Michael J.

Wallach II
10/13/2009

Classification of
Various Bacteria
through
Observations of
Growth on
Various Media
Types.

1
 INTRODUCTION

Microbes can be identified and classified based on a few factors: their metabolic processes, growth
requirements, and structural and functional morphology. These experiments investigated the techniques of several
tests in various media types to identify bacteria. All tests performed as well as media type descriptions are
summarized in Table 1. Classification of bacteria involves analysis and organization based on structural similarities.
Identification of bacteria uses several characteristics to enable sorting into taxonomic groups.(). Identification
requires information obtained from various tests and often relies on help from dichotomous keys to direct the order
of testing. A dichotomous key is a hierarchal flow chart that enables scientists to identify bacteria in a systematic
way. Each level of testing is accompanied by yes or no questions regarding the results. The key ends with
individual organisms listed on the bottom row. ().
Microbes require consideration of many environmental factors to grow successfully, including: water
availability, salt concentration, pH, temperature, concentration of oxygen (O2), pressure, and radiation.(). Bacteria
can be divided into groups based on where they derive an energy source and a carbon source. Photoautotrophs
derive energy from light and utilize the carbon from carbon dioxide (CO2). Chemoautotrophs also utilize the carbon
from CO2 but derive their energy from chemical compounds, such as hydrogen or sulfur. Photoheterotrophs derive
energy from light but utilize carbon from organic compounds other than CO2. Chemoheterotrophs, the group
containing most animals and bacteria, derive their energy from chemical compounds and utilize the carbon from
organic compounds other than CO2.().
Bacteria can be called obligate aerobes, obligate anaerobes, facultative anaerobes, aerotolerant anaerobes,
and microaerophiles. Obligate aerobes must utilize O2 for respiration and obligate anaerobes must rely on
fermentation or find another final electron acceptor in respiration. Facultative anaerobes are able to switch back and
forth from fermentation and aerobic respiration depending on the surrounding environment’s oxygen supply.
Facultative anaerobes are able to produce more energy when undergoing aerobic respiration. In aerobic respiration,
O2 acts as the final electron acceptor and CO2 is created as a gaseous byproduct. In fermentation lactic acid or
alcohol are main products that are formed. During alcohol fermentation, pyruvate is decarboxylated yielding CO2
release. During lactic fermentation, no CO2 is released. Fermentation products are acidic. Aerotolerant anaerobes
are able to detoxify O2 in its reduced form but cannot participate in aerobic respiration. These organisms rely on
enzymes such as catalase to break down hydrogen peroxide into water and oxygen. Microaerophiles live in defined
oxygen level environments and require less than a 10% environmental oxygen level.().
Bacterial cells can also be classified based on individual cell shape (spherical/cocci, rod-like/bacilli, or
helical/spirilla) and in the groups the individual cells form. For example, cocci cells can either be diplococci
(attached in pairs), streptococci (attached in chains), tetrads (groups of four cells), sarcinae (in a cuboidal
arrangement), or staphylococci (attached in clusters). While rod-like or bacilli cells are usually found as single cells
but can sometimes attach in pairs (diplobacilli) or chains (streptobacilli).
Media can be broken down into two of five categories: defined or complex; and selective, differential, or
both. In defined media, all the components are known and are very specific. In complex media, not all the

2
 ingredients are specifically known. Complex media are often derived from extracts. Consequently, scientists are
able to have a general idea of the components but not an exact inventory. Selective media allows or inhibits the
growth of one type of group, or in some cases specific microbe. Differential media allows the growth of multiple
groups and allows for identification of each through different appearances, often through a color change in response
to metabolic products. Media classified as both selective and differential are able to simultaneously select for or
against growth of one group while allowing scientists to differentiate between growths of two other groups. Media
can also be liquid or solid. (). Liquid media are called broths and solid media are generally agar. By understanding
the demand for certain conditions by a species, or even genus, of bacteria, scientists are able to create a series of
tests on various media used to replicate microbial environments. Enzymes tested for on the various media types
used were: catalase, DNase, tryptophanase, gelatinase, cysteine desulfurase, and urease. Catalase is an enzyme that
is used by obligate aerobes, facultative anaerobes, and microaerophiles to break down and detoxify hydrogen
peroxide into oxygen and water. DNase is an enzyme that hydrolyzes the DNA of the host a bacterium infects.
Tryptophanase is an enzyme that hydrolyzes tryptophan into pyruvate and indole. Gelatinase is an enzyme that
hydrolyses gelatin to extract peptides and amino acids for energy. This stops gelatination from occurring. Cysteine
desulfurase breaks down cysteine and methionine resulting in hydrogen sulfide (H2S) as by-product. Urease is an
enzyme that hydrolysis urea into ammonia and CO2. The ammonia reacts with water to form ammonium hydroxide
which causes a rise in pH of the broth culture. ().
Indicators used in differential media to produce the visual change are most often methyl red and phenol red.
These indicators react to changes in pH. Methyl red is red at a pH below 4.4 and at pH 4.4 begins turning yellow.
As the pH increases, red becomes increasingly yellow until reaching a full yellow color at pH 6.0 and beyond.
Phenol red is yellow at a pH below 6.8 and begins to turn red at pH 6.8. As the pH increases, yellow becomes
increasing red until reaching a full red color at pH 8.4 and beyond. As the pH increases still, the color red will
deepen. (). These sensitive pH indicators can be used in conjunction with media types to select for and differentiate
between bacteria with different metabolic pathways.

Table 1: The media types used in addition to their explanation and definition of a positive and negative test.
().

Negative Test
Media Reaction Rational for Results Positive Test (+)
(--)
Organisms that utilize citrate also
Citrate is the sole carbon
convert ammonium phosphate
source and ammonium
Simmon’s into ammonium. This reaction Blue Color/ Citrate Green/ No Citrate
phosphate as the sole
Citrate causes an increase in pH and Oxidized Oxidized
nitrogen source of this
changes the bromothymol blue
defined medium.
pH indicator from green to blue.
This media is composed of
Organisms that produce
gelatin (derived from
gelatinase will hydrolyze the
collagen), peptone and
Nutrient gelatin in the media, causing it to Not Solidified/ Solidified/ No Gelatinase
beef extract. It is used for
Gelatin liquefy. Organisms that do not Gelatinase Present Present
the identification of
possess the enzyme will not be
organisms that produce
able to liquefy the media.
the enzyme gelatinase.
Urea Broth This differential media is Organisms that produce the Red/ Urease Yellow-Orange/ No Urease
used to distinguish rapid enzyme urease will be able to Present Present

