Gram Negative Unknown Lab Report

# 31

BIOL 3444-007
Diana Monroe
TA: Madhab Sapkota

Abstract
In this experiment an unknown gram-negative sample was obtained randomly to identify the possible
microorganisms. Using comparative analysis several biochemical tests were performed to determine
which bacterium out of the six potential unknowns was given. The biochemical tests carried out
included; triple-sugar iron agar (TSIA), sulfur indole motility (SIM), citrate, urease, gelatinase, methyl red
(MR) and voges-proskaeur (VP). In order to determine the microorganism characteristics the sample
was first isolated using a t-streak and the colonies were gram stained to reveal its shape and
morphology and then inoculated into several sequences of media corresponding with the proper
biochemical test. After allowing the corresponding time for each biochemical test, data was collected to
determine the unknown bacteria. The broth culture in this experiment was determined as Escherichia
coli.
Introduction
All organisms are divided into three domains; bacteria, archaea, and eukarya. The organisms making up
domain Bacteria and domain Archaea are all prokaryotes. Although bacteria and archaea look the same,
archaea is more closely related to eukarya (Madigan et.al 2009). The ability to adapt to a broad range of
habitats helps to explain why prokaryotes are the most abundant organism on earth. The main
characteristics of a prokaryote include, no nucleus, circular DNA, and no membrane bound organelles. A
key feature of nearly all prokaryotic cells is the cell wall, which maintains cell shape, and provides
physical protection. Most bacterial cell walls contain peptidoglycan, a network of modified-sugar
polymers cross-linked by short polypeptides.
All known pathogenic bacteria fall under prokaryotes, but not all bacteria are pathogenic (Madigan et.al
2009).
Using a differential staining technique bacteria can be divided into two groups; gram positive or gram
negative. The gram-positive bacteria have thick cell wall made of peptidoglycan. Gram-negative bacteria
have thinner layer of peptidoglycan, and are structurally more complex, with an outer membrane that
contains lipopolysaccharides. Gram staining is a valuable tool to determine whether an unknown
bacterium is gram negative or gram positive. Samples are first stained with crystal dye and iodine, then
rinse in alcohol, and finally counterstain with safranin. In gram-positive bacteria the crystal violet
adheres to the peptidoglycan layer staining it purple,. In gram-negative the crystal violet is easily
removed due to the thinner layer of peptidoglycan making the cell appear pink or red. (Leboffe 2010).
The domain bacteria is very diverse, making it impossible to identify it without the use of staining and
biochemical tests. Biochemical tests can test for a variety of characteristics such as shape, morphology,
pH, etc. that are specific to a certain type of bacteria. Tests such as Methyl Red and Voges- Proskauer
check for glucose fermentation. The Citrate Test is a nutrient utilization test that determines whether a
bacterium produces the enzyme citrate- permease to be utilized as its sole source of carbon. Urease test
is used to indicate if a bacterium can hydrolyze the intracellular enzyme urea. The Gelatinase test
indicates whether or not a bacterium contains this exoenzyme which hydrolyzes the proteins and allows
the gelatin to be converted into solid when is chilled.
Two types of combination differential media biochemical tests were used; SIM (Sulfur Indole Motility)
and TSIA (Triple Sugar Iron Agar). SIM gives three test results; Sulfur reduction, which determines
whether a bacterium can use enzymes to reduce sulfur to hydrogen sulfide, Indole production used to
determine if the bacteria can hydrolyze tryptophan to pyruvate, ammonia, and motility when in the
semisolid media a motile bacteria can diverge from the stab line. TSIA is designed to differentiate
bacteria with reference to glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur
reduction (Leboffe 2010).

