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

Michael J. Wallach II 10/13/2009

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

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INTRODUCTION

Microbes can be identified and classified based on a few factors: their metabolic processes, growth requirements, and structural structural and functional morphology. These experiments investigated investigated the techniques of several tests in various media types to identify bacteria. bacteria. All tests performed as well as media type descriptions descriptions are summarized in Table 1. Classification of bacteria bacteria involves analysis and organization based on structural similarities. similarities. Identification Identification of bacteria uses several characteristics to enable sorting into taxonomic taxonomic groups.(). Identification 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 scientists to identify bacteria in a systematic way. Each level of testing is accompanied accompanied by yes or no questions questions regarding the results. results. The key ends with individual organisms listed listed on the bottom row. (). Microbes require consideration of many environmental factors to grow successfully, including: water  availability, availability, salt concentration, pH, temperature, concentration of oxygen (O 2), pressure, and 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, compounds, such as hydrogen or sulfur. Photoheterotrophs derive energy from light but utilize carbon from organic compounds other than CO2. Chemoheterotrophs, 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. microaerophiles. Obligate aerobes aerobes must utilize O2 for respiration and obligate anaerobes must rely on fermentation or find another final electron electron acceptor in respiration. Facultative anaerobes anaerobes are able to switch back and forth from fermentation and aerobic respiration depending on the surrounding environment’s oxygen supply. Facultative anaerobes 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. byproduct. In fermentation fermentation lactic acid or  alcohol are main products that are formed. During alcohol fermentation, fermentation, pyruvate is decarboxylated yielding yielding CO2 release. During lactic fermentation, no CO2 is released. released. Fermentation products are acidic. Aerotolerant anaerobes are able to detoxify O 2 in its reduced form but cannot participate in aerobic respiration. respiration. These organisms rely on enzymes such as catalase to break down hydrogen peroxide into water and oxygen. Microaerophiles 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) 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 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). (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 components are known and are very specific. specific. In complex media, not all the the

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ingredients are specifically specifically known. Complex media are often often derived from extracts. extracts. Consequently, scientists scientists are able to have a general idea of the components but not an exact inventory. Selective media allows or inhibits inhibits the growth of one type of group, or in some cases specific microbe. Differential 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 differential are able to simultaneously select for or  against growth of one group while allowing scientists to differentiate differentiate between growths of two other groups. Media can also be liquid or solid. (). (). Liquid media are called called broths and solid media are generally generally agar. By understanding 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. environments. Enzymes tested for on the various media types used were: catalase, DNase, DNase, tryptophanase, gelatinase, cysteine 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 hydrolyzes the DNA of the host a bacterium infects. Tryptophanase Tryptophanase is an enzyme that hydrolyzes tryptophan into pyruvate pyruvate and indole. Gelatinase is an enzyme that hydrolyses gelatin gelatin to extract peptides and amino amino acids for energy. This stops gelatination gelatination from occurring. Cysteine desulfurase breaks down cysteine and methionine resulting in hydrogen sulfide (H2S) as by-product. by-product. Urease is an enzyme that hydrolysis urea into ammonia and CO 2. The ammonia reacts reacts with water to form ammonium 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 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 types to select for and differentiate  between bacteria with different metabolic pathways.

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

Simmon’s Citrate

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

 This media is composed of  gelatin (derived from collagen), peptone and Nutrient beef extract. It is used for Gelatin the identification of  organisms that produce the enzyme gelatinase. Urea Broth  This differential media is used to distinguish rapid

Negative Test (--)

Rational for Results

Positive Test (+)

Organisms that utilize citrate also convert ammonium phosphate into ammonium. ammonium. This reaction 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

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

Organisms that produce the enzyme urease will be able to

Red/ Urease Present

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 Yellow-Orange/ No Urease Present

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

DNase Methyl Green

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

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.

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 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. 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 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 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.

Decolorized ring around growth/ DNase Produced

No decolorized ring/ No DNase Produced

+ mild sized ++ large sized

 Yellow/ Mannitol Fermented

No Color Change/ No Mannitol fermented

Black Centers/ Colorless or Light Pink/ No Lactose Fermented Lactose Fermented

Organisms that are Gram negative are able to grow on this media. The degree 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

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

Green-Dark/ α-hemolysis Or Clear Zone/ ß-hemolysis

No Discoloration/ γ-hemolysis

Organisms that are Gram negative will be able to grow on

Pink to Red/ Colorless/ No Lactose Lactose Fermented Fermented

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media. It contains lactose bile salts, neutral red and crystal violet. The bile bile salts and crystal violet inhibit the growth of Grampositive 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 βgalactosidase. The pH indicator in the media is azolitmin.

 Triple Sugar Iron

VJ Agar

MR–VP Broth (Methyl Red Test) MR–VP Broth (VogesProskauer  Test))

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

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

SIM (H2S) SIM (Indole)

SIM (Motility)

Catalase  Test

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

 This test can be done on 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 this media. If the pH decreases as a result of lactose fermentation, then the media will turn pink. An alkaline alkaline reaction reaction results in a blue color. Other results include curdling of the 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 of the media associated with changes in pH. Acid production production is indicated by a yellow color, alkaline reactions by a red color and hydrogen sulfide by a black color. Coagulase positive and Gramnegative organism can grow on this media. media. Those organisms organisms that reduce tellurite form black colonies that contain a black precipitate from tellurite.  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. 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. sulfide. Upon addition addition of Kovac’s reagent, an organism that produces the enzyme tryptophanase will test positive (pink ring) for indoles. Hydrogen peroxide is applied to the colony and if the organism is

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

No Color Change/ No Reaction

Many Reactions Possible (See Figure 2)

Black Spots – yellow/ tellurite produced – Mannitol fermented Red/ Acid Production, Glucose Fermented

No Growth or No Color Change

 Yellow, No Color Change/ No Glucose Fermented

Red/ Acetoin Produced

Other color/ No Acetoin Produced

Black Precipitate/ H2S Produced

No Color Change/ No H 2S Produced

Red/ Indole Produced

No Color Change/ No Indole Produced

Cloudy/ Growth Not Restricted to Stab Line

Clear/ Immotile

Bubbles/ Catalase Present

No Bubbles/ No Catalase Present

colonies or it can be done by transferring a colony to a slide. 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 Lactose PR Mannitol

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

catalase positive, there will be production of gas bubbles (from the oxygen gas).