3
 urease positive bacteria
from slow urease-positive
or urease negative
organisms. The media
contains phenol red
indicator, peptone, glucose
and urea.
This media identifies
organisms that produce
DNase
the exoenzyme DNase.
Methyl
This enzyme catalyzes the
Green
depolymerization of DNA
in the media.
This media is both
selective and differential.
The high salt
concentration selects for
Mannitol organism that are
Salt Agar halophiles, while the
mannitol in the media
differentiates between
organisms ability to utilize
this sugar source.
EMB is a selective and
differential media. It
contains lactose in
Eosin addition to eosin and
Methylene methylene blue dyes. The
Blue (EMB) dyes inhibit the growth of
Agar Gram-positive organism.
The media differentiates
the degree of lactose
fermentation.
Endo agar is a selective
and differential media. It
contains lactose in
addition to sodium sulfite
and basic fuchsin dye. The
Endo Agar sodium sulfite and basic
fuchsin inhibit the growth
of Gram-positive organism.
The media differentiates
the degree of lactose
fermentation.
Blood agar is trypticase
soy agar supplemented
with 5% sheep blood. The
Blood Agar blood allows for
differentiation of bacteria
based on their hemolytic
properties.
MacConkey MacConkey agar is a
Agar selective and differential
convert urea to ammonia, thus
increasing the pH and changing
the color of the pH indicator in
the media to a bright pink.
DNA fragments in the media are
conjugated to the dye methyl Decolorized ring
green. Organisms that produce around growth/
this enzyme will cleave the DNA DNase Produced No decolorized ring/ No
in the media into smaller pieces DNase Produced
and uncouple the DNA-dye + mild sized
complex resulting in a clearing of ++ large sized
the media.
Organisms that have the ability to
grow on this media can withstand
high salt concentrations, termed
halophiles. The differential
component of this media uses
phenol red to detect the ability of
the organism to utilize mannitol. Yellow/ Mannitol No Color Change/ No
If the organism converts mannitol Fermented Mannitol fermented
into an acid product, the media
will turn yellow. Organisms that
utilize the peptone in the media
will produce ammonia and
change the color of the media to
pink.
Organisms that are Gram
negative are able to grow on this
media. The degree to which
lactose is fermented is measured
by a color change of the colonies. Black Centers/ Colorless or Light Pink/ No
Colorless or light pink colonies Lactose Fermented Lactose Fermented
indicate slight lactose
fermentation while purple and
metallic green colonies indicate
heavy lactose fermentation.
Organisms that are Gram
negative are able to grow on this
media. The degree to which
lactose is fermented is measured
by a color change of the colonies. Red/ Lactose Colorless/ No Lactose
Colorless or light pink colonies Fermented Fermented
indicate slight lactose
fermentation while purple and
metallic green colonies indicate
heavy lactose fermentation.
Hemolytic reactions are classified Green-Dark/
as alpha (showing partial α-hemolysis
No Discoloration/
destruction of RBCs), beta Or
γ-hemolysis
(complete destruction of RBCs) Clear Zone/
and gamma (no hemolysis). ß-hemolysis
Organisms that are Gram Pink to Red/ Colorless/ No Lactose
negative will be able to grow on Lactose Fermented Fermented
4
 media. It contains lactose
bile salts, neutral red and
crystal violet. The bile
salts and crystal violet
inhibit the growth of Gram-
positive organism. The
media differentiates the
degree of lactose
fermentation using neutral
red as a pH indicator.
This differential media
identifies an organism’s
ability to breakdown milk
products and lactose using
Litmus Milk the enzymes rennin,
casease and β-
galactosidase. The pH
indicator in the media is
azolitmin.
TSI agar is a medium
designed to differentiate
organisms based on their
Triple ability to ferment glucose,
Sugar Iron lactose, sucrose and
produce hydrogen sulfide.
Phenol red is the pH
indicator.
VJ agar is selective for
coagulase positive
staphylococci and Gram-
VJ Agar
negative bacteria. The
media also differential for
tellurite reduction.
MR–VP
Broth MR-VP broth is a
(Methyl combination media used
Red Test) for both the methyl red
MR–VP and Voges-Proskauer tests.
Broth The media contains a
(Voges- buffer, glucose and
Proskauer peptone.
Test))
SIM (H2S)
SIM SIM media is used to
(Indole) identify the motility of an
organism, the production
of indole and hydrogen
sulfide.
SIM
(Motility)
Catalase This test can be done on
Test any agar surface that has
this media. Organisms that
produce acid end products from
lactose fermentation will
decrease the pH of the media, as
indicated by a red color produced
from the neutral red indicator.
A variety of results is possible
with this media. If the pH
decreases as a result of lactose Color Change,
fermentation, then the media will Curdling, Acid
No Color Change/ No
turn pink. An alkaline reaction Clots, Gas
Reaction
results in a blue color. Other Production (See
results include curdling of the Figure 1)
media, acid clots and gas
production.
The agar is prepared as a slant
with a deep butt, providing both
aerobic and anaerobic
environments. A variety of results
is expected with this media all
based upon changes in the color
Many Reactions Possible (See Figure 2)
of the media associated with
changes in pH. Acid production is
indicated by a yellow color,
alkaline reactions by a red color
and hydrogen sulfide by a black
color.
Coagulase positive and Gram-
Black Spots –
negative organism can grow on
yellow/ tellurite
this media. Those organisms that No Growth or No Color
produced –
reduce tellurite form black Change
Mannitol
colonies that contain a black
fermented
precipitate from tellurite.
Red/ Acid
The MR test is designed to detect
Production, Yellow, No Color Change/
an organism that under goes
Glucose No Glucose Fermented
mixed acid fermentation. Upon
Fermented
addition of methyl red, an acidic
environment will change the color
to red. The VP test detects an Red/ Acetoin Other color/ No Acetoin
organism’s ability to convert acid Produced Produced
products to acetonin.
An organism that shows a Black Precipitate/ No Color Change/ No H2S
radiating or diffusion pattern out H2S Produced Produced
of the inoculation stab tests
positive for motility. The Red/ Indole No Color Change/ No
production of a black precipitate Produced Indole Produced
is indicative of reduction of
hydrogen sulfide. Upon addition
of Kovac’s reagent, an organism Cloudy/ Growth
that produces the enzyme Not Restricted to Clear/ Immotile
tryptophanase will test positive Stab Line
(pink ring) for indoles.
Hydrogen peroxide is applied to Bubbles/ Catalase No Bubbles/ No Catalase
the colony and if the organism is Present Present
5
 colonies or it can be done
by transferring a colony to
a slide. Bacteria that
produce the enzyme
catalase can convert
hydrogen peroxide to
water and oxygen.
This media is selective for
Gram-positive organisms.
Phenylethyl
It contains phenylethyl
Alcohol
alcohol that is
Agar
bacteriostatic against
Gram-negative organism.
PR Glucose
PR Sucrose PR broths are differential
media that contain phenol
PR Lactose
red pH indicator and a
specific sugar.
PR
Mannitol
catalase positive, there will be
+few
production of gas bubbles (from
++medium
the oxygen gas).
+++high
Gram-positive organism show
normal colony morphology on this No Growth/ Gram-
Growth/ Gram-
media. Gram-negative organisms Negative or Gram-Positive
Positive Cocci
either do not grow or are severely Rods
limited on this media.
Acid production from the
fermentation of the carbohydrate
will lower the pH of the media
and change the color of the
See Key from Table 2
media to yellow. Organisms that
undergo deamination of amino
acids will turn the media alkaline
and pink or red in color.

MATERIALS AND METHODS

All biochemical tests were performed on solid agar plates, liquid growth media, agar deeps and/or agar
slants. Inoculation of the solid agar media involved the sterilization of a transfer loop using the flame from a
Bunsen burner prior to sampling from the pure culture and then aseptically streaking onto the solid agar plate.
Inoculation of liquid media was done in a similar manner, sterilizing the transfer loop before sampling from the
original pure culture and transferring the culture to the liquid media. Agar deeps were inoculated using an
inoculating needle that was sterilized using the flame from a Bunsen burner prior to sampling from the pure culture.
After sampling from the pure culture, the inoculating needle was stabbed into the center of the agar deep until the
needle reached the bottom of the tube and then the needle was carefully withdrawn from the agar. The agar slants
were inoculated in a similar manner as the agar deeps. The inoculating needle was flame-sterilized, then used to
sample from a pure culture. The needle was inserted into the agar in the deep end and then carefully withdrawn
from the deep and gently streaked across the surface of the slant. All agar plates where inverted before incubation
and labeled on the bottom with group members’ initials, inoculation date, laboratory section number, media type,
test to be performed, and the inoculated specimen. Media in test tubes were labeled with the same information but
written on tape and then wrapped around the test tube.

