1. EXERCISE 20. EFFECT OF TEMPERATURE ON GROWTH OF MICROORGANISMS

2. Cardinal temperatures EFFECT OF TEMPERATURE ON GROWTH OF MICROORGANISMS Cardinal temperatures Minimum temperature: : It is the lowest temperature which will support the growth of the microorganism. Below the minimum temperature the microorganisms survive for a long time and such low temperatures may be used for their storage. Optimal temperature: It is the temperature, or more usually the range of temperatures, which supports the fastest growth rate. Maximum temperature: It is the highest temperature which will support the growth of the microorganism. Above the maximum temperature the microorganism is usually killed and such high temperatures may be used in sterilization.

3. Types of microorganisms classified by optimum temperature: Psychrophiles: Psychrotrophs (facultative psychrophiles): Mesophiles: Thermophiles: Thermotolerant and thermoduric microorganisms: Hyperthermophiles:

4. Types of microorganisms classified by optimum temperature:

5. Adaptations to cold temperatures Membrane composition (increased membrane fluidity) – Increased number of double bonds in membrane fatty acids – Increased amount of hopanoids (in bacteria) or cholesterol (in eukarya) in membrane – Decreased fatty acid chain length Enzymes – Must function efficiently at lower temperatures Adaptations to high temperatures Membrane composition – Decreased number of double bonds in membrane fatty acids – Decreased amount of hopanoids or cholesterol – Increased fatty acid chain length – Archaea – use ether linkages instead of ester linkages – greater stability at high temperatures. Some archaea use tetraether lipids that form a monolayer instead of a lipid bilayer. Proteins and enzymes – More cross-linking or altered composition (change the hydrophilic and hydrophobic interactions, increased charge interactions between amino acids) for greater protein stability Ribosomes and other molecular machines need to be very stable

6. Method Serratia marcescens and Escherichia coli 1. Label the tubes as follows Slants: label one 25 o C (RT), and the other 37 o C, Broths: 5 o C, 25 o C (RT), and 42 o C 2. Inoculate the tubes as follows Slants: one with E. coli, other one with S. marcescens Broths: E. coli or S. marcescens 3. Place the tubes in racks and incubate at proper temperatures for 24 hours. Evaluation: Slants: Compare the nutrient agar slants of S. marcescens. Using colored pencils, draw the appearance of the growths on the laboratory report. Broths: 1. Calibrate the spectrophotometer with sterile nutrient broth according to the instructor’s direction. 2. Shake each culture sufficiently to completely disperse the organisms and pour 2 ml into cuvette in given order. 3. Measure the absorbance (O.D.) of your cultures and record these values on the laboratory report. 4. Plot the O.D. values on the graph-paper.

7. EXERCISE 21. LETHAL EFFECT OF TEMPERATURE

8. The factors affecting the heat resistance of a microorganism: The species and strain, How it is grown, The medium in which it is heated (food vs. buffer), The environment in which it will recover after heating.

9. Measurements used in order to compare the susceptibility of different organisms to increased temperatures Thermal Death Point (TDP): lowest temperature required to kill all microbes in liquid suspension in 10 minutes. Thermal Death Time (TDT): Minimal time required to kill all microorganisms keeping temperature (or bacteriocidal agent) constant; i.e. at a specific temperature. Decimal Reduction Time (DRT or D value): Used for heat resistant bacteria; Time required to kill 90% of the population at a given temperature (Log growth and log death concept). * Moisture, pH, composition of medium and age of cells will greatly influence these values

10. Materials: 1.24 h nutrient broth culture of the following organisms: a. Escherichia coli DH5α b. Bacillus thuringiensis 2. Nutrient agar plates (five for each group) Method: Label five agar plates as; control, 10 min., 20 min., 30min., and 40 min., respectively. Each group is assigned one culture of organisms to be heated at a specific temperature (55-65-75-85 o C) Shake the culture of organisms and transfer 0.1 ml of the organism with a 1 ml pipette to control plate. Place the culture of your group into the water bath. As soon as the temperature reaches the desired one, record the time. Watch the temperature carefully to make sure it does not vary appreciably. After ten minutes have elapsed, transfer 0.1 ml from the culture to the 10 min. plate with a fresh 1 ml pipette. Repeat this operation at ten minute intervals until all the plates that have been inoculated. Use fresh pipettes each time and be sure to shake the culture. Spread the culture over the medium by rotating the glass spreader. Incubate at 37oC for 24 – 48 hours.

11. Evaluation

12. EXERCISE 22. OSMOTIC PRESSURE AND BACTERIAL GROWTH

13. Osmosis: the net movement of H 2 O across a semipermeable membrane (i.e. cell membrane). Osmotic pressure: the force that drives osmosis. It is caused by different solute concentrations in the cell and its environment.

15. Osmophiles – Love high osmotic pressure – Salts or sugars Saccharophiles: – Osmophiles which love high sugar Halophiles: – Osmophiles which love high salt Xerophiles: – Osmophiles which love very dry environments

16. Material 1. 24 h nutrient broth cultures of following organisms: a. Escherichia coli b. Staphylococcus aureus c. Bacillus subtilis 2. Following plates for each group: 1 nutrient agar plate (0.5 % NaCl) 1 nutrient agar plate (5 % NaCl) 1 nutrient agar plate (10 % NaCl) 1 nutrient agar plate (15 % NaCl) Method 1. Mark the bottoms of the four Petri plates as indicated in below 2. Streak each organism in a straight line on the media, using a wire loop. 3. Incubate all the plates at 37 o C for 48 h

17. Evaluation