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Hands-On Labs SM-1 Lab Manual
60
EXPERIMENT 6: Microbes Everywhere
Read the entire experiment and organize time, materials, and work space before beginning.
Remember to review the safety sections and wear goggles when working with chemicals.
Part 1 takes 3 to 6 days to incubate. Allow 15-20 minutes per slide for Part 2.
Objective: To demonstrate the growth and characteristics of microorganisms.
Materials: Student Provides: Paper towels Timer or clock
Distilled water Clear tape
Candle, matches, or lighter
From LabPaq: Safety goggles
Clinical face mask
Disposable gloves
Test tube clamp
Thermometer
Wax marker pencil
Microscope slides, 5
Field microscope
Hand magnifying lens
Metal spatula
Staining tray
Experiment Bag: Sterile slant tubes of agar, 6
Sterile swabs, 6
Gram Stain Kit: Crystal violet solution
Gram Iodine stain
Safranine stain
Decolorizer
Discussions and review: Microbes are microorganisms such as bacteria, molds, and
yeasts. They are around us all the time, and no matter how hard we try to clean and
disinfect our environment, they always come back. Some microbes are carried in the
air and then multiply when they find a hospitable surface. Aerobic microbes need
oxygen to live, anaerobic microbes do not. Microbes tend to be found more often in
some places than in others. Most microbes, especially those found around the home,
grow best in warm areas like the kitchen and bathroom and the warm folds of the
human body. They also grow best where there are ample nutrients for them such as
body oils or food residue and dirt that accumulate from frequent handling and
insufficient cleaning.
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Since microbes can cause infectious diseases, it is important to know where they are
most often found and to work to limit their numbers. In this laboratory exercise you will
discover and compare the types of microbes found in your personal environment.
PROCEDURES: Part 1 – Finding Microbes
You will need: 6 sterile agar slant tubes, 6 sterile swabs, safe, warm incubation area. Note: Cultures
need 3 to 6 days incubation period. Plan accordingly.
Because this experiment involves culturing microorganisms from a human environment,
it is possible that unknown microbes may become incorporated into the sample. Any
culture that may contain an unknown organism should be treated as potentially
pathogenic. Therefore, be certain to wear the gloves and mask provided when handling
the cultures to protect yourself from unintended exposure. Handle the cultures carefully
and maintain an organized, clutter free work space to prevent spills. As with all materials
in your LabPaq, use and store the cultures out of the reach of children and pets. DO
NOT CULTURE YOUR OWN THROAT OR OTHER BODY FLUIDS AS THEY MAY
CONTAIN BACTERIA THAT WHEN CULTURED COULD INFECT SOMEONE ELSE.
1. Consider where might be the best conditions in your home for microbes to thrive.
Make a list and prioritize it based upon the above information about conditions
favorable to microbe growth. Some places to consider are:
electric light switches
door knobs
residue around a bathtub or shower
inside the end of a water faucet
exhaust hood over the kitchen range
drain pan under the refrigerator
inside the garbage disposal (be sure to turn it off first!)
anywhere around the kitchen where food might spill
2. Select six sources from your prioritized list that are from different areas of your
home. Set up a data table similar to the one shown below to number your microbes
and record information and observations about them for Part 1 and Part 2 activities.
Location Of
Microbes Colony Growth Notes …. Gram Stain Notes ………
# Description Temp Shape Color Size Type Shape Color Size Type Observations
1
2
3
6
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3. Select a place where you can incubate cultures of the microbes you will collect.
Chose a warm spot where the cultures will not be disturbed and cannot be reached
by small children and pets. An ideal site is often the top of a hot water heater. If you
choose to use your hot water heater, first place a multi-folded bath towel on top of
the heater surface, and then place the incubation tubes on the towel. The towel will
keep the tubes from rolling off plus provide insulation to keep them from overheating.
Electrical equipment tends to generate heat and makes another potential incubation
site. Consider areas around electronics that run most of the time such as a
transformer, computer, or stereo. (As far as we know noise does not bother
microbes.) There are usually lots of warm spots around a kitchen. However, if you
incubate cultures in your kitchen, NEVER open the tubes around where food is
prepared or eaten. Rather, the culture tubes should be sealed when you take them
into the kitchen and not opened until you leave the kitchen.
4. Mark the six sterile slant tubes of agar with a wax marker pencil, numbered 1
through 6. These numbers will correspond to the microbes you will collect from the
sources you numbered 1 through 6 in Step B above. Do not open the agar
incubation tubes until you are ready to place specimens inside them.
