Biological Characteristics of Bacteria

1. Biological Characteristics of Bacteria • The Growth, Survival & Death of Microorganism • Cultivation of Microorganism • Microbial Metabolism

2. The growth of microorganisms Growthis the orderly increase in all the components of an organism such as size and/or population number. The rule for bacteria growth can be described as single cell dynamicsandpopulation dynamics

3. Growth: single cell dynamics c a d a. Bacterial cell first can been seen to enlarge or elongate. b b. Then followed by the formation of transverse membrane and new cell wall. c. The new membrane and cell wall grow inward from the outer layers. d. The cell divided into two daughter cells. Bacteria multiply by binary fission, the process in which a parent cell splits into two daughter cells with approximately equal size.

4. Growth: single cell dynamics • Under optimal conditions, the average time required for a population of bacteria to double in number (for complete cell division) is called as generation time or doubling time. • The generation time for many common bacteria is 20-30 min, for a few of slow-growing bacteria such as Tuberculosis bacteria might be up to 18-20 h.

5. Growth: population cell dynamics • When microorganisms are grown, due to some factors such as nutrient limitation and waste accumulation, growth rate cannot maintain for a long time. • If a liquid medium is incubated with microbial cells, and the number of viable bacterial cells per milliliter is measured and plotted, we can obtain a curve that called as growth curve. • Normally characterized by the phases lag, log (or exponential) growth, stationary growth, and death.

6. How to Graph Bacterial Growth (i) • Measuring the numbers of bacteria. • Common methods include: • Turbidity: to measure the total bacteria (live and dead) in liquid cultures. This is usually quantitated with a spectrophotometer, the absorption wavelength at 600 nm will be measured. • Colony counting method:that means countingthecolonynumbers on a medium plate after inoculated with a known volume of bacterial liquid culture.

7. Measuring total bacteria (live + dead) in liquid culture——Turbidity (Cloudiness) The cloudiness of a liquid media caused by bacteria growth that are generally invisible to the naked eye, similar to smoke in air.

8. Measuring viable bacteria colony A visible cluster of bacteria growing on the surface of or within a solid medium, presumably cultured from a single cell

9. How to Graph Bacterial Growth (ii) (II) Plotting the logof turbidityornumber of living cellsversustime is referred to as the growth curve (four or six phases): GrowthCurve (four phases)

10. Growth curve (six phases) A: lag phase, B: acceleration phase, C: log (exponential) phase, D: deceleration phase, E: stationary phase, F: death phase Y-axis presents the log number of living cells X-axis presents the period of time (usually in hours) The curve can be divided into six phages represented by the letters A-F

11. What are the characteristics of bacteria in each phase?

12. Growth curve (four phases) • The Lag Phase (A and B): Bacteria are becoming "acclimated" to the new environmental conditions (pH, temperature, nutrients, etc.) (A). Enzymes and intermediates are formed and accumulate until they are present in concentrations that are permit growth (B). An increase in bacterial mass per unit of volume, but no increase in cell count. The metabolism of the bacteria adapts to the conditions of the nutrient medium.

13. Growth curve (four phases) • The Exponential/log Phase (C): Conditions are optimal for growth. The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. The bacteria are suitable for biochemical and morphological identification, The bacteria are suitable to use for drug sensitivity test

14. Growth curve (four phases) • The Maximum Stationary Phase (D and E): With the exhaustion of nutrients and accumulation of metabolic wastes, the growth rate has slowed to the point where the growth rate equals the death rate (D). Effectively, there is no net growth in the living bacteria population (E). The bacteria produce spores, toxins (e.g. exotoxin) and antibiotics.

15. Growth curve (four phases) • The Decline Phase (F): The living bacteria population decreases with time, due to a lack of nutrients and toxic metabolic by-products. In many cases (but not always) the bacteria autolyse (during the death phase) and the turbidity decreases.

