Biology Concepts
Wild Microbes Lab Project, Page 1
Where are the wild microbes?
OVERVIEW
The goal of this lab is to test for the quantity and diversity of bacteria and other
environmental (wild?) microbes from three different locations. We’ll start by choosing
locations and hypothesizing as to which location will harbor more wild microbes. Then,
we’ll sample (or swab) from those locations, culture our samples, and gather data on the
diversity and quantity of microbes that we have found at each location. Finally, we’ll
analyze that data to determine which location harbors the greatest quantity and diversity
of wild microbes.
SCHEDULE
1ST lab day: CHOOSING AND SAMPLING LOCATIONS (STEPS ONE, TWO)
2nd lab day: GETTING DATA FROM PLATES (STEP THREE)
3rd lab day: LOOKING AT MICROBES IN MICROSCOPE (STEP FOUR)
At home: LAB WRITE-UP POSTER
STEP ONE. CHOOSING LOCATIONS (1st LAB DAY)
Choose three locations that you would like to sample for bacteria and other microbes. Try
to choose one location where you believe there may be no microbes, one location that
might be typical, and another location that you believe might have lots of microbes.
LOCATION
Level of microbial
presence
1.
Little or none
2,
Medium or typical
3.
Lots
Reason for
guessing/hypothesizing
this level of microbes
STEP TWO. SAMPLING FROM EACH LOCATION (1st LAB DAY)
Materials needed:
 Permanent ink marker
 Three Petri dishes
 Three cotton swabs
 Vial of sterile water
 Incubator (if available).
1. Label the bottom of each Petri dish with your name, date and the location to be
sampled using a permanent ink marker
2. Use a new sterile cotton swab for each location so that you are not contaminating
your culture with microbes from anywhere else.
3. Dip the swab in sterile water and then rub it over the sample site
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Wild Microbes Lab Project, Page 2
4. Then gently slide your swab back and forth all across the surface of the agar in the
Petri dish.
5. Discuss what temperature the incubator should be set at. What is the best
temperature for bacteria? What is the best temperature for rapid growth? How
long should our cultures be left to grow before we interpret them?
STEP THREE. INTERPRETING YOUR COLONIES OF BACTERIA (2ND LAB DAY)
1. Count the total number of colonies you have from each location
2. Describe the different kinds of colonies from each location according to the
following characteristics:
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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.
Chromogenesis (pigmentation) - For example, white, buff, red, purple, etc.
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Wild Microbes Lab Project, Page 3
Science Buddies: Interpreting Plates
(http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_Interpreting_Plates.shtml)
(great website for seeing what you might find on your plates and how to interpret it)
Example of a colony description:
Size: 2mm diameter
Shape: Circular
Margin: Entire
Elevation: Convex
Surface: Smooth
Density: Opaque
Pigment: Yellow, nondiffusible pigment
From: http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Colony%20Description%20result.html
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3. Now define particular colony types (for example jagged yellow, smooth white, fuzzy
white, etc.) and then count how many of each colony-type you have at each
location. Make a table in your notebook to do this…it might look something like
this…
NAME OF
COLONY
Yellow jagged
(example)
DESCRIPTION
Small round colonies,
ragged edge, ridges
on surface, bump in
middle of profile
DRAWING
NUMBER OF COLONIES
Location
1
0
Location
2
6
Location
3
3
STEP FOUR. LOOKING AT MICROBES IN MICROSCOPE (3rd lab day)
1. Choose three or four different types of bacteria to look at in the microscope.
2. When handling bacteria and other microbes, assume they could be infectious and
always use your best sterile technique. Never directly touch the bacteria. Always
sterilize your inoculating loop in the Bunsen burner after it’s had bacteria on it.
3. To see your microbes in a microscope, first you have to prepare a smear. This is how
to make a bacterial smear:
Place an almost invisible speck of bacterial growth in the center of a clean slide Be sure the
slide is clean. Use your marker to draw a line down the slide so you know which side is up.
Add a tiny drop of water to your specimen with your inoculating loop.
Now, with your inoculating loop, mix the specimen with the water
completely and spread the mixture out to cover about half of the
total slide area.
Gently pass the slide through the Bunsen burner to fix the bacteria so they won’t fall off. Slide
should just feel hot to touch.
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4. Gram Staining. Follow these procedures for gram-staining so you can see your
bacteria in the microscope (demo to be given in lab):

A heat-fixed bacterial smear is first covered completely with a few drops of a solution of crystal violet, a purple
basic dye.
 After 30-60 sec the smear is rinsed with water by squirting the slide above the smear and letting the water
wash over it until the water runs clear.
 Several drops of iodine (the mordant) are applied to cover the smear and left for 60 sec., then rinsed again.
 A few drops of an isopropanol-acetone mixture or similar solvent are added at a time until the wash is
colorless, then the slide is rinsed again.
 A red basic dye, aqueous safranin, is applied for 30-60 sec.followed by a rinse.
 The smear is blotted (not wiped) to remove excess water, using bibulous (absorbent) paper or a paper towel.
The slide is then air dried the rest of the way.
How to screw up a Gram stain
 Screw up the smear to start with
 Apply stain to the wrong side of the slide
 Stain or rinse the smear incompletely
 Allow stain to dry up before rinsing
 Forget to decolorize or decolorize incompletely
 Rub off the material when you blot it
5. Observing stained bacteria in the microscope
To look at bacteria in the microscope requires patience and practice. Never put a
cover slip on and always use the highest-power oil immersion lens with a drop of
immersion oil on the slide. The hardest part is finding the bacteria. Focus first on the
black line down the middle of the slide. The bacteria should be off to one side.
STEP 5. MAKING YOUR LAB WRITE-UP POSTER (at home).
Your poster write-up should include the following. It will be presented and graded
during our evaluation week. Be as creative as you like. Use a big piece of poster-board
or butcher-paper so everyone will be able to see your poster easily.
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Title of project and your name.
Hypothesis or proposal with explanation of different sites where bacteria were
collected.
Materials and methods. This should have text and images—drawings or
photographs—of how we collected bacteria, how we analyzed the plates and how
we made stained smears to look at in the microscope.
Results/Interpretation of bacterial colonies. This should clearly show how many
colonies, and how many of each type of colony you found at different locations. It
should show these results in a table and also describe them with words. You also
might include how some of the different bacteria looked different in the microscope
Analysis. This section MUST then use the results to decide if your original
hypothesis or proposal about where the most bacteria would be found is correct. It
does not matter whether it was right or not. You should then have a few sentences
about why you think it was right or wrong.