MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #25: FOOD MICROBIOLOGY

NAME, ID #:_______________________________________________.

REVISION DATE: 08/04/99

INTRODUCTION

Microbial contamination of food has been a problem for humans throughout history. During most of the 5 million years of our evolution hominids ate grossly contaminated food; food covered with dirt, chewed on by rats and mice, infested with maggots and various microbes, rotten to various degrees etc. Clearly we are not as delicate as we may imagine ourselves. On-the-other-hand spoiled and rotten food probably did in a large number of our ancestors and surely made a lot more of them often extremely uncomfortable. We are only the third generation to have the benefits of safe and secure food preservation methods. As I've discussed with you in lecture, at 65 I can recall when we had to depend on the "ice-man" and a wooden ice-box to keep our food fresh and that was HIGH-TECH. Prior to the end of WWII (1945) frozen food was a rarity and far too expensive for the common folk (who didn't have a freezer to put it in anyway). While canning had been around since the early 1800's, its mechanism of preservation was not understood nor was the significance of the canning temperature appreciated until the late 1800s. The bottom line is that until my lifetime a lot of people, even in the US, ate a lot of rather bad food; stuff we would today gag at and toss in the nearest dumpster.

Maintaining a safe food supply is a never ceasing struggle. It was only since the 1930s when a book by Sinclair Lewis exposed the filthy conditions in the country's slaughter houses that the Federal Government began to regulate food safety. Even today there are those who feel that our food supply is over regulated and that there is little reason for concern about the safety of our food supply. What do you think? Many of you have worked in the food service industry. Do you think that removing the safety rules and regulations would result in a significant change in the quality of our food?

In the past few year the Northwest region has suffered more than its share of serious food borne bacterial disease caused by a new strain of E. coli, O157:H7. This virulent pathogen has killed several people and have left a number severely injured for life. Even Pullman has not been spared an epidemic of this bacterium, but I won't name names. This bacterium has been found most commonly in ground meats like hamburger. However, it has also been detected in a variety of other foods including apple cider, fruit juices and produce. Although a lot of research has been done to pinpoint the source of this organism, it is still not known. Beef cattle seem to be one of the prime carriers of E. coli, O157:H7, but other animals may also be involved. The danger is such that anyone who eats poorly cooked ground meat must be considered at risk for contracting this disease.

Most of you have been told by your mother to store foods in the refrigerator and to wash your hands before handling food, either for eating or in preparing them. In this exercise you will estimate the bacterial contamination in some hamburger purchased at a local supermarket. You will see the effect on improper storage on the numbers of bacteria in the food and you will observe how high salt or sugar concentrations inhibit the growth of many bacteria.

Below are some microbes associated with food; some good, some very bad characters.

PURPOSE OF LABORATORY:

1. To learn the spread-plate count technique.
2. To learn how to estimate the bacterial contamination of hamburger and the effect of its storage conditions upon the number of bacteria.
3. To understand the preservative nature of high osmotic pressure.

RELATIONSHIP TO LECTURE MATERIAL

• NetText: CHAP. XVIX , Food microbiology lectures.

GENERAL INSTRUCTIONS & MATERIALS:

1. Please place all appropriately labeled drawings on the back of the manual so the instructor can identify them.
2. Samples of hamburger that have been stored properly and improperly.
3. Media containing a high concentration of salt or sugar.

PROCEDURE: Bacterial Counts in Variously Stored Meat

1. Read pg. 83-85 in A Photographic Atlas for the Microbiology Laboratory. Study the details of the dilution procedure on pg. 83-84 so that you understand "dilutions" as it relates to the tubes or bottles and then to the plates.
2. NOTE: The following dilutions may not work and your instructors may change to procedure to insure that you obtain suitable results. For example, they may supply you with a 1:100 dilution of the sample, in which case all the dilutions would be increased 10-fold.
3. Obtain a 1:10 dilution of the sample of the hamburger from the instructor (20 gm + 180 ml of sterile water). Record the history of the sample. One half of the lab will test a sample that has been stored in the refrigerator since its purchase and the other half will test a sample that was left out at room temperature overnight.
4. Obtain, per pair, 4 plates of count media, a 100 ml dilution blank, disposable plastic pipettes, a spreading rod and a beaker of ethanol.
5. Label the plates as follows:
   • Undiluted, 1 ml, 1:10
   • Undiluted, 0.1 ml, 1:100
   • Diluted, 1 ml, 1:1000
   • Diluted, 0.1 ml, 1:10,000
6. Remove 1.0 ml from the 1:10 sample and transfer it into the dilution bottle and mix thoroughly. This will give you a 1:1000 dilution of the original sample.
7. Place the dilutions on the respective plates. Work backwards starting with the MOST HIGHLY diluted sample first.
   • Add 0.1 ml of the sample from the dilution bottle (1:1000 dilution) to the center of the #4.4 plate (1:10,000 dilution) and then 1.0 ml of the same sample to the #4.3 plate (1:1,000 dilution). Using the same pipette repeat the process with the UNDILUTED sample to the #4.2 & #4.1 plates respectively.
8. Remove the glass spreading rod from the ethanol breakers and pass it through the flame. DO NOT HOLD IT IN THE FLAME. The ethanol will catch fire. Allow it to burn off AWAY FROM THE BEAKER. Hold it for about 30 seconds until it cools.
9. Successively spread the plates in EXACTLY the following order: the 1:10,000 plate first; the 1:1,000 plate second; the 1:100 plate third and the 1:10 plate last. Do not flame the spreading rod between plates.
   • Allow the plates to sit a room temperature until the liquid soaks into the agar, then place them in the incubator.
10. Incubate the cultures at 37^oC until the next lab and then count the number of colonies per plate. Plates with greater than 300 isolated colonies are to be labeled TNTC (= Too Numerous To Count). Prepare a table relating the dilution to the number of colonies and calculate the number of bacteria per gm in the original sample. Place your result on the board with your team names under the appropriate sample heading. Compared your results with others testing the same sample and with the results from the other sample.
11. Write down your conclusions; the TA will discuss the results.

PROCEDURE: Effect of High Osmotic Conditions on the Growth of Bacteria

1. Obtain 4 broth tubes per pair with the following concentrations of salt or sugar: Salt- 0, 1%, 5%, 15% or sugar- 0, 5%, 25%, 50% (percentage by weight; i.e. for a 50% sugar solution add the dried medium (for 100 ml of medium) to 50 gm of sucrose and add enough water to bring the solution to 100 ml final volume).
2. Label each tube.
3. Inoculate each tube with 10 drops of the original meat suspension (allow the large chunks of meat to settle out first).
4. Mix well.
5. Incubate as described by the Instructor.
6. Estimate the amount of growth in each tube following incubation, assuming that the growth in the control tube was 4+ and no growth is (-).
7. Record these results in the table below.
8. Gram stain a sample form a tube showing growth.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• Explain why cuts of meat like steak are not considered as dangerous for bacterial infection as is hamburger.
• What are the symptoms of E. coli, O157:H7 infection?
• What is the treatment for an E. coli, O157:H7 infection?
• How can you tell if your hamburger is cooked properly? (Sending it to a lab for testing is not the correct answer)
• Using information LEARNED PREVIOUSLY in the course, determine how you might test specifically for E. coli, O157:H7 bacteria in the meat so that you could rapidly detect only a few 100 bacteria per gram?

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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