Materials and Method

To prevent errors in any procedure personal protective and equipment aseptic technique was strictly
used. These techniques included sterilizing the loop on the Bunsen burner between inoculations and
flaming the opening of the test tube before inserting in the loop with the bacteria. To isolate pure single
colonies the unknown # 31 microorganism was T-streaked on a TSA plate and a TSA slant for backup.
The plate was incubated for 24 hours in a in a 37 degree Celsius room and then transferred to a cold
room 4degree Celsius room.
After incubation, a gram stain was performed on the isolated single colonies to determine if the
unknown microorganism was indeed gram negative. First, the organism was smeared onto a slide with a
drop of distilled water. The smear was allowed to air dry and heat fixed right after to provide bacteria to
stick onto the slide. Staining started with crystal violet, which is used to flood the slide completely for a
minute and once again rinsed for about 10 seconds with DI water. The slide was blotted dry with
bibulous paper and was observed using the microscope under 100x oil immersion. The color, shape, and
morphology of the bacteria were recorded.
The first and easiest test conducted was an oxidase test, which is use to test for the presence of
cyrochrome c oxidase. A small amount of the culture was transferred onto a reagent paper, and rinsed
with a drop of DI water. Data was recorded within 20 seconds.
Next the TSIA test was performed. The test proves whether a bacterium can ferment carbohydrate,
produce gas and produce Hydrogen sulfide. The bacterium was swiped on to the needle and was stab
two thirds down the TSIA media then streaked along on the TSIA slant. It was later placed in the hot
room for 24 hours. After 24 hours the TSIA and SIM test medium were taken out to observe the results.
For the TSIA, the butt and slant were inspected for color change. Yellow indicates positive for glucose
and lactose fermentation with acid accumulation, and black precipitate (sulfur reduction), and gas
production.
The SIM test was performed to test for sugar fermentation, indole and motility. The semisolid medium
contains casein, amino acids, an iron-containing compound, and sulfur in the form of sodium thiosulfate.
The SIM tube was first inspected for back precipitate indicating sulfur reduction and cloudiness around
stab line indicating motility. Kovacs reagent was added in the medium (a depth of 2-3 mm) with a red
color change in the alcohol layer. Red color is positive for indole production.
After 48 hours the MR and VP test mediums were taken out to read the results. This particular test
determines if the organism has the ability to ferment. Both tests consist of peptone, glucose and a
phosphate buffer. Three drops of Methyl Red were added to the MR test tube. As the solution becomes
more acidic with the added methyl red indicator a positive MR would turn read immediately. Negative
results would have no color change in the media. For the VP test, 15 drops of reagent A, followed by 5
drops of reagent B were added and mixed well together. The tube was checked for color change every
10 minutes for 60 minutes. Red color change indicated VP (+), whereas no color change indicated VP (-).
To determine the capability of the microorganism to use citrate as its source of carbon a citrate
utilization test was used. The citrate test was inspected for color change after 24 hours and then 48
hours. If the bacterium survives in the medium and utilizes the citrate it will also converts the
ammonium phosphate to ammonia and ammonium hydroxide, both of which tend to alkalinize the agar.
Even a small amount of blue color change meant citrate was utilized.
Gelatinase test is utilized to establish the ability of a microbe to produce gelatinase, an extracellular
enzyme secreted by some microorganisms to hydrolyze gelatin, which causes the media to liquefy even
after getting chilled. The Gelatin media was placed in the 4 degree Celsius room for one week. After 24
hours, the gelatin media was checked to see if it solidified. If it did, it meant no gelatinase was present. If
the solid in the tube remained for the initial time, the process of incubation is repeated for eight days.
After the eight days, if the solid still continued then it is considered negative (Leboffe and Pierce 171).
Urease test is used to differentiate organisms based on their ability to hydrolyze urea with enzyme
urease. The Urea test was inspected for color change every 24 hours for 1 week. If the orange/ yellow
medium changed to a pinkish color, this meant urea was produced or hydrolyzed. If initial observation
did not incur a pink color change; considered as a positive result, then it is then placed back into the hot
room and the process was repeated for eight days. If no change then the test was concluded to be
negative (Leboffe and Pierce 166).
Results
TSIA H2S Indole Motility MR VP Citrate Urease Gelatin
Unknown # 31 A/A - + + + - - - -

TSA plate was observed after 24 hours of incubation; the individual colonies appeared to be white and
small. After placing a smear under the microscope, the bacteria appeared pink color and rod-shaped.
TSIA was observed after 24 hours and cracks in the media were observed. Both the butt and slant were
yellow indication that the bacteria fermented glucose and lactose. The SIM was also observed after a 24-
hour period but there was no change in the slants color however there was murkiness away in the slant.
Citrate test results were negative with the added bromthymol blue indicator.
After 48 hours the MR and VP tests were inspected. The MR test was confirmed to be positive due to
the immediate color change to red indication of mixed acid fermentation. The VP test media had no
change in color when the reagents were added to the broth. The urease broth media had no change of
color after one-week incubation. The gelatin test concluded as negative since the media remained solid
in the cold room (after one week in the hot room) indication that no gelatinase was present in the
unknown sample.

Discussion
The organism was confirmed to be negative using gram staining. The TSIA test was intended for testing
for fermentation, hydrogen sulfide utilization, and gas production. The yellow slant and yellow butt
indicated the organism was able to ferment glucose and lactose along with buildup of acid.
The SIM test showed some signs of cloudiness away from the stab line. A color change of Kovacs
reagent to a red color showed the bacteria was positive for indole production, indicating that the
organism had the ability to hydrolyze tryptophan to pyruvate. In addition the organism was able to
move in medium, which indicated motillty (Leboffe and Pierce 177).
The Citrate test resulted negative due to no change in color. This indicating that the organism did not
have the ability to convert the ammonium phosphate to ammonium and ammonium hydroxide and the
organism did not use citrate as its carbon source.
The MR test show a low pH indication and the medium turned slightly red after adding methyl red
indicator drops to the tube. This indicates the organism can indeed carry out a mixed acid fermentation.
For the VP test, the medium remains yellow after adding Barritts Reagent A and Barritts Reagent B to
the tube. This indicates that the organism was not able to convert acid by-product to acetoin and 2, 3-
butanediol, in other words, glucose fermentation.
The Gelatinase test was negative due to the gelatin remaining solid indicating that the organism did not
have the ability to hydrolyze gelatin. Lastly, the Urease test did not undergo color change resulting
negative when the organism is not able to hydrolyze urea with enzyme urease.
After comparing the data and results from the biochemical tests along with the colony morphology,
shape and gram- negative unknown chart, it was clear that the unknown sample #31 was Escherichia
coli.
Some of the results had similar characteristics to other gram-negative bacterium. TSIA test was helpful
to narrowing down the search from six to three unknown organisms. To be able to differentiate the
three organisms, MR test was issued to narrow down once again to two unknown organisms,
Escherichia coli and Klebsiella pneumoniae. Left with two organisms, Citrate and urease tests finally
indicated the distinction between the two organisms.
Escherichia coli are a Gram- negative bacterium commonly found in the human digestive tract, which
helps to break down food and helps absorb many necessary vitamins, like vitamins B12 and K (Hu 2002).
While some strain of Escherichia coli are helpful, there are other strains that are harmful that can cause
stomach cramps, vomiting, and diarrhea when a person is infected.
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Neil A. Campbell, Jane B. Reece, 2008, Biology: Eighth Edition. Pearson Benjamin Cummings