Access Excellence Classic Collection

Demonstrating An Epidemic

M. Beth Powel
1993 Woodrow Wilson Biology Institute

Introduction

Target Group:

Class Time Required:

Teacher Materials:

• 1 24 hour nutrient broth culture of Micrococcus roseus or Serratia marcescens
  (see teacher preparation guide)
• Sterile water
• Sterile serological pipette
• Piece of hard red candy per student (peppermints work well) $1.00
• Sterile Petri dish per student
  (74-1348 $7.84/pkg of 30)

Student Materials:

• 1 sterile cotton swab per student
  (70-3033 $14.30/box)
• 1 plate sterile nutrient agar per student
  (82-1860 $13.50/pkg of 10 or 82-1861 $109.00/pkg of 100)
• If you can prepare these yourself it is MUCH cheaper!
• 1 numbered Petri dish containing a piece of red candy treated with 2 mL of unknown liquid
• Disinfectant hand soap
• Disinfectant cleaner
• Disposable gloves (if desired, medical/surgical supply about $13.00/ 100)
• Laboratory marker
• Lab coats
• Incubator, 32-37degC

Safety Precautions:

Teacher Preparation:

1. Broth culture: Both Serratia marcescens (15- 5450 $6.95) and Micrococcus roseus (15-5160 $6.95) are provided as agar/cultures from which a broth culture must be made. Sterile nutrient broth tubes (82-6120 $10.60/10) are available but it is much cheaper if you can prepare your own. To prepare a broth culture you will need a sterile tube of nutrient broth, a stock plate culture of Micrococcus roseus or Serratia marcescens, an inoculating loop and a Bunsen burner, alcohol burner or incinerator.

   a. Sterilize the inoculating loop by holding the wire portion in the flame until it glows red. Allow to cool briefly and test for coolness by stabbing the wire into an uncontaminated portion of the agar plate.

   b. Gently stroke across a colony to obtain a small amount of inoculum on the loop.

   c.While holding the contaminated loop in one hand, unscrew the top of the broth tube and pass the open mouth of the tube quickly through the flame to prevent air contaminants from entering.

   d. Place the loop into the broth and agitate to dislodge the inoculum. Again pass the mouth of the tube through the flame and screw the lid loosely in place. Sterilize the loop as previously described. Incubate the tube upright in a 32-37degC incubator overnight.

2. Unknown candy samples: Prepare at least 30 minutes prior to class to allow sufficient dissolving to occur.

   a. Carefully unwrap and drop a single piece of red candy into a sterile Petri dish, one per student. Avoid handling the candy directly to prevent unwanted contamination. Cover immediately.

   b. Treating only one plate at a time, lift the lid of the dish and pipette 2 mL of sterile water onto the surface of the candy. Reclose the dish. Treat all candy plates with water except one. On this final plate pipette 2 mL of broth culture.

   c. Number the plates, noting the number of the plate containing the bacterial contaminant. All the plates should appear to have identical contents.

3. Data collection: On the day of the experiment record the number of the students as they are called to shake hands. On the second day each student will record the number of red colonies counted on that plate so that the entire class may share data.

4. Sterilization of contaminated materials: All contaminated materials must be sterilized before discarding. This may be accomplished by steam sterilization at 15 psi for 15 minutes.

Student Instructions:

1. Obtain all materials. Lab coats must be worn whenever dealing with microbes. Label the sterile nutrient agar plate on the bottom portion with the experimenter's name, date, and the number indicated on the candy dish.

2. If gloves are being used, place glove on left hand. Alternately, spray both hands with disinfectant spray and wait one minute. Then wash hands with disinfectant soap and rinse in sterile water. Do not dry. Keep clean or gloved hand slightly closed to avoid outside contamination.

3. Carefully pour the liquid and the candy into the left hand. Roll the candy around until the hand is thoroughly moistened. Avoid allowing any liquid to escape. Return the candy to the original dish and close the hand.

4. When all experimenters are similarly exposed the instructor will direct a sequence for shaking left hands based on the numbered dishes. Making firm hand contact is essential to the success of the experiment. The first individual will shake hands with the next one called, then return to inoculate the nutrient agar plate. The second individual called will then shake hands with the third before inoculating. Each individual, except the first and last called, will shake with two others before inoculation.

5. Remove a sterile cotton swab from its package with the right hand. Thoroughly moisten the swab with candy liquid by rolling the cotton tip across the contaminated hand. Cover all of the cotton surface.

6. Carefully lift the lid of the agar plate to limit outside exposure. Using a rolling motion, gently rub the swab over the surface of the agar in a close zig-zag pattern; completely cover the face of the agar. Close the lid of the dish. Return the swab to the empty package and discard to be sterilized.

7. If a glove was used, place it in a container to be sterilized. If not, repeat the disinfection procedure described earlier.

8. Place the inverted agar plate into the incubator for 24 hours. Disinfect the areas used, taking particular care to clean any drops of liquid on tables or the floor. Remove lab coats. Record the sequence of contact for the entire group.

9. After 24 hour incubation, remove the plate from the incubator. Count the number of red colonies on the plate and record with class data. If there are a large number of red colonies, divide the bottom of the plate into 6 or 8 equal wedges. Count one half of the wedges, choosing those that have a representative distribution and multiply the total number counted by two. Arrange the plate with those of the other experimenters so that they represent the contact sequence and observe the general trend in numbers of red colonies. Prepare a data chart indicating the sequence of contact and the number of red colonies per plate for the entire class. Indicate those individuals who got the "disease" by a + and those who were disease free by a -. Place a = by the individual who started the epidemic.

10. Discard plates in sterilization containers and disinfect all areas used.

Questions:

1. Which individual began the "epidemic? How can this be determined from the class data?

2. Were there individuals who appear to have passed on the organism without having the disease? How does this appear in the data? Does this occur in an actual epidemic?

3. Describe at least two ways in which this model epidemic differs from a real one.

Reference:

Additional activity to simulate vaccination:

What would happen if some individuals had been vaccinated against this epidemic? You can simulate this situation by selecting some students to receive "vaccinations." These students simulate vaccination by covering their left hands either with a second glove placed over the first, or with a piece of sterile foil (you can sterilize foil squares with steam sterilization as above.) Vaccinated students place the glove or foil "cover" in the proper waste receptacle after having their hands shaken, but before shaking the next person's hand. Thus, the vaccinated individual shakes the next person's hand with a "clean" hand. Complete the experiment as above. What happens to the spread of the epidemic? Vary the number of people receiving the vaccination? How do more (or fewer) vaccinated people effect the outcome?

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