PURPOSE
The purpose of this experiment was to determine which heating or freezing temperature is a better method to kill bacteria.
I became interested in this idea when I froze bacteria at extreme temperatures last year and wondered whether heating or freezing would kill more bacteria because freezing bacteria was a very effective way to either slow down the growth rate or kill bacteria at extreme temperatures such as –60∞C.
The information gained from this experiment could help scientists and doctors understand how freezing or heating bacteria can be helpful.
My first hypothesis was that the highest heating temperature would kill more bacteria than lower heating temperatures.
My second hypothesis was that the lowest
freezing temperature would kill more bacteria of each type than less extreme
freezing temperatures.
I based my
second hypothesis on the results of my experiment from last year, although I
tested only Staph and E. coli.
The constants in this study were:
The testing procedure
The time the bacteria was frozen and heated
The amount of bacteria
The way they were measured after freezing and heating
The four types
of bacteria
The vials the bacteria were in
The temperatures of the refrigerators were
always –20°, -30°, and -60° Celsius
The heating temperatures were always 5°, 35°,
and 40° Celsius
The manipulated variable was the freezing and heating temperatures during storage.
The responding variable was the survival rate.
To measure the responding variable, I counted the bacteria colonies.
QUANTITY
|
ITEM DESCRIPTION
|
20 micro
liters
|
Escherichia
Coli
|
20 micro
liters
|
Staphylococcus
Aureus
|
20 micro
liters
|
Pseudomonas
Aeruginosa
|
20 micro
liters
|
Straptococcus
Agalactiae
|
Lots
|
Cotton Swabs
|
Lots
|
Disposable
Pipette Tips
|
Lots
|
Sterile
Saline solution
|
120
|
Test Tubes
|
120
|
Blood Agar
Plates
|
120
|
Inoculating
Loops
|
1
|
10 Micro
liter Pipette
|
1
|
200 Micro
liter Pipette
|
1
|
Colorimeter
(turbidity meter)
|
1
|
Lab Coat
|
PROCEDURES
1. On day obtain the following bacteria from hospital lab.
- Escherichia Coli
- Staphylococcus Aureus
- Pseudomonas Aeruginosa
- Streptococcus Agalactiae
2. Next, make
80% turbidity suspension in sterile saline solution for each bacteria type.
3. Divide one solution of bacteria between 30 vials, with 200 micro-liters per vial.
4. Select five of these vials and label them with the name of the bacteria, the temperature of storage, and the vial number. This is an example for Staphylococcus Aureus at 35° C.: SAU V.1 35°, SAU V.2 35°, SAU V.3 35°, SAU V.4 35°, SAU V.5 35°
5. Repeat steps 3-4 with all the bacteria and all temperatures.
6. Store bacteria labeled 5°, 35°, and 40° at those Celsius temperatures.
7. Store bacteria labeled –60°, -30°, and –20° C. at those temperatures.
8. On day one, take all vials marked “V.1” out and let them sit for 10 min. at room temperatures.
9. Refreeze or reheat all these vials where they belong.
10. On day two take out all V.1 and V.2 vials, and let them sit for 10 min. at room temperature.
11. Refreeze or reheat all these vials where they belong.
12. Repeat steps 8-9 for three more days and additionally removing V.3 on the third day, V.4 on the fourth, and finally V.5 on the last day.
13. On day 6, pipette 10 micro-liters from each vial to its own blood agar plate.
14. Spread the bacteria with inoculating loop over the surface of the blood agar plate. Label plates exactly as the vials were labeled.
15. Incubate bacteria overnight at 37° Celsius.
16. Remove plates from incubator.
17. Do a colony count for each plate under fluorescent light using the unaided eye. Record this number.
18. Destroy all bacteria on equipment contaminated with bacteria in autoclave-using hospital’s procedures.
RESULTS
The original purpose of this experiment was to determine whether heating or freezing is a better method to kill bacteria.
The results of
the experiment were that the bacteria that was frozen had way more bacteria
than the ones put in 35∞ and 40∞ heaters.
My first hypothesis was that the highest heating temperature would
kill more bacteria than lower heating temperatures.
My second hypothesis was that the lowest freezing temperature
would kill more bacteria of each type than less extreme freezing temperatures.
The results indicate that
both hypothesis should be should be accepted.
Because of the results of this experiment, I wonder if time
affects the number of bacteria.
If I were to conduct this
project again I would freeze and heat the bacteria longer than five days.
Researched by - Welson L
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