Running head: IDENTIFICATION OF UNKNOWN BACTERIA LAB REPORT 1
Identification of Unknown Bacteria Lab Report
Rosalind Belser
Grand Canyon University: BIO-205 L
November 3, 2019
IDENTIFICATION OF UNKNOWN BACTERIA LAB REPORT 2
Identification of Unknown Bacteria Lab Report
Aims
Activity1
The purpose of this lab is to determine the unknown bacteria’s species through
performing the gram test, streak plate method while learning more methods and tests such as the
lactose fermentation, oxidase, UTI membrane, EMB plate for gram negative organisms and the
Catalase test, Esculin rapid membrane, pigmentation, Blood agar plate, and Mannitol salt agar
plate for gram positive organisms.
Materials
Activity 1
24-hour unknown bacterial culture broth
Glass microscope slides
Bunsen burner
Inoculating loop
Permanent marker
Gloves
NA plate
MAC plate
EMB plate
Blood agar plate
Hydrogen peroxide
Oxidase test strip
MSA plate
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Bile esculin filter
UTI filter
Method
Day 1 Activity 1
1. Streak the organism onto one NA plate for colony isolation and incubate at 37oC
for 24-48 hours.
2. Streak the organism onto one MAC plate for selection and differentiation
analysis.
Day 2
1. Once isolated colonies are achieved from the streak plate procedure, do a Gram
stain (confirm with the instructor). Be sure to note the Gram reaction and the
morphology and arrangement of the cells.
2. If the organism grows on the MAC plate and is Gram-negative, proceed with the
various tests by following the Gram-Negative Rods portion of the flow diagram
(see Figure 14.7). Record results of the four different tests in the lab notebook.
3. If the organism does not grow on the MAC plate and is Gram-positive, proceed
with the various tests by following the Gram-Positive Cocci portion of the flow
diagram (see Figure 14.7). Be sure to record results of all tests in the lab
notebook.
Day 3
1. For Gram-negative bacteria observe and record the results from the EMB
plate.
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2. For Gram-positive bacteria observe and record the results from the Blood agar
plate or the MSA plate.
Results
Streak plate
MAC plate (left side bacteria #23, right side bacteria #9)
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EMB plate
Table for Unknown
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Analysis
Bacteria identification is important in order to determine what type of treatment to give to others
and to determine if it is resistant to antibiotics. To determine the bacteria you can either use
selective or differential media. The selective media allows an organism to grow while it stops
certain organisms from growing, while differential media you use different organisms but they
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all have different reactions so you can tell the difference between the organisms on one plate.
Biochemical test identifies the enzyme a bacteria produces, which would be the oxidase test. The
unknown bacteria was Enterobacter aerogenes based on the results up above. The test for the
lactose fermentation was positive meaning that the colonies that was on the MacConkey plate
was pink which means that the bacteria produces lactose., The oxidase test was negative, which
when performed lets you know if the bacteria does or does not have the enzyme cytochrome c
when it is oxidized. UTI membrane was positive, which means that someone has a UTI
infection, it is the fastest way in a clinical setting to determine if someone has a UTI and you can
determine the bacteria type by the color the UTI membrane turns (also known as a rapid IDD
membrane) , and the last test was EMB plate, which was negative, and tells you how much
lactose there is. Since E.coli and Enterobacter are both Gram-negative rods that look similar
under the microscope you have to perform some test to differentiate them. The tests that would
be performed to differentiate them would be the UTI membrane and the EMB plate tests to see
how they react with each test. In the UTI membrane the Enterobacter should turn a blue/ purple
color while the E.coli would turn a purple pink. In the EMB plate the two bacteria would have a
different color compared to each other, the E.coli would turn the plate green, while the
Enterobacter would be a blue dot on a pinkish background. The other two tests weren’t
mentioned because they both have the same end result. The MacConkey agar lets you know
whether or not lactose fermentation is produced from the bacteria. Possible errors for results that
require repeated attempts to identify the unknown bacteria are contamination, the wrong bacteria
was analyzed, and maybe the gram stains were incorrectly performed (like the decolorization
process was done too long or too short).
Conclusion
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The objective of this lab was to determine what the unknown bacteria was. In this lab we
ran through multiple test ranging from looking at the organism under a microscope to completing
the four tests, which include, the lactose fermentation test, the oxidase test, the UTI membrane
test, and the Eosin Methylene Blue plate after identifying that the bacteria was a gram-negative
bacteria. Based on the flow chart for the gram-negative bacteria, the bacteria should be
Enterobacter , however the EMB plate was the only thing to remain colorless in all the test,
which leads me to believe that there was an error somewhere. So if I were to do this experiment
again, I would probably do the different test multiple times for the very reason of errors. The
reason for performing these test is to determine the type of bacteria a patient might and these test
help to narrow down the bacteria because each bacteria reacts to each test differently and as a
nurse it is very likely that I will be the one performing or assisting in these test. It is important to
know what type of bacteria a patient has so you know what type of medication to give them to
treat the bacteria.
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