Carbohydrate Metabolism and Fermentation

For carbohydrate metabolism and fermentation tests, the following media were used: six black capped
phenol red (PR) glucose broths (with Durham tubes to collect gas), six red capped phenol red (PR) sucrose broths,
six green capped phenol red (PR) lactose broths, six yellow capped phenol red (PR) mannitol broths, and two
Simmon’s citrate agar Y plates (plates subdivided into three sections). Escherichia coli, Salmonella typhimurium,
Staphylococcus epidermidis, Enterococcus faecalis, Proteus vulgaris and Bacillus subtilis were inoculated into each

6
type of media. All organisms were incubated at 37°C, except B. subtilis which was incubated at 30°C, for 18-24
hours. After incubation, the plates were placed in a 4°C refrigerator until observation around 48 hours post
inoculation. The results were recorded.

Microbial Enzymes
For microbial enzymatic tests, the following media were used: six red capped 16×100 nutrient gelatin agar
deeps, three red marked brain heart infusion (BHI) agar split plates (plates subdivided into two sections), six red
capped 16×125 SIM agar deeps, two brown marked DNase methylene green agar Y plates, and six yellow capped
16×125 urea broth. E. coli, S. typhimurium, S. epidermidis, E. faecalis, P. vulgaris, and B. subtilis were inoculated
into each type of media. For DNase agars, the organisms were inoculated by using an inoculating loop to aseptically
streak 1 thick line down the middle of the agar. All organisms were incubated at 37°C, except B. subtilis which was
incubated at 30°C, for 18-24 hours. After incubation, the media were placed in a 4°C refrigerator until observation
around five days post-inoculation. A single drop of 30% hydrogen peroxide was applied to the growth on the BHI
agar plates in order to determine the results of a catalase test. Ten drops of Kovac’s reagent was added to the SIM
agar deep to test for the presence of indole. Observations were also taken on the SIM agar deeps, urea broth, and
DNase agar. Liquefied gelatin agar deeps were placed in an ice bucket along with an uninoculated control. After
the control had solidified, observations were taken. All results were recorded. It was noted that the nutrient gelatin
tubes needed additional incubation time and were returned to their respective temperatures to incubate for an
additional five days. A DNase methylene green agar Y plate was streaked for P. vulgaris and B. subtilis, and P.
mirabilis then incubated for 37ºC for 18-24 hours and placed in a 4ºC refrigerator until observation 48 hours post-
inoculation. This was due to lack of indicator added the first time to the two DNase methylene green agar Y plates
prior to inoculation. Results were recorded.

Selective and Differential Media

For observations on selective and differential media, the following media were used: two mannitol salt agar
plates, three Trypticase soy agar (TSA) with 5% sheep blood plates, two MacConkey agar plates, two phenylethyl
alcohol agar plates, and two eosin methylene blue (EMB) agar plates. Each TSA blood agar plate was divided into
two sections using a black permanent marker to define halves from underneath. E. coli, S. typhimurium, S.
epidermidis, E. faecalis, Proteus mirabilis, and Staphylococcus aureus were inoculated into each type of media. All
organisms were streaked for isolation on the mannitol salt agar, EMB agar, MacConkey agar, and phenylethyl
alcohol agar (see Figure 1). This was done by first placing a thick, short, S-shaped streak in the corner of one split.
After sterilizing the inoculating loop, the thick S-shaped streak was streaked across the entire surface of the split.
All organisms were incubated at 37°C for 18-24 hours. After incubation, the plates were placed in a 4°C refrigerator
until observation around 48 hours post inoculation. The results were recorded.

7
 2nd Streak

1st Streak

Figure 1: Streaking for Isolation on Split plates ()

Gram Positive Bacteria

Gram positive rods were inoculated on the following media: two TSA with 5% sheep blood plates (one
divided in halves and one divided in fourths with a permanent marker), four red capped nutrient gelatin agar deeps,
two BHI agar split plates, and four green capped 16×100 litmus milk liquid media. Corynebacterium
pseudodiptheriticum, Bacillus cereus, B. subtilis, and Mycobacterium smegmatis were inoculated on each type of
media. Following streaking of each species on the TSA blood agar, the inoculating loop was stabbed in the agar 3-4
times. All organisms were incubated at 37°C for 18-24 hours. After incubation, the plates were placed in a 4°C
refrigerator until observation five days post inoculation. A single drop of 30% hydrogen peroxide was applied to
each growth on the BHI agar plate in order to determine the results of a catalase test. Liquefied gelatin agar deeps
were placed in an ice bucket along with an uninoculated control. After the control had solidified, observations were
taken. The results were recorded.
Gram positive cocci were inoculated on the following media: two TSA with 5% sheep blood plates (one
divided in halves and one divided in thirds with a permanent marker), 4 BHI agar split plates, seven black capped
phenol red glucose broths (with Durham tubes), seven green capped phenol red lactose broths, seven yellow capped
phenol red mannitol broths, three pink mannitol salt agar Y plates, and three orange VJ agar Y plates. S. aureus, S.
epidermidis, Micrococcus luteus, Micrococcus roseus, Streptococcus salivarius, Streptococcus pyogenes and E.
faecalis were inoculated on each type of media, making sure to incubate M. luteus and M. roseus alone together on
split plates and Y plates. All organisms were streaked for isolation (see Figure 1) on the VJ agar and mannitol salt
agar plates. Following streaking of each species on the TSA blood agar, the inoculating loop was stabbed in the
agar 3-4 times. All organisms, except M. luteus and M. roseus, were incubated at 37°C for 18-24 hours. M. luteus
and M. roseus were incubated at 30°C for 96 hours. After incubation, the plates were placed in a 4°C refrigerator
until observation five days post inoculation. A single drop of 30% hydrogen peroxide was applied to each species
growth on the BHI agar plate in order to determine the results of a catalase test. All results were recorded.

Gram Negative Rods

Gram negative rods were inoculated on the following media: seven black capped phenol red glucose broths
(with Durham tubes), seven green capped 16mm×125mm phenol red lactose broths, seven yellow capped

8
16mm×125mm phenol red mannitol broth, seven red capped SIM agar deeps, seven blue capped 16mm×125mm
triple sugar iron (TSI) agar slants, seven yellow capped 16mm×100mm urea broths, two pink endo agar X plates
(plates subdivided into four sections), three dark purple EMB agar Y plates, three green Simmon’s citrate agar Y
plates, three tan BHI agar X plates, 14 16mm×100mm tubes of MR-VP broth (seven with blue caps and seven with
green caps), and three light purple MacConkey agar Y plates. E. coli, Enterobacter aerogenes, Klebsiella
pneumonia, S. typhimurium, P. mirabilis, P. vulgaris, and Serratia marcescens were inoculated on each type of
media. No streaking for isolation was performed. All organisms were incubated at 37°C for 18-24 hours. After
incubation, the plates were placed in a 4°C refrigerator until observation 48 hours post inoculation. A single drop of
30% hydrogen peroxide was applied to each species’ growth on the BHI agar plate in order to determine the results
of a catalase test. Ten drops of Kovac’s reagent was added to each SIM agar deep and was allowed to sit for several
minutes to test for the presence of indole. Five drops of methyl red indicator was added to one MR-VP tube for each
species and allowed to sit for 20 minutes to observe color changes. To the other MR-VP tube for each species, 1.2
mL α-Naphthol Reagent and 0.5 mL 40% KOH were added. These tubes were allowed to sit for 30 minutes to
observe color changes. The results for all tests were recorded. (Christine Simmons, 2009)

RESULTS

Carbohydrate Metabolism and Fermentation

Following incubation, E. coli was observed to produce acidic products in the PR glucose, PR lactose, and
PR mannitol broths, changing the phenol red to yellow. In PR sucrose, the phenol red was darkened, and on
Simmon’s citrate agar the green color was preserved. Also, the presence of bubbles was noted in the Durham tube
in the PR glucose broth. For S. typhimurium, a yellow color change was observed in PR mannitol and PR glucose,
while a darkened red color was observed in PR sucrose and PR lactose. Also, bubbles were observed in the Durham
tube in the PR glucose broth. S. typhimurium caused a color change to blue in the Simmon’s citrate. For both E.
faecalis and S. epidermidis, a yellow color changed was observed in all PR sugar broths and no color change was
observed on the Simmon’s citrate agar. For P. vulgaris, a color change was observed to yellow in PR glucose and to
blue in Simmon’s citrate agar. For B. subtilis, color changes were observed in PR glucose, PR sucrose, and PR
mannitol only. These results are summarized in Table 2.

