Download (Effect of Tears on Bacterial Growth).

Name:
Date:
Period:
Teacher’s Guide:
Effect of Tears on Bacterial Growth
Objectives:
Students will learn:
 how to perform a controlled experiment
 how to make serial dilutions
 how the skin and bodily fluids, such as tears, contain substances
that kill microorganisms
 the skin is the first line of defense, an example of innate immunity
 lab skills: micropipetting, streak plate, inoculation, incubation of
bacteria
 purpose of a control
Before passing out lab:
1. Go over innate vs. adaptive immunity
3. Go over anatomy of bacteria including description of the peptidoglycan
cell wall- a large polymer with many covalent bonds (“chain link fence”).
Show image below.
4. Mention that penicillin (extracted from mold, Penicillium) acts by
preventing the cross-linking in the cell wall of many types of bacteria, which
leads to cell lysis. This interference with cell wall synthesis makes penicillin
an antibiotic.
5. Go over Ecology standard 10a. Not all bacteria are harmful; they are part
of the ecology of the human body, particularly the large intestine. In many
cases, bacteria exist in a mutualistic relationship to humans. In the large
intestines, for example, bacteria synthesize Vitamins K, and some B
vitamins)
Introduction: Bacteria can survive in almost every environment on Earth. Why is
it, then, that we don’t get sick all the time? There are two kinds of immunity:
innate and adaptive. Innate immunity refers to immediate, non-specific defense
which does not depend on antigens. Adaptive immunity, on the other hand,
entails a specific antigen and results in immunological memory. In this lab, you
will explore the effects of tears, which contain the enzyme, lysozyme, on the
growth of bacteria.
Science Standards:
6a. Students know biodiversity is the sum total of different kinds of
organisms and is affected by alterations of habitats
10a. Organisms have a variety of mechanisms to combat disease. As
basis for understanding the human immune response:
Students know the role of the skin in providing nonspecific defenses
against infection.
Vocabulary:
Lysozyme- enzyme in human tears, egg white,saliva, and other body fluids
Bactericidal-anything that kills bacteria
Bacteriostatic- anything that limits the growth of bacteria by interfering with protein
synthesis, replication, or any aspect of bacterial metabolism
Serial dilution- stepwise reduction in concentration- “watering down”- of a particular
substance in a neutral agent
Inhibition- the suppression of bacterial growth
Innate immunity-inborn, immediate, non-antigen based immunity, which does not
confer immunological memory; response is the same every time
Adaptive immunity- antigen-specific immunity in which exposure results in
immunological memory (clones of B lymphocytes, specific antibodies)
State the Problem: How do tears affect bacterial growth?
Read through this lab and formulate your hypothesis.
Hypothesis: _____________________________________________________
Any answer is acceptable
Materials:
onions
E.coli: HB101 K–1lyophilized on LB/agar
inoculated filter paper discs saturated with dilutions of tears
micropipettes
microcentrifuge tubes
DI water
Incubator
Permanent markers for labeling
Procedure
1. Slice several onions in an attempt to stimulate tear formation in your own
eyes.
2. Swab corner of ears with filter paper. Place soaked filter paper into a tube
with 10uL DI water.
3. Label 4 microcentrifuge tubes with the 4 serial dilutions you will make:
1/.1/.01/.001
4. Make 4 serial dilutions of tears (1, .1, .01, .001) Transfer 5uL of tear
solution into 50 uL DI water. Make three dilutions total.
5. Label petri dishes so that there are 5 “pie slices” with tear dilutions
labeled.
6. Soak filter paper discs into each prospective solution
7. Add inoculated filter paper disc into appropriate quadrant
8. Incubate plates for 48 hours at 37C.
OP TIONAL; students may use saliva, which also contains lysozyme
Making serial dilutions:
5uL
5uL
5uL
Add 5uL from each tube to
the adjacent tube to make
10-fold dilutions of tears
45uL
water
Tube # 1
10 uL tearsremove 5uL and
put into next test
tube
Tube #2
45uL
water
Tube #3
45uL
water
Tube #4
#4
4. Observations:
In the diagram below, record the 4 tear dilutions, one per “slice.” Indicate
approximate number of bacterial colonies in each quadrant. Show “zones of
inhibition” in each quadrant.
Control (DI
water )
No inhibiton
No inhibition
Pure tears Maximum zone of
inhibition expected
Dilution = .1
Smaller zone of inhibiton
expected
Dilution = .001
Smallest zone of
inhibition with tears
Dilution = .01
The amount of bacterial inhibition going from least to most is as follows:
1. Control (pure DI water)
2. .001 tear solution
3. .01 tear solution
4. .1 tear solution
5. pure tears
Analysis Questions:
1. Why was the control in this lab?
Answer: The control is the plate of DI water- not containing the variable
you are testing.
2. Why is a control group necessary in this lab?
Answer: A contol plate is a point of comparison (guide) to help you interpret the
experimental results. Without the control, you would not know that it was the
tears that caused increasing levels of bacterial death.
3. If you wanted to make 2-fold dilutions of tears (doubling the dilution each
time) with a starting quantity of 10uL tears, how would you make the
dilutions? Make a labeled drawing below.
Possible answer: dilutions showing 1:2, 1:4, 1:8, 1:16 ratios of tears to DI
water.
4. Explain how this lab relates to innate or adaptive immunity.
Answer: This lab relates to innate immunity, a generalized, all or none
immune response; tear response is the similar with every exposure, no buildup of specific white blood cells and antibodies result as with adaptive
immunity.
5. Conclusion
In 5 paragraphs, answer the following questions:
1.
2.
3.
4.
5.
Write a summary of the lab’s purpose, materials, and procedure
Re-state your hypothesis and explain whether it was right or wrong.
Summarize the lab’s result
What does the data indicate about the role of skin in human immunity?
What were some avoidable and unavoidable sources of error? How could
you extend this lab in an effort to explore the effects of tears on bacterial
growth?