2. Bacterial Growth and Physiology

3. Growth: increase in size of organisms and increase in their number, the net effect is increase in the total mass of the culture. In the laboratory: growth is need as technique for detection and identification and for assessment of antibiotic effects.

4. Types of growth: 1- development of colonies, the macroscopic product of 20-30 cell divisions of a single cell. 2- by transformation of clear broth medium to a turbid suspension Media for bacterial growth: → could be solid (agar) or liquid (broth) →the aim is achievement of pure culture; only one type of growing cell and single colonies of this type.

5. Media could be : 1.Nutrient 2.Supportive 3.Selective or enrichment 4.Examples on media : Blood agar, MacConkey agar and Chocolate agar

6. Bacterial Nutrition Bacteria need nutrients for 1- Maintenance of their metabolism 2- Cell division. Sugars, proteins and lipid macromolecules are broken down outside the cell into smaller molecules that are transported across the membrane, in the cytoplasm the metabolite are converted into pyruvic acid which is either used for energy or to synthesis of new carbohydrate, protien and lipid, and nucelic acid

7. Elements needed: 1. Hydrogen and Oxygen------obtained from Water 2. Carbon Source: external source must be found e.g. carbohydrate (sugars) degraded by either oxidation or fermentation and this provide ATP 3. Nitrogen source: main source is ammonia or produced by the bacterium by deamination of amino acids released from proteins.

8. 4. Minerals 5. Growth factors: that a cell must have for growth but cannot synthesize itself. Such as amino acids, purines, pyrimidines, and vitamins Nutrient uptake by diffusion and active transport.

9.  In this lecture, we will talk about:  Growth requirement of the bacteria: o Nutrients o Growth factors o Oxygen o Carbon dioxide o Temperature o Hydrogen ion concentration  Bacterial growth curve

10. Nutrients Heterotrophic bacteriaAutotrophic bacteria o Require complex organic materials o Use simple inorganic materials like CO2 as a source of carbon and ammonium as a source of nitrogen o Can not form organic compounds from the simple inorganic materials o They form complex organic compounds from the simple inorganic materials o Derive their energy: Oxidation or fermentation of organic compounds such as glucose o Derive their energy: From light (photosynthetic bacteria) Oxidation of inorganic materials (chemolithotrophic bacteria)

11. Most bacteria of medical importance are heterotrophic bacteria

12. Growth factors  These are organic compounds which bacteria must contain to grow.  But, they are unable to synthesize them  So, they must be added ready formed to the culture medium  Examples: amino acids, vitamins, purines and pyrimidines

13. Gas Requirements Bacteria are classified according to O 2 requirement into 1- aerobic 2- obligate anaerobic 3- facultative anaerobic 4- microaerobic Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycollate broth: 1- Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest. 2- Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest. 3 - Facultative anaerobes can grow with or without oxygen because they can metabolise energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration. 4: - Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube but not the very top. 5: - Aerotolerant organisms do not require oxygen as they metabolise energy anaerobically. Unlike obligate anaerobes however, they are not poisoned by oxygen. They can be found evenly spread throughout the test tubebacteriathioglycollate brothObligate aerobesObligate anaerobesFacultative anaerobesMicroaerophilesAerotolerant

15. Aerobic bacteria produce three enzymes in order to protect themselves from oxygen-free radicals which are highly toxic. These enzymes: superoxide dismutase, catalase peroxidase 2O 2 - + 2H + SOD H 2 O 2 +O 2 2H 2 O 2 Catalase 2H 2 O +O 2 H 2 O 2 +AH 2 peroxidase 2H 2 O+A

16. Oxygen ExamplesDefinitionClass Mycobacterium tuberculosis Grow only in the presence of O2 Obligate aerobe Clostridia Can not grow in the presence of O2 Obligate anaerobe Most bacteria of medical importance Can grow in the presence or absence of O2 Facultative anaerobe Campylobacter Require low O2 tension Microaerophilic bacteria

17. In the presence of oxygen, two toxic substances to the bacteria are produced which are hydrogen peroxide and superoxide anion. In obligate aerobes and facultative anaerobes: Catalase and peroxidase enzymes degrade hydrogen peroxide. Superoxide dismutase enzyme degrades superoxide anion. BUT In obligate anaerobes: These enzymes are not present. So, the presence of oxygen is toxic to them.

