Designing Lab Exercises to Simulate Pathogen

Designing Lab Exercises to Simulate Pathogen Transmission
Elizabeth Ingram, Presenter: Valencia College, Orlando, FL
eingram@valenciacollege.edu
Objectives:
After completing this exercise, the students should be able to:
1. understand the difference between infection and disease;
2. understand the manner by which infectious agents are transmitted from person to person;
3. understand the manner by which infectious agents are transmitted from food and water;
4. perform serial dilutions, calculate dilution factors, and determine bacterial density of the sample using a standard
formula;
5. perform the spread plate technique and count colonies on plates showing appropriate growth;
6. evaluate the applications of the viable plate count as a quantitative procedure in microbiology;
7. analyze various food items for the presence of potential pathogens;
8. extrapolate collected data to illustrate the importance of sanitation and safe handling practices;
9. show that they can relate the information to public health safety challenges.
Background:
Infection is defined as the entry of a microorganism into the body of the host. Disease results only when the infectious
agent causes the appearance of typical signs and symptoms in the host. In 1876, Robert Koch established the link
between the causative agent and infectious disease in his famous Koch’s Postulates. These postulates still hold true
today. The public health practice that involves the study of the occurrence of diseases in terms of time, place and
distribution is called epidemiology.
Infectious agents can be transmitted by direct contact between hosts by skin to skin contact, body fluid contact, or by
droplet nuclei (e.g. cough, sneeze). They can also be spread indirectly by contact with contaminated inanimate objects
called fomites (e.g. beddings, towels). Sometimes pathogens are ingested as contaminants of food or water. Others are
transmitted from one host to another by means of arthropod vectors, which serve either as mechanical (e.g., houseflies)
or biological (e.g. mosquitoes) carriers of pathogens. Another interesting aspect of epidemiology is the fact that some
human or animal hosts serve as continual sources of infection, and they are called reservoirs. Those individuals who
harbor pathogens without exhibiting signs and symptoms are called carriers.
Food microbiology deals with the study of the role of microbes in human enteric disease and food spoilage. As we know,
foods and drinks are common vehicles by which bacterial diseases of the digestive system are transmitted. During
processing and preparation, food may be contaminated with microbes from the soil, animals, food handlers and
machinery. Sanitary standards have been set by government agencies so that the quality of food and drink can be
controlled and regulated. In this way consumers are protected from food infections and food intoxications. The general
public frequently refers to the resulting illness as food poisoning. Public health laboratories routinely test for the
presence of coliforms (gram-negative, non-spore-forming bacilli that ferment lactose with gas production) in foods and
drinks because these organisms serve as indicators of fecal contamination.
To test foods for the presence of enteric pathogens, the standard or viable plate count is used to determine the total
number of bacteria in a food sample. Large numbers of bacteria in food are usually equated with the presence of
pathogens or increase the potential for food spoilage. The standard plate count has some clinical and diagnostic
applications as well. For instance, it is useful in determining the clinical significance of urinary tract infections and severity
of bacteremic and septicemic infections.
A serial dilution of the original sample is performed and an aliquot of each dilution is inoculated on agar plates using the
spread plate technique. After incubation, only plates containing 30-300 colonies are counted. Plates containing fewer
than 30 colonies will give unreliable counts and those containing more than 300 are too crowded to provide accurate
counts. Counts are expressed in terms of CFU (Colony Forming Units) per mL. CFU replaced the term colony to
account for the fact that a single growth on the plate sometimes results from a clump of cells attached together rather than
a single cell. The original bacterial density of the sample can be calculated using the following formula:
Number of colonies counted
Original cell density =
= CFU/mL
Volume plated x Dilution Factor
Dilution Factors:
Basically, dilutions can be expressed in the form of a fraction, a ratio or an exponential figure. Either one can be used.
The following general formula may be used:
Volume of the sample
Dilution Factor =
Total Volume (Sample Volume + Diluent Volume)
0.1 mL
For example in Tube 2 =
0.1 mL
=
0.1 mL + 9.9 mL
Dilution factor for Tube 2 is 1:100 or 1/100 or 10
10.0 mL
-2
Sometimes it is not necessary for food microbiologists to know the exact number of bacteria in order to predict a viable
health threat by a bacterial pathogen. The occurrence of a new strain of E. coli, E. coli O157:H7, in the late 1980s
precipitated the labeling of meat packaging with safe handling practices. This strain has the potential to cause illness and
death in very small numbers. In addition, the exponential growth rates of bacteria by binary fission can result in very high
numbers of bacteria in food and drink in a short time given the perfect conditions provided by the lack of refrigeration.
