The Effect Of Extreme Freezing On Bacteria

Researched by Welson L.
2003-04



PURPOSE

The purpose of this experiment was to determine how extreme freezing would affect the subsequent growth rate of bacteria.

I became interested in this idea when my dad started freezing different types of commercial food at different freezing levels and getting different results.  

The information gained from this experiment could help scientists and doctors understand how freezing bacteria can be helpful.



HYPOTHESIS

My hypothesis was that the colder the temperature, the fewer the bacteria colonies would subsequently be.  

I based my hypothesis on the fact that freezing will stop bacteria growth for a while.

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EXPERIMENT DESIGN

The constants in this study were:

  • The testing procedure
  • The time the bacteria is frozen
  • The amount of bacteria 
  • The way they were measured after freezing
  • The two types of bacteria
  • The same vials the bacteria was in
  • The temperatures are always -60, -30, -20,and -10 degrees Celsius 


The manipulated variable was the different freezing levels.

The responding variable was the colony growth.

To measure the responding variable in this experiment, I counted the bacteria colonies.  

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MATERIALS
QUANTITY ITEM DESCRIPTION
10 micro liters Escherichia Coli
10 micro liters Staphylococcus Aureus
Lots Cotton Swabs
Lots  Disposable Pipette Tips
Lots  Saline
40 12X75 Test Tubes
40 Blood Agar Plates
40 Inoculating Loops
1 10 Micro litter Pipette
1 200 Micro litter Pipette
1 Colorimeter
Lab Coat
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PROCEDURES

1. First, gather the following bacteria on day 0.
a. Escherichia coli
b. Staphylococcus aureus 
2. Next, make suspension for the bacteria.
3. Divide the bacteria between 20 vials, with 200 micro-liters per vial.
4. Label the vials.  
  a. Label five vials, S V. 1, V. 2, V. 3, V. 4, and V. 5 -20 Celsius 
  b. Label five other vials S V. 1, V. 2, V. 3, V. 4, and V. 5 - 30 Celsius
  c. Label five other vials S V. 1, V. 2, V. 3, V. 4, and V. 5 - 60 Celsius
  d. Label the five remaining vials S V. 1, V. 2, V. 3, V. 4, and V. 5 -10 Celsius
5. Repeat step 4, but label the vials with an E instead of an S for Escherichia Coli
6. Freeze the bacteria at the four temperatures,
  -20 Celsius
  -30 Celsius
  -60 Celsius
  -10 Celsius
7. On day one, take vial one out, thaw, and let it sit for 10 min. at room temperature.
8. Refreeze vial one.  
9. On day two take out vial 1 and 2, thaw, and let it sit for 10 min. at room temperature.  
10. Refreeze vial one and two.
11. Repeat steps 6-7 with vials 3, 4, and 5.
12. On day six, thaw all the bacteria
13. Pipette 10 micro-liters to a blood agar plate.
14. Spread the bacteria with inoculating loop over the surface of the blood agar plate.
15. Incubate bacteria overnight at 37 degrees Celsius
a. Do a colony count.  
16. Destroy bacteria in autoclave-using hospital’s procedures.  

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RESULTS

The original purpose of this experiment was to determine how extreme freezing would affect the subsequent growth rate of bacteria.

The results of the experiment were that the colder the temperature the bacteria were kept frozen, there were less bacteria colonies.

See my Data tables and graphs.


CONCLUSION

My hypothesis was that the colder the temperature, the fewer the bacteria colonies would subsequently be.

The results indicate that this hypothesis should be accepted

Because of the results of this experiment, I wonder if there would be a difference if I froze different types of bacteria.

If I were to conduct this project again, I would do a couple of things differently. First, I would try different types of bacteria. Second, I would try different freezing levels. Third, I would freeze them longer. Fourth, I would incubate more blood agar plates of plates.

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RESEARCH REPORT

Introduction
Freezing food is very important to human health. It helps because freezing foods that contain bacteria will stop bacteria growth rate for a while. If you leave foods unfrozen for a long period of the time, bacteria will grow in the foods and if you eat then, it could make you ill.  

Bacteria
Bacteria are one-celled organisms that exist almost everywhere. They exist in water, soil, air, even the human body. There are bacteria inside you, there are bacteria around you. Bacteria are so small, they measure about 0. 3 to 2. 0 microns (1 Micron=0. 001 millimeter. ) Scientist classify bacteria as prokaryotes. Some bacteria are scrounging which means parasitic. They cause diseases by producing poisons called toxins. Others are either harmless or even helpful. There are many types of bacteria. Here are a few examples of commonly known bacteria. Cocci, are round looking bacteria, Bacilli, which are rod shaped bacteria, Vibrios, which look like bent rods, and Spirilla, which look like spiral- shaped bacteria.

Helpful Bacteria
Some kinds of bacteria live in either the insides of human beings or animals. The bacteria help produce vitamins needed in the body, destroy harmful organisms, and help digestion. Some bacteria even help recycle carbon, nitrogen, sulfur, and other elements used by living things such as animals. “Many bacteria help decompose dead organisms and animal wastes into chemical elements. ” World Book Encyclopedia

Harmful Bacteria
Some bacteria cause diseases in humans such as cholera, leprosy, pneumonia, syphilis, typhoid fever, and whooping cough. Bacteria enter a person’s body either through the nose, mouth, or through breaks in the skin. “Certain bacteria produce toxins, which cause diseases such as diphtheria, scarlet fever, and tetanus. ” World Book Encyclopedia. Some bacteria toxins are produced by living bacteria, but others are released only after a bacterium dies. Some toxins cause a type of food poisoning called botulism from bacteria in improperly canned foods. Bacteria that are harmless that usually live in the body may cause diseases or infections when a person’s resistance to disease is low. Some bacteria in the throat reproduce faster than a body can dispose them. This can result in a sore throat. Bacteria also cause diseases in animals and plants such as anthrax, which is a disease that infects many cattle, sheep, and other animals. Two bacteria plant diseases are fire blight, and soft rot. Fire blight occurs in apple and pear trees. Soft rot decays some fruits and vegetables. Some bacteria cause growths called crown galls. Crown galls attack various plants.  
 

