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

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

Michael J. Wallach II10/13/2009

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. (Simmons, 2009).

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. (Simmons, 2009).

Microbes require consideration of many environmental factors to grow successfully, including: water

availability, salt concentration, pH, temperature, concentration of oxygen (O2), pressure, and radiation. (Kennell,

2009). 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. (Kennell, 2009).

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. (Kennell, 2009).

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

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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. (Kennell, 2009). 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. (Simmons, 2009).

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. (Simmons, 2009). 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. (Simmons, 2009).

Media Reaction Rational for Results Positive Test (+)Negative Test

(--)

Simmon’s Citrate

Citrate is the sole carbon source and ammonium phosphate as the sole nitrogen source of this defined medium.

Organisms that utilize citrate also convert ammonium phosphate into ammonium. This reaction causes an increase in pH and changes the bromothymol blue pH indicator from green to blue.

Blue Color/ Citrate Oxidized

Green/ No Citrate Oxidized

Nutrient Gelatin

This media is composed of gelatin (derived from collagen), peptone and beef extract. It is used for the identification of organisms that produce the enzyme gelatinase.

Organisms that produce gelatinase will hydrolyze the gelatin in the media, causing it to liquefy. Organisms that do not possess the enzyme will not be able to liquefy the media.

Not Solidified/ Gelatinase Present

Solidified/ No Gelatinase Present

Urea Broth This differential media is used to distinguish rapid urease positive bacteria from slow urease-

Organisms that produce the enzyme urease will be able to convert urea to ammonia, thus increasing the pH and changing the

Red/ Urease Present Yellow-Orange/ No Urease Present

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positive or urease negative organisms. The media contains phenol red indicator, peptone, glucose and urea.

color of the pH indicator in the media to a bright pink.

DNase Methyl Green

This media identifies organisms that produce the exoenzyme DNase. This enzyme catalyzes the depolymerization of DNA in the media.

DNA fragments in the media are conjugated to the dye methyl green. Organisms that produce this enzyme will cleave the DNA in the media into smaller pieces and uncouple the DNA-dye complex resulting in a clearing of the media.

Decolorized ring around growth/ DNase Produced

+ mild sized++ large sized

No decolorized ring/ No DNase Produced

Mannitol Salt Agar

This media is both selective and differential. The high salt concentration selects for organism that are halophiles, while the mannitol in the media differentiates between organisms ability to utilize this sugar source.

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. If the organism converts mannitol 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.

Yellow/ Mannitol Fermented

No Color Change/ No Mannitol fermented

Eosin Methylene Blue (EMB) Agar

EMB is a selective and differential media. It contains lactose in addition to eosin and methylene blue dyes. The dyes inhibit the growth of Gram-positive organism. The media differentiates the degree of 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. Colorless or light pink colonies indicate slight lactose fermentation while purple and metallic green colonies indicate heavy lactose fermentation.

Black Centers/ Lactose Fermented

Colorless or Light Pink/ No Lactose Fermented

Endo Agar

Endo agar is a selective and differential media. It contains lactose in addition to sodium sulfite and basic fuchsin dye. The sodium sulfite and basic fuchsin inhibit the growth of Gram-positive organism. The media differentiates the degree of 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. Colorless or light pink colonies indicate slight lactose fermentation while purple and metallic green colonies indicate heavy lactose fermentation.

Red/ Lactose Fermented

Colorless/ No Lactose Fermented

Blood Agar

Blood agar is trypticase soy agar supplemented with 5% sheep blood. The blood allows for differentiation of bacteria based on their hemolytic properties.

Hemolytic reactions are classified as alpha (showing partial destruction of RBCs), beta (complete destruction of RBCs) and gamma (no hemolysis).

Green-Dark/ α-hemolysisOrClear Zone/ ß-hemolysis

No Discoloration/γ-hemolysis

MacConkey Agar

MacConkey agar is a selective and differential 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.

Organisms that are Gram negative will be able to grow on 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.

Pink to Red/ Lactose Fermented

Colorless/ No Lactose Fermented

Litmus Milk This differential media identifies an organism’s ability to breakdown milk products and lactose using the enzymes rennin, casease and β-galactosidase. The pH indicator in the media is azolitmin.