Some of these bacteria also produce toxins, which are not inactivated by the reheating of food. The following is the safe
handling label that now appears on all meat products. It appears in red to draw the public’s attention to it.
SAFE HANDLING INSTRUCTIONS
THIS PRODUCT WAS PREPARED FROM INSPECTED AND PASSED MEAT AND/OR POULTRY. SOME FOOD
PRODUCTS MAY CONTAIN BACTERIA THAT COULD CAUSE ILLNESS IF THE PRODUCT IS MISHANDLED OR
COOKED IMPROPERLY. FOR YOUR PROTECTION, FOLLOW THESE SAFE HANDLING INSTRUCTIONS. KEEP
REFRIGERATED OR FROZEN. THAW IN REFRIGERATOR OR MICROWAVE. KEEP RAW MEAT AND POULTRY
SEPARATE FROM OTHER FOODS. WASH WORKING SURFACES (INCLUDING CUTTING BOARDS), UTENSILS,
AND HANDS AFTER TOUCHING RAW MEAT OR POULTRY. COOK THOROUGHLY. KEEP HOT FOODS HOT.
REFRIGERATE LEFTOVERS IMMEDIATELY OR DISCARD.
There are several methods in place in the food industry to reduce or eliminate pathogenic bacteria from food. One
important practice that has been in effect for over a hundred years is pasteurization. There are now several variations on
the original method but the outcome is the same – the maintenance of food quality with a reduction in bacteria that cause
spoilage and disease.
This exercise is composed of two experiments designed to provide students with active learning experiences pertaining to
food and water safety. The first experiment involves sampling of meat purchased from a local grocery store for potential
pathogens and the importance of using safe handling practices when handling raw meat. Second, a scenario is provided
that investigates the effects of a water main break on the contamination of water by Escherichia coli. Students will
determine if water has been contaminated at six locations and if the levels of contamination warrant intervention by the
health department.
Materials:
Body Fluid Transmission
TSB culture of Micrococcus luteus (mystery tube)
Trypticase Soy Broth (TSB)
Trypticase Soy Agar (TSA) plates
Meat Experiment:
Samples of beef, chicken, fish, and pork
Sterile Pasteur pipettes
Sterile cotton swab
Media: TSB, LSB, PA, MSA, BESC
Water Experiment Standard Plate Count:
Water samples seeded with Escherichia coli
Eosin Methylene Blue Agar (EMB) plates
Two 9.9 mL Dilution tubes
Three 9.0 Dilution tubes
Sterile 1 mL pipettes and Pipettor
Beaker containing ethanol
Bent glass rod (“Hockey Stick”)
Colony counter
Part A: Pathogen Transmission by Body Fluid Exchange
This experiment is designed to demonstrate how quickly a pathogen can be spread through contact with body fluids. Your
results will have a real-life application, especially if you associate it with the rapid increase in the number of individuals
infected with sexually transmitted diseases each year. One “mystery” tube has been inoculated with Micrococcus luteus.
Will you be the one to spread the infection?
1. Obtain a TSA plate and a mystery TSB tube from your instructor (each student will work with a set of TSA and
TSB).
2. Label your TSA plate by drawing a small circle at the top center and writing your tube # (as assigned by your
instructor) inside the circle.
3. Draw 4 separate parallel lines across the plate below your number. These will eventually be labeled with the
number of each person you exchange your sample with.
4. Soak a sterile cotton swab in your mystery TSB tube and then touch the swab to the circled area on your plate.
5. Leave your mystery tube on your bench top while you are move around the room carrying your swab with you.
6. To exchange “body fluids” with another student, introduce your swab into TSB tubes of your classmates. Your
classmates will go around and randomly sample others too.
7. Swirl the swab twice in their tubes, and then roll your swab across the TSA plate on the first, second, third or
fourth line depending on which level of exchange is involved.