+few ++medium +++high

Gram-positive organism show normal colony morphology on this Growth/ Grammedia. Gram-negative organisms Positive Cocci either do not grow or are severely 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 media to yellow. Organisms that undergo deamination of amino acids will turn the media alkaline and pink or red in color.

No Growth/ GramNegative or Gram-Positive Rods

See Key from Table 2

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 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 incubation and labeled on the bottom with group members’ initials, inoculation date, laboratory section section number, media type, test to be performed, and the inoculated specimen. Media in test tubes were labeled with the same information information but written on tape and then wrapped around the test tube.

Carbohydrate Metabolism and Fermentation

For carbohydrate metabolism and fermentation 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, typhimurium , and Bacillus subtilis were inoculated into each Staphylococcus epidermidis, epidermidis, Enterococcus faecalis faecalis,, Proteus vulgaris and Bacillus

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type of media. All organisms were were incubated at 37°C, except B. except B. subtilis which was incubated at 30°C, for 18-24 hours. After incubation, the plates were 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 coli, S. typhimurium, typhimurium, S. epidermidis, epidermidis , E. faecalis , P. vulgaris, vulgaris , and B. 16×125 urea broth. broth. E. coli, and B. subtilis were inoculated into each type of media. For DNase agars, the organisms were inoculated inoculated by using an inoculating loop to aseptically streak 1 thick line down the middle of the agar. All organisms were incubated incubated at 37°C, except B. except B. subtilis which was incubated at 30°C, for 18-24 hours. After incubation, the media were placed in a 4°C refrigerator 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 agar deeps were placed in an ice bucket along along with an uninoculated control. control. After  the control had solidified, solidified, observations were were taken. All results were were recorded. It was noted that the nutrient nutrient gelatin tubes needed additional incubation time and were returned to their respective temperature temperaturess to incubate for an  P. vulgaris and B. additional five days. days. A DNase methylene methylene green agar Y plate was streaked streaked for  P. and B. subtilis , and P. and P. mirabilis then incubated for 37ºC for 18-24 hours and placed in a 4ºC refrigerator until observation 48 hours postinoculation. inoculation. This was due to lack of indicator added the first time to the two DNase methylene green agar Y plates  prior to inoculation. 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 agar plates. Each TSA blood agar plate was divided into coli , S. typhimurium, typhimurium, S. two sections using a black permanent marker to define halves from underneath.  E. coli, epidermidis, epidermidis, E. faecalis, 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 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 incubated at 37°C for 18-24 hours. After incubation, the plates were placed in a 4°C refrigerator  refrigerator  until observation around 48 hours post inoculation. The results were recorded.

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2nd Streak 1st Streak

Figure 1 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,  pseudodiptheriticum, Bacillus cereus , B. subtilis , and Mycobacterium smegmatis were inoculated on each type of  media. Following streaking streaking of each species on the TSA blood agar, the inoculating loop was stabbed in the agar 3-4 times. All organisms were were incubated at 37°C for 18-24 hours. hours. After incubation, the plates were placed in a 4°C refrigerator until observation 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 observations were taken. The results results were recorded. 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, aureus, S. and E. epidermidis, epidermidis, Micrococcus luteus, luteus, Micrococcus roseus, roseus , Streptococcus salivarius, 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 streaking of each species on the TSA blood agar, the inoculating loop was stabbed in the agar 3-4 times. times. All organisms, organisms, except M. luteus and M. roseus, 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

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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. plates. E. coli, coli , Enterobacter aerogenes , Klebsiella  pneumonia,  pneumonia, S. typhimurium, typhimurium, P. mirabilis , P. vulgaris, vulgaris , and Serratia marcescens were inoculated on each type of  media. No streaking for isolation was was performed. All organisms were incubated incubated at 37°C for 18-24 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 (Christine Simmons, 2009)

RESULTS

Carbohydrate Metabolism and Fermentation

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

Table 2: 2: Results from Carbohydrate Metabolism and Fermentation with accompanying symbol key

Growth Medium PR  Glucose PR Sucrose PR Lactose PR  Mannitol

 Escherichia coli

Salmonella typhimurium

 Enterococcus  faecalis

Staphylococcus epidermidis

 Proteus vulgaris

 Bacillus  subtilis

AG

AG

A

A

A

A

K A

K K

A A

A A

K K

A K

A

A

A

A

K

A

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Simmon’s Citrate

Symb ol A AG K NC + --

--

+

--

--

Observation

Meaning

Yellow Yellow with Bubbles

Acid Production Acid & CO2 gas Produced Alkaline Oxidative Respiration Citrate Oxidized No Citrate Oxidized

Deep Red Little to No Color Change (Remains Red) Blue No Color Change (Remains Green)

+

--

 Microbial Enzymes

Analyzing the SIM deep agar showed blackening only for S. 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. only E. coli. coli . Growth beyond the inoculated stab indicating motility was observed in both E. both  E. coli and S. typhimurium (Figure 2).