INTRODUCTION
Our goal, in this experiment, was to successfully isolate and observe
three pure cultures and determine their optimal growth requirements. Once
classified, it was important that we share our results with those in the
scientific world.
Through streaking on a TSA
plate we were able to isolate three pure cultures: Coccus helenus ,
Coccobacillus hemephile, and Spirochaeta halotolerans. Coccus helenus
is in the Coccus genus and is named after Helen Chin one of the discoverers.
Coccobacillus hemephile is in the Coccobacillus genus and is distinguished
by its blood lying ability. Spirochaeta halotolerans is named for
its ability to survive in high salt concentrations. This conventional naming
system is important because it shows how the organism fits in the microbial
world, and it gives a very general description of the organism’s somewhat
unique characteristics.
These cultures were easily distinguishable by their
diverse shapes, as their names suggest, and their very distinct growth
patterns. Coccus helenus is a small, round organism and grows in
a very condensed pattern, which felt dry and rough when touched with a metal
inoculating loop. Coccobacillus hemephile are small and rod
shaped (which were found usually in pairs) and grow forming white, cloudy
colonies. Spirochaeta halotolerans are small and had a small, loose
spiral shape. These grew in very small colonies that did not always
connect, giving the appearance of a very “spotty” growth.
RESULTS
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Figure 1. Blood Agar Plates
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Figure 2. The Control Plate
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Shown here are 3 organisms cultivated
on Blood Agar are displaying hemolytic patterns. The agar contains sheep's
blood and was incubated for 48 hours.
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A close look at the three isolated
cultures on Nutrient Agar. This is our control plate that was incubated
for 48 hours at 37 degrees Celsius.
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Bergey’s Manual Description:
Coccobacillus
hemephile gen. Coccobacillus sp.
hemephile (heme = blood, phile = loving) Coccus helenus
is a gram positive obligate aerobe and grows best at temperatures between
25◦C to 37◦C. Coccus hemephile will not grow in salt concentrations
over 0%. It secretes an exoenzyme as seen through the amylase
test and oxidase tests but it is not a fermenting organism. Able
to completely lyse blood cells.
Coccus helenus gen. Coccus sp. helenus
(named after one of the organism’s discoverers) Coccus helenus is a gram
positive obligate aerobe and grows best at temperatures between 25◦C to
37◦C. C. helenus will grows very slowly in concentrations over 0%,
does not grow in salt concentrations over 10%. Oxidase and catalase negative.
Spirochaeta halotolerans gen. Spirochaeta sp.
halotolerans (halo = salt, tolerans = tolerant) Spirochaeta halotolerans
is a non-sporing, gram negative facultative anaerobe. Grows
best at temperatures between 25◦C to 37◦C. S. halotolerans
is able to survive in areas of very high concentration (~15%). Spirochaeta
halotolerans is able to use ferment lactose and glucose. Oxidase
and catalase negative.
DISCUSSION
Using the streak plate method
we were able to successfully isolate three pure ultures. At first glance
these organisms looked very similar but with close observation to differences
in growth pattern and color were used to differentiate the cultures.
Under the microscope, the three isolates have very distinct shaped as their
name implies.
Through the results from gram staining procedures
we were able to check for an initial difference. We saw that the Coccobacillus
hemephile and Coccus helenus were gram positive, while Spirochaete
halotolerans was gram negative. Unfortunately none of the organisms
produced spores or capsules, so we could not use these results to distinguish
the organisms.
The tests that were most helpful were those that
tested for optimal growth environment and metabolic patterns. All tests
produced consistent results for each organism. Both Coccus helenus
and Coccobacillus hemephile failed to grow in the OF tube or
on the medium placed in the anaerobe chamber. Spirochaeta halotolerans
grew in the medium placed in the anaerobe chamber. Spirochaeta halotolerans
also produced lactic acid when exposed to lactose and glucose, consistent
with previous results, showing that it has the ability to use fermentation
to produce energy. The metabolic tests made a clear distinction between
Spirochaeta halotolerans and our two isolates. The differentiating
test between Coccus helenus and Coccobacillus hemephile
were the iodine and the oxidase tests. In both tests only Coccobacillus
hemephile showed clearing to indicate the production of an exoenzyme.
Coccobacillus hemephile was also the only organism with the ability
to completely lyse blood cells.
Spirochaeta halotolerans of its ability
to survive in both high and low salt concentrations it is probably found
in an estuary. Estuaries have combinations of fresh and salt water flowing
into them. Coccus helenus and Coccobacillus hemephile
, because of their need for oxygen, are probably found at the top of
fresh water.
Phenotypic analysis is observation of physical
traits that are expressed through the genes. Phylogenic analysis
looks at the DNA of an organism. In phylogenetic analysis more unique
observations can be made. Where phenotypic observation can be limited
because of similarities with other organisms, phylogenetic analysis would
reveal very distinct differences between organisms that are not expressed
as observable traits.
Have any questions or comments? All you have
to do is to e-mail us!
Helen Chin
Adrienne Smith
Michele Thomas-Kunjoo