A variety of results is possible with this media. If the pH decreases as a result of lactose fermentation, then the media will turn pink. An alkaline reaction results in a blue color. Other results include curdling of the media, acid clots and gas production.

Color Change, Curdling, Acid Clots, Gas Production (See Figure 1)

No Color Change/ No Reaction

Triple Sugar TSI agar is a medium designed to The agar is prepared as a slant with a deep Many Reactions Possible (See Figure 2)

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Iron

differentiate organisms based on their ability to ferment glucose, lactose, sucrose and produce hydrogen sulfide. Phenol red is the pH indicator.

butt, providing both aerobic and anaerobic environments. A variety of results is expected with this media all based upon changes in the color 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.

VJ Agar

VJ agar is selective for coagulase positive staphylococci and Gram-negative bacteria. The media also differential for tellurite reduction.

Coagulase positive and Gram-negative organism can grow on this media. Those organisms that reduce tellurite form black colonies that contain a black precipitate from tellurite.

Black Spots –yellow/ tellurite produced – Mannitol fermented

No Growth or No Color Change

MR–VP Broth (Methyl Red Test)

MR-VP broth is a combination media used for both the methyl red and Voges-Proskauer tests. The media contains a buffer, glucose and peptone.

The MR test is designed to detect an organism that under goes mixed acid fermentation. Upon addition of methyl red, an acidic environment will change the color to red. The VP test detects an organism’s ability to convert acid products to acetonin.

Red/ Acid Production, Glucose Fermented

Yellow, No Color Change/ No Glucose Fermented

MR–VP Broth (Voges-Proskauer Test))

Red/ Acetoin ProducedOther color/ No Acetoin Produced

SIM (H2S)

SIM media is used to identify the motility of an organism, the production of indole and hydrogen sulfide.

An organism that shows a radiating or diffusion pattern out of the inoculation stab tests positive for motility. The production of a black precipitate is indicative of reduction of hydrogen sulfide. Upon addition of Kovac’s reagent, an organism that produces the enzyme tryptophanase will test positive (pink ring) for indoles.

Black Precipitate/ H2S Produced

No Color Change/ No H2S Produced

SIM (Indole) Red/ Indole ProducedNo Color Change/ No Indole Produced

SIM (Motility)

Cloudy/ Growth Not Restricted to Stab Line

Clear/ Immotile

Catalase Test

This test can be done on any agar surface that has 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.

Hydrogen peroxide is applied to the colony and if the organism is catalase positive, there will be production of gas bubbles (from the oxygen gas).

Bubbles/ Catalase Present

+few++medium+++high

No Bubbles/ No Catalase Present

Phenylethyl Alcohol Agar

This media is selective for Gram-positive organisms. It contains phenylethyl alcohol that is bacteriostatic against Gram-negative organism.

Gram-positive organism show normal colony morphology on this media. Gram-negative organisms either do not grow or are severely limited on this media.

Growth/ Gram-Positive Cocci

No Growth/ Gram-Negative or Gram-Positive Rods

PR Glucose

PR broths are differential media that contain phenol red pH indicator and a specific sugar.

Acid production from the fermentation of the carbohydrate will lower the pH of the media and change the color of the media to yellow. Organisms that undergo deamination of amino acids will turn the media alkaline and pink or red in color.

See Key from Table 2PR Sucrose

PR Lactose

PR Mannitol

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

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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

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-

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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.

Figure 1: Streaking for Isolation on Split plates (Simmons, 2009)

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.

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2nd Streak

1st Streak

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

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

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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 Medium

Escherichia coli

Salmonella typhimurium

Enterococcus faecalis

Staphylococcus epidermidis

Proteus vulgaris

Bacillus subtilis

PR Glucose

AG AG A A A A

PR Sucrose

K K A A K A

PR Lactose

A K A A K K

PR Mannitol

A A A A K A

Simmon’s Citrate

-- + -- -- + --

Symbol Observation Meaning

A Yellow Acid ProductionAG Yellow with Bubbles Acid & CO2 gas

ProducedK Deep Red AlkalineNC Little to No Color Change (Remains

Red)Oxidative Respiration

+ Blue Citrate Oxidized-- No Color Change (Remains Green) No Citrate Oxidized

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

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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.

