Unknown used to experiment on this unknown bacterium were

 

 

 

 

 

Unknown Bacteria 228

Adisney Pino

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Professor Dowding

Miami Dade College

Section T/R 7:05PM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introduction

            Enterobacter aerogenes is rod-shaped bacteria that belongs in the
family of Enterobacteriaceae. This rod-shaped bacterium is gram negative which
makes it a facultative anaerobe which grew perfectly in 37-degree temperature.
This species was motile and therefore it can synthesize an enzyme known as
ornithine decarboxylase. This bacterium is prevalent in the intestines of animals,
there presence in the intestines of animals makes it easier to get in the soil,
water and sewage. In humans, this bacterium is known as an opportunistic
pathogen which can cause multiple conditions which include, meningitis,
bacteremia, pneumonia, endocarditis and urinary tract infections.

            Many
times, exposure to this bacterium is the result of hospital stays and nursing
homes. Long hospitalizations and invasive procedures increase your chances of
getting this bacterium.   Recently some Enterobacter organisms have become drug resistant which is making
it harder to make a treatment to fight the infection. The usual treatment for Enterobacter infections consist of
antimicrobial therapy. Some of these treatments include Aminoglycoside, Fluoroquinolone
and Cephalosporin, however in some recent cases the enzyme that is produced by this
bacterium when exposed too much to these drugs will cause the bacteria to
become resistant to the treatment. Therefore, newer and more modern treatments
are being put into place, such combining multiple therapies at once to force
the bacteria to go away.

            Some
signs and symptoms one might have when infected include, fever, hypotension,
tachycardia, and even hypothermia when it comes to respiratory tract
infections. This bacterium is diagnosed by performing laboratory studies such
as urine analysis, fluid analysis (cells and differential analysis for example)
and electrolyte evaluation. Furthermore, Enterobacter
are capable of nitrogen fixation. Some species in specific E. cloacae, this bacterium has been found to be involved in
symbiotic nitrogen fixation in plants and have even been separated from the
roots of certain types of plants.

 

Materials

            The
materials used to experiment on this unknown bacterium were as follows:

Microscope, agar slants, Phenol red broth, Durham
tubes, NH3, Iodine, Bromothymol blue dye, Kovac’s reagent, Enterotube 2, transfer
pipette, loop, Bunsen burner, agar plates, slants, distilled water, tube racks,
tape, sharpie, metric ruler, Methyl Red reagent, test tubes, hydrogen peroxide,
microscope slides, Nutrient agar, cotton tipped applicator, zinc powder,
nitrate powder, gelatin stabs, Simmons citrate slants.

Methods

            The
following steps were taken to discover what bacteria was in the original
randomly given plate. When the original agar plate was given, the first step
was to look at the bacterial colony features. Here we looked for the elevation,
margin and the way the colony looked. After the agar was observed, a sample was
taken from the original plate and it was put in different temperatures. Two
samples went in a nutrient broth and two more samples went in a slant. The two
samples of the nutrient broth went on different temperatures, one went to 25
degrees Celsius, while the other went at 37 degrees Celsius. The following lab,
we observed the slant with the best growth. This slant became the “working
stock” which was used as a sample for the rest of the labs and the slant with
the least growth was left as a backup in case of contamination. With this very
same samples, we made a gram stain. For this gram stain, we heat fixed our
unknown bacteria and covered it with crystal violet stain for a minute, after
this we rinsed the slide with distilled water and removed the excess of stain.
After, we covered the smear with Iodine stain and after a minute we rinsed the
excess of stain and then we decolorized the smear using acetone. This procedure
was done until most of the stain was gone, then we used the counterstain safranin
for another minute and once the minute passed we rinsed it with distilled water
and used bibulous paper to dry the smear and after it was fully dried we
observed the smear under the microscope.

            The
next step done was to inoculate broths with our unknown organism and using
Fluid Thioglycolate tubes we inoculated three broths with our unknown organism.
Then we stored these away at 37 degrees Celsius, the next class we looked at
the growth pattern and decided if it was aerobe or facultative anaerobe. After
this procedure, we tested our unknown bacteria in glucose, lactose, mannitol
and arabinose broths. Once again, we inoculated each broth with our unknown
organism and when done we put it away at our optimal temperature of 37 degrees
Celsius. The broths used for this test were PR broths, some of this had a
smaller tube inside to test for gas. Once done with the PR broths you will obtain
three MR-VP broths, and we will inoculate two broths with our cultures, we did
not inoculate the control which was the third tube. The following lab day we
grabbed test tube one (Methyl Red Test) and we added three drops of Methyl Red
agent and we observed it for red color change, this change had to happen
immediately.

