The Effect of Various Temperatures on the Type and Growth Rate of Bacteria in Meat
 Photo of Researcher

Researched by SARAH S.
2005-06



Purpose
The first purpose of this experiment was to determine the effect of temperature on the growth rate of bacteria in meat.

The second purpose of this experiment was to determine the effect of temperature on the types of bacteria that grow in meat.

I became interested in this idea when my mom would thaw out the meat in the sink then leave it there until we cooked it. I wondered how long it took the meat to spoil in different temperatures.

The information gained from this experiment could help homemakers and the food service industry protect people from food poisoning. Hospitals, schools, and restaurants would all benefit.


 Hypothesis
My first hypothesis was that the most bacteria would grow in the temperatures between 21° and 37° Celsius.

I based my first hypothesis on a statement made my the U.S Department of Agriculture's Meat and Poultry Hotline that said  “Bacteria grow most rapidly in the range of temperatures between 21° and 37°C. If the temperature is above 32°C, food should not be left out more than one hour.”

My second hypothesis was that the four main types of bacteria that would grow were Staphylococcus aureus, Salmonella enteriditis, Escherichia coli O157:H7, and Campylobacter.

I based my second hypothesis on another statement made by the U.S. Department of Agriculture’s Meat and Poultry Hotline which said “leaving food out too long at room temperature can cause bacteria (such as Staphylococcus aureus, Salmonella enteriditis, Escherichia coli O157:H7, and Campylobacter) to grow to dangerous levels that can cause illness.”

 Experiment Design
The constants in this study were:
  •  Amount of Meat
  •  Temperatures Used
  • Sterile knives and grinder
  •  Autoclave
  •  Incubator and incubation temperature
  •  Petri Dishes
  •  General test method
The first manipulated variable was the storage temperatures:
  •  Freezing (0ºC)
  •  Refrigeration (4ºC)
  •  Room Temperature (20ºC)
  •   Body Temperature (36ºC)
The second manipulated variable was the types of meat used.
  •  Hamburger
  •  Sausage
  •  Steak
  •  Chicken
The first responding variable was the amount of bacteria in the meat after storage.

The second responding variable was the type of bacteria that grew after storage.

To measure the responding variable I took the saline/meat "slurry" and spread it on the blood agar plates and incubated them for 48 hours. Then I took a colony count and identified the bacteria using the API 20 E procedures (see appendix.)
 

Materials

Quantity Items
15
 Set of gloves
2
 Incubators
1
 Autoclave
1
 Vortex
78
 Petri dishes
4
 Disposable Grinders
1
 Saline Fluid
1
 Pound of Beef
1
 Pound of Chicken
1
 Pound of Steak
1
 Pound of Pork
80
 Loop  Spreading tips
1
 Freezing Temperature (0°C)
1
 Refrigeration (4°C)
1
 Body Temperature (36°C)
1
 Room Temperature (20°C)
1
 Lab coat
4
 Sterile knifes
 



Procedures
1.  Purchase approximately 5 ounces of four different types of meat
  •  Hamburger
  •  Steak
  •  Pork
  •  Chicken
2. Go to the Yakima Valley Memorial Hospital
3. Wash hands
4. Put on lab coat and gloves
5. Prepare the sterile knife, grinder and scissors.
6. Cut one square inch meat out with sterile knife.
7. Cut meat into one square cm pieces and place in sterile grinder.
8. Grind for two minutes
9. Fill grinder with 50 ml. saline fluid
10. For one minute vortex the saline/meat “slurry”
11. Divide slurry into 4 different ten ml. containers
12. Discard leftover slurry in autoclave
13. Label each container with type of meat and number
14. Place four containers in each temperature at hour zero
  •  Freezing (0ºC)
  •  Refrigeration (4ºC)
  •  Room Temperature (20ºC)
  •  Body Temperature (36ºC)
15. Take one slurry and add 10 micro L onto blood agar.
16.  Return slurry to storage temperature.
17. Spread with loop and place in incubator at time zero.
18. Label agar
19. Repeat steps 5 through 18 with each type of meat.
20. At hour 12 repeat steps 15 though 18.
21.  Repeat step 19 with each meat from each temperature at each hour
  •  12 hours
  •  24 hours
  •  36 hours
  •  48 hours
22. After the 48 hour incubation period remove plates from incubator
23. Count colonies with unaided eye under a white light and record.
24. Autoclave all contaminated items
25.  Follow procedures for Gram Stain from appendix on page
26.  Identify types under microscopes
27.  Follow procedures “API 20 E” from appendix on page
27. Wash hands.



