Name_____________________________________________________________________________ Date_______________________
Investigating the Effect of _________________________ on Bacterial Growth
Developed by Ana Caldeira and Amanda Tsoi; HHMI Summer Workshop 2013
Introduction
You can find bacteria everywhere, even on your desk and on your skin. Although they are
much too small to be seen individually without the help of a powerful microscope, you
can see evidence of their presence by growing colonies in this lab.
There are many variables that can affect the growth of bacteria. In this lab, you will be investigating the
effect of a natural product on B. Subtilis bacterial growth.
B. subtilis is rod-shaped bacteria found in soil and in the human gut. When it is in
extreme environmental conditions, it has the ability to form a tough, protective
endospore, allowing it to “hibernate” safely. When favorable environmental
conditions return, the endospore can convert to a growing
bacterial cell. Within 24 hours, one bacterial cell will divide by
binary fission and produce over a million. This population of
cells is called a colony.
Because we only want to investigate B. Subtilis bacterial growth, we do not want
to contaminate our experiment with other species of bacteria. In this lab, we will
be using sterile technique, which means you will need to:
o wear gloves, goggles, and lab coats
o spray lab station with 70% ethanol and wipe clean before and after lab
work
o do not touch the inside of the Petri dish (including the inside of the Petri
dish lid)
o only remove the Petri dish cover when absolutely necessary
o whenever the lid is removed, it should be held over the plate as a shield to
prevent dust and bacteria from falling into the Petri dish
In order to form your hypothesis and title your lab, you will need to decide what variable you would like
to test. It needs to be a naturally occurring substance. Some examples are spices (i.e. cinnamon,
turmeric), food plants (i.e. garlic, ginger), outdoor plants (i.e. pine needles, bark), etc.… Some of these are
provided in the lab; if you would like to test something else, you may bring it to class.
Hypothesis
If bacteria are exposed to ______________________________ (experimental variable), then the
population of bacteria will _______________________________________________(increase or decrease).
Materials
o 70% ethanol spray bottles
o 2 petri dishes (plates)
o LB agar (algae-based gelatin with
nutrients for the bacteria to grow on)
o B. Subtilis bacterial spores
o parafilm (to seal plates)
o
o
o
o
o
150mL beaker
hot hands
stirring rod
pipette
*your experimental variable*
Procedure – Day 1
1. Put on goggles, gloves and lab coat.
2. Spray down work area with 70% ethanol and wipe clean.
3. Obtain two Petri dishes and on the bottom label one “Control” and one
“Experimental Variable”. Put your initials and date on both. Refer to the
diagram on the right.
4. Using the hot hands, obtain a 150mL beaker with 30mL hot LB agar.
5. Using the hot hands, carefully pour half of the LB agar into the “Control”
Petri dish. Cover the plate immediately after.
6. Add a small, grape-sized amount of your experimental variable to the remaining LB agar in the
beaker. Ensure that your experimental variable is in a homogenous (uniform), mushy or liquid
form, without chunks. Stir this into your LB agar.
7. Using the hot hands, carefully pour the rest of the LB agar-mixture into the “Experimental
Variable” Petri dish. Cover the plate immediately after.
8. Bring your plates to the storage area. Make sure they are properly labeled.
Procedure – Day 2
1. Put on goggles, gloves and lab coat.
2. Spray down work area with 70% ethanol and wipe clean.
3. Bring your plates from last class to your teacher. He/she will pipette 1mL of B. Subtilis
bacterial spores onto your plate for you.
4. With the plate covered, gently tilt the plate from side to side to make sure the entire surface of
agar is covered with the liquid containing the spores.
5. Seal both plates with parafilm and bring them back to the storage area.
Procedure – Day 3
1. Put on goggles, gloves and lab coat.
2. Spray down work area with 70% ethanol and wipe clean.
3. Bring your plates from last class to your station.
4. Observe your bacterial colonies. Use the key below to write your observations in your
qualitative data chart.
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•
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•
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Criteria Used to Describe Bacterial Colonies
Form - What is the basic shape of the colony?
For example, circular, filamentous, etc.
Elevation - What is the cross sectional shape of
the colony? Turn the Petri dish on end.
Margin - What is the magnified shape of the
edge of the colony?
Surface - How does the surface of the colony
appear? For example, smooth, glistening, rough,
dull (opposite of glistening), rugose (wrinkled),
etc.
Opacity - For example, transparent (clear),
opaque, translucent (almost clear, but distorted
vision, like looking through frosted glass),
iridescent (changing colors in reflected light),
etc.
Pigmentation - For example, white, buff, red,
purple, etc.
From: http://www.sciencebuddies.org/science-fair projects/project_ideas/MicroBio_Interpreting_Plates.shtml
5. Tape your plate to graph paper.
6. Count the colonies of bacteria from top right to bottom left, as shown
in the diagram below. If you have more than ~200 colonies, divide
your plate into 4 quadrants, only count one quadrant, and multiply
this number by 4.
7. Record your data in the quantitative data chart.
8. Clean up (keep gloves, goggles and lab apron on):
a. With gloves on, submerge one plate at a time in the bleach-water container. While
under water, remove the cover to expose the bacteria to the bleach. This will kill the
bacteria. Take the plate out of the water and put it in the trash.
b. Return all materials to their appropriate storage spaces.
c. Spray down work area with 70% ethanol and wipe clean.
d. Throw away gloves, return goggles and lab apron.
Data
Qualitative Data Chart
Observations
Control Plate
Experimental Plate
Form
Elevation
Margin
Surface
Opacity
Pigementation
Smell
Other
Quantitative Data Chart
Plate
Control
Experimental Variable
Number of Colonies of Bacteria
Analysis
Complete the bar chart below based on your quantitative data (for your lab report, you will need to insert
a bar chart designed in Microsoft Excel). Title your graph and label the axes. The x-axis should be
labeled with your independent variable (this rate/value remains the same regardless of the other
variable). The y-axis should be labeled with your dependent variable (the value depends on the
independent variable).
Write three factual statements about your data.
1.
2.
3.
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Conclusion
Answer the following question in paragraph form:
A. Use your quantitative data to explain whether or not your experimental variable impacted
bacterial growth.
B. How does this compare to your hypothesis?
C. Provide an idea for why you obtained the results that you did. Use at least one website or other
source.
D. Explain why you are unable to make a definitive conclusion about the impact of your
experimental variable on bacterial growth based on this experiment alone.
E. Did you make any mistakes? Explain.
F. At what points during the lab could you have been more accurate (measuring, counting, following
procedures, etc..)?
G. If you performed this lab again, what would you do differently to obtain more accurate results?
Bibliography
For all websites used, include the title, author, date and URL. For all other resources, include the title,
author and date.