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BIO
BIO
BIO
Introduction:
The purpose of this lab is to determine the kinds of bacteria present in a given tube, that
are unknown to the student. All of the possible bacteria were discussed in the lab. The unknown
test tube contained at least three bacteria, which could be gram negative and gram positive. In
order to determine the specific types of bacteria present, a series of tests were performed which
The unknown test tube of bacteria selected was unknown B48. After selecting the
unknowns, they were stored in a refrigerator. To begin, the unknown was streaked onto several
plates including tryptic soy agar (TSA), mannitol salt agar (MSA), MacConkey (MAC), nutrient
agar NA, eosin-methylene blue (EMB). For streaking plates, an inoculating loop was heated in
an open flame until it was a bright orange color to make it sterile. In order to not kill bacteria’s,
the loop was cooled for 60 seconds before dipping it in the unknown test tube. Throughout the
process of finding the unknown bacteria, any time a test tube was opened it was followed by
flaming the opening of the test tube, and the opening of the test tube was also flamed prior to re-
capping all test tubes. This technique prevented contamination of bacteria from hands and
environment. After the inoculating loop had been cooled it was dipped in the unknown bacteria
test tube and then spread onto each individual plate in a specific manner. The purpose of this
streaking was to isolate the bacterial colonies growing on plate. After the streak plates were
done, they were placed in an incubator for a period of at least 24-hours. After that bacteria grew
on the streaked plates, the plates were viewed. The objective was to find individual colonies of
bacteria growing that were not touching any other colonies. Shown below in the figure 1 are the
Figure 1.
Using a sterile inoculating needle, isolated colonies from each plate was transferred onto a TSA
slant. TSA slants were labelled according to what plate the colony came from. After the bacteria
were inoculated in slants, they were kept in incubators for at least 24 hours for bacteria to grow
on slants. After the bacteria had grown onto the various TSA slants, they were all transferred
onto clean glass slides using sterile inoculating loop. The smear was allowed to dry on glass slide
and then was heat fixed. All the slides were then gram stained and then viewed under a
microscope. The gram stain gave some clue to determine the bacteria weather the bacteria was
gram positive or gram negative, and also allowed us to view the shape of the bacteria. To gram
stain, gram’s crystal violet was used as the primary stain and then after 1 minute was rinsed off
with deionized water. Then grams iodine which is mordent was flooded over the slides and
rinsed with alcohol. After rinsing with alcohol, it was quickly removed and then rinsed with
deionized water. Then lastly slides were flooded with safranin and again rinsed with water. This
resulted in the gram-positive bacteria being purple and the gram-negative being stained with pink
color. After determining the shape of the bacteria under microscope, the next step was to
determine what biochemicals tests needed to be performed. To determine that the following flow
Figure 2
The flow chart was a good source to start and to determine what biochemical tests needed to be
performed. For one of the gram-positive bacteria by looking at the MSA plate, the nitrate and
catalase test were performed. The purpose of nitrate test is to determine if the enzyme nitrate
reductase is present. To perform this test, the bacteria is transferred to nitrate broth using a sterile
inoculating loop. For the best results the bacteria were in incubator for 48-72 hours. After two
days, 7 drops of nitrate solution A and B were added to the nitrate broth. If the solution turns red,
it means it’s positive for the enzyme nitrate reductase. A catalase test was also performed by
placing small amount of bacteria onto NA plate and flooding the plate with H2O2. If the bacteria
produced bubbles when flooded with H2O2, then it is positive for catalase. For my gram-negative
bacteria, by looking at my MAC plate I inoculated bacteria from MAC slant into SIM stab to see
if a black precipitate is formed. Then, I inoculated my bacteria into urea broth to test for the
enzyme urease. If urease was present the broth will turn from a light orange to hot pink. Next, I
inoculated bacteria from EMB and MAC into lactose broth to differentiate between gram (-)
lactose fermenters and non-fermenters. After allowing them to grow for 24-48 hours in lactose
broth it was determined that the bacteria were lactose fermenter or not and further biochemical
tests for gram negative bacteria were performed like IMVIC. Based on the biochemical tests and
due to some wrong techniques performed I was only able to determine two unknown bacteria in
my unknown B48.
Results:
By looking at MSA plate I was aware of what could be the two possible bacteria. So, to
confirm that I performed nitrate and catalase test as mentioned above. which came out to be
positive for both tests and so it was determined that the bacteria was staph aureus and not
micrococcus. Bubbles were formed for the catalase test and nitrate broth turned red after nitrate
solution A and B were added. Figure 3 shown below is positive result for nitrate test and figure 4
Next, for my second bacteria due to amber color colonies on MAC plate, I tested it for
SIM stab. Which came out to be negative and confirmed that it’s not proteus. Then I performed
urea test which also came out to be negative. Figure 5 shown below is negative test for SIM stab.
Figure 5
Next, I inoculated bacteria in lactose broth and the bacteria fermented sugar with gas
produced in durham tube. It was a strong lactose fermentation so, I performed citrate test from
IMVIC tests. The citrate test results were negative. So, if citrate is negative and SIM stab is also
Conclusion:
Due to failure in performing some technique and getting wrong results I was able to
determine only two bacteria for my unknown B48, which are Staph aureus and E. coli.