 Results
The original first purpose of this experiment was to determine the effect of temperature on the growth rate of bacteria in meat.

The second purpose of this experiment was to determine the effect of temperature on the types of bacteria that grow in meat.

The results of the experiment were that Hamburger had the most bacteria at 0 hours and chicken had the least. Freezing all of the meats was more effective in limiting the growth rate of bacteria than storing the meats in any of the other temperatures. All meats grew the most in the 37 degree temperature at 48 hours and the least in 0 degrees centigrade temperature.

Pork had on average about 13 bacteria colonies at freezing temperature, 135 colonies at refrigeration temperature, 6000 colonies at room temperature, and 7563 colonies at body temperature. The types of bacteria on the meat were Aeromonas, Hydrophilia, Serratia, Non fermenting GNR, Serratia plymothica, and Staph Coagulant.

Chicken had, on average, about 13 bacteria colonies at freezing temperature, 27 colonies at refrigeration temperature, 1200 colonies at room temperature, and 4600 colonies at body temperature. Overall, the chicken had the least amount of bacteria colonies. The types of bacteria were Bacillus, Diptheroids-Skin flora, Staph Coagulent, and Escherichia Coli.

Steak had, on average, about 90 bacteria colonies at freezing temperature, 260 colonies at refrigeration temperature, 9000 colonies at room temperature, and 9875 colonies at body temperature. The types of bacteria were Diphtherioids/Cat P, enterococci/Cat WR, Micro Coccus, and Serratia.

Hamburger had about 852 bacteria colonies at freezing temperature, 7275 colonies in refrigeration temperature, 12,125 colonies at room temperature, and 14,000 colonies at body temperature. Overall, Hamburger grew the most bacteria in any temperature. The types of bacteria were Micro coccus, Enterococci, Streptococcus ap, Serratia sp, Enterobacter cloacae, and Acinetobacter baumanii


 Conclusion
My first hypothesis was that the most bacteria would grow in the temperatures between 21° and 37° Celsius. The results indicate that my first hypothesis should be accepted. All the meats grew at a steady rate and had the most growth at 37°.

My second hypothesis was that the four main types of bacteria that would grow were Staphylococcus aureus, Salmonella enteriditis, Escherichia coli O157:H7, and Campylobacter.The results indicate that my second hypothesis was partially correct. We did find campylobacter, staphlylococcus and a strain of Escherichia coli but not Escherichia coli 0157:H7 or Salmonella. I rejected this hypothesis.

After thinking about the results of this experiment, I wonder what would happen if I did my procedures again with cooked meat. Would the bacteria content be completely different? Would we still find pathogenic bacteria?

If I were to conduct this project again I would make sure all my meats had the exact same “pull” dates. Also when I counted my bacteria colonies I would make sure I had more exact numbers instead of some estimations. I would use a more exact counting system for that, perhaps a microscope with a millimeter grid.



Research Report

Bacteria
Bacteria are very important single-celled organisms that do not have distinct cell structures. Without bacteria we could not live and our earth could not function. Bacteria can be harmful but most are helpful and found everywhere from our intestines and mouth to our skin.  They are in dirt, plants and food. They have been around for as long as the earth has. There is an uncountable amount of different types of bacteria on the earth. Certain types can withstand temperatures ranging from millions of degrees above boiling to thousands of degrees below zero.

Bacteria are found in four different shapes. Cocci, which are shaped like little round balls.  Rods are rectangle or stick shaped. Sprilla are usually long and squiggly. And Vibrio  are commonly known as a comma shape.  Some bacteria can also have flagella, which are long whip like structures to help them move. Different bacteria are identified by the way they behave, if they clump or chain. They can have spores (little holes) or may create a slime layer for protection or to help with movement.