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Figure 2: SIM agar deeps after addition of Kovac’s reagent. Label from left to right is B. subtilis, P. vulgaris, S. epidermidis, S. typhimurium, E. faecalis, and E. coli

Figure 3: BHI agar after addition of H2O2. Notice large P. vulgaris bubble formation on top compared to B. subtilis on bottom.

Figure 4: BHI agar after addition of H2O2. E. faecalis is on top and S. epidermidis is on bottom.

Figure 5: BHI agar after addition of H2O2. Notice S. typhimurium bubble formation 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 Performe

d

Escherichia coli

Salmonella typhimurium

Enterococcus faecalis

Staphylococcus epidermidis

Proteus vulgaris

Bacillus

subtilis

Proteus mirabilis

Gelatin Hydrolysi

s-- -- -- -- -- +

SIM Deep:

Hydrogen Sulfide

-- + -- -- -- --

SIM Deep: Indole

+ -- -- -- -- --

SIM Deep:

Motility+ + -- -- -- --

Urea Hydrolysi

s-- -- -- -- -- --

Catalase + ++ -- + +++ +DNase ++ -- +

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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.

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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 8: Mannitol salt agar with bottom up and top up views. Inoculated with, starting at the top yellow colony moving clockwise, S. epidermidis, P. mirabilis, and E. faecalis.

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 10: MacConkey agar with bottom up and top up views. Inoculated with, starting from the purple colonies and going clockwise, E. coli, S. aureus, and 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.

MediaEscherichia

coliSalmonella

typhimuriumEnterococcus

faecalisStaphylococcus

epidermidisProteus mirabilis

Staphylococcus aureus

Mannitol Salt Agar

No Growth No Growth No Growth+

(small yellow)No Growth +

MacConkey + + No Growth No Growth + No Growth

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

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

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

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

Figure 15: Blood agar with top and bottom views. From left to right, E. coli and S. aureus.

Figure 16: Blood agar with top and bottom views. From left to right, E. faecalis and S. epidermidis.

Figure 17: Blood agar with top and bottom views. From left to right, P. mirabilis and S. typhimurium.

Agar(Pink w/Dark

Centers)(Light Pink) (Light Pink)

Phenylethyl Alcohol Agar

+ -- + + -- +

EMB Agar+

(Metallic Green)

+ (Pink)

--(Colorless/ Light Pink)

--(Colorless)

+(Pink)

--(Colorless)

Blood Agar+

(α-hemolysis)--

(γ-hemolysis)--

(γ-hemolysis)--

(γ-hemolysis)+

(α-hemolysis)+

(ß-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

13

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

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.

14

Figure 18: Blood agar with top and bottom views. From top to bottom, S. epidermidis and S. aureus.

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

Figure 20: Litmus milk broths following incubation. From left to right: Control broth, B. cereus, C. pseudodiptheriticum, M. smegmatis, and B. subtilis.

TABLE 6-2 Litmus Milk Results and Interpretations

TABLE OF RESULTS

Result Interpretation Symbol

Pink Color Acid reaction A

Pink and solid (white in the lower portion if the litmus is reduced); clot not movable

Acid clot AC

Fissures in the clot Gas G

Clot broken apart Stormy fermentation S

White color (lower portion of medium)

Reduction of litmus R

Semisolid and not pink; clear to gray fluid at top

Curd C

Clarification of medium; loss of “body”

Digestion of peptone; peptonization

P

Blue medium or blue band at top

Alkaline reaction K

No change None of the above reactions NC

15

Figure 21: Phenol Red Glucose Broths. From left to right: M. roseus, M. luteus, S. epidermidis, E. faecalis, S. salivarius, and S. aureus.

Figure 22: Phenol Red Lactose Broths. From left to right: M. luteus, M. roseus, S. pyogenes, S. epidermidis, 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 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.