Table 2: Results from Carbohydrate Metabolism and Fermentation with

accompanying symbol key

Growth Escherichia Salmonella Enterococcus Staphylococcus Proteus Bacillus

Medium coli typhimurium faecalis epidermidis vulgaris subtilis
PR
AG AG A A A A
Glucose
PR Sucrose K K A A K A
PR Lactose A K A A K K
PR
A A A A K A
Mannitol

9
Simmon’s
-- + -- -- + --
Citrate
Symb Observation Meaning
ol
A Yellow Acid Production
AG Yellow with Bubbles Acid & CO2 gas
Produced
K Deep Red Alkaline
NC Little to No Color Change Oxidative
(Remains Red) Respiration
+ Blue Citrate Oxidized
-- No Color Change (Remains No Citrate Oxidized
Green)

Microbial Enzymes
Analyzing the SIM deep agar showed blackening only for S. typhimurium indicating H2S production.
Reaction with the Kovac’s reagent yielding a pink ring indicative of indole production was observed in only E. coli.
Growth beyond the inoculated stab indicating motility was observed in both E. coli and S. typhimurium (Figure 2).
All organisms, except for E. faecalis, produced bubbles when the hydrogen peroxide was added to the BHI agar
plates, with S. typhimurium and P. vulgaris showing medium and high bubble production respectively (Figures 3-5).
The nutrient gelatin agar remained solid at room temperature for all organisms except for B. subtilis, which was also
able to resist solidification following cooling in an ice bucket. All organisms tested negative for urease, resulting in
no color change in the urea broths. The first round of DNase plates were unable to be observed due to lack of
indicator added prior to inoculation. For the second attempt with indicator added, only B. subtilis was unable to
produce a colorless ring around growth in the agar. P. vulgaris produced a large decolorized ring and P. mirabilis
produced a medium sized decolorized ring (see Figure 6). These results are summarized in Table 3.

Figure 2: SIM agar deeps after addition of Figure 3: BHI agar after addition of H2O2. Notice
Kovac’s reagent. Label from left to right is B. large P. vulgaris bubble formation on top
subtilis, P. vulgaris, S. epidermidis, S. compared to B. subtilis on bottom.
typhimurium, E. faecalis, and E. coli

10
Figure 5: BHI agar after addition of H2O2. Notice
Figure 4: BHI agar after addition of H2O2. E. S. typhimurium bubble formation on bottom
faecalis is on top and S. epidermidis is on bottom. compared to E. coli on the top.
 Figure 6: 2nd attempt DNase agar plate post inoculation
viewed from both sides. Statrting at the top and moving
clockwise on the bottom up view is B. subtilis, P. mirabilus,
and P. vulgaris.

Table 3: Results from Lab 6 – Microbial Enzymes. It includes only the second DNase test performed.
Positive and Negative test descriptions can be found in the last two columns of Table 1.

Test Escherichia Salmonella Enterococcus Staphylococcus Proteus Bacillus Proteus

Performed coli typhimurium faecalis epidermidis vulgaris subtilis mirabilis
Gelatin
-- -- -- -- -- +
Hydrolysis
SIM Deep:
Hydrogen -- + -- -- -- --
Sulfide
SIM Deep:
+ -- -- -- -- --
Indole
SIM Deep:
+ + -- -- -- --
Motility
Urea
-- -- -- -- -- --
Hydrolysis
Catalase + ++ -- + +++ +
DNase ++ -- +

11
 Selective and Differential Media
Growth on the mannitol salt agar was only observed for S. epidermidis and S. aureus, both creating a
yellow color change. This color change was lightly defined in S. epidermidis (see Figures 7 and 8). On the
MacConkey agar, no growth was observed for E. faecalis, S. epidermidis, and S. aureus. Light pink colonies where
observed for both S. typhimurium and P. vulgaris. Pink colonies with dark centers were observed for E. coli (see
Figures 9 and 10). On phenylethyl alcohol agar, growth was observed for all organisms except for S. typhimurium
and P. mirabilis (Figures 11 and 12). On EMB agar, E. faecalis, S. epidermidis, and S. aureus were observed to
have colorless colonies, although E. faecalis had some colonies that we very light pink. Pink colonies were
observed for S. typhimurium and P. mirabilis. E. coli produced colonies with a metallic sheen (Figures 13 and 14).
On blood agar, hemolytic activity through either a darkening or clearing of the blood agar was witnessed with only
E. coli, P. mirabilis, and S. aureus colonies. E. coli and P. mirabilis showed a darker, almost green color when
viewed from the bottom of the agar indicating α-hemolytic activity. S. aureus caused a clear ring to surround each
colony indicating ß-hemolytic activity. All non-color changing organisms where recorded as γ-hemolytic (Figures
15, 16, and 17). These results are summarized in Table 4.

Figure 7: Mannitol salt agar

with bottom up and top up
views. Inoculated with, starting
at the top yellow colony moving
clockwise, S. aureus, E. coli, and
S. typhimurium.

Figure 9: MacConkey Agar

inoculated with, starting from the
top and going clockwise, E. faecalis,
P. mirabilis, and S. epidermidis.
Viewed from the bottom up

Figure 8: Mannitol salt agar with bottom up and Figure 10: MacConkey agar with bottom up
top up views. Inoculated with, starting at the top and top up views. Inoculated with, starting
yellow colony moving clockwise, S. epidermidis, P. from the purple colonies and going clockwise,
mirabilis, and E. faecalis. E. coli, S. aureus, and S. typhimurium.

12
 Figure 11: Phenylethyl alcohol agar viewed Figure 12: Phenylethyl alcohol agar viewed
from the bottom. From the top left moving from the bottom. From the top left moving
clockwise there is P. mirabilis, E. faecalis, and clockwise there is S. aureus, E. coli, and S.
S. epidermidis. typhimurium.

Figure 13: EMB agar with top and bottom views. From Figure 14: EMB agar with top and bottom views. From
the top section on the right plate moving clockwise, we the top section on the right moving clockwise, we have:
have: P. mirabilis, E. coli, and S. typhimurium. S. aureus, S. epidermidis, and E. faecalis.

Figure 15: Blood agar with

top and bottom views. From Figure 16: Blood agar with Figure 17: Blood agar with
left to right, E. coli and S. top and bottom views. From top and bottom views. From
aureus. left to right, E. faecalis and left to right, P. mirabilis and
S. epidermidis. S. typhimurium.

Table 4: Observations from Lab 7 – Differential and Selective Media. Positive and Negative test
descriptions can be found in the last two columns of Table 1.