Of the four types of bacteria, all are either gram negative or gram positive. The difference is mainly between the wall layers.  The gram-positive cells tend to be more pathogenic and have thicker walls than the gram negative.  To identify the difference the bacteria are taken through a number of simple steps (staining) to find the gram reaction. The gram reaction was named after a Danish physician Christian Gram. He created the staining technique in 1884. He took the bacteria that had been dried on slides and applied a series of liquids and water to the slide. Then after rinsing the purple liquid (Crystal Violet) away with water he determined the type by its colorization; purple is gram positive and pink is gram negative.

Escherichia Coli
 E. coli was discovered by a German Biologist named Theodor Escherich, thus the name Escherichia Coli. He found the bacteria in the human colon and discovered it caused diarrhea and other digestive problems.  Science has found over a hundred strains of E. coli. Not all E. coli is harmful and almost all types are naturally found in our body. After the discovery of E. coli scientists found that it was easy to make this organism grow. Because of this they could use it to find the results for many tests sooner.

Escherichia Coli 0157:H7 is the most dangerous type of E. coli.  When first discovered E. coli 0157:H7 was only found in humans who had recently eaten hamburger. Since then it has been found in fruits, vegetables, milk and milk products and sometimes in lake water. In 1982 the first two cases of E. Coli 0157:H7 were found in Oregon and Michigan. In 1998 in a small town in Wyoming it took only a few days to infect more than sixty people. Their symptoms were terrible stomach cramps and bloody diarrhea. If bloody Vvvvvvdiarrhea occurse it is sign to get immediate stool testing done for E. coli 0157:H7.  E. Coli 0157:H7 infects about 73,000 humans annually in the United States and sends 2,100 people to the hospital. It causes around 61 deaths each year in the United States. Since the first case was detected in 1982 E. coli infections have been reported in more than thirty countries from six continents.

Salmonella
Salmonella much like E. coli causes severe diarrhea, stomach cramps, vomiting and fever. Salmonella can be found in almost all foods such as fruits, vegetables, milk and a majority of meats but it is most likely found in raw poultry and eggs. Cooking usually kills this organism.  It can be passed from the feces of animals and humans to other living creatures. Salmonella can be contracted from reptiles such as pet iguanas, lizards, turtles and snakes.

Salmonella only affects about 40,000 victims in the United States every year. When it infects children, infants, the elderly or people with weak immune systems it causes a more severe illness and they are more likely to need hospitalization. The infection may travel into the blood stream, then into the joints which could cause irritation in the eyes and also painful urination. If this is the case then it can lead to chronic arthritis that may last for months or even years, this is called Reiter’s syndrome and is very difficult to treat.

After you have been infected with salmonella the symptoms will come into process anywhere from twelve hours to three days. Most people usually improve around four to seven days but can be in progression for up to two weeks. People who have been infected with the illness can be a carrier for up to a year.

Bacillus
Bacillus are gram positive rods. There are many types of Bacilli, all are harmless except for two types; B. anthracis, which causes anthrax in sheep, cows and sometimes humans, and B. cereus which causes types of food poisoning that usually infects milk, cereal and stew but recently it has been found in fried rice. The symptoms of vomiting and diarrhea are similar to those of staphylococcus food poisoning. Since the bacteria grow in the infected food after it has been cooked it is very important to keep the food cold immediately after cooking.

 Most types of Bacillus are found in the soil, water and air borne dust and are sometimes used for testing. When the conditions are stressful a type of bacillus can create endospores so they can stay dormant for long periods. There is only one other known species of bacteria able to do that.

Skin Flora
Skin Flora is a very common gram-positive bacterium that is found almost everywhere.  It differs from age, body location, gender, temperature, humidity also what kind of soaps and disinfectants you use too. Gram-positive bacteria have peptidoglycan. Peptidoglycan is a polymer that enables the cell wall to be considerably strong under many pressures.  It is usually not harmful and is found in or on skin or meat. Some species are closely related to the type of bacillus flora that live in the human intestine. Humans naturally have flora in and on their bodies, it is difficult to find the difference of a infective flora and a natural flora. When a normal flora is transferred from its analyzed site to another a infection may occur. The infection is usually not very harmful but can cause hospitalization if not treated correctly.
        