16. Growth curve

17. Biological Characteristics of Bacteria • The Growth, Survival & Death of Microorganism • Cultivation of Microorganism • Microbial Metabolism

18. What are the requirements for bacterial growth?

19. Bacterial chemical components • Water: free water and compound water. • Inorganic salt: phosphus, potassium, magnesium, calcium, nitrium, etc. • Protein:50-80% of dry weight according bacterial kinds and age. • Sugar:mainly distributing in cell wall and capsule. • Lipids:composed of lipid, fatty acid, wax, etc. • Nucleic acid: RNA and DNA.

20. Sources of metabolic energy • fermentation • An enzyme-induced chemical change in organic compounds that takes place in the absence of oxygen. The change usually results in the production of organic acids and energy • respiration • The process cells use to convert the energy in the chemical bonds of nutrients to ATP energy. Aerobic respiration Anaerobic respiration

21. Sources of metabolic energy • photosynthesis • Reduction of an oxidant via a specific series of electron carriers establishes the proton motive force. Respiration:the energetically favorable oxidation of organic matter by an electron acceptor such as oxygen Photosynthesis: the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane

22. Bacterial Nutrition and Growth • Nutrient Requirements: • Water • Carbon source (C) • Nitrogen source (N) • Inorganic salts • Growth factors • Sulfur source (S) • Phosphorus source (P) • Environmental factors for bacteria growth: • Temperature • Gas (oxygen) • pH • Osmotic pressure

23. Nutrient Requirements • Based on their source of carbon including nitrogen, bacteria are divided into two groups : Autotrophs and Heterotrophs. • Autotrophs: This group of bacteria has a more complete enzyme system so they can obtain energy and various nutrientsfrom simple compounds such as carbon from CO2. None of these organisms are pathogenic ! 1) Carbon source

24. Heterotrophs: This group of bacteria has a simpler set of enzymes so they must use organic carbon for growth. It can be further divided into: Parasitic bacteria: grow in and feed on a different organism. Saprophytic bacteria:obtain their nutrients from dead or decaying organic matter. Most of pathogenic bacteria are parasitic bacteria. Some saprophytic bacteria cause disease by acting on food to produce toxins.

25. Nutrient Requirements • A major component of proteins, nucleic acids, and other compounds. • Nitrogen fixation:The ability to assimilate N2 reductively via NH3. This process requires a large amount of metabolic energy and is readily inactivated by oxygen. It is a property unique to prokaryotes, especially in divergent bacteria, that have evolved quite different biochemical strategies to protect their nitrogen-fixing enzymes from oxygen. 2) Nitrogen source

26. Nutrient Requirements In formulating a medium for the cultivation of most microorganisms, it is necessary to provide sources of potassium, magnesium, calcium, and iron, usually as their ions (K+, Mg2+, Ca2+, and Fe2+). Required for appropriate osmotic pressure. Additionally, also needed to stabilize or activate certain enzymes. 3) Mineral source

27. Nutrient Requirements • A growth factor is an organic compound which a cell must contain in order to grow but unable to synthesize. • A number of different growth factors are required for bacterial growth such as amino acids, purines, pyrimidines, and some certain vitamins. 4) Growth factors

28. Environmental factors for bacteria growth • Most bacteria have a narrow optimal pH range. • Neutralophiles: grow best at neutral pH (pH 6.0-8.0, 7.5) • Some can survive/grow • - Acidophiles (pH 1.0-6.5 ) • - Alkaliphiles (pH 9.0-11 ) Internal pH is regulated by a set of proton transport systems in the cytoplasmic membrane, including a primary, ATP-driven proton pump. 5) pH

29. Environmental factors for bacteria growth • Different microbial species are vary widely in their optimal temperature ranges for growth: • Mesophilic forms30-37 ℃ • All human microbial pathogens belong to this forms • Psychrophilic forms15-20 ℃ • Thermophilic forms 50-60 ℃ 6) Temperature

30. High Temperature static action cidal action

31. 6) Temperature Heat-shock response: a transient synthesis of a set of "heat-shock proteins," when exposed to a sudden rise in temperature above the growth optimum. These proteins appear to be unusually heat-resistant and to stabilize the heat-sensitive proteins of the cell. Cold shock response: the killing of cells by rapid as opposed to slow cooling.