            The
next tube was for the VP test and we added 15 drops of VP reagent A, we then
vortexed the tube and added 5 drops of VP reagent B. Once again, we vortexed
and we observed for red color formation, this process took 10 minutes. After
these tests were done we looked at the tables in our lab manuals and depending
on the color change we had, we decided if we had fermentation or not. On the
next lab date, we used test tubes we had arranged with our unknown bacteria and
grabbing a good portion of bacteria we dropped a mass on an oxidase slide test.
A color change had to happen within the first 20 seconds, after this we grabbed
the same tube and added hydrogen peroxide directly into the test tube and we
observed the tube for bubbles/reaction. This lab we also got four Nitrate
broths and inoculate the broths and after 48 hours in our optimal temperature
we examined the tubes for evidence of gas production. After the observations
were done, we added eight drops of reagent A and reagent B to each test tube
and using our control we observed and compared it to our control. The following
step done was to grab a starch agar plate, a milk agar, and a Tributyrin agar where
we spot inoculated the agar plates and then incubated them at our optimal
temperature.

            The
following lab date we observed the growth on the starch agar and then added
iodine and looked for a halo, after that we picked up the milk agar and looked
for a halo without adding any agent. Then, we looked at the Tributyrin agar and
looked for a clearing/halo around the bacteria. After this procedure, we
inoculated three urea broths and after storing it at our optimal temperature for
48 hours we brought it out and examined them for color change. After these
tests had been run and observed we grabbed three nutrient gelatin stabs, and we
inoculated two tubes, this time we stabbed the gelatin once with an inoculating
needle and left one more nutrient stab for control purposes. The following lab
we observed the gelatin, if it was solid or soft and watery. We also watched
for any formation where we stabbed the gelatin. After these observations were
done, we obtained a Simmons Citrate tubes, and made a zigzag pattern on the
slant, lightly inoculating with a needle and incubated them all at our optimal
temperature. The following lab we will watch the slant for any color change and
growth. The following lab date we had a Phenylalanine Agar slant and inoculated
heavily doing a zigzag motion and the lab class after that we added 2 or 3
drops of 12% ferric chloride solution to the tube and observed for color
change. The reaction we were looking for should have happened immediately.

            Additionally,
we grabbed a SIM tube and inoculated three tubes with the unknown organism and
we stabbed the tube using a needle, wherever originally stabbed we had to try
and stay in the same line. We then inoculated these tubes. The following lab
class we examined the tubes for spreading away from the stab line and if any
formation of black precipitate in the medium had formed. We then added Kovac’s
reagent to each tube and waited for about 5 minutes and observed for any red
color formation at the top, by now we were able to make a presumptive
identification of our unknown bacteria. The following test would then have
proved or disproved our identification. We grabbed an Enterotube 2 and removed
the blue and white cap, being careful not to contaminate the sterile wire. Then
we grabbed our original plate and removed a lar amount of growth from one of
the colonies in my agar and then slowed turned the wire through the Enterotube
2 compartment. We continued to turn until we reached the bottom where we will
then roll it back inside and leave the tip of the needle in the Glucose
compartment and break of the bottom part that is left out. This was then get
stored at our optimal temperature where the following lab we compared the
results from this tube and the ones we had been getting throughout the class.

 

Results and Conclusions

In order to get the exact bacteria in our
unknown agar plate, we had to run a series of test and observe what the reactions
were. We started with the characteristics of the bacteria, for example we did a
gram-negative stain and noted that unknown bacteria 228 was a gram-negative
bacterium. We then did an oxygen test where we watched for an aerobe or
facultative anaerobe and my unknown bacteria tested facultative anaerobe,
furthermore 37 degrees Celsius was this unknown bacteria’s optimal growth
temperature because when the test tubes were observed it showed the most growth
between the two temperatures. This bacterium was also a motile bacterium because
after doing a wet mount the bacteria although difficult to tell, was moving
around the slide which made it motile and the growth pattern on the nutrient
broth was sediment like. The next couple of test would have given us this
unknown bacteria’s fermentation and respiration. We did a series of test with
glucose, lactose, mannitol and arabinose this was done to be able to
differentiate members of Enterobacteriaceae
and to distinguish them from other gram-negative rods. The glucose test
tested positive for both acid and gas, as did the mannitol and arabinose
however the lactose tested positive for acid but negative for gas. These tests
were done using a series of PR broths that had an inverted Durham tube inside
to test for gas production, when these tests were compared to our controls we
were able to know whether these organisms had gas production and to see if
there was any acid production.

 On these
very same lab days we ran a Methyl red test and a Voges-Proskauer test, which
for our bacterium tested for negative on both tests. Furthermore, we ran a
catalase and oxidase test and the catalase once the hydrogen peroxide was added
it immediately formed bubbles making it a positive test. Yet, for the oxidase
slide test it tested for negative since no color change happened within the
first 20 minutes. The next step was to run a nitrates reductase test which for
our bacterium was a positive test.

Additionally, the next series of test were
Hydrolysis test and we tested for starch, urea, casein, gelatinase and lipase
presence. The starch and urea tested negative however the casein and gelatin as
well as the lipid test all tested positive. By now we could assume what our
bacteria might have been. The next couple of biochemical test would have
defined what our bacteria truly was. Here we tested for citrate permease and we
got a positive reaction there, the next test was a Phenylalanine deaminase test
and our results were negative as well as our hydrogen sulfide test and indole
test. By now we could make a presumptive assumption that our bacterium was an Enterobacter bacterium.