 Staphylococcus aureus 
Staphylococcus aureus (occasionally called the “Golden Staph”) are gram-positive circular shapes a millimeter in diameter. They are the main cause for “staph infections” which hospitalize over 500,000 Americans a year. Many healthy people carry S. aureus in their nostrils naturally. IT can also enter the body if the skin is punctured, cut or broken. Surface areas or dirty skin can also transfer it easily from human to human.

Staphylococcus aureus causes a variety of pathogenic infections. Illnesses such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections occur from S. aureus. S. aureus can cause skin infections including boils, styes and furunculosis. Also it is a likely cause of food poisoning, osteomyelitis, endocarditis and can cause toxic shock syndrome by releasing superantigens into the blood stream. Finally it is also a major factor for hospital acquired (nosocomial) infection caught from surgical injuries.   
               
History
A Dutch tradesman named Antony Van Leeuwenhoek was not an educated man. Yet he was very curious and discovered many things on his own. He found bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes, tooth plaque, rotifers, and much more. He invented his own microscopes that magnified up the object up to two hundred times its natural size. His magnifying glass had only one lens and was about four or five inches long. It was very simple and required little knowledge and a lot of patience. It was said he had about five hundred of these, only thirty still remain. Robert Hooke’s compound microscope that had been built about forty years before only magnified the object thirty times its original size. it required little knowledge and a lot of patience. 

Leeuwenhoek never wanted to be a great discoverer, he was just curious. In June of 1716 he said “My work, which I've done for a long time, was not pursued in order to gain the praise I now enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in most other men. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof.” Antony van Leeuwenhoek lived from 1632-1723 and started the discoveries that have turned into the medical world we have today

Meat
Meat is one of the most consumed foods in the US.  Over 8 billion pounds of beef alone are sold in America each year. Out of all meat consumed, beef and poultry make up 33% each. 25% of all the meat sold and consumed is pork leaving 8% for fish and 1% for lamb and mutton.  There are two ways to cook meat. Dry heat and moist heat depending on the tenderness and the cut of the meat. More specific methods are broiling; pan broiling, roasting, pan-frying, deep-frying and more.

Meat Inspection
In 1906 comprehensive legislation was introduced.  This meant that 60 % of all the meat including cattle, hogs, sheep, goats and even reindeer had to be inspected before death or after death if they were going to sold nationally or internationally in the US.  In 1967 all the meat had to meet federal inspection.


Summary
It is important to know about meat and the bacteria inside meat for more that one reason. Mainly, so we can know when and how to cook our food so we can be safe and healthy. 


Bibliography

"Bacteria that Causes Human Disease."  Microsoft Encarta. 2005

Blackburn, C. " Food Spoilage." Food spoilage Microorganisms. October 27th, 2005. <http://www.woodheadpublishing.com/en/book.aspx?bookID=835>

 Chavarria, Gabriel. "Causes of Food Spoilage." Spoilage October 27th, 2005 <http://www.arches.uga.edu/~gchava/Causes.html>

 Clark, Marie. Mentor, October-January, 2005.

  Fracklam, Howard and Margery. Bacteria. Canada: Fitzhenery and Whiteside Ltd. 1994

  Hayhurst, Chris. E. Coli New York: Rosen Publishing Group Inc., 2004

 Marguis, Robert E. "Bacteria" Microsoft Encarta. 2005.

"Meat." World Book Encyclopedia. 2002. Volume 13

 Medic8® Family Health Guide. "Patients Guide." Salmonella. December 1st  2005. <http://www.medic8.com/healthguide/articles/salmonella.html>

 National Center for Infectious Diseases. "Division of Bacterial and Mycotic Diseases." Escherichia coli O157:H7. October 27th, 2005. <http://www.cdc.gov/ncidod/dbmd/diseaseinfo/escherichiacoli_g.htm>

 Purdue University Animals Sciences. "Meat Quality and Safety." Meat Spoilage. October 27th 2005. <http://ag.ansc.purdue.edu/meat_quality/spoiled_meat.html>

 Stone, David and Martha B. Talor, Stephanie. "Food Processing and
 Preservation." Microsoft Encarta. 2002

 Williams, Willard F. Miller, Markus F, "Meat Packing Industry." Microsoft  Encarta Standard. 2007. 2002.
 

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