5. To collect smears of your six microbe sources, carefully follow these instructions for
each one. Systematically collect only one microbe source at a time.
a. First measure and record the temperature at the collection site.
b. Open the cap of the correspondingly numbered sterile agar slant incubation tube
for the source number to be tested. Let the opened cap rest over the mouth of
the tube until you are ready to use it. Your objective is to have the sterile tube
ready to accept a smear while at the same time limiting its exposure to
contaminants
c. Unwrap a sterile swab and tightly grip its stem in your dominant hand, usually the
right hand. Wipe only the end tip of the swab firmly across a small area of the
selected surface. Do not swirl the swab or touch anything else with it. You
should perform this and the next step fairly quickly, yet very carefully.
d. Remove the cap of the incubation tube and hold the open tube at eye level in the
hand opposite the one grasping the swab. Very, very carefully insert the swab
into the incubation tube and firmly touch the contaminated end of the swab tip to
the center of the agar slant tube. Ensure the swab tip contacts nothing but this
designated site on the agar.
e. Recap the incubation tube; place the used swab in the trash, and repeat the
above steps a. through d. for the next of your five microbe sources.
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6. Place the six tubes containing microbe sources in the site previously selected for
incubation. Make sure everyone in your home knows what the incubation tubes are
and where they are located and that they should never be disturbed and
especially that they should not be opened. Leave a note stating “Experiment in
Progress – Do NOT Disturb” with the tubes while they incubate for three full days.
7. Keep the culture tubes closed! Use the hand magnifying lens to
observe the growths of colonies within the tubes. Note their shape,
size, color, and anything else distinguishing. Bacteria will grow in small
circular colonies, whereas molds will spread out more and may look
fuzzy. Yeasts tend to grow initially in tight, compact colonies and their
color is somewhat darker than that of bacteria. Record what you see in
the data table. Draw what you see for each. Try to deduce the types of
microbes each cultured colony contains: bacteria, mold, or yeast. Some
cultures may contain more than one type of microbe.
8. If some of the cultures are not yet well developed let all of the cultures
continue to incubate for up to three additional days until you see visible
growth in them all. Record final observations for each tube..
Questions:
A. From which samples did you observe the most and least microbial growth?
B. Why do you think there were differences in the number and types of microbes at the
different sampling sites?
C. Did microbes from warmer or cooler sites multiply faster? Hypothesize why.
D. How could the information gained in this experiment be useful in your home?
Part 2 – A Closer Look
You will need: safety goggles, disposable gloves, face mask, paper towels, microscope slides, metal
spatula, gram stain experiment bag containing crystal violet stain (#1), PVP iodine stain (#2), decolorizer
(#3) and safranine stain (#4), staining tray, clean water to rinse slides, candle or matches.
Most of the microbes that you have cultured are very small, much smaller than you can
see individually with a hand lens and even a 150-power field microscope. However, at
150-power magnification you will be able to see the culture’s structure and possibly
some individual microbes. That is why you will now prepare microscope slides and take
a closer look at your home’s microbes.
To make the microorganisms stand out in relief from the background light it is necessary
to stain them. A common way of doing this is with Gram stain, a process using a
sequence of stains that divide all microorganisms into two categories, either gram-
positive or gram-negative. Gram-positive microorganisms will hold the blue color of
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the first stain in the sequence and will appear blue at the end of the process. Gram-
negative microorganisms will lose the blue color of the first stain in the decolorizing
step, but will hold the color of a subsequent stain, and will finally appear red in color.
Past research has identified certain bacteria or pathogens as gram-positive or gram-
negative; thus gram staining is often used in medical laboratories as a first step in
diagnosis. The color results from gram staining quickly narrows down the possibilities
during the microbe identification process. For example, the Streptobacillus from which
we get “strep throat” is a gram-negative microorganism. When the medical laboratory
cultures and stains the swab from your throat and the final color of the stain is blue, they
know that you do not have strep throat.
Important: Wear a clinical face mask, disposable gloves, and safety goggles at all
times during this part of the experiment. Some microbes can become airborne and
you do not want to breathe them in. Do not touch your face, especially your mouth and
eyes, during this part of the lab exercise. At the end of the experiment, place the face
mask and gloves in the trash and wash your hands and equipment thoroughly. It is
wise to perform this experiment in an area where food is not prepared or eaten. While
most of the cultures will be harmless, it is always possible to produce one that is
contagious, so don’t take chances! Put on your mask, gloves, and goggles!
