Download Experiment 4: Bacteria in the environment

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BACTERIA
All bacteria are members of the kingdom Monera, the simplest organisms.
Monerans are referred to as prokaryotes, "pro" meaning before and "kary" meaning nut.
This is a reference to the lack of a nucleus in bacteria. The four other kingdoms of
organisms are made up exclusively of organisms that have a membrane bound nucleus.
These nucleated cells are referred to as eukaryotic, "eu" meaning true, a reference to their
"true" nucleus. Other characteristics that can be used to distinguish bacteria from other
organisms include:
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Typically smaller size than other
cells
Division by binary fission instead
of mitosis
Peptidoglycan cell wall
All bacteria are single celled rather
than multicelled.
Four general characteristics are
commonly used to identify specific species
of bacteria:
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Colony morphology - Colonies
may vary in color, texture and
shape. Figure 1 shows some of the
different shapes and textures
colonies may have.
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Cell morphology - Cells may vary in shape and also in the number of other cells
they are joined to (Figure 2). The term cocci, meaning berry, describes spherical
bacteria. Rod shaped bacteria are called, somewhat logically, rods. Spirillum
describes spiral shaped bacteria. When two cells are joined together the prefix
"diplo" meaning two is added. For example a "diplococcus" is a bacteria
commonly found as two cocci joined together. A bacteria commonly found as a
chain of cocci is called a "streptococcus," "strep" meaning twisted chain.
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The outer covering of the cell Bacteria may secrete capsules
that protect them from their
environment. This was true of
the Streptococcus bacteria used
by Griffith in his study of the
genetic material. All bacteria
can be classified on the basis of
whether they are Gramm
positive or negative. The
Gramm staining procedure
takes advantage of the fact that
Crystal Violet will stain the
peptidoglycan cell wall of
bacteria, but not their cell
membrane. Bacteria that are
Gramm positive have no
membrane on the outside of
their cell wall. Gramm
negative bacteria have a
membrane that covers their cell
wall, so it is not stained by
crystal violet and a negative
test results. Figure 3 shows
the different outer coverings of bacterial cells.
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Biochemical pathways - Different bacteria can
make different biochemicals and may used
different carbon and energy sources. For example,
some bacteria are anaerobic and lack the ability to
break down hydrogen peroxide to oxygen and
water while other bacteria can do this.
2 H 2 O2 _2 H 2 O + O2
Other bacteria can use manitol or lactose as their carbon and energy source, while
others can only use glucose.
When working with bacteria it is essential to maintain aseptic conditions. This
means that everything that could possibly come into contact with your bacterial cultures
must be sterile. Before doing anything else, you should wash your hands and the bench
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area you will be working on with disinfectant. A Bunsen burner flame is used to sterilize
the wire loops that are used to transfer bacteria between cultures. All glass culture tubes
are "flamed" at the lip before and after a sample is removed. Care must be taken to not
get hair or skin in contact with any of the culture media both before and following
inoculation with bacteria. Always wash your hands with bacteriocidal soap after working
with bacteria.
WARNING: The bacteria you are working with are not typically pathogenic, but every
precaution should still be taken to avoid coming into contact with them. This is
particularly true in the case of individuals with weak immune systems. If you get cultures
on yourself or the bench tell your instructor immediately, and wash the contaminated area
thoroughly with bacteriocidal soap.
WARNING: When working with open flames use extreme caution. Keep hair and loose
clothing away from the flame. If your hair or clothing catches on fire smother the fire,
DO NOT RUN, this will only fan the flames. If you are burnt, run the burn under cold
tap water, do not put ice on the burn. Inform your instructor immediately if you are
burned.
Exercise 1: Colony morphology
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A number of different bacteria are available in broth cultures at the front of the
lab, select one culture per student (3 per group)
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Obtain a nutrient agar petri plate and clearly mark it with the type of bacteria you
have chosen and your name.
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Sterilize a transfer loop by heating it to red hot then letting it cool. Be careful to
avoid touching anything with the loop before it is cool enough to use in the
transfer.
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Open the broth tube and flame the top for a couple of seconds.
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Dip the loop into the broth culture then lift the lid of the petri dish and streak
along one side of the nutrient auger.
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Flame the opening to the broth culture and replace the cap.
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Flame your transfer loop
then open your petri plate
again and streak the loop
across the plate at 90o to the
original streak. The object
of this is to get a few
bacteria from the original
streak and spread them out
so that colonies resulting
from one individual bacteria
can be observed later. To
spread out the bacterial even
more, repeat this step twice
more, flaming your loop between each streak. Each time rotate 90o from the
previous streak. This is depicted in Figure 4.
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Place your plate auger side up in a 37 oC incubator and leave it for 24 hours.
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Check the morphology of your colonies the next day using a dissecting
microscope and record the results.
Experiment 2: Biochemistry
Once you have checked the morphology of your colonies, place a drop of
hydrogen peroxide solution on one of the colonies. If the enzyme catalase is present,
bubbles of oxygen will appear. If the enzyme is absent, no bubbles will appear.
Experiment 3: The Gramm Stain
You will want to do a Gramm stain on all the live bacterial cultures available in
this lab.
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Place one loop-full of broth culture on a clean slide and smear it around in a small
area. If you are using a colony from an agar plate, put a drop of water on the slide
then get some bacteria on a loop and smear them around in the drop.
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Allow the smear to air dry.
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Pass the slide two or three times through a flame to heat fix the bacteria. This will
cause them to stick to the slide and not be washed off. Do not over heat the slide
or your bacteria will go up in smoke.
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Be careful when using the following stains as they will stain you and your
clothing just as well as they will stain bacteria. Take the slide to the sink and
place several drops of crystal violet stain on your smear. Leave the stain on the
smear for exactly 1 minute then wash it off gently with water. The crystal violet
stains the peptidoglycan cell walls of Gramm positive bacteria, but cannot do the
same to Gramm negative bacteria as the outer membrane prevents the stan from
getting to the cell wall in these bacteria.
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Cover the smear with Gramm's iodine and allow it to stand for 1 minute before
rinsing off the iodine with water. The iodine helps to fix the stain to the cell wall.
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This step is crucial. Flood the smear with decolorizing solution (95 % ethanol)
for 10 - 20 seconds. The time should be just long enough to get the decolorizing
solution to run clear, but not so long that everything is washed off the slide.
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Stain the smear for 20 - 30 seconds with safranin. Safranin stains membranes so
it will stain the outer membrane of Gramm negative bacteria, so that they will be
visible as red bacteria on your slide.
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Examine your smear under the oil immersion lens of your microscope. Gramm
positive bacteria should appear dark purple while Gramm negative bacteria should
appear red.
Experiment 4: Bacteria in the environment
Each individual in your group will be provided with a nutrient agar plate which
you can expose to the surface, liquid, or environment of your choice. For example, you
may want to compare water from the faucet with water from a puddle. Yogurt contains
excellent cultures of lactobacillus. You may also want to check out the effect of washing
your hands with soap versus not washing them. Use your own imagination, inoculate
your plate and place it in the incubator to be checked in the morning.
Materials
Equipment
Bunsen burners
Flints or matches to light burners
Inoculating loops
Microscopes: Compound and Dissecting
Prepared slides of bacteria
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37 oC Incubator
Supplies
Gramm stain chemicals
Lysol to sterilize benches
Nutrient agar plates (2 per student)
Three different bacterial broth cultures