Rev 1/16
Bacteriology Exercise
Name ____________________________________
Observing Bacterial Structure: Gram Stain
To observe the structure of bacteria, we often use a process called the Gram stain developed in Europe
by Hans Christian Gram (1884). You might choose to check off  each step as you complete it.
 1. Place one very small drop of water on a clean slide. Using a sterile loop, touch the edge of a healthy colony of
bacteria in the stock culture. Stir the loop in the drop of water to make a very thin suspension of cells on the
slide. Spread the cell suspension on the slide, keeping it away from the ends of the slide but covering a large
area. Flame the loop to clean it. Allow the cell suspension to air dry…you can apply gentle heat from the
Bunsen burner to speed this process. After the film is completely dry, pass the slide about three times through
the flame. You are just trying to attach the cells to the glass…NOT trying to cook them!
 2. Flood the dry smear with a drop or two of Crystal violet (aka gentian violet) stain. Use the tip of the dropper to
spread the stain to cover the entire smear. Allow the stain to stand for 10 seconds. The dye will pass into the
cells attached to the glass.
 3. Wash with water into a waste beaker using the wash bottle…you should see a purple smear on the glass.
 4. Flood the entire purple smear with Gram iodine for another 10 seconds. This will also pass into the cells and
form complexes with the crystal violet stain inside the cells.
 5. Wash with water into a waste beaker using the wash bottle…the purple smear should now look black.
 6. Flood the slide with 95% alcohol to destain the entire blackish smear. Rinse the smear with more alcohol, until
what is dripping into the beaker are colorless drops. Gram+ bacteria will likely retain a lot of purple-black
color…Gram– bacteria will destain rapidly and completely (seem to disappear!).
 7. Wash with water into the waste beaker using the wash bottle…the slide may appear blank if Gram–.
 8. Flood the entire previously smeared area with Safranin to counterstain the cells for 10 seconds.
 9. Wash with water into a waste beaker using the wash bottle. Blot dry the bottom and edges with a towel.
 10. Label your slide with your group ID and the species abbreviation (BT, EF, ML or EC) and place it in the
designated tray to observe in the microscope next week.
If your staining technique is good, Gram+ bacteria will appear to be purple and Gram– bacteria will be
pink. If the Gram– species are too pale to see, you can do a second staining but stop after the water
wash of the iodine; this leaves Gram– bacteria purple and easier to observe.
Cell Shape
Cell Grouping
Gram
Bacillus megaterium
coccus bacillus spirillum vibrio
uni diplo strepto staphylo
+ –
Enterococcus faecalis
coccus bacillus spirillum vibrio
uni diplo strepto staphylo
+ –
Micrococcus luteus
coccus bacillus spirillum vibrio
uni diplo strepto staphylo
+ –
Escherichia coli::GFP
coccus bacillus spirillum vibrio
uni diplo strepto staphylo
+ –
Use a BioRad UV/Blue penlight to observe fluorescence of the green fluorescent protein gene product that
was cloned into normally-white E. coli.
Describe the color you observe:_________________________
Using good logic, which of these genera have…
thick peptidoglycan (aka: murein) walls? Bacillus
Enterococcus
Micrococcus
Escherichia
Gram, H. C. 1884. Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten. Fortschritte der
Medizin 2: 185–189.
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Document © Ross E. Koning 1994. Permission granted for non-commercial instruction.
Koning, Ross E. 1994. Bacteriology Exercise. Plant Information Website.
http://plantphys.info/organismal/labdoc/bacteria.doc
A Pigment Biosynthesis Pathway: Prodigiosin
Serratia marcescens D1 is a well-known pigmented bacterium synthesizing the red pigment, prodigiosin. This pigment
is the result of a biochemical pathway (below) in which portions of amino acids are assembled into the final prodigiosin
chemical structure. One intermediate, MAP, is a volatile gas and another, MBC, is water soluble in the medium. Diffusion
in gas is rapid; diffusion in water is comparatively very slow. The details of the pathway are still not completely understood,
but you will learn a few things about it. Biochemistry majors should have a smile on their faces!
