristics Some are both!

17. Adjusting atmospheric requirements CO2 levels (capnophiles) Candle jars (microaerophiles) Anaerobe jars/chambers (guess) reducing agents

18. Why would you use selective enrichment medium?

19. Measuring bacterial growth Considerations Rapidity Viable vs. total bacteria Large numbers or small numbers of bacteria Growth in progress?

20. Direct counting Rapid Can count organisms that can’t grow in medium Can’t distinguish live from dead cells Motile organisms are hard to count! Special instruments needed

21. Viable counts Of living (and growing) bacteria requires some skill and time Can be performed on special media Need fairly concentrated culture Dilute samples membrane filtration MPN (most probable number)- statistical estimate, NOT direct count

25. Measuring cell mass Most common method- turbidity (spectrophotometry) Cells grown in liquid medium Can measure “progress” of cell growth Changes (in turbidity) correlate with increase in cell numbers Growth can be measured either by turbidity or by counting to determine growth curve

26. Metabolic activities change in late log/stationary phase

27. Metabolites are often of research/commercial interest Bacteria can be grown to enhance production

28. How do bacteria actually grow in nature? Are nutrients always available, and in constant amounts? They may elaborate protective structures when necessary glycocalyx endospores biofilm (protects communities of microbes) Interactions may be required (one organism produces substances required by another