32. 7) Gas Requirements Environmental factors for bacteria growth According to the requirement of O2 during bacteria growth, bacteria can be divided into four groups: Aerobic Anaerobic 1. Obligate aerobe: Growth No growth 2. Microaerophile: Growth at low O2 No growth 3. Obligate Anaerobe: No growth Growth 4. Facultative aerobe: Growth Growth

33. Obligate aerobes • grow in presence of oxygen • no fermentation • oxidative phosphorylation • (Electron transfer through the cytochrome system liberating free energy which is transformed into high-energy phosphate bonds)

34. Microaerophile (Microaerophilic bacteria) grow • low oxygen (2-5%) • killed • high oxygen

35. Obligate anaerobes superoxide dismutase peroxidase H2O2 O2-+2H+ H2O /NAD NADH catalase H2O + O2 • no oxidative phosphorylation • fermentation • killed by oxygen • lack certain enzymes：

36. Facultative anaerobes • fermentation and oxidative phosphorylation • aerobic respiration • survive in oxygen

37. Cultivation methods Concerning two parts: I. Choice of suitable medium II. Isolation of bacteria for pure culture

38. I. Choice of suitable medium

39. Medium (I) (1) raise a crop of cells of a particular species that is on hand; (2) determine the numbers and types of organisms present in a given material; (3) isolate a particular type of microorganism from a natural source.

40. Medium (I) Basic medium Enrichment medium Selective medium Differential medium A classification of media based on their respective usages:

41. Enrichment medium: Nutrient broth, nutrient agar, peptone water are commonly used in enrichment media. e.g. blood agar plate Basic medium: supplies only the minimal nutritional requirements of a particular microorganism. e.g. broth

42. Supports the growth of desired bacteria while inhibiting the growth of many or most of the unwanted ones, either by adding one or more selective agents which is a "poison" to the unwanted bacteria but not harmful to desired bacteria, or by including certain nutrients for the desired ones and deleting certain nutrients for the unwanted ones. e.g. L-G medium for M. tuberculosis Selective medium:

43. This medium allows two or more different bacteria to grow, but it contains dyes and/or other components upon which different bacteria act in various ways to produce a variety of end products or effects (usually by showing different colors). e.g. SS agar Differential medium

44. Medium (II) liquid medium Solid medium Semi-solid medium Classification according to physical condition (according to the content of solidifying agent):

45. The major solidifying agent used in bacteriological media. • An polysaccharide gum that extracted from certain red algae. • Agar can be dissolved at 100 C, and solidified at about 43 C. • Added 1.5-2.0% of Agar for solidplates or slanted media, 0.1-0.5% for semisolid media. Agar

46. Usage of different media • Used to obtain a large number of bacteria, and to perform drug sensitivity test and bacterial growth assay. • The bacteria grown in liquid medium will display some certain characteristics of bacteria (alignment and clustering) that can't be seen easily in solid media. liquid medium

47. i ii iii • forming cloudiness in broth • (growth with uniform turbidpattern), • forming a ringat the top of broth (growth with suspension pattern) • forming sediment at the bottom of broth • (growth with sedimentary pattern) Phenomena of bacterial growth in liquid medium

48. Used to obtain a large number of bacteria, isolate identical clones of bacteria (colony), and to perform drug sensitivity test. • A colony is a bacterial cluster which propagated (multiplied) from a single initial bacterial cell(So a colony is a pure bacterial culture). • Colony can be used to determine the original bacterial numbers by counting colonies and to evaluate viability of bacteria (colony forming units, CFU). Solid medium

49. Test the motility of bacteria (a bacterium has a flagellum or flagella whether or not ) Positive:bacteria grow into the medium give cloudiness to the medium. Negative:bacteria grow in situ. Semisolid medium

50. Cultivation methods II. Isolation of bacteria in pure culture