Escherichia Salmonella Enterococcus Staphylococcus Proteus Staphylococcu

Media
coli typhimurium faecalis epidermidis mirabilis s aureus
Mannitol Salt +
No Growth No Growth No Growth No Growth +
Agar (small yellow)
+
MacConkey + +
(Pink w/Dark No Growth No Growth No Growth
Agar (Light Pink) (Light Pink)
Centers)
Phenylethyl + -- + + -- +

13
Alcohol Agar
+ --
+ -- + --
EMB Agar (Metallic (Colorless/
(Pink) (Colorless) (Pink) (Colorless)
Green) Light Pink)
+ -- -- + +
--
Blood Agar (α- (γ- (γ- (α- (ß-
(γ-hemolysis)
hemolysis) hemolysis) hemolysis) hemolysis) hemolysis)

Gram Positive Bacteria

For the gram positive rods tested, the nutrient gelatin agar was liquefied following cooling in an ice
bucket for only B. cereus. On blood agar, hemolytic activity through either a darkening or clearing of the blood agar
was witnessed with C. pseudodiptheriticum, B. cereus, and B. subtilis colonies. C. pseudodiptheriticum and B.
subtilis showed a darker, almost green color when viewed from the bottom of the agar indicating α-hemolytic
activity. B. cereus caused a clear ring to surround each colony indicating ß-hemolytic activity. The non-color
changing organism M. smegmatis was recorded as γ-hemolytic. C. pseudodiptheriticum and B. cereus produced
medium sized bubbles when the hydrogen peroxide was added to the BHI agar. Small bubbles where observed for
M. smegmatis. No growth of B. subtilis was observed on the BHI agar. For the litmus milk test C.
pseudodiptheriticum was observed to have a semisolid grey curd at the top and be pink in overall color. B. cereus
was observed to have no overall color change and a semisolid grey curd at the top with a blue band. B. subtilis was
observed to have no color change. M. smegmatis was observed to have a grey curd at the top and no overall color
change (see figure 20). Litmus milk symbols are in Table 6.2. These results are summarized in Table 5.
For the gram positive cocci species tested, a yellow color change was observed in all species in the PR
glucose broth, with the addition of bubbles at the top of the Durham tube for S. aureus, M. luteus, and M. roseus (see
figure 21). For the PR lactose broths, S. aureus, S. epidermidis, and E. faecalis all produced acidic products,
causing the phenol red to turn yellow. M. luteus and S. salivarius caused a deeper red color change of the phenol red
indicating an increase in pH. M. roseus and S. pyogenes resulted in no color change (see figure 22). For PR
mannitol broths, no color change was observed for M. roseus, S. epidermidis, and S. pyogenes. S. aureus and E.
faecalis produced acidic products causing a yellow color change, and M. luteus and S. salivarius caused an increase
in pH creating a deeper red color change (see figures 23). For the catalase test, bubbles were produced on the BHI
agar when hydrogen peroxide was added for S. aureus, S. epidermidis, S. pyogenes, M. luteus, and S. salivarius; the
latter two producing a larger amount of bubbles at a faster pace. No growth was observed of M. roseus. On blood
agar, hemolytic activity through either a darkening or clearing of the blood agar was witnessed with S. aureus, M.
luteus, S. salivarius, and E. faecalis colonies. M. luteus, S. salivarius, and E. faecalis showed a darker, almost green
color when viewed from the bottom of the agar indicating α-hemolytic activity. S. aureus caused a clear ring to
surround each colony indicating ß-hemolytic activity. The non-color changing organisms, S. epidermidis and S.
pyogenes, were recorded as γ-hemolytic. No growth was observed for M. roseus (see Figures 18 and 19). For
mannitol salt agar plates, a yellow color change was observed for S. aureus and E. faecalis. S. epidermidis also
produced a yellow color change but was less significant. No growth was observed for S. salivarius. For the VJ agar
plates, no growth was observed for M. roseus, S. epidermidis, and S. salivarius. Both S. pyogenes and E. faecalis

14
 showed little growth of pink colonies. M. luteus grew pink colonies and S. aureus grew yellow colonies and both
had many black centers (see Figures 24, 25, and 26). These results are summarized in Table 6.

Figure 18: Blood agar with top and bottom views. Figure 19: Blood agar with top and bottom views.
From top to bottom, S. epidermidis and S. aureus. From the top section on the right plate moving
clockwise, S. salivarius, S. pyogenes, and E. faecalis.

15
 TABLE 6-2 Litmus Milk
Results and Interpretations
TABLE OF RESULTS
Result Interpretation Symb
ol
Pink Color Acid reaction A
Pink and solid Acid clot AC
(white in the
lower portion if
the litmus is
Figure 20: Litmus milk broths following reduced); clot
incubation. From left to right: Control broth, B. not movable
cereus, C. pseudodiptheriticum, M. smegmatis,
and B. subtilis. Fissures in the Gas G
clot
Clot broken apart Stormy fermentation S
White color Reduction of litmus R
(lower portion of
medium)
Semisolid and Curd C
not pink; clear to
gray fluid at top
Clarification of Digestion of peptone; P
medium; loss of peptonization
“body”
Blue medium or Alkaline reaction K
blue band at top
No change None of the above NC
reactions

Figure 21: Phenol Red Glucose Broths. From left to

right: M. roseus, M. luteus, S. epidermidis, E. faecalis,
S. salivarius, and S. aureus.

Figure 23: Phenol Red Mannitol Broths. From

left to right: M. roseus, S. pyogenes, E. faecalis,
S. aureus, S. salivarius, and S. epidermidis.

Figure 22: Phenol Red Lactose Broths.

From left to right: M. luteus, M. roseus, S.
pyogenes, S. epidermidis, S. salivarius, and
S. aureus.

16

Figure 24: VJ agar with views
of bottom and top. From the
top left section going
clockwise: E. faecalis, and S.
salivarius.
Figure 25: VJ agar with views of
bottom and top. From the top
left section going clockwise: M.
luteus and M. roseus.
Figure 26: VJ agar with views of
bottom and top. From the top
yellow section going clockwise:
S. aureus, S. pyogenes, and S.
epidermidis.

Table 5: Gram Positive Rod Bacterial Tests. Positive and Negative test descriptions can be found in
the last two columns of Table 1. Litmus milk observation symbols can be found in Table 6-2.

Corynebacterium Mycobacterium
Media/ Test Bacillus cereus Bacillus subtilis
pseudodiptheriticum smegmatis
Gelatin
-- + -- --
Hydrolysis
+ + + --
Blood Agar
(α-hemolysis) (ß-hemolysis) (α-hemolysis) (γ-hemolysis)
+
Catalase + + No Growth
(Very Little)
CKNC
Litmus Milk CA (darker than NC CNC
control)

17
 Table 6: Gram Positive Cocci Bacterial Tests. Positive and Negative test descriptions can be found in
the last two columns of Table 1. Phenol Red Broth symbol can be found in the key of Table 2.

Staphylococc
Staphylococc Micrococc Micrococcus Streptococc Enterococcu Streptococc
Media us
us aureus us luteus roseus us pyogenes s faecalis us salivarius
epidermidis
Phenol
AG A A A
Red AG AG A
(Cloudy) (Slightly) (Clear) (Slightly)
Glucose
NC
Phenol
K A (Very A K
Red A NC
(Cloudy) (Clear) Slight (Clear) (Clear)
Lactose
Yellow)
Phenol NC
NC
Red K (Very
A NC (Very Slight A K
Mannito (Cloudy) Slight
l Yellow)
Yellow)
Catalase + ++ No Growth + + -- ++
+ -- + +
+ No Growth --
Blood (α- (γ- (α- (α-
(β- (Contamin (γ-
Agar hemolysi hemolysis hemolysis hemolysis
hemolysis) ated) hemolysis)
s) ) ) )
Mannito
+ No
l Salt + -- -- -- +
(½ Yellow) Growth
Agar
+
+ + +
(Pink
(Yellow w/ (Little (Little No
VJ Agar with No Growth No Growth
black Growth, Growth, Growth
black
centers) Pink) Pink)
centers)

