1. Bacterial Physiology Medical Microbiology L3 Dr. Saleh M Y OTH PhD Medical Molecular Biotechnology and Infectious Diseases 05/10/2011 MBBS-Phase II-IMS - MSU

7. - Nutritional - Environmental - Pure culture Culture of microorganisms Culture media Dependence on oxygen - Bacterial growth in laboratory conditions Growth curve - Microbial metabolism Factors for microbial growth: Bacterial Growth

9. 1-Nitrogen source; Ammonium (NH 4+ ) is used as the sole N source by most microorganisms. Ammonium could be produced from N 2 by nitrogen fixation, or from reduction of nitrate and nitrite. 2-Sulfur source; Most microorganisms can use sulfate (SO 4 2- ) as the S source. 3-Phosphate source (PO 4 3- ) is usually used as the P source. Bacterial Growth Nutritional factors

10. 4-Mineral source; For most microorganisms, it is necessary to provide sources of K + , Mg 2+ , Ca 2+ , Fe 2+ , Na + and Cl - . Many other minerals (e.g., Mn 2+ , Mo 2+ , Co 2+ , Cu 2+ and Zn 2+ ) can be provided in tap water or as contaminants of other medium ingredients. 5-Growth factors; Compounds that bacteria require but cannot synthesize Bacterial Growth Nutritional factors

11. 1-Temperature Psychrophile (15 o C - 20 o C) Mesophile (30 o C - 37 o C) Thermophile (50 o C - 60 o C) 2-pH Neutrophile (pH 6 - 8) Acidophile (pH 1-5) Alkaliphile (pH 9-11) Environmental factors 3-Oxygen availability Obligate aerobe (O 2 ) Obligate anaerobe (CO 2 ) Facultative anaerobe (O2 /CO 2 ) Microaerophile (5-10% O 2 ) 4-Water availability Osmophile Halophile Bacterial Growth

12. Obligate aerobe Facultative anaerobe Obligate anaerobe Microaerophile 1 2 3 4 O 2 O 2 or Co 2 Co 2 Superoxide dismutase

13. Obtaining a pure culture A solid medium is required for obtaining a pure culture of microorganism. Agar: an algae extract, polysaccharide in nature, which very few bacteria can degrade. The agar plate contains 1.5% of agar. Colony: population of bacterial cells arising from a single cell. Cultivating bacteria on a solid medium (bacterial isolation)

16. Streak-plate method

17. Pour plate method

18. Culture of microorganisms Complex (rich) media nutrient agar or broth; blood agar or chocolate agar for more fastidious bacteria. Chemically defined (minimal media) Selective media Inhibitors for organisms other than the one being sought are added. Culture media Differential media Substances that certain bacteria change in a recognizable way are added. Nutrient broth Glucose-salt Peptone Glucose Meat extract Dipotassium phosphate Water Monopotassium phosphate Magnesium sulfate Ammonium sulfate Calcium chloride Iron sulfate Water

19. MacConkey agar plate Blood agar plate Back

20. Principles of bacterial growth -Bacteria multiply by binary fission . -Microbial growth is defined as an increase in the number of cells in a population. Bacterial growth curve Bacterial growth in laboratory conditions Generation time E. coli : 20 min M. tuberculosis : 12-24 h

21. Bacterial growth curve Bacterial growth in laboratory conditions

22. A balance between slow loss of cells through death and the formation of new cells through growth and division. The bacteria die off rapidly, the curve turns downward, and the last cell in the population soon dies. Bacteria synthesize macromolecules required for multiplication. The length of lag phase depends on the conditions in the original culture and the medium into which they are transferred. The doubling time is measured during this period. The bacteria are most susceptible to antibiotics during this time. Bacteria stop growing due to decrease of nutrients and O 2 supply, and accumulation of toxic metabolites.

23. Assimulation (anabolism): energy-requiring Dissimulation (catabolism): energy-acquiring Bacterial Metabolism Focal metabolites: metabolic intermediates that link anabolic and catabolic pathways. Glycolysis Pentose phosphate pathway TCA cycle Respiration (aerobic and anaerobic) Fermentation

24. Saccharomycetes E. coli Clostridium Propionebacterium Enterobacter Streptococcus Lactobacillus

25. Increased CO 2 Candle jar; CO 2 incubator Microaerophilic Culture methods Anaerobic Anaerobic jar; anaerobic chamber; reducing agents

26. Enrichment cultures Isolating an organism from natural sources

27. Maintaining stock cultures Agar slant Store agar slant cultures in a refrigerator. Stock at –70 to -80 o C Store a pure culture in the presence of 17% glycerol. Lyophilization (freeze drying) Dry a pure culture with a lyophilizer. This can be stored at room temperature for years.

28. Direct cell count Count under a microscope; cell-counting instrument Measuring biomass Turbidity; total weight; chemical constituents Viable cell count Plate counts(MPN); membrane filtration; Detecting cell products Methods to detect and measure bacterial growth

30. Overview of Medically Important Bacteria Rickettsia Chlamydia Mycoplasma* Clostridia Actinomycetes Obligate Intracellular Extracellular & Facultative Gram-positive Gram-negative Mycobacteria Spirochetes Cocci Bacilli Staphylococci Streptococci Enterococci Listeria Bacillus Corynebacteria Cocci and coccobacilli Bacilli Haemophilus Bordetella Neisseria Enterobacteriaceae Pseudomonads Legionella Campylobacter Helicobacter Borrelia Leptospira Treponema Brucella Francisella Pasteurella Aerobes Strict Anaerobes Vibrio

31. Topics for reading: Using of Microscope and its parts Classification of Bacteria Bacterial structure Bacterial multiplication Bacterial genetic

32. Topics for reading: Catabolism and anabolism ATP Generation and energy conservation Fermentation

33. Thank you Study Questions Besides chemical nutrients, what are 4 other factors you would consider when trying to grow a bacterium for the first time? Why do you need to sterilize bacterial media? What are some ways you could do this? What would happen if you didn’t sterilize the media? What are the four phases of growth curve? What is happening in each?