18.  Most bacteria require CO 2 in small concentration as that in the air.  However, some bacteria require higher concentrations of CO 2.  These bacteria are called capnophilic bacteria.  Example:  Neisseria requires 5-10% CO 2 Carbon dioxide

19. CO 2 N. meningitidis N. gonorrhoeae

20. Temperature Optimum temperature Range of temperature Bacteria 3718 - 42Mesophilic 15 - 205 - 30Psychrophilic 50 - 6025 - 80Thermophilic

21. Most bacteria of medical importance are mesophilic bacteria

22. Temperature Psychrophilic forms (15-20°C) Mesophilic forms (30-37 °C) include all human pathogens and opportunists. Thermophilic forms (50-60 °C)

25.  Hydrogen ion concentration is called pH.  Most bacteria grow within pH range 7.2-7.6  However, some bacteria require different pH.  Examples:  Vibrio cholera needs alkaline pH  Lactobacilli need acidic pH Hydrogen ion concentration

26. pH Neutrophiles (6.5-7.5) almost all pathogen Acidophiles (3-5) ex. Lactobacillus Alkaliphiles (pH > 8) ex. Vibrio cholera

27. Bacterial Growth Bacteria multiply by binary fission

28.  Binary fission:  The bacterial cell divides into two equal daughter cells similar in genetic characters to the mother cell. 1) Elongation of the bacterial cell. 2) Duplication of the chromosome. 3) The two sister chromosomes attach to the mesosome. 4) Cell membrane and cell wall will form a transverse septum that divides the mother cell into 2 equal daughter cells. 5) The daughter cells may remain attached to each other.  Doubling time: the time required by the bacterial cell to double its number. Bacteria growth (reproduction)

29. BACTERIAL REPRODUCTION

30. Bacterial Growth Bacteria undergo exponential growth (Logarithmic growth): Number of cells 1 2 4 8 16

31. Generation : the time it takes for a population of bacteria to double in number many common bacteria ： 20~60 min most common pathogens in the body ： 5-10 hours

32.  A small number of bacteria is inoculated in a liquid medium and the number of living bacteria is counted at intervals.  Growth curve with 4 phases is obtained: Bacterial growth curve

33. 1)Lag phase:  No cell division.  The bacteria form the enzymes and molecules needed for replication.  Clinical significance: this phase = incubation period of a disease. 2)Logarithmic phase:  Rapid cell division occurs.  The number of living bacteria increases by time.  Clinical significance: this phase = symptoms and signs of the disease.

34. 3)Stationary phase:  Nutrients are exhausted.  Waste products are accumulated.  The number of dying cells = number of new cells.  The number of living bacteria remains constant. 4)Decline phase:  Nutrients are more exhausted.  Waste products are more accumulated.  The number of dying cells > number of new cells.  The number of living bacteria decreases by time.  Clinical significance: this phase = recovery and convalescence.

35. the growth curve: a. The Lag Phase: The lag phase represents a period during which the cells, adapt to their new environment. High metabolic activity occurs but cell do not dived. Enzymes and intermediates are formed and accumulate until they are present in concentrations that permit growth to resume. b. The Log or Exponential Phase:rapid division and the mass increases in an exponential manner. This continues until one of two things happens: either one or more nutrients in the medium become exhausted, or toxic metabolic products accumulate and inhibit growth.

36. the growth curve c. The Maximum Stationary Phase: nutrient depletion or toxic product cause growth to slow until number of new cells produced balances the number of cells that die, resulting in a steady state ( equilibrium between cell division & death) d. The Death or decline Phase: decline in the number of viable bacteria. Number of death exceeds the number of new cells formed

37. PhaseGrowth Rate LagZero ExponentialConstant Maximum stationaryZero DeclineNegative (death) Table 3. Phases of the Microbial Growth Curve

38. GOOD LUCK