Microbial Growth

1. Microbial Growth Chapter 6 Dr. G. López-de-Victoria

2. Growth Requirements • Physical • Temperature • Optimum growth temperature- temperature at which the organism grows its best • Psychrophiles- cold loving- range from –10 ºC to < 20 ºC; optimum 15 ºC • Mesophiles- moderate-temperature-loving- optimum 25-40 ºC; most common • Thermophiles- heat-loving- optimum 50-60 ºC • Psychotrophs- range from 0 ºC to < 40 ºC; responsible for food spoilage GLV

3. Growth Requirements (cont.) • pH- most bacteria grow best between pH 6.5 and 7.5; fungi prefer more acidic environments, optimum pH, 5-6 • Acidophiles- acid loving; can live as low as pH 1, e.g. coal mines • Osmotic pressure • Halophiles- salt-loving- some can grow in as much as 30% NaCl GLV

4. Fig. 6.1 Typical growth rates… GLV

5. Fig. 6.2 Food Spoilage Temperature GLV

6. Fig. 6.3 The effect of the amount of food on its cooling rate… GLV

7. Fig. 6.4 Plasmolysis GLV

8. Chemical • Water • Carbon sources • Heterotrophs-organics • Autotrophs- CO2 • Sources of • Nitrogen- amino acids, nucleic acids • Sulfur- amino acids, vitamins • Phosphorous- ATP GLV

9. Chemical (cont.) • Trace elements- cofactors; iron, copper, zinc • Oxygen- Obligate aerobes • Anaerobes • Facultative anaerobes- use oxygen but can grow without it by fermentation or anaerobic respiration; have catalase and SOD, e.g. E. coli • Obligate anaerobes- lack enzymes to neutralize toxic forms of oxygen • Aerotolerant anaerobes- tolerates oxygen; SOD • Microaerophilic aerobes- low oxygen concentration • Enzymes- • Organic growth factors- e.g. vitamins, purines GLV

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11. Enzymes • Superoxide dismutase- SOD- convert the superoxide free radical into molecular oxygen and hydrogen peroxide • Catalase- converts hydrogen peroxide into water and oxygen • Peroxidase- breaks down hydrogen peroxide into water GLV

12. Culture Media • Types • Agar- solid or semisolid • Plates, test tubes (deep or slanted) • Broth- liquid- NO agar • Chemically defined media- media whose exact composition is known • Useful for “fastidious” organisms • Complex media- nutrients derived from a variety of extracts; can vary slightly from one batch to the next • Nutrient broth- NB • Nutrient agar- NA GLV

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16. Anaerobic Growth Media • Reducing media- contains ingredients such as sodium thioglycolate to help deplete the oxygen from the culture medium • Anaerobe jar- contains chemicals in a packet that generate hydrogen and carbon dioxide; when mixed with water the hydrogen then reacts with the oxygen present in the air and forms water, thus removing oxygen from the jar • Anaerobe chamber- equipped with air locks and gases; used when handling a lot of cultures GLV

17. Fig. 6.5 Anaerobic jar GLV

18. Fig. 6.6 An anaerobic chamber GLV

19. Special Culture Techniques • Capnophiles- microorganisms that grow better at high CO2 concentrations • Carbon dioxide incubators • Candle jar GLV

20. Fig. 6.7b CO2 generating packet GLV

21. Fig. 6.7a Candle Jar GLV

22. Selective Media- used to suppress the growth of unwanted bacteria and encourage the growth of the desired organism • Bismuth sulfite agar • Inhibits Gram+ and most G- bacteria • Used to isolate Salmonella typhi • Sabouraud glucose agar • has a pH of 5.6 which inhibits most bacteria • used to isolate fungi • Brilliant Green Agar • inhibits G+ • Used to isolate Salmonella spp. GLV

23. Differential Media • Medium that makes it easier to distinguish colonies of the desired organism • Blood agar • used to identify organisms that lyse RBCs • E.g. Streptococcus pyogenes GLV

24. Fig. 6.8 Blood agar, a differential medium containing RBCs. GLV

25. Selective and Differential Media • Mannitol salt agar (MSA) • contains 7.5% NaCl- inhibitory • contains mannitol- carbon source • contains pH indicator that changes color if acid is produced from the fermentation of mannitol • organisms that can tolerate high concentrations of salt (selective component) and ferment mannitol (differential component) are likely to be Staphylococcus aureus GLV

26. MacConkey agar • Contains bile salts and crystal violet which inhibits G+ bacteria (selective) • Contains lactose (differential) • lactose fermenters appear as pink colonies; e.g. E. coli • non-lactose fermenters appear as colorless colonies GLV

27. Fig. 6.9 Bacterial colonies on several different media. GLV

28. Enrichment Media • Provides nutrients and environmental conditions that favor the growth of a particular microorganism and not others; encourages the growth of all bacteria present in a sample GLV

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30. Cultures • Pure cultures • Colony- arises from a single cell or groups of cells • Streak Plate method- used to isolate colonies • Preservation • Deep-freezing- liquid suspension frozen –50 ºC to –70 ºC; lasts for years • Lyophilization- freeze-drying- suspension is quickly frozen and then sublimated- powder like product lasts for years and the suspension can be hydrated to “revive” the microorganism • Growth • Bacterial division- binary fission • Generation time- the time required for a cell to divide • Logarithmic growth GLV

31. Fig. 6.10 Streak Plate Method GLV

32. Fig. 6.11 Binary Fission in Bacteria GLV

33. Fig. 6.12 Cell Division GLV

34. Growth Curve • Lag phase- period of little or no division • Log phase • logarithmic increase in the number of bacteria; exponential • Stationary phase • The number of bacteria being produced equals the number of bacteria dying • Death or decline phase • Logarithmic decrease in the number of bacteria GLV

35. Fig. 6.14 Bacterial Growth Curve GLV

36. Measurement of Growth • Plate counts- measures viable cells; CFU- colony forming units • Serial dilutions- uses diluted samples • Pour plates and spread plates- • Filtration- used for samples with low microbial numbers; filter concentrates the microorganism • Most Probable Number (MPN) • Direct microscopic counts- direct enumeration of the cells GLV

37. Fig. 6.15 Plate counts and serial dilutions GLV

38. Fig. 6.16 Methods for Plate counts GLV

39. Fig. 6.17 Counting bacteria by filtration. GLV

40. Fig. 6.18 MPN Method GLV

41. Fig. 6.19 Direct microscopic counts GLV

42. Estimating Bacterial Numbers by Indirect Methods • Turbidity • Metabolic activity • Dry weight • Urine culture colony estimates’ • 10,000 or below- contamination • 10,000-100,000- not conclusive • > 100,000 - infection GLV

43. Fig. 6.20 Turbidity estimation of bacterial numbers. GLV