Miniexam : Bacterial nutrition & factors that effect bacterial growth.

1. Miniexam: Bacterial nutrition & factors that effect bacterial growth. A. Intro all bacteria (and all other living things) require certain nutrients and environmental conditions for growth and replication.

2. B. Nutrients required for growth of bacteria 1. Energy sources (external) a. Organic or inorganic chemicals or visible light b. uses: required for biosynthesis or ATP production 2. C source (external) a. organic chemicals (carbos, lipids, proteins, etc.) or an inorganic source (CO2) b. uses: required for biosynthesis or ATP production

3. B. Nutrients required for growth of bacteria Classification of life, based on C metabolism C source Energy source

4. B. Nutrients required for growth of bacteria (cont) 3. Nitrogen source (external) a. Organic or inorganic chemicals b. uses: required for biosynthesis of aa and proteins, nucleic acids, ATP and other biochems 4. Sulfur source (external) a. organic chemicals or inorganic sources b. uses: required for biosynthesis of certain aa’s, others

5. B. Nutrients required for growth of bacteria (cont) 5. Phosphorous source (external) a. inorganic PO4= b. uses: required for biosynthesis of nucleic acids, ATP and P-lipids

6. B. Nutrients (cont) 6. source (external) of metallic ions a. inorganic b. uses 1) trace elements (e.g. Fe, Mg, Cu, Mn, Co, etc.) are used in enzyme prosthetic groups 2) trace elements are often parts of vitamins, which are often enzyme cofactors 3) Ca is used to stabilize the CW 4) Na, K, Cl are used as part of the osmoticum

7. B. Nutrients, etc (cont) 7. H2O uses a) medium in which all biochemical reactions take place. b) hydration of nearly all biochemicals c) part of the osmoticum d) solution in which nearly all of the transport within the cell takes place

8. B. Nutrients, etc (cont) 8. Variety of growth factors a. examples: preformed aa’s, vitamins b. reasons that growth factors are required by some mo’s 1) some mo’s lose ability to make certain chems through mutation and that chem now must be provided by the growth medium. 2) fastidious mo’s, through long association with a host, have lost the ability to make certain chem that are provided by the host.

9. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth a. intro 1) The environment effects growth of all organisms 2) Understanding how the environment effects growth can lead to methods for controlling bacterial growth.

10. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) b. temperature 1) all organisms have temps that are optimal, but also minimum and maximum temp for survival as well.

11. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) b. temperature (cont) 2) organisms can be classified by their temp requirements a) psychrophiles: -10 to +20°C b) psychrotrophs: 0 to +30°C c) mesophiles: 10 to 50°C d) thermophiles: 40 to 70°C

12. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) b. temperature (cont) 3) how temp affects organisms a) all biochemical reactions are controlled by enzymes b) temp affects enzymes: enzymes are denatured by temps that are too hot or cold. c) non-optimal temps slow or stop enzyme activity, causing the death of the organism. d) non-optimal temps can also cause CM instability.

13. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) b. temperature (cont) 4) How do extremophiles survive extreme temps? a) their enzymes are adapted to the extremes. e.g. the enzymes of a thermophile will operate well at 60-70°C but not at 37°C b) thermophiles are restricted to extreme temp environments

14. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) c. pH 1) review of pH pH = - log [H+] (therefore each full number change is a change of 10x) pH 7 is neutral pH > 7 is basic pH < 7 is acidic

15. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) c. pH (cont) 2) all organisms have a pH which is optimal for growth (and other pH’s are not suited for their growth) •acidophiles that live at acid pH’s (mine runoff and hot spring bacteria, many fungi) •basophiles that live at basic pH’s •most organisms are happy at neutral pH’s (acidic solutions are used to kill most mo’s)

16. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) d. mo’s and osmosis 1) all cells contain solutes such as ions and proteins 2) the concentration of the solutes in a cell is important as they must be balanced with the surrounding environment for osmotic stability. 3) Def. Osmosis: Diffusion of water across a membrane from an area of low [solute] to an area of high [solute]

17. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) d. mo’s and osmosis (cont) isotonic hypertonic hypotonic

18. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) d. mo’s and osmosis (cont) 4) Halophile bacteria are adapted for growth in saline environments 5) How can halophiles grow in saline environments? 6) Can salt be used to preserve food?

19. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) e. mo’s and oxygen 1) mo’s are often grouped by their ability to use or tolerate oxygen. a) aerobes: use ox. resp. only and require oxygen b)anaerobes: use fermentation only and often can not tolerate oxygen c) facultative organisms: use ox. resp. if oxygen is available or fermentation without oxygen d) others

20. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) e. mo’s and oxygen (cont) 2) oxygen can be a problem for organisms of many types a) during ox. resp., reactive oxygen species (ROS) are generated (e.g. H2O2, OH• (hydroxyl radical), singlet O, O2- (superoxide) b) ROS can tear up CM and enzymes, killing the mo c) therefore all respiring organisms must inactivate ROS

21. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) e. mo’s and oxygen (cont) 3) How do respirers rid themselves of ROS? peroxidase a) H2O2 H2O + O2 catalase b) OH• : Vitamin D? superoxide dismutase c) O2-  H2O2 + O2

22. B. Nutrients, etc (cont) 9. Variety of physical and environmental factors that effect bacterial growth (cont) e. mo’s and oxygen (cont) 4) How mo’s should theoretically growing in broth based on their oxygen requirements/tolerance? Facultative Anaerobe (strict) Anaerobe (e.g. E. coli) (e.g. Clostridium) (aerotolerant) (e.g. some Strept.) Aerobe (strict) Microaerophile (e.g. Pseudomonas) (e.g. Neisseria)

23. 9. Variety of physical and environmental factors that effect bacterial growth (cont) e. mo’s and oxygen (cont) 5) methods for growing anaerobes will be covered in the lab.

24. C. Bacterial Growth 1. Intro a. Importance of understanding cell growth 1) Cells must make copies of themselves for reproduction. 2) Genetically engineered cells can produce desired products. Optimizing growth of the organism increases yield of the product. 3) Control of growth requires an understanding of growth.

25. C. Bacterial Growth 1. Intro (cont) b. Def of Growth: increase in cell number or cell mass 2. Cell Growth a. What does a cell do when it grows? 1) cells are biosynthetic machines: cells make nearly all the compounds necessary for growth.

26. C. Bacterial Growth 2. Cell Growth (cont) a. What does a cell do when it grows? (cont) 2) Cells make or absorb small molecules (e.g. aa’s, monosaccharides, nucleotides) and link them together in polymers (e.g. proteins, polysacc. and nucleic acids) 3) Then cells assemble polymers into new cell components and structures (e.g. ribosomes, chromosomes, peptidoglycan CW). a) assembly is usually directed by enzymes. b) few components self-assemble (e.g. CM)

27. C. Bacterial Growth 2. Cell Growth (cont) a. What does a cell do when it grows? (cont) 4) Then the cell divides into 2 by Binary Fission a) cell grows to about 2x normal length b) while the cell elongates, the DNA replicates c) CM invaginates d) cross-wall forms, splitting the cell into 2

28. C. Bacterial Growth 3. Growth of a population of bacterial cells (i.e. an increase of the number of cells) a. binary fission leads to this: 1 cell  2 cells  4 cells  8 cells, etc. b. definitions 1) generation time or gt (AKA doubling time): time required for cell number to double gt varies among organisms E coli: 15 to 20 min. Mycobacterium: 24hrs 2) growth rate: increase in cell number or mass per unit of time.

29. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations 1) build up numbers quickly because the growth rate is exponential (or logarithmic) Time (hrs)* Number of mo’s *gt= 30min. 0 1 0.5 2 1 4 2 16 3 64 4 256 5 1024 10 1,048,576

30. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 2) Typical growth curve for bacteria

31. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 3) Phases of growth a) lag phase: cell #’s increase slowly (1) cells are adapting to new medium (need time to express new genes and make new proteins) (2) cellular repairs are made (3) start with low #’s (4) doubling of low #’s = slow increase in #’s

32. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 3) Phases of growth (cont) b) log or exponential phase (1) cells are in optimal condition (2) #’s have increased to the level that doubling increases #’s significantly (3) ample nutrients

33. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 3) Phases of growth (cont) c) stationary phase (1) cell #’s stable (2) cell “births” = cell deaths (3) nutrients starting to run out and/or wastes building up

34. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 3) Phases of growth (cont) d) exponential death phase (1) cell #’s decrease exponentially (2) many cell deaths, few cell “births” (3) nutrients have run out and wastes levels are toxic

35. C. Bacterial Growth 3. Growth of a population of bacterial cells (cont) c. Characteristics of growth of cell populations (cont) 4) What if cells continued log phase without going to stationary phase (i.e. nutrients don’t run out)? E coli with a gt of 20min., starting with 1 cell (=1x10-12 gm.), growing for 2 days, would make a mass of cells equal to 4000x weight of the earth

36. C. Bacterial Growth 4. Methods for counting cells a. Petroff-Hauser chamber (direct, combined live and dead count)

37. C. Bacterial Growth 4. Methods for counting cells (cont) b. Coulter Counter™ (direct, combined count) note: can be adapted to count any small particle cells laser

38. C. Bacterial Growth 4. Methods for counting cells (cont) c. Spectrometry (indirect, combined count) note: can be adapted to count any small particle or colored liquid

39. C. Bacterial Growth 4. Methods for counting cells (cont) d. Standard Plate Count (direct, live count)

40. C. Bacterial Growth 4. Methods for counting cells (cont) e. MPN and others

41. D. Bacterial Media: How do you provide bacteria with what they need to grow? 1. Intro a. Do you try to exactly duplicate the natural environment from which the mo comes? b. Use a medium which provides the minimum nutritional requirements for that organism (C, energy source, N, P and sometimes there are many more required compounds).

42. D. Bacterial Media (cont) 2. There are 2 basic types of Media (p.), Medium (s.) a. Undefined (complex) media: contents are not known exactly 1) advantages a) broad applications b) easy to make c) cheap d) often support good growth 2) disadvantage: don’t know exact composition

43. D. Bacterial Media (cont) 2. There are 2 basic types of Media (cont) a. Undefined (complex) media (cont) 3) contents: crude digests of nutritious substances e.g. milk, yeast, beef heart 4) example of these media: Tryptic soy agar (TSA), Blood agar, Beef Heart infusion

44. D. Bacterial Media (cont) 2. There are 2 basic types of Media (cont) b. Defined (synthetic) media: contents are known exactly 1) advantages a) contents known b) can change composition c) free of contaminants (?)

45. D. Bacterial Media (cont) 2. There are 2 basic types of Media (cont) b. Defined (synthetic) media (cont) 2) disadvantages a) difficult to make b) expensive to make c) often doesn’t support good growth 3) uses a) nutritional studies b) mutant isolation

46. D. Bacterial Media (cont) 3. Making Media a. Methods to sterilize media, etc. 1) Autoclaves a) Large, overpriced pressure cookers b) autoclave environment: 125°C, 2 atm pressure (~15lb./in2) for 20 mins. (designed to kill endospores) c) use: to sterilize media, dispose of bacterially contaminated trash

47. D. Bacterial Media (cont) 3. Making Media a. Methods to sterilize (cont) 2) Other methods a) UV: hard, flat surfaces b) radiation: food mostly c) poison gas (e.g. ethylene oxide): porous or complex surfaced objects d) filtration: labile liquids

48. D. Bacterial Media (cont) 3. Making Media (cont) b. Media making steps 1) measure out components, mix 2) check pH 3) + add solidifying agent (usually agar) a) clear b) non-nutritional c) melts at a high temp. but solidifies at a low temp. d. few mo’s digest it.

49. D. Bacterial Media (cont) 3. Making Media (cont) b. Media making steps (cont) 4) + aseptically add supplements (e.g. blood, aa’s, vitamins) 5) dispense media (e.g. pour plates) 6) let cool

50. D. Bacterial Media (cont) 4. Media classification by purpose a) Selective media: a media with added compounds that inhibit the growth certain mo’s while allowing other mo’s to grow 1) examples of added components: antibiotics, cationic dyes 2) use: when looking for a specific bacterial species in a specimen that is likely to contain lots of mo’s of no interest, it helps to eliminate some of the mo’s of no interest