Gram Negative Rods

For the gram negative rod bacteria tested, in PR glucose broths all, except for P. vulgaris which created
no color change, caused a yellow color change indicative of acidic products being produced. Of these yellow color
changing organisms it was observed that all, except for S. marcescens, caused a gas bubble to form in the top of the
Durham tube. For PR lactose broths, E. aerogenes, P. vulgaris, S. typhimurium, P. mirabilis, and S. marcescens
caused a deeper red color change. K. pneumoniae, and E. coli produced acidic products causing a yellow color
change. For PR mannitol broths, all organisms tested caused a yellow color change from acidic products being
produced, except P. vulgaris and P. mirabilis which caused a deeper red color change.
When BHI cultures were tested for catalase production, all organisms with growth created bubbles upon
addition of hydrogen peroxide, with M. mirabilis producing high amount of bubbles and S. marcescens and S.
typhimurium producing medium amount of bubbles. E. aerogenes had a notably small amount of bubbles form. P.
vulgaris had no growth observed.
For EMB agar plates, light pink colonies of S. marcescens and P. mirabilus were observed indicating no
lactose fermentation. E. aerogenes and S. typhimurium both had pink colonies grow, indicating lactose fermentation

18
 occurred. Metallic colonies where observed in K. pneumoniae and E. coli. No growth was observed in P. vulgaris.
For endo agar plates, colorless colonies of S. marcescens, P. mirabilus, and S. typhimurium were observed
indicating no lactose fermentation. E. aerogenes had pink colonies grow, indicating lactose fermentation occurred.
Metallic colonies where observed in K. pneumoniae and E. coli. No growth was observed in P. vulgaris (see
Figures 27 and 28).
For Simmon’s citrate agar, no growth was observed in P. vulgaris, E. coli, P. mirabilus, and S. marcescens.
E aerogenes, K. pneumoniae, and S. typhimurium all tested positive changing the agar color to blue.
On the MacConkey agar, no growth was observed for S. typhimurium and P. mirabilus. Dark Pink colonies
were observed for E. coli, K. pneumoniae, and E. aerogenes. No growth was observed for P. vulgaris and S.
marcescens.
For the TSI slant agars, observations were taken for the bottom or butt of the agar tube and for the slant of
the agar tube. E. aerogenes had a red slant and a yellow butt with lifting of the agar in numerous places. K.
pneumoniae and E. coli had a yellow slant and a yellow butt with lifting of the agar in numerous places. P. vulgaris
and S. marcescens had a red slant and red butt. S. typhimurium had a red slant, black precipitate in the butt, and
lifting of the agar. P. mirabilus had a red slant and black precipitate in the butt (see Figure 29 and Table 6-6).
Analyzing the SIM agar deeps showed blackening only for S. typhimurium (see Figure 30). Reaction with
the Kovac’s reagent in the SIM agar deeps yielded a pink ring in only E. coli. Growth beyond the inoculated stab in
the SIM agar deeps was observed in E. coli, S. typhimurium, P. mirabilus, and E. aerogenes.
In the urea broths, only the P. mirabilus culture caused a color change to pink from the original yellow
color (see Figure 31).
In the set of MR-VP tubes with methyl red added, all organisms changed the broth color from yellow to
red, except for P. vulgaris and E. aerogenes which remained yellow (see Figure 32). In the set of MR-VP tubes
with 1.2 mL α-Naphthol Reagent and 0.5 mL 40% KOH added, a red color was only observed in E. aerogenes and
S. marcescens cultures (see Figure 33). These results are summarized in Table 7.

Figure 27: Endo Agars, bottom-top and top- bottom Figure 28: Endo Agars, bottom-top and top-bottom
views. From the top section on the right plate going views. From the top section on the right plate going
clockwise, we have: E. coli, P. mirabilus, and S. clockwise, we have: K. pneumoniae, P. vulgaris, S.
marcescens. typhimurium, and E. aerogenes.

19
 TABLE 6-6 TSI Results
and Interpretations
TABLE OF RESULTS
Result Interpretation Sym
bol
Yellow slant/ Glucose and lactose A/A
yellow butt and/ or sucrose
fermentation
Red slant/ Glucose fermentation; K/A
Figure 29: Triple Sugar Iron (TSI) Slants. From left to
yellow butt Peptone catabolized
right: E. aerogenes, K. pneumoniae, P. vulgaris, S. Red slant/ red No fermentation; K/K
typhimurium, E. coli, P. mirabilus, and S. marcescens. butt Peptone catabolized
aerobically and/ or
anaerobically. Not from
Figure 30: H2S precipitate Enterobacteriaceae
formed in S. typhimurium
Red slant/ no No fermentation; K/NC
culture grown in SIM agar
deep change in the Peptone catabolized
butt aerobically; Not from
Enterobacteriaceae
No change in Organism is growing NC/N
slant/ no change slowly or not at all; Not C
in butt from
Enterobacteriaceae
Black Sulfur reduction H2S
precipitate in
agar
Cracks in or Gas production G
lifting of agar

Figure 31: Urea broth test for urease production. From the left: E.
aerogenes, K. pneumoniae, P. vulgaris, S. typhimurium, E. coli, P.
mirabilus, and S. marcescens.

Figure 32: Methyl Red Test in the MR-VP Tubes.

From Left to right: E. aerogenes, K. pneumoniae, P. Figure 33: Voges-Proskauer (VP) Test in MR-VP tubes.
vulgaris, S. typhimurium, E. coli, P. mirabilus, and S. From Left to right: E. aerogenes, K. pneumoniae, P.
marcescens. vulgaris, S. typhimurium, E. coli, P. mirabilus, and S.
20 marcescens.
 Table 7: Lab 9 - Gram Negative Enterobacteriaceae Test Results. Positive and Negative test
descriptions can be found in the last two columns of Table 1. Phenol Red Broth symbols can be found
in the key of Table 2. TSI symbols are in Table 6-6.

Media/ Test Enterobacter Klebsiella Proteus Salmonella Escherichia Proteus Serratia

aerogenes pneumoniae vulgaris typhimurium coli mirabilus marcescens

Phenol Red AG AG NC AG AG AG A
Glucose

Phenol Red K A K K A K K
Lactose (slight) (slight)

Phenol Red A A K A A K A
Mannitol (slight)

Catalase + + No ++ + +++ ++
(small) Growth
EMB Agar + + No + + -- --
(pink) (metallic) Growth (pink) (metallic) (light (light
pink) pink)
Endo Agar + + No -- + -- --
(pink) (metallic) Growth (metallic) (No (No Color)
Color)
Simmon’s + + No + No Growth No No Growth
Citrate Agar Growth Growth

MacConkey + + No -- + -- No Growth
Agar Growth

TSI Slant K/A,G A/A,G K/K K/H2S,G A/A,G K/H2S K/K

Hydrogen -- -- -- + -- + --
Sulfide

Indole -- -- -- -- + -- --

Motility + -- -- + + + --

Urease -- -- -- -- -- + --
(pink)
MR- Methyl -- + -- + + + +
VP Red
Voges- + -- -- -- -- -- +
Proskau
er

21
 DISCUSSION

Carbohydrate Metabolism and Fermentation

The yellow color change is a result of the production of acidic products following fermentation of sugar.
Bubble formation in the Durham tube is indicative of CO2 production as a byproduct of fermentation. From the
results shown in Table 2 it is clear that all organisms actively ferment glucose. Sucrose fermentation seemed to only
occur in E. faecalis, S. epidermidis, and B. subtilis. While the indicator turned deeper red indicating deamination of
amino acids in E. coli, S. typhimurium, and P. vulgaris. This deamination created a rise in the pH of the broth.
Lactose was fermented by E. coli, E. faecalis, and S. epidermidis. Amino acid deamination occurred in S.
typhimurium, P. vulgaris, and B. subtilis. Mannitol was fermented by all organisms except P. vulgaris, which
deaminated amino acids. Regardless of the carbohydrate source, no organisms were observed to cause a color
change, an observation that indicates oxidative respiration. Color change to blue on the citrate agar for S.
typhimurium and P. vulgaris indicated that these organisms were capable of “using citrate as the only source of
oxidizable carbohydrate” ().