8. Label the first, second, third or fourth line on your plate with the other students’ tube#.
Note: Make “fluid exchanges” with 4 other students that belong to different groups. You will be moving
throughout the lab so be especially careful not drip your sample on the floor or touch your swab to other objects and
items.
9. Place your swab back into the original paper wrapping. Then break and discard the swab in the biohazard
container.
10. Incubate your plates in an inverted position at 37C for 24 to 48 hours.
11. After incubation, examine your plates for growth and enter your results in the Observation and Results section in
Table 1.
Part B: Safe Food Handling of Meat
1. Each group will be assigned to work on a specific food item by the instructor. (1-beef, 2-chicken, 3-fish, 4-pork, 5beef, 6- chicken). These food items were purchased from a local grocery store.
2. Each group will obtain the following media: TSB, LSB, PA, MSA, and BESC.
3. The food sampling technique is as follows:
a. Soak a sterile cotton swab in TSB for 5 seconds.
b. Vigorously scrub a 2.5 cm area (postage stamp size) of the sample for 30 to 60 seconds, rotating the
swab as you scrub.
Note: This is the only time you will scrub to sample the food.
4. Aseptically inoculate a tube of LSB (Lauryl Sulfate Broth) using the dip and swirl method.
5. Using the same swab, inoculate your plates and BESC tube in this order: PA (Pseudomonas Agar), MSA
(Mannitol Salt Agar), and BESC (Bile Esculin Agar).
6. Roll the swab across plate in 5 parallel lines and use a fish tail for BESC.
7. Place swab back in the original paper, break and discard the swab in the biohazard container.
8. Mark your plates as recommended. Incubate them at 37C for 24 hours.
9. After incubation, read your plates and enter your results and those of other groups as well in the Observations
and Results section in Table 2.
Part C: Pathogen Transmission of Escherichia coli in Water
D
F
C
B
E
A
Case Study:
Sophia T. was driving home from work in the early morning hours and noticed “a lot of water on the road”. Her concern
prompted her to contact the authorities, who in turn notified the water department at which you are employed as the
manager of the water quality division. When the emergency response team arrived on the scene, they traced the hazard
to a break in the water main and started working to repair the water lines. You have advised the local authorities to issue a
boil water warning for the area. Your assistant has just handed you six water samples and now it is your job to test the
water and determine which samples indicate a potential health threat if any to the residents in the affected area.
In the previous map, the star represents the flooded area that Sophia had encountered. Water samples were taken from
other parts of the water lines at the points identified by the letters A through F.
Note: The success of this procedure depends on strict adherence and careful performance of dilutions, plating and
counting techniques. Even the slightest deviation from the protocol or errors made through careless performance of the
procedures and techniques can yield grossly inaccurate results.
Day 1: [Work in your assigned groups.]
1. Obtain one of the water samples.
2. Prepare serial dilutions of this sample as follows:
-2
-4
-5
-6
-7
a. First label 5 test tubes containing sterile saline solution as follows: 10 , 10 , 10 , 10 , and 10 .
b. The first two tubes will contain 9.9 mL of saline solution. The last 3 tubes will contain 9.0 mL of sterile
saline solution.
-5
-6
-7
-8
c. Next label 4 EMB plates as follows: A, B, C, D, E, or F (water sample); 10 , 10 , 10 , and 10 (final
dilution factor); your group; date of inoculation;
d. Using a sterile 1 mL pipette, transfer a 0.1 mL aliquot of the sample to the first tube and 1mL to the second
-2
-4
-5
-6
-7
tube (10 , 10 ), and then 1.0 mL aliquots to the each of the next 3 tubes (10 , 10 , and 10 ).
Caution: Use a different pipette for each transfer!
e. Mix these tubes well by tapping the bottom of each tube before transferring the sample to the next tube.
(Alternately you may use your Pipettor to mix the samples by aspirating and expelling the fluid a few times
inside the tube)
Aseptic technique precautions and reminders:

DO NOT set the pipette down on the desk. Maintain sterility at all times.

DO NOT handle the part of the pipette to be inserted into the tube. Only grasp the pipette from the end.



DO NOT leave the canister of pipettes open to the unsterile environment. Replace the cap of the canister
after aseptically removing each pipette.
