Download Inquiry Lab: Identifying Bacteria using Morphology, Physiology, and

Inquiry Lab: Identifying Bacteria using
Morphology, Physiology, and Molecular
Data
Objective:
Bacteria is a prokaryotic organism that can be found almost anywhere on earth.
Identifying prokaryotic species can be difficult in a high school lab. The purpose of this
lab it to use information that is known about bacteria cell wall composition, size, shape,
arrangements, enzymatic activity, colony growth, and molecular data (DNA) to identify
species of known and unknown bacterial samples.
Background:
Colony Morphology:
Bacteria grow tremendously fast given the right type and amount of nutrients. Different
types of bacteria will produce different-looking colonies. The characteristics of a colony
(shape, size, pigmentation, etc.) are termed the colony morphology. Colony morphology
is a way scientists can identify bacteria. In fact there is a book called Bergey's Manual of
Determinative Bacteriology (commonly termed Bergey's Manual) that describes the
majority of bacterial species identified by scientists so far. This manual provides
descriptions for the colony morphologies of each bacterial species. You can access the
pdf by typing “Bergey's Manual of Determinative Bacteriology” on an Internet search
engine.
Although bacterial colonies have many
characteristics, there are a few basic
elements that you can identify for all
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.
Shapes and Arrangements:
Prokaryotic organisms are
single-celled organisms but
come in a variety of shapes
and arrangements. Bacterial
cell shape is determined
primarily by a protein called
MreB. MreB forms a spiral
band around the interior of
the cell just under the
membrane. It is thought to
define shape by recruiting
additional proteins that then
direct the specific pattern of
bacterial cell growth. Bacterial
arrangement is determined by the interaction or separation of bacterial cells after
division. Common shapes include coccus, bacillus, and spiral. Common arrangements
include chains (strepto), clumps (staphylo), pairs (diplo), and single (mono).
Cell wall composition:
Most prokaryotic cell walls are made up of a polymer
called peptidoglycan. The peptidoglycan layer in
bacteria varies in thickness. This allows scientists to
classify bacteria into two major subgroups- gram
positive and gram negative. In gram-positive bacteria,
the peptidoglycan layer is thick. In gram-negative
bacteria, the peptidoglycan layer is thinner, and the cell
wall also consists of an outer membrane made of
lipopolysaccharides. The names “gram-positive” and
“gram-negative” come from the staining procedure used
to identify the bacteria type. Gram-positive stain purple
and gram-negative stain pink.
Catalase
Catalase is an enzyme found in some bacteria that can break down hydrogen peroxide
into oxygen and water. A simple test can be performed to test for catalase in bacteria.
*see procedure
Molecular Data (DNA):
In Bacteria, there are many genes and other sections of DNA that are preserved from
one species to another. One section of DNA known as the 16S-23S intergenic space
varies among bacterial species. Because of this, it can be assumed that the number of
nucleotides in this section of DNA is different in different bacterial species. The best
way to estimate the number of nucleotide base pairs that are in the intergenic space of
a bacterial species is to run a gel electrophoresis with a marker (standard or ladder).
Once the base pair length is estimated, various resources can be used to determine the
prokaryotic group the bacteria is found in. Before running a gel electrophoresis, the
section of DNA of interest (in this case the intergenic space of the 16S and 23S rRNA
genes) needs to be amplified using PCR. Specific primers need to be used to make sure
just that section of DNA is amplified. This entire process of amplifying the intergenic
region with PCR and running the sample through a gel to determine base pair length is
called ARISA.
Amplify!
Procedure:
1. As a class, practice identifying the characteristics of Ecoli (a known) using the
information from the Background section. The class will perform a PCR, gram
stain (to determine cell wall type, shape, and arrangement), colony analysis, and
a catalase test.
2. Lab groups will then be required to design a lab using these lab techniques to
compare and contrast bacteria species on different surfaces, or analyze species
diversity in a particular environment. Students must also come up with one
other test using the information from “Bergey's Manual of Determinative
Bacteriology” Some suggestions include identifying bacterial species found in
the mouth before and after using mouthwash, and identifying bacteria on the
hands vs. what is found on a computer keyboard.
a. Objectives, Hypotheses, Procedure, and a plan for data collecting must
be approved by the teacher before testing can occur. It is important to check
with the teacher about proper ways to collect and grow bacteria.
b. Bacteria collecting will occur on a day designated by the teacher.
Testing will begin the day after collection occurs to allow bacterial colonies to
grow overnight.
c. Students will be required to complete a lab write-up or a poster
presentation of their results.
Lab protocols:
Gram Staining:
1. Put a one drop of water on a
microscope slide
2. Using an inoculating loop, scrape
a small portion of a colony from
an agar plate (other portion of
colony needs to be used for other
tests)
3. Swirl colony in water, spreading it
out on your slide
4. Heat fix
5. Add a few drops of crystal violet
and let is sit for 1 minute
6. Rinse off the crystal violet with a pipette and water (do not complete this step at
the sink with the faucet)
7. Add a few drops of iodine and let it sit for 30 seconds
8. Rinse off with water the same way you rinsed the crystal violet
9. Decolorize by washing the slide with ethyl alcohol at the sink
10. Add a few drops of safranin and let it sit for 1 minute
11. Rinse off with water the same way you rinsed the crystal violet
12. Dab the slide dry with a paper towel
13. Using a cover slip, (and possibly oil if you have an oil immersion microscope) look
at your bacteria under the microscope or keep for viewing next day. Label your
slide so you know what sample you are looking at.
PCR (ARISA)
1. put 20 uL of master mix into each PCR tube
2. using a sterile toothpick, inoculating loop, or pipette tip, take one colony off
the plate and stir in PCR tube (with master mix)
3. repeat step 2 for each PCR tube.
4. Run PCR with the following protocol
94 degrees Celcius – 5 minutes
Cycle: 35 times
94 degrees Celcius – 30 seconds
53 degrees Celcius – 30 seconds
72 degrees Celcius- 1 minute
Final: 72 degrees Celcius – 5 minutes
Master mix: 20 uL total for each PCR tube
2uL of PCR buffer
Possible primers to use:
.8uL of MgCl2
1.
.4uL of dNTP
Card_ITSF
.4uL of forward primer
Card_ITSReub
.4uL of reverse primer
OR
.2uL of Taq polymerase
2.
15.8uL of water
SDBact1522bS20
LDBact132aA18
*the volumes listed above are for one PCR tube. If you plan on running 10 samples, for
example, take the master mix volumes from above and multiply them by 10.
Gel Electrophoresis:
1. Micropipette 4uL of ladder into the first well
2. Micropipette 5ul of PCR product and 2uL of loading dye into other wells. Make
sure to mix the loading dye and PCR sample on Parafilm or well plates with a
micropipette before loading
3. Use teaching instructions for setting up the electrophoresis chamber
Information obtained from:
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Science Buddies. Copyright 2002-2012
http://www.sciencebuddies.org/science-fairprojects/project_ideas/MicroBio_Interpreting_Plates.shtml
Doc Kraisers Microbiology page. 28 June 2006.
http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/shape/shape.html
What are Bacteria, Yeasts and Molds?. University of Georgia, Department of
Agriculture and Environmental Sciences
http://www.google.com/imgres?q=bacteria+shape+and+arrangement&um=1&h
l=en&client=safari&sa=N&rls=en&biw=1154&bih=709&tbm=isch&tbnid=sUaMOtfmDpPGM:&imgrefurl=http://www.caes.uga.edu/publications/pubDetail.cfm%3
Fpk_id%3D6018&docid=sAVDrcmgsdeK3M&imgurl=http://www.caes.uga.edu/a
pplications/publications/files/html/B817/images/bacterial%252520cell%252520s
hapes.jpg&w=915&h=603&ei=AFj_T4HkGYHo0QGw-u3dBA&zoom=1
The Gram Stain.
http://www.google.com/imgres?q=gram+stain+procedure&hl=en&client=safari
&rls=en&biw=1154&bih=731&tbm=isch&tbnid=z5es4o4v5hXzM:&imgrefurl=http://www.highlands.edu/academics/divisions/scipe/bi
ology/labs/rome/gram_stain.htm&docid=2_DxGiXLUnBcUM&imgurl=http://ww
w.highlands.edu/academics/divisions/scipe/biology/labs/rome/gram_s1.gif&w=
526&h=442&ei=eHj_TvwIcLw0gGN8OT0BA&zoom=1&iact=hc&vpx=285&vpy=394&dur=303&hovh=16
3&hovw=211&tx=139&ty=98&sig=107095031745811268909&page=1&tbnh=16
1&tbnw=209&start=0&ndsp=15&ved=1t:429,r:6,s:0,i:94
Bergey's Manual of Determinative Bacteriology