Microbial Enzymes
Production of the enzyme gelatinase allows those organisms to hydrolyze gelatin for the release of soluble
peptides and amino acids. (). This prevents gelatination at temperatures below 25ºC. Only B. subtilis was observed
to have this capability. H2S production created a black precipitate when reacting with metals in the SIM agar deeps
in only the S. typhimurium culture. This indicates that S. typhimurium produces cysteine desulfurase used to
breakdown cysteine and methionine. (). Tryptophanase presence is determined by observing the formation of a red
color when Kovac’s reagent is added to the top of SIM agar deeps. Tryptophanase hydrolyzes tryptophan into
pyruvate which is in turn used in metabolism. (). Indole reacted with the reagent in E. coli creating a pink layer on
top of the SIM agar deep. The presence of urease in a culture of bacteria in urea broth allows the organism to
hydrolyze urea into ammonia and carbon dioxide. Ammonia forms ammonium hydroxide in water which increases
the pH of the urea broth and phenol red indicator, thus turning red. (). Urease was not observed as present in any
organism tested. However, it should be noted that P. vulgaris is known to produce urease. (). This inconsistency is
explained by an unknown error that occurred with the laboratory’s stock culture of P. vulgaris that caused it to
behave in a strange manner. This behavior remained despite repeated attempts to obtain a pure and proper culture of
P. vulgaris to derive the cultures from. E. faecalis was the only organism not observed to utilize the enzyme
catalase upon addition of hydrogen peroxide to the BHI agar plates. Catalase is present to break down hydrogen
peroxide into water and oxygen in facultative anaerobes and aerobic bacteria. (). Bubble formation is indicative of
the O2 product formation. DNase methyl green agar plates are used to test for the presence of the DNase enzyme.
This enzyme breaks down DNA in other host organisms and thus in the agar, releasing the methyl green and causing
discoloration of the agar. (). Methyl green was not included upon first inoculation of the DNase agar plates. This
resulted in no available observations. The test for DNase was re-performed on P. vulgaris, B. subtilis, and P.

22
mirabilus. Only B. subtilis lacked discoloration around the colony streak. However, DNase activity should have
been observed in this specimen. (). Perhaps longer incubation would have yielded an observable discoloration.

Selective and Differential Media

Mannitol salt agar was used to test for fermentation of mannitol. S. aureus produced a prominent yellow
color change indicating fermentation. S. epidermidis also produced a yellow color change region, but was less
prominent. This was an error perhaps caused by contamination or varying pH regions in the media prior to
inoculation. Mannitol salt agar is often used to distinguish these two organisms, with S. epidermidis able to grow
but not ferment mannitol like S. aureus. Also, this media favors growth of the staphylococcus species. (). No other
organisms were able to ferment Mannitol. MacConkey agar was used to test for lactose fermentation in
enterobacteriaceae while inhibiting growth of gram positive bacteria due to the presence of bile salts. (). E. coli, S.
typhimurium, and P. mirabilis were all observed to ferment lactose. Gram positive organisms are typically favored
on phenylethyl alcohol agar. This media contains phenylethyl alcohol which inhibits DNA synthesis in gram
negative bacteria. (). A positive test, indicated by colony growth, was observed for E. coli, E. faecalis, S.
epidermidis, and S. aureus. However, E. coli is a gram negative bacterium and should not have grown. All those
organisms shown to ferment lactose on the MacConkey agar, also was shown to ferment lactose when grown on
EMB agar. These organisms also were observed to be gram negative. E. coli on this agar developed a reflective
metallic surface, a typical reaction. All blood agar results for hemolytic activity were as expected.

Gram Positive Bacteria

For the gram positive rods tested, gelatinase activity was observed in only B. cereus. This differed from
previous lab results (see Table 8) which showed gelatinase activity in both B. cereus and B. subtilis. On blood agar,
C. pseudodiptheriticum was classified as α-hemolysis which differed from previous labs’ consensus of γ-hemolysis.
The litmus milk tests were used to further classify the bacteria based on ability to metabolize the components of
lactose and casein. this test can produce many different results and should therefore only be used to confirm results
of another test. (). All organisms tested had different results with the data from previous labs. C.
pseudodiptheriticum was not observed to have fissures in the clot, a result of gas production. B. cereus was not
observed to have a white color in the lower part of the medium, but instead have no overall color change and a
semisolid grey curd at the top. B. subtilis was observed to have no color change while previous labs observed a dark
blue medium or band at the top. M. smegmatis was observed to have a grey curd at the top and no overall color
change, while previous labs observed a blue medium color or blue band located at the top. The blue band indicated
an alkaline reaction had occurred. These variations are typical of a litmus milk test.
For the gram positive cocci tested, in PR glucose CO2 production was observed in the Durham tubes of
the S. aureus, M. luteus, and M. roseus broths. This differed from previous lab observations of no gas produced,
only acidic products. For PR lactose, previous lab observations found all organisms tested to produce acidic
production. This differed from the observations of M. luteus, M. roseus, S. pyogenes, and S. salivarius. M. luteus
and S. salivarius were both observed to deaminate amino acids and raise the pH of the broth. M. roseus and S.
pyogenes were both observed to have no little to no color change, indicating oxidative use of lactose. PR mannitol

23
broths had many different results than previous labs. M. luteus and S. salivarius were both observed to deaminate
amino acids rather than use mannitol oxidatively and ferment mannitol to produce acid byproducts respectively. M.
roseus, S. epidermidis, and S. pyogenes all were observed to use mannitol oxidatively. However, prior labs had
observed M. roseus and S. epidermidis as fermenting mannitol, and S. pyogenes as deaminating amino acids.
A few differences were also noted when comparing results of the catalase test. M. roseus was observed to
have no growth. However, previous labs observed bubble formation when hydrogen peroxide was added to M.
roseus growths. Bubble formation was observed on S. pyogenes and S. salivarius cultures. However, previous labs
observed no bubble formation on these organisms.
Blood agar culture for hemolytic activity classification also had many differences with previous lab
observations. M. luteus, E. faecalis, and S. salivarius all were observed to have α-hemolytic activity, while previous
labs observed these organisms to be γ-hemolytic, or have no hemolytic activity. S. pyogenes was observed to have
no hemolytic activity (γ-hemolysis). Previous lab observations greatly contrast this result listing S. pyogenes as
having had ß-hemolytic activity. The blood agar plates where split inaccurately with a permanent marker. Proof of
this possibility is best demonstrated in the culture of M. roseus. On this agar plate, M. roseus colonies were
overgrown by colonies of M. luteus.
Mannitol salt agar plates held more consistent with the past and present lab observations. Previous labs
observed M. roseus and E. faecalis as having no growth. However, M. roseus was observed to have colorless
growth indicating no fermentation of mannitol. This is a correct observation as M. roseus is a member of a smaller
group of gram positive cocci able to grow, but not ferment, on mannitol salt agar. (). It is interesting to note that
mannitol salt agar in both the past lab observations and this lab observations was unable to act as a differential
media for S. aureus and S. epidermidis.
VJ agar had more overall growth and tellurite production when compared to past lab observations. S.
aureus grew as expected matching past lab observations; it produced tellurite and fermented mannitol. M. roseus, S.
epidermidis, and S. salivarius produced no growth on the agar. The same result was true of previous lab
observations. For M. luteus, no mannitol fermentation but tellurite production was observed to have occurred; this
differs from previous labs which observed no growth of M. luteus. No fermentation was observed to have occurred
by the pink colonies in either S. pyogenes or E. faecalis. These growths were small. Previous lab observations
recorded S. pyogenes as having no growth and E. faecalis as fermenting mannitol. These many variations, most
likely caused by some level of contamination, stress the importance of careful practice of aseptic techniques
throughout inoculating.