Protection Against Harmful Bacteria
A lot of bacteria live in the mouth, intestines, and on skin. The rest of the body tissues are free of bacteria. “The skin and the membranes that line the digestive and respiratory systems, prevent most harmful bacteria from entering the rest of the body. ” World Book Encyclopedia. When some bacteria enter the body, white blood cells surrounds the bacteria and attack them. The blood produces antibodies that kill or at least weaken them. A certain type of antibody called antitoxins destroys toxins. If the body cannot make its own antitoxins fast enough, a physician may inject an antitoxin from another person or from an animal such as a rabbit. Dead bacteria are used in making vaccines, which can prevent the disease caused by those types of bacteria. Vaccines are injected into the body. The vaccines cause the blood to produce antibodies that attack the bacteria. Some vaccines protect the body from infection for several years.

The Structure of Bacteria
Nearly all kinds of bacteria are surrounded by a tough protective layer called a cell wall. This wall gives the bacteria its shape and lets it live in a wide range of environments. Some bacteria are also enclosed by a capsule, a slimy layer outside the cell wall. The capsule enables the cell to resist certain types of chemicals. All bacteria have a cell membrane. The cell membrane is an elastic, baglike structure just inside the cell wall. “Small molecules of food enter the cell through pores in this membrane, but large molecules cannot pass through. ” World Book Encyclopedia. The cytoplasm is inside the membrane. Cytoplasm contains chemicals called enzymes, which helps break down food and build cell parts. Like the cells of all living things, bacterial cells contain DNA, which controls cells activities such as growth, and reproduction. The DNA of a bacterial cell forms on area of the cytoplasm called the nucleoid. In all other organisms except the blue-green algae, the DNA is in the nucleus, a part of the cell separated from the cytoplasm by a membrane.  

Where Bacteria Live
Bacteria live almost everywhere, including places where other life forms cannot survive. Bacteria are always present in the respiratory systems and the digestive systems and on the skin of human beings and other animals. Some bacteria need oxygen to live and are called aerobes. Other bacteria such as anaerobes can survive without it. Some anaerobes can exist with or without oxygen. Other anaerobes cannot live with even a trace of oxygen. “Some bacteria protect themselves against a lack of food, oxygen, or water by forming a new, thicker cell membrane inside the old one. ” World Book Encyclopedia. The cell material surrounding the new membrane dies and the remaining organism becomes inactive and is called a bacterial spore. The bacterial spore may live for years and years because it can resist harsh conditions and extremely high or low temperatures. The spores change back into active bacteria if food, water, and oxygen become available.

How Bacteria Move
Bacteria are carried long distances by water currents, air, clothing, utensils, and animals. Some kinds of bacteria have thin hairs called flagella that let them “swim”. Some species that do not have flagella move by wriggling.  

How Bacteria Obtain Food
Heterotrophic bacteria are a kind of bacteria that feed on other organisms. Autotrophic bacteria, manufacture their own food. Photosynthetic bacteria make food from water, sunlight and carbon dioxide. Some bacteria may be either heterotrophic or autotropic, depending on the food available. Some heterotrophic bacteria are parasites. A majority of heterotrophic bacteria feed on dead organisms. Some parasitic bacteria cause diseases, but others cause little or no harm.

How Bacteria Reproduce
Most Bacteria divide into two identical cells by a process called binary fission. Most bacteria reproduce quickly. Some double their number every twenty minutes. In ten hours the bacteria would produce over a billion bacteria if it were given enough food. Industrial and laboratory process often produce enormous numbers of bacteria, but in nature, bacteria lack an adequate food supply to maintain such a high rate of reproduction. Scientist have created methods that allow them to cut off fragments of DNA and then insert these fragments into different bacteria.  

History
Bacteria were probably the first living things on the earth. The oldest known fossils belong to bacteria that lived about 31/2 billion years ago. Bacteria were first explained in the mid-1670 by a scientist named Anton van Leeuwenhock, a Dutch scientist. Scientists believed that bacteria came from nonliving matter, until 1800 when French chemist Louis Pasteur showed that only living things can produce living things. Louis Pasteur and Robert Koch, a German physician, helped develop the science of bacteriology.


Summary

Bacteria are tiny organisms much smaller than a millimeter. They have a huge affect on health, both helpful and harmful.

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BIBLIOGRAPHY

“Bacteria,” Britannica Intermediate Encyclopedia, December 9, 2002. October 29, 2003

"Bacteria" December 3, 2003, 

“Bacteria,” ENCARTA Encyclopedia Deluxe, 2001.

Bacteria (singular bacterium) Helicon Publishing Ltd, 1998. 1

Clark, Marie. Personal Interview. January 5, 2004

Marquis, Robert E. Bacteria November 18, 2003 http://www. encarta. msn. com

Schlessinger, David. “Bacteria,” World Book Encyclopedia, 2002.

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ACKNOWLEDGEMENTS

I would like to thank the following people for helping make my project possible:

  • My mom for driving me to Yakima Memorial Hospital.  
  • Mr. Newkirk for helping me correct my report.  
  • Marie Clark for helping me conduct my experiment on such short notice.  
  • Mrs. Helms for helping me correct my computer problems.

 
 


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