## Extreme temperatures and its effect on the growth of bacteria |

You’ll need the following materials to carry out this experiment:

- 7500 micro liters Bacillus Cereus

- 7500 micro liters Clostridium Perfringens

- 7500 micro liters Mycobacterium Tuberculosis

- 7500 micro liters Salmonella

- Cotton buds

- Normal saline solution

- 120 Vials / Test Tubes with stoppers

- 120 Petri dish filled with blood agar

- A box of disposable plastic pipette tips

- A 10 micro liter pipette

- A 250 micro liter pipette

- Calorimeter

- Goggles

- Lab coat

1. The independent variable of this experiment is the temperature of storage of the bacteria. The dependent variable of this experiment is the number of bacteria colonies killed. The few constants in this experiment include: the amount of bacteria used, the type of blood agar used and the length of time used.

2. Make 70% turbidity suspension in the normal saline solution for each different bacterium.

3. Separate one solution of bacteria between 30 vials, with 250 micro liters per vial.

4. Choose 5 of these vials and label them according to their names, their temperature of storage, and their vial number. For example, the 5 vials for Salmonella would read

a. At 40 degrees Celsius:- SAL V.1 (40°), SAL V.2 (40°), SAL V.3 (40°), SAL V.4 (40°), SAL V.5 (40°)

b. At 35 degrees Celsius:- SAL V.1 (35°), SAL V.2 (35°), SAL V.3 (35°), SAL V.4 (35°), SAL V.5 (35°)

c. At 5 degrees Celsius:- SAL V.1 (5°), SAL V.2 (5°), SAL V.3 (5°), SAL V.4 (5°), SAL V.5 (5°)

d. At -20 degrees Celsius:- SAL V.1 (-20°), SAL V.2 (-20°), SAL V.3 (-20°), SAL V.4 (-20°), SAL V.5 (-20°)

e. At -30 degrees Celsius:- SAL V.1 (-30°), SAL V.2 (-30°), SAL V.3 (-30°), SAL V.4 (-30°), SAL V.5 (-30°)

f. At -60 degrees Celsius: - SAL V.1 (-60°), SAL V.2 (-60°), SAL V.3 (-60°), SAL V.4 (-60°), SAL V.5 (-60°)

5. Repeat steps 3-4 with the other bacteria and all different temperatures.

6. Store the respective vials at the respective temperatures. Note the time.

7. At the same time on day one (24 hours after you have put the vials in the respective temperatures), remove all vials labeled “V.1” and let them sit for 15 minutes at room temperatures.

8. Reheat or refreeze these vials respectively.

9. At the same time on day two, remove all V.1 and V.2 vials and let them sit for 15 minutes at room temperature.

10. Reheat or refreeze these vials respectively.

11. Repeat steps 6-7 for 3 more days and eventually taking out V.3 on the third day, V.4 on the fourth day and so on and so forth for 7 days.

12. On day 8, remove 10 micro liters from each vial using a pipette. Transfer each specimen to a clearly labeled blood agar Petri Dish. The naming should follow that of step 4 as above.

13. Spread the bacteria using a cotton bud (sterile) onto the surface of the blood agar.

14. Put the Petri dishes into an incubator for 24 hours at a pre-set temperature of 37 degrees Celsius.

15. After that, count the number of colonies in each dish under fluorescent light and record the number in the table below.

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Complexity level:

9

Project cost ($):

50

Time required:

2 hours to prepare (Day 0),
7 days incubation period (Day 1 to Day 7),
15 minutes each day (from Day 1 to Day 7),
30 minutes to transfer samples on Petri Dish (Day 8)
24 hours for inoculation,
Observation on Day 9

Material availability:

The school biology laboratory should provide all the necessary materials.

Safety concerns:

Goggles and gloves must be worn at all times to prevent contamination as we’re dealing with biohazardous materials. Adult supervision encouraged.

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