1. Select four of the six cultured microbes from which to prepare microscope slides.
Ideally you should choose the culture with the largest growth plus, assuming they
exist, one each of the cultures that appears to be bacteria, yeast, and mold per your
earlier identification. In your data table, note the four culture selections next to their
corresponding numbers, source names, and suspected types. Label four microscope
slides with the corresponding numbers of the four sources selected.
2. As detailed below, successively process the four selected cultures onto separate
microscope slides. These instructions are not complex, but they are long with many
steps in this process. Read the instructions and fully understand all the steps before
you begin. Complete all work for each slide before going on to the next culture.
a. Light a candle or have a match or lighter ready to ignite.
b. Place a clean microscope slide on a flat surface and arrange it to hang over the
side of the surface so you can later grasp it with the test tube clamp.
c. Open the culture tube containing your selected culture. Carefully insert the
LabPaq’s metal spatula into the culture tube and scrape off a small portion of the
targeted culture. Recap the culture tube.
d. Grasp the clean microscope slide with the test tube clamp and smear the
removed culture onto the center of the slide.
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e. Sterilize the metal spatula tip by slowly passing it through the flame of a candle,
match or lighter flame for five seconds. Lay the spatula on a clean paper towel
that can be used to wipe off any soot or other material collected on it.
f. Pass the underside of the smeared
microscope slide back and forth a few inches
above the tip of a candle, match, or lighter
flame. You want to heat the smear until it is
dry, but not burn it. The heat fixes the culture
on the slide so it will not wash off during the
staining process
g. Hold the slide over the staining tray from the LabPaq as
shown in the photo on the right, place three drops of
crystal violet solution (Gram stain #1) on the smear, and
let it stand for one minute. Crystal violet is a very strong
stain. Do not let it contact anything that can be
damaged. If you must set the stained slide down, set it in
the tray.
h. After one minute, lightly rinse the stained slide by briefly passing it through a thin
stream of running
tap water.
Gently shake the excess tap water from the slide
into the sink and rinse the excess down the drain. Note: these slides can also
be rinsed by dunking them into a glass of clean water several times and then
shaking the excess water into the staining tray or onto a paper towel.
i. While holding the slide over the staining tray, place three drops of PVP Iodine
(Gram stain #2) on the smear and let it stand for one minute. Iodine is also a
strong stain so do not let it contact anything that can be damaged.
j. After one minute, again lightly rinse the stained slide by
and gently shake off excess tap water.
k. While holding the stained slide over the staining tray,
slowly and repeated drop single drops of decolorizer
(Gram stain #3) onto the smear, as shown, until the
drops running off the slide no longer have any color.
Briefly rinse the slide again r and gently shake off the
excess tap water.
l. Place three drops of safranine (Gram stain #4) on the smear and let it stand for
30 seconds. Safranine is also a strong stain so do not let it contact anything that
can be damaged.
m. After 30 seconds, briefly rinse the smear again and gently shake off the excess
tap water.
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n. Gently blot the unsmeared areas of the slide with a paper towel or tissue, but
NOT the actual smear area. Let the slide air-dry for a minute or two.
o. Note the color of the stained material on the slide. Examine the slide under your
microscope. Describe and draw what you see. Do not wash the slide yet. Keep
it for now so you can compare it to the other slides.
p. Repeat steps a. through o. above to successively stain and examine slides for
each of the other cultures selected.
3. After you have finished examining all the slides and answered the following
questions, wash the slides well with detergent, rinse them with distilled water, and
allow them to air-dry before returning them to your LabPaq. Properly clean all other
equipment. Place the sealed incubation tubes and used paper towels in the trash.
Questions:
A. Was the structure or arrangement of the colonies of microorganisms different among
what you identified as bacteria versus yeast versus mold?
B. Four slides are, of course, a very small sample, but regardless of this limitation, what
can you hypothesize about differences in the microbe growth patterns? Does your
hypothesis match the descriptions at the beginning of the experiment?
C. Were you able to see any individual microorganisms? If so what would you guess
they are (e.g., mold, yeast, etc.)?
D. Do any of the slides appear to have more than one type of microorganism? Did you
determine this by physical appearance of the culture or by color of stain?
- SM-1 Manual COLOR 105 08-17-07
Pretend that you will be conducting the Microbes Everywhere lab with elementary students (you do NOT need to develop a formal lesson plan for purposes of this discussion). Given this scenario, compose a reflective piece that addresses the following points?
1. What are some questions you would pose to students during the activity?
2. What do you want students to understand about the activity from your questioning?
3. Why do you think these questions would promote further discussion?