Media/ TestCorynebacterium

pseudodiptheriticumBacillus cereus

Bacillus subtilis

Mycobacterium smegmatis

Gelatin Hydrolysis

-- + -- --

Blood Agar+

(α-hemolysis)+

(ß-hemolysis)+

(α-hemolysis)--

(γ-hemolysis)

Catalase + + No Growth+

(Very Little)

Litmus Milk CACKNC

(darker than control)

NC CNC

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.

MediaStaphylococcus aureus

Micrococcus luteus

Micrococcus roseus

Staphylococcus

epidermidis

Streptococcus

pyogenes

Enterococcus faecalis

Streptococcus

salivariusPhenol Red Glucose

AGAG

(Cloudy)AG A

A(Slightly)

A(Clear)

A(Slightly)

Phenol Red Lactose

AK

(Cloudy)NC

A(Clear)

NC(Very Slight

Yellow)

A(Clear)

K(Clear)

Phenol Red Mannitol

AK

(Cloudy)NC

NC(Very Slight

Yellow)

NC(Very Slight

Yellow)

A K

Catalase

+ ++ No Growth + + -- ++

Blood Agar

+(β-

hemolysis)

+(α-

hemolysis)

No Growth(Contaminat

ed)

--(γ-

hemolysis)

--(γ-

hemolysis)

+(α-

hemolysis)

+(α-

hemolysis)Mannitol Salt Agar

+ -- --+

(½ Yellow)-- + No Growth

16

VJ Agar

+(Yellow w/

black centers)

+(Pink with

black centers)

No Growth No Growth

+(Little

Growth, Pink)

+(Little

Growth, Pink)

No Growth

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

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.

17

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.

18

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

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

Figure 29: Triple Sugar Iron (TSI) Slants. From left to right: E. aerogenes, K. pneumoniae, P. vulgaris, S. typhimurium, E. coli, P. mirabilus, and S. marcescens.

Figure 30: H2S precipitate formed in S. typhimurium culture grown in SIM agar deep

TABLE 6-6 TSI Resultsand Interpretations

TABLE OF RESULTS

Result Interpretation Symbol

Yellow slant/ yellow butt

Glucose and lactose and/ or sucrose fermentation

A/A

Red slant/ yellow butt

Glucose fermentation; Peptone catabolized

K/A

Red slant/ red butt No fermentation; Peptone catabolized aerobically and/ or anaerobically. Not from Enterobacteriaceae

K/K

Red slant/ no change in the butt

No fermentation; Peptone catabolized aerobically; Not from Enterobacteriaceae

K/NC

No change in slant/ no change in butt

Organism is growing slowly or not at all; Not from Enterobacteriaceae

NC/NC

Black precipitate in agar

Sulfur reduction H2S

Cracks in or lifting of agar

Gas production G

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 aerogenes

Klebsiella pneumoniae

Proteusvulgaris

Salmonella typhimurium

Escherichia coli

Proteus mirabilus

Serratia marcescens

Phenol Red Glucose

AG AG NC AG AG AG A

Phenol Red Lactose

K A K (slight)

K A K K(slight)

Phenol Red Mannitol

A A K A A K A(slight)

Catalase +(small)

+ No Growth

++ + +++ ++

EMB Agar +(pink)

+(metallic)

No Growth

+(pink)

+(metallic)

--(light pink)

--(light pink)

Endo Agar +(pink)

+(metallic)

No Growth

-- +(metallic)

--(No

Color)

--(No Color)

19

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. vulgaris, S. typhimurium, E. coli, P. mirabilus, and S. marcescens.

Figure 33: Voges-Proskauer (VP) Test in MR-VP tubes. From Left to right: E. aerogenes, K. pneumoniae, P. vulgaris, S. typhimurium, E. coli, P. mirabilus, and S. marcescens.

Simmon’s Citrate Agar

+ + No Growth

+ No Growth No Growth

No Growth

MacConkey Agar

+ + No Growth

-- + -- No 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-VP

Methyl Red

-- + -- + + + +

Voges-Proskauer

+ -- -- -- -- -- +

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” (Simmons, 2009).

