BIO 1112 Bacteria

Portions of this lab adapted from Cultivating of Bacteria from Commercial Yogurt, from
University of Missouri-St. Louis Microbes in Action
http://www.umsl.edu/~microbes/activities.html.

Learning Objectives
• Find bacteria on a prepared slide
• Observe bacterial movement
• Observe multiple bacterial types
• Understand some ways that bacteria can be beneficial

Introduction
Bacteria are microscopic organisms that were among the first life forms to inhabit earth. There
are about 50 bacterial phyla that show diversity in shape, habitat, metabolism etc. While some
are known to be harmful, many also form symbiotic relationships with eukaryotic organisms.

Endospores and Clostridia

Members of the genus Clostridium (phylum Firmcutes) form endospores, which are protein-
coated, dormant forms that can survive a wide variety of environmental conditions (including
high temperatures, radiation, lack of water). This genus includes both Clostridium botulinum
and Clostridium tetani, the causative agents of botulism and tetanus, both of which are caused
by toxins secreted by the bacteria rather than by direct infection. Clostridia are prevalent in the
environment, particularly in soil. They are mostly aerotolerant anaerobes, which means that
they do not use oxygen but have the enzymes to handle its oxidative effects. However, they
usually cannot form endospores in the presence of oxygen. Clostridia are generally obligate
fermenters, and some species are used industrially for the organic solvents they produce
through fermentation.

We will attempt to grow and examine Clostridium pectinovorans, a species that breaks down
pectin, a saccharide polymer found in plant cell walls. Pectin-degrading bacteria are used
industrially in the production of fibers such as linen from plant materials (such as flax), where
the bacteria break down the pectin, allowing the cellulose fibers of the plant to separate and be
used to make cloth. These pectin-degrading bacteria are common in soil. Their fermentation
produces carbon dioxide along with formic, butyric, and acetic acids.

Bacteria and Food

The Clostridia experiment emphasizes the negative view we often have of bacteria as spoilers of
food and agents of disease. However, bacteria are key to your life- bacteria in the environment
help recycle key nutrients, and bacteria in your gut help digest your food and provide you with
vitamins, and many of the foods we enjoy are made by bacteria. While there are a variety of
bacteria (and fungi) that help make our food and drinks, the main players are lactic acid
bacteria (LAB), which are generally aerotolerant anaerobes that ferment our food and produce
flavor compounds we enjoy as a product of their fermentation. Many yogurts, cheeses,

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fermented pickles, sausages, olives are produced or improved by LAB. LAB also live in your gut –
although the strains in your gut and food are generally different, and research is supporting the
concept that consuming LAB and some other “probiotic” bacteria can help support your
digestive and immune systems. In this portion of lab we will look at the bacteria in our food.

PreLab
1. Choose a small sample of food to bring in with you for Day 1 of your lab. You will need
to choose a food that you would expect to find bacteria living in. What food did you
choose and do you expect bacteria to be present?
Brought an onion, expecting soil bacteria

2. Do some research with the manufacturer, what bacteria would you expect in the food
you used? What purpose is it serving in the food?
Expected soil bacteria due to onions being a ground vegetable. Soil bacteria do a variety of things,
recyling nuterients and breaking down organic material, as examples

3. You will be observing prepared/stained slides of bacteria. What are the common
staining techniques used? (Use your book to answer this).
Gram staining, colored molecules for electron microscopy, stained bands

4. You will be submerging a dirty potato in water after you have stabbed it several times.
After a few days, the potato will float. What causes the potato to begin to float?
air pockets created by the bacteria cause the potato to become lighter then the water surrounding it.

Materials – Day 1
Prepared Slides
• Mixed shape bacteria slide

Clostridia from Potato (materials may be shared by multiple groups)

• Dirty potato (may be shared by multiple groups)
• Tap water
• Knife/fork/other sharp implement
• Beaker of water
• Aluminum foil

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Bacteria in Food
• Yogurt, cheese, kombucha, sauerkraut, or any other food that you wish to examine (liquids
are easier)
— Cooking will kill the bacteria of interest, so no breads, pasteurized cheese, etc.
• Microscope slides and cover slips
• Disposable pipettes
• Sterile water
• Scoopulas in ethanol
• Petri dish with bacteria agar
• Sterile swab

Procedures– Day 1
Prepared Slides of Bacteria
1. Obtain a prepared slide of bacteria.
2. Use the compound light microscope to find and observe the bacteria. What makes
organisms on the slides look different from one another?

Clostridia from Potato

1. Wash your potato with tap water. Leave some dirt on!
2. Stab the potato a few times with a knife/fork.
a. Note that any oxygen introduced by stabbing will be rapidly consumed, mostly by
enzymes in the potato’s cells.
3. Place the potato in a beaker and add enough water to cover the potato.
4. Cover the potato with aluminum foil. Your instructor will incubator the potato at 37 ⁰C.
5. After several days, the potato will begin to float. When the potato begins to float, proceed
to day 2 of the lab.

