1. LAB NO 8 LAB NO 8 Environmental Factors Affecting Microbial growth

2.  There are many factors in the environment of the microorganism that can dramatically affect the rate and the extent of the microbial growth. 1. Hydrogen Ion Concentration (PH). 2. Moisture content. 3. Solute Concentration (osmotic pressure). 4. Oxygen. 5. Temperature.

3. 1.Moisture content  Humidity is absolutely necessary for metabolic reactions and multiplication of the microorganism.  However, drying does not kill microorganism, but only prevent them from further multiplication. ⁂ generally, endospores and relatively small cells are more resistant to drying than vegetative and large cells.

4. 2. Hydrogen Ion Concentration (pH)  The enzymes, electron and nutrient transport system found in the cell membrane are sensitive to the concentration of hydrogen ions (H + ).  The optimum pH for most bacteria is 6.5 - 7.5  Fungi & acidophilic bacteria [e.g; Lactobacillus sp.] can tolerate acidic pH up to 4.

5.  while basophilic bacteria [e.g;Vibrio cholera] can grow at pH up to 9. ※ That’s why certain food are not easily contaminated with bacteria due to their acidity like citrus fruits.

6. 3. Solute Concentration (osmotic pressure) 3. Solute Concentration (osmotic pressure)  The conc. of solutes dissolved in the aqueous environment of the bacterial cell can influence the passage of water and nutrient into the cell.  Some microorganism inhibited or killed by different concentration of solute such as NaCl or sugars as a result of their effect on the osmotic pressure of the medium.

7.  Most microorganisms can grow well at isotonic environment.  In Hypertonic environment, cells have the tendency to lose water to their environment, as the conc. outside the cell is greater than inside the cell. 3. Solute Concentration (cont.) 3. Solute Concentration (cont.)

9.  Under this condition, most bacteria can’t reproduce due to dehydration of the cells leading to concentration of solute & electrolytes within the cells which cause toxic effects. 3. Solute Concentration (cont.) 3. Solute Concentration (cont.)

10.  In general microorganisms are classified into two major groups according to their oxygen requirement: 1. Aerobes: In which oxygen is absolutely necessary for growth. 2. Anaerobes: They can reproduce in the absence of oxygen. In fact oxygen inhibit their growth & in most cases kill them. 4.Oxygen 4.Oxygen

11. 5.Temperature  Microorganisms can be classified according to the optimum temperature for growth into: a) Psychrophilic (cold-loving);  Which can grow best at temperature between 0 - 20ºc. b) Mesophillic (middle-loving);  which can grow best at temperature between 20 - 40ºc. c) Thermophilic (heat-loving);  which can grow best at temperature between 40 – 80ºc.

13. Temperature (cont.)  The lethal effect of high temperature on microorganisms can be attributed to: 1. Irreversible denaturation of intracellular proteins such as enzymes and other vital components of the cell. 2. Water loss due to evaporation leads to increase concentration of electrolytes within the cell causing toxic effect.

14. Temperature (cont.)  Generally, bacterial endospores are more resistant to adverse environmental conditions such as drying or high temperature due to: A. Low water content of spores and low metabolic activities. B. Low permeability due to thick spores coat. C. Presence of dipicolinic acid and dicarboxylic acid in the spore coat which are complex with divalent ions particularly calcium to give a heat resistant complex.

15. Temperature (cont.) NOTICE:  Bacterial endospores are heat and chemical resistant as they serve a protective function.  However, the spores of yeast and molds are not resistant to environmental conditions as they serve a reproductive function.

16. DeterminationofThermalDeathTime Determination of Thermal Death Time  Thermal death time:  of a given species defined as the time required to sterilize (kill) a standardized pure culture of that species at a specified temperature.  Thermal death point:  of a given species defined as the lowest temperature required to sterilize (kill) a standardized pure culture of that species at a specified time.

17. DeterminationofThermalDeathTime Determination of Thermal Death Time  Determination of thermal death time is useful in sterilization processes, preparation of heat killed vaccines, canning of food and many other application.  Materials:- Culture of Bacillus subtilis and E. coli. Nutrient agar plate. Thermometer and water bath.

18. DeterminationofThermalDeathTime Determination of Thermal Death Time 5 10 1520 B E 5 min 10 min 50 ºc 70ºc 95 ºc Invert & incubate for 48 hrs

19. Result 1.E. coli Temp. time 5 min 10 min 15 min 20 min 50 ℃ 70℃ 95℃ 2- B. subtilis; Compare the result of spore forming microorganism against non-spore forming one. give your comment.

21. MIC: It is the lowest concentration of the antimicrobial agent that inhibits the growth of the test organism but not necessarily kills it. MBC (minimum bactericidal conc.) It is the lowest concentration of the antimicrobial agent that kills the test organism.

22. Bactericidal drugs have MBC 2-4 times MIC. Bacteriostatic drugs have MBC much higher than MIC.

23. The breakpoint of an antimicrobial agent: It is the concentration of the drug which can be achieved in the serum with optimal dose.

24. Susceptible organisms : are those with MICs at/below the breakpoint. Resistant organisms: are those with MICs above the breakpoint.

25. Material:  Culture of staph. aureus  Augmentin  Saline  Melted nutrient agar bottle  Sterile peri dish  4 sterile vials  2 sterile pipette 1ml  1 sterile pipette 5 ml

26.  Procedure: 45°c S o.2 ml

27. AB 12 3 4 1 ml 1 ml sterile water

28. 4 Incubate the plate Uninverted at 37°c for 24h 3 2 1 4 3 2 1

29. Results:  Measure the diameter of each inhibition zone The diameter of the inhibition zones are directly proportional to the conc. of the antibiotic. Zone diameter Well diameter

30. Agar Diffusion Method  Results: AB concLog conc Zone diameter Well diameter X X² 100 50 25 12.5 X = Zone diameter – Well diameter 2 If Original Antibiotic Conc. = 200 µg/ml

31. x² Log conc Anti-log = MIC x x x x

32. conc MIC If use semi-log paper, then No need to calculate Log conc. x² x x x x

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