Place all used pipettes in a steel beaker with disinfectant, point down.
Flame the mouth of each tube after removing the cap and before replacing it.
3. Aseptically transfer 0.1 mL from the last four of the diluent tubes to the 4 agar plates as follows:
-4
-5
a. Transfer 0.1mL from the tube marked 10 to 10
-5
-6
b. Transfer 0.1mL from the tube marked 10 to 10
-6
-7
c. Transfer 0.1mL from the tube marked 10 to 10
-7
-8
d. Transfer 0.1mL from the tube marked 10 to 10
Remember: The final dilution factor (FDF) incorporates the 0.1 mL aliquot as if it were an additional tenfold
-5
dilution. For example, the FDF for the first plate would be 10 .
4. Aseptically spread the drop of inoculum transferred over the surface of the EMB plate using a glass rod (hockey
stick) by rotating the plate while the glass rod is touching the surface of the plate.
5. Place all plates in the incubator under the appropriate laboratory section shelf at 37C for 24 to 48 hours.
Day 2:
1. After incubation, examine all the plates and select those that show between 30 and 300 colonies. Set aside and
dispose of all plates that are not countable.
2. Count the colonies using the Colony Counter with the aid of a thin marking pen to avoid miscounting and
duplicate counting of colonies. Counts of two plates of the same dilution factor should be averaged out before
using in the calculations.
3. Determine the cell density of the original sample by using the standard formula.
4. Enter your results in the Observation and Results section in Tables 3 and 4.
Observations and Results:
Part A: Pathogen Transmission by Body Fluid Exchange
1. Examine your plate for growth starting with your own spotted sample, and then read the spaces for the first, second,
third and fourth exchanges. Enter your individual result and tally it for the entire class in Table 1.
Table 1 – Bacterial Transfer Patterns Observed on Individual Plates and Collated as a Group Experience
Your Data
Growth
(+)
Class Data
First Exchange
First Exchange
Second Exchange
Second Exchange
Third Exchange
Third Exchange
Fourth Exchange
Fourth Exchange
Number of
Positives
% Positive
2. Draw a graph by plotting the number of students infected on the Y-axis versus the number of “fluid exchanges” on the
X-axis in Figure. Analyze the graph and draw your conclusions about the manner and ease by which disease can be
transmitted from one host to another.
Part B: Safe Food Handling of Meat
Read your tubes and plates for the presence or absence of bacterial growth. Refer to the Table of Culture Media in the
Appendix for proper interpretation of results. [LSB-look for coliforms; PA - look for Pseudomonas; MSA - look for
Staphylococcus; BESC-look for Enterococcus]. Enter your results in Table 2.
Table 2 – Bacterial Contaminations in Meat, Fish and Poultry Tested
LSB
Beef
Chicken
Fish
Pork
PA
MSA
BESC
Part C: Pathogen Transmission of Escherichia coli in Water
Standard Plate Count:
1. Examine your plates and determine or estimate the number of colonies observed for each dilution and enter your
results in Table 3.
Table 3 – Number of Colonies Counted on EMB Plates Inoculated With Serially Diluted Water
Plate
Final Dilution
Factor (FDF)
Colony Forming Units (CFU)
Per Individual Plate
1
2
3
4
2. Calculate the approximate number of bacteria present in the water sample using your average count data
obtained from Table 3 and the formula below:
Average CFU
Original Cell Density =
= [expressed in CFU/mL]
Final Dilution Factor
Your computations:
Your answer: ____________________
Table 4 – Final Colony Counts for Each Water Sample Location
Samples
OCD (CFU/mL)
A
B
C
D
E
F
Questions:
NOTE: Answers to some of the questions below can be found at the Centers for Disease Control and Prevention website:
http://cdc.gov
Part A: Pathogen Transmission by Body Fluid Exchange
1. In the disease transmission lab, how many students were infected? Is this about the number you expected?
2. According to your graph, how many body fluid exchanges would need to occur for the entire class to be infected?
3. What would be the quickest way for a pathogen to spread through any given population?
4. How would the dynamics be different if we would be investigating the outbreak associated with a food-borne
pathogen?