Bacteria in Food
1. Use a disposable pipette to take a small sample of your food. Save some for the next test!
a. If your food is thick or opaque, you may want to add some distilled water to thin it – you
can put some in a beaker, add the water, and then use the pipette to mix it.
b. If your food is solid you will have to mix it enough to make a puree, so put some in a
beaker with a little sterile water and use the scoopula to crush it as much as possible,
then suck up a sample.
2. Put a dot of this sample on a microscope slide.
3. Gently drop a coverslip on top, minimizing air bubbles.
4. Look at your food sample under the microscope. Bacteria are generally small (need the 40X
objective to see them) and either sphere- or rod- shaped. Foods may also contain fungi
(larger ovoids (yeast) or long threads with regular segments (molds)). You may also see

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 blobs of fat or crystals of sugar, which are generally easy to see on the 10X objective. Draw
and identify what you see:

5. When you have found a food with bacteria, we will test to see if they are lactic acid
bacteria. You will spread the bacteria out on a plate – the following steps allow you to dilute
the amount of bacteria on each area of the plate.
a. Label the bottom (jelly half) of a plate with your name and the date.
b. Take a sterile swab and rub it in your food.
c. Rub the swab on one part of your
plate (Start 1 in Figure 1). Start #2
d. Get another sterile swab and rub it
on the plate so that you cross your Start #1
first streak and then spread a small
portion of the original bacteria
across another region of the plate
(Start 2) as indicated in Figure 1. Start #3
e. Get a third sterile swab and then
spread a small portion of the
bacteria from region 2 across
another region of the plate (Start 3)
as indicated in Figure 1. Figure 1. How to make a triple-
6. Your instructor will incubate your plate. streak plate (plating for colonies).

Materials – Day 2
Clostridia from Potato (materials may be shared by multiple groups)
• Floating potato from Day 1
• Cutting board
• Knife
• Disposable pipette(s)
• Microscope slides and cover slips
Bacteria in Food
• Plate from day 1
• Toothpicks
• Dissecting microscope

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Procedures– Day 2
Clostridia from Potato
1. Put on gloves. You may also want to protect your clothes (lab coat).
2. Once the potato begins to float, pour the water out of the beaker.
3. Wash and then dissect the potato with a knife.
a. From what you can see, how does the inside of the potato differ from how it probably
started?

b. What has happened to cause these differences?

4. Poke the tip of the pipette into the inside of the potato and suck up a bit of tissue.
5. Drop this tissue onto a new, clean slide. Use the tip of the pipette to spread it out until you
have a thin layer on the slide. You may need to add a drop of water if it is dry.
6. Gently drop a coverslip onto the slide, minimizing air bubbles.
7. Examine the tissue under the microscope, looking for endospore-bearing rod- or spindle-
shaped cells.
a. Clostridia form terminal endospores. Identify clostridia in the microscope and draw
what you see. Label the endospores. How does it look under the microscope?

b. Draw and identify at least two other bacterial types that you see. If you do not see two
other types, obtain a prepared and stained slide and draw and identify the types seen
on that slide.

c. Note any interesting behaviors that you see. What are the bacteria doing?
(Note: bacteria may all be moving due to Brownian motion or the flow of the water on the
slide- but you may see individual bacteria moving on their own, and that is what you need
to look for.)

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Bacteria in Food
1. Look at your plate. If you spread the bacteria out sufficiently, individual bacteria were able
to reproduce into populations that form dots on the plate, called colonies – each colony
started from a single bacterium, and thus they should all be genetically similar!
2. There are two common bacteria in yogurt that are easy to identify and may also be seen
from other foods:
a. Lactobacillus bulgaricus forms wide, flat colonies that can have a crystal-like structure. If
you touch them with a toothpick they will be sticky.
b. Lactobacillus thermophilus forms smaller, yellowish colonies (these may be hard to see
with the naked eye, you can use the dissecting scope to get a closer look). If you touch
them with a toothpick they may appear to dissolve.
3. Identify the bacteria on your plate as best you can.

4. You may have used plates that differentiate the bacteria in other ways – if so, your
instructor will explain them to you. Note what they tell you about your bacteria here, and
discuss whether that makes sense given your food.

Questions
1. Which Domain include the Gram positive and Gram negative organisms?

2. What are two ways that bacteria are characterized?

3. Clostridium are anaerobes, so how could they survive on the outside of the potato?

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4. Species of Clostridium are obligate anaerobes and capable of producing endospores.
Oxygen kill Clostridium. When were endospores formed during this activity? What is the
adaptive advantage for formation of endospores at that time?

5. Colonies were used to identify the bacteria in your plate rather than identifying an
individual bacterium? What was the reason for this?

6. Using your textbook or other reliable sources, what are three other ways that bacteria can
be beneficial? Be sure to cite your sources!