Microbial Enzymes

Production of the enzyme gelatinase allows those organisms to hydrolyze gelatin for the release of soluble

peptides and amino acids. (Simmons, 2009). 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

20

desulfurase used to breakdown cysteine and methionine. (Simmons, 2009). 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. (Simmons, 2009). 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. (Simmons, 2009). Urease was not observed as present in any organism tested. However, it should be noted that

P. vulgaris is known to produce urease. (Deacon). 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. (Simmons, 2009). 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. (Simmons, 2009). 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.

mirabilus. Only B. subtilis lacked discoloration around the colony streak. However, DNase activity should have

been observed in this specimen. (Wolinowska, Ceglowski, Kok, & Venema, 1991). 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. (Simmons,

2009). 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.

(Simmons, 2009). 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. (Simmons, 2009). 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.

21

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. (Kibota). 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

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

22

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. (Huggins, 2009). 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

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

23

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). (Leboffe & Pierce, 2005)

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.

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

Media/ TestCorynebacterium

pseudodiptheriticumBacillus cereus

Bacillus subtilis

Mycobacterium smegmatis

Gelatin Hydrolysis -- + + --

Blood Agar--

(γ-hemolysis)+

(ß-hemolysis)

+(α-

hemolysis)

--(γ-hemolysis)

24

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

MediaStaphylococcus

aureusMicrococcus

luteusMicrococcus

roseusStaphylococcus

epidermidisStreptococcu

s pyogenesEnterococcus

faecalisStreptococcus

salivariusPhenol Red Glucose

A A A A A A A

Phenol Red Lactose

A A A A A A A

Phenol Red Mannitol

A NC A A K A A

Catalase + + + + -- -- --

Blood Agar

+(β-hemolysis)

--(γ-

hemolysis)

--(γ-

hemolysis)

--(γ-hemolysis)

+(β-hemolysis)

--(γ-hemolysis)

--(γ-hemolysis)

Mannitol Salt Agar

+(Yellow)

--(Pink)

No Growth+

(Yellow)--

(Pink)No Growth No Growth

VJ Agar+

(Black)No Growth No Growth No Growth No Growth

--(Yellow)

No Growth

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 aerogenes

Klebsiella pneumoniae

Proteusvulgaris

Salmonella typhimurium

Escherichia coli

Proteus mirabilus

Serratia marcescens

Phenol Red Glucose

AG AG A AG AG AG A

Phenol Red Lactose

A A K K A K K

Phenol Red Mannitol

A A K A A K A

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

+ + -- + -- -- +

25

MacConkey Agar +(Pink)

+(Pink)

+(Pink)

--(Colorless)

+(Pink)

--(Colorless)

+(Pink)

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

A/K, H2S A/A,G A/K, H2S A/K

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

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

Motility + -- + + + + --

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

MR-VP

Methyl Red

-- + + + + + +

Voges-Proskauer

+ -- -- -- -- -- --

26

REFERENCES

Deacon, J. (n.d.). The Microbial World: Proteus vulgaris and clinical diagnostics. Retrieved October 11,

2009, from Institute of Cell and Molecular Biology, The University of Edinburgh:

http://www.biology.ed.ac.uk/research/groups/jdeacon/microbes/proteus.htm

Huggins, J. (2009, June 17). Bacterial Characteristics Sheet. Retrieved October 12, 2009, from Arkansas

State University: http://www.clt.astate.edu/jhuggins/pet_characteristics.htm

Kennell, J. (2009). Nutrition, Culturing, and Growth. Microbiology 464-01. Saint Louis: Saint Louis

University.

Kibota, T. (n.d.). Litmus Milk. Retrieved October 12, 2009, from Unknowns:

http://web.clark.edu/tkibota/240/Unknowns/LitmusMilk.htm

Leboffe, M. J., & Pierce, B. E. (2005). A Photographic Atlas for the Microbiology Laboratory (3rd ed.). (D.

Ferguson, Ed.) Englewood, Colorado, United States: Douglas N. Morton.

Simmons, C. (2009). General Microbiology Laboratory Manual. Saint Louis.

Wolinowska, R., Ceglowski, P., Kok, J., & Venema, G. (1991). Isolation, sequence and expression in

Escherichia coli, Bacillus subtilis and Lactococcus lactis of the DNase (streptodornase)-encoding

gene from Streptococcus equisimilis H46A. Pubmed.

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