Part B: Safe Food Handling Of Meat
1. What is the purpose of using the set of differential and selective media in this experiment?
2. Do you think it would be a greater health threat to have high numbers of bacteria growing on MSA or PA? Why?
(Hint: What organisms are selected for by each medium?)
3. What might be the source of contamination (i.e. where did the bacteria come from) if the LSB tube is coliforms (+) or
the BESC tube is Enterococcus (+)?
4. What might be the source of contamination (i.e. where did the bacteria come from) if there are a large number of
organisms growing on MSA?
5. What might be the source of contamination (i.e. where did the bacteria come from) if there are a large number of
organisms growing on PA?
6. How would cooking alter the results of this food microbiology experiment?
7. What are the most common pathogens associated with food-borne illness?
8. Which pathogen and food was associated with the most recent outbreak of food-borne illness?
9. What is the difference between infection and intoxication as it pertains to food-borne illness? How do the symptoms
vary?
10. Have you ever read the “safe handling” label on the meat package? (Refer to the Background Section.)
11. How does performing this experiment affect how you handle the meat that you are serving at home?
Part B: Pathogen Transmission of Escherichia coli in Water
1. Which location(s) showed evidence of water contamination by E. coli? What pattern of contamination was evident
from where the break in the water main was reported?
2. Did the data show that there was more than one point source contributing to the contamination?
6
3. If an infectious dose of 10 CFU/mL is needed to cause illness, which water sample(s) would have had high enough
levels of E. coli to be of concern?
4. Infection by E. coli O157:H7 may occur with only 10 CFU/mL. Which sample(s) would be of concern if the
contaminated water contained strain O157:H7?
5. What are the symptoms of an intestinal infection caused by E. coli? How do the symptoms differ if the infection is
caused by the strain O157:H7?
6. If a “boil water advisory” is issued, how should you treat the water to make it drinkable? Once the boil water advisory
is over, what advice does the CDC give?
7. Name three other water-borne infectious agents.
Appendix – Table of Culture Media
Abbreviation
Purpose
C
Bile Esculin
Agar (BESC)
Isolation of
Enterococcus
S
&
D
Eosin
Methylene
Blue Agar (EMB)
Isolation of
Gram-negative
Enterics
Lauryl Sulfate
Broth (LSB)
Special
Ingredients
Preparation
Inoculation
Reading Criteria
SA = Bile
DA = Esculin
Typical
Fish Tail
1.Black =
Enterococcus +
2. Not black =
Enterococcus -
S
&
D
SA = Eosin and
Methylene Blue
DA = Lactose
Typical
Quadrant
Streak
1.Metal green sheen,
black, or pink mucoid
= coliforms +
2.Not as above =
coliforms -
Detect/ID
coliforms
in foods
S
&
D
SA = Sodium
Lauryl Sulfate
DA = Lactose
SP = Durham
tube
Place Durham
tube in test
tube before
autoclaving
Dip & Swirl
1.Gas bubble in
Durham tube =
coliforms +
2.No gas =
coliforms -
Mannitol
Salt
Agar (MSA)
Isolates and
differentiates
Staph species
S
&
D
SA = 7.5%
NaCl;
DA = Mannitol;
pH Indicator =
Phenol Red
Typical
Quadrant
Streak
Pseudomonas
Isolation Agar
(PA)
Isolation of
Pseudomonas
species
S
SA = Irgasan®
ES = Glycerol
Glycerol
added prior
to autoclaving
Quadrant
Streak
Trypticase
Soy Agar (TSA)
Growth of wide
range of bacteria
G
None
Typical
Varies
Trypticase
Soy Broth (TSB)
Growth of wide
range of bacteria
G
None
Typical
Dip & Swirl
1.Growth, medium is
lemon-yellow =
mannitol
fermentation +
2. Growth, medium is
pink = mannitol
fermentation3. No growth =
Staphylococcus –
1. Growth on
streaked line =
Pseudomonas +
2. No growth=
Pseudomonas –
Growth of wide range
of bacteria; making
smears & lawns
Growth of wide range
of bacteria; making
smears & lawns
Legend:
Category (C):
B = Biochemical
D = Differential
S = Selective
G = General
Special Ingredients:
ES = Energy Source
DA = Differential Agent
SA = Selective Agent
SP = Selective Property