Gram Negative Rods

The enterobacteriaceae tested for glucose fermentation all tested positive, yielding acidic products with the
exception of P. vulgaris which showed no growth. This was a result of the P. vulgaris stock culture impurity.
Much like the past lab observations, all organisms besides P. vulgaris and S. marcescens produced CO2. Both PR
lactose and PR mannitol shared similar observations with the previous lab observations. The only different is E.
aerogenes was observed to ferment lactose in past lab observations (see Table 10). P. vulgaris should have also
showed presence of catalase activity, but instead yielded no growth. All other organisms had catalase present, much

24
like the past lab observations. EMB agar showed more variation with past lab results, showing a metallic surface on
the colonies of K. pneumoniae rather than just appearing pink as in the past lab observations. This indicated much
higher lactose fermentation. S. marcescens showed little lactose fermentation, contrasting the higher amount
observed in the past lab indicated by a pinker color. P. vulgaris showed no growth showing more proof of a
possible stock culture contamination
Differentiation of different enterobacteriaceae was unreliable when using Simmon’s citrate agar, TSI slant
agar, and endo agar to test for citrate oxidation, acidic vs. alkaline metabolic reactions, and lactose fermentation
respectively. In endo agar K. pneumoniae was observed as having heavy lactose fermentation. P. vulgaris was
observed as having no growth. P. mirabilus and S. marcescens where observed as having growth but no lactose
fermentation. All these organisms in the past lab results were observed as having moderate lactose fermentation. In
Simmon’s citrate agar, no growth was observed in P. vulgaris, E. coli, P. mirabilus, or S. marcescens. This
contrasts with past lab results in that growth but no citrate oxidation was observed for P. vulgaris, E. coli, and P.
mirabilus cultures; and citrate oxidation was observed in S. marcescens. The TSI slant agar observations appear to
be easily variable due to the greater amount of qualities to observe. E. aerogenes, P. mirabilus, and S. marcescens
all had a red slant, rather than a yellow slant as observed in the past lab results. S. typhimurium had a red slant,
heavily darkened from H2S butt, and gaseous pockets in the agar. In the past lab results, S. typhimurium lacked the
gas pocketing and had a yellow slant. P. vulgaris once again showed inconsistences, having a red slant and red butt
observed, compared to the past lab observations of a yellow slant, a yellow butt, and H2S precipitate formed.
Yellow color change in the slant and butt is indicative of glucose and lactose fermentation and possible sucrose
fermentation. A red slant and a yellow butt indicate glucose fermentation and peptone catabolism. A red slant and
red butt indicate no fermentation and the catabolism of peptone, either aerobically or anaerobically. A red slant and
no color change in the butt indicate no fermentation and the aerobic catabolism of peptone. No change in the slant
or the butt indicates slow or no microbial growth. Black precipitate is the result of sulfur reduction and cracks,
pockets, or lifting of the agar indicates CO2 gas production (see Table 6-6). ()
MacConkey agar tests also differed from the past lab observations in that P. vulgaris and S. marcescens had
no growth. In the past lab results, both were observed fermenting lactose. All SIM deep agar tests matched that of
the past lab results except for P. vulgaris. P. vulgaris tested negative for H2S production, indole production, and
motility. P. vulgaris was observed as positive for these tests in the past lab results. Further, P. vulgaris should have
tested positive for urease presence and caused a color change in the methyl red test in the MR-VP tubes. All past lab
results are summarized in Tables 8, 9 and 10.
A few inconsistencies also occurred between microbial testing groups. E. coli was tested for citrate
oxidation two times. The first time, growth occurred and no citrate was oxidized. The second time, no growth
occurred. P. vulgaris was tested two times in PR glucose, for catalase, and in Simmon’s citrate agar. The first time,
glucose was fermented, many O2 bubbles were produced by catalase, and citrate oxidation was observed. The
second time, glucose was used oxidatively, and no growth was observed for both the catalase test and on the
Simmon’s citrate agar. These inconstancies bring into question not only the purity of the stock P. vulgaris culture
but also its consistency over time. S. typhimurium was tested for lactose fermentation on MacConkey agar two
times. The first time, it produced pink colonies indicating lactose fermentation had occurred. The second time, S.

25
 typhimurium produced colorless colonies indicating no lactose had been fermented. P. mirabilus produced light
pink colonies on EMB agar one time, and darker pink colonies another time. This change in colony color indicated
a change in rate or amount of lactose fermentation between both tests. E. faecalis appeared to be α-hemolytic and be
fermenting mannitol in one test period, and then appeared to be γ-hemolytic and not be fermenting mannitol in
another test period. S. epidermidis appeared to not have fermented mannitol in one test in PR mannitol broth, and to
have fermented mannitol in another PR mannitol broth test. These isolated inconsistencies, those separate from P.
vulgaris, are most likely the result of contamination during inoculation and/or transferring post-inoculation. Further
testing should be performed in addition to careful practice of aseptic technique.
Table 8: Past Lab Results for Gram Positive Rod Bacterial Tests Performed. The highlighted
observations indicates data that differed with this lab’s observations

Corynebacterium Bacillus Mycobacterium

Media/ Test Bacillus cereus
pseudodiptheriticum subtilis smegmatis
Gelatin Hydrolysis -- + + --
+
--
-- + (α-
Blood Agar (γ-
(γ-hemolysis) (ß-hemolysis) hemolys
hemolysis)
is)
Catalase + + + +
Litmus Milk AC,G KR DK K

Table 9: Past Lab Results for Gram Positive Cocci Bacterial Tests Performed. The highlighted
observations indicates data that differed with this lab’s observations

Staphylococcus Micrococcus Micrococcus Staphylococcus Streptococcus Enterococcus Streptococcus

Media
aureus luteus roseus epidermidis pyogenes faecalis salivarius
Phenol
Red A A A A A A A
Glucose
Phenol
Red A A A A A A A
Lactose
Phenol
Red A NC A A K A A
Mannitol
Catalase + + + + -- -- --
-- --
+ -- + --
Blood (γ- (γ- --
(β- (γ- (β- (γ-
Agar hemolysis hemolysis (γ-hemolysis)
hemolysis) hemolysis) hemolysis) hemolysis)
) )
Mannitol + -- No + --
No Growth No Growth
Salt Agar (Yellow) (Pink) Growth (Yellow) (Pink)
+ No No --
VJ Agar No Growth No Growth No Growth
(Black) Growth Growth (Yellow)
Table 10: Past Lab Results for Gram Negative Rod Bacterial Tests Performed. The
highlighted observations indicates data that differed with this lab’s observations

Media/ Test Enterobacter Klebsiella Proteus Salmonella Escherichia Proteus Serratia

aerogenes pneumoniae vulgaris typhimurium coli mirabilus marcescens

26
 Phenol Red AG AG A AG AG AG A
Glucose

Phenol Red A A K K A K K
Lactose

Phenol Red A A K A A K A
Mannitol

Catalase + + + + + + +

EMB Agar + + + + + -- +
(Pink) (Pink) (Pink) (pink) (metallic) (light (Pink)
pink)
Endo Agar + + + -- + + +
(Pink) (Pink) (Pink) (Light Pink) (metallic) (Pink) (Pink)
Simmon’s + + -- + -- -- +
Citrate Agar

MacConkey + + + -- + -- +
Agar (Pink) (Pink) (Pink) (Colorless) (Pink) (Colorless (Pink)
)
TSI Slant A/A,G A/A,G A/A, A/K, H2S A/A,G A/K, H2S A/K
H2S
Hydrogen -- -- + + -- + --
Sulfide

Indole -- -- + -- + -- --

Motility + -- + + + + --

Urease -- + + -- -- + --

MR- Methyl -- + + + + + +
VP Red
Voges- + -- -- -- -- -- --
Proskaue
r

27
 REFERENCES

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Retrieved October 11, 2009, from Institute of Cell and Molecular Biology, The
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http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/proteus.htm
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