In showing a biochemical pathway, the intermediates are shown between arrows representing the chemical reactions
converting one intermediate to the next. These reactions are catalyzed by enzymes. Enzymes are proteins that are coded by
genes in the DNA of the nucleoid in the bacterium. The D1 strain of Serratia marcescens has the wild-type (normal)
versions of all the genes coding for enzymes in this pathway and therefore colonies of this strain appear dark red with lots of
prodigiosin.
alanine
proline
enzyme x
enzyme y
MAP(g)
[MAP=2-methyl-3-pentyl-n-amyl-pyrrole]
enzyme z
MBC(ws)
prodigiosin
[MBC=4-methoxy-2,2-bipyrrole-5-carbaldehyde]
Scientists have irradiated wild-type Serratia marcescens D1 with UV light resulting in two mutants that are less
colorful. We will observe two mutants known as 933 and WCF. Record the color of the three strains when grown alone on
 PGA medium: D1 wild type:_______________ 933 mutant:_______________ WCF mutant:_______________
Both mutants produce less-colorful colonies rather than the dark red colonies of strain D1 because changes in their
DNA result in a defective enzyme in the pathway shown above. If any of the three enzymes is defective, the dark-red
prodigiosin cannot be produced. But which enzyme is defective in each of these mutant strains? We shall soon see by
growing the three strains together.
Streak the three strains pairwise onto three plates of PGA medium using the template provided at your lab
station. Remember, if you can see the bacteria on the gel after you have streaked it, you have applied too much!
Bag your dishes in separate ZipLock™ bags and incubate them with the cover down (medium up) to retain gases
released, and at room temperature, until next week.
Sketch the outline of each streak-colony. Shade the areas where prodigiosin (orange-red) is produced, being
careful to have the shading correlate with the intensity of the red color. Use your color words! (8 points)
PGA
D1
PGA
933
D1
PGA
WCF
933
WCF
Decide what your results mean about the pathway to prodigiosin…where the 933 and WCF mutations block the
path…recalling diffusion rates and the effect of closeness of a neighbor. (4 points)
Which of the strains…
…lacks MAP, the gaseous intermediate, that the other two can provide? D1 933 WCF
…lacks MBC, the water-soluble intermediate, that the other two can provide? D1 933 WCF
…has a mutation rendering enzyme x non-functional? D1
933 WCF
…has a mutation rendering enzyme y non-functional? D1
933 WCF
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Interaction of Light and Genes: UV Light Exposure
Now, that we know a bit about the pigmentation of Serratia marcescens D1, we will see if we can
mutate the bacteria with exposure to ultraviolet light to create our own colorless mutant bacteria. You
will make your plate by spreading bacteria from a broth culture over solid medium.
Use a sterile yellow pipette tip and a pipetter set for 100 µL. Place a 100 µL drop of the culture
in the center of each of two LB agar plates. A spreader is standing in a fingerbowl of alcohol. Remove
the spreader and ignite the wet end in the flame of a Bunsen burner. CAUTION: do not put the spreader
back in the alcohol because there may be flames still on the spreader that could start a very LARGE
fire! When the alcohol has burned off, gently lower the spreader onto the agar and DO NOT push
down! Instead let the weight of the spreader rest on the agar. Rotate the plate so that the spreader
completely coats the entire surface of the agar with the 100µL of broth culture.
Label one plate as the control and put it in a ZipLok™ bag. Tape the cover to the bottom of the
dish for UV treatment. Trace around a sunglasses lens on the cover of the plate, then tape the lens in
place. Place this plate with the bagged control in the designated place. The treatment is exposure to the
analytical UV lamp for 10 minutes. After exposure, remove the lens and place the dish into the
ZipLok™ bag with the corresponding control. Make a sketch of the results here: after a week of
incubation at room temperature:
Shade the colonies and label if they are red/orange/purple. Be sure to show where the sunglass lens
was located by drawing and labeling its outline with a very dark smooth line on your sketch too!
LB
LB
No UV Control
UV Exposed
What do these results tell you?
Most of the bacteria spread and irradiated with UV light ___________________________________
and therefore produced no colonies.
The bacteria sufficiently shielded from the UV light by the sunglass lens ______________________
and therefore were able to grow into vibrant red colonies.
Any colonies that are less than dark red may have mutations in genes
for enzymes leading to the production of the red pigment, named: __________________________ .
Why do you seem to find no colonies lacking prodigiosin? __________________________________
______________________________________________________________________________
Some bacteria that were spread but grew into a colony outside the protected area of the sunglass lens
may have been protected by _______________________________________________________ .
Why were colonies larger outside the lens edge than colonies inside the lens edge? _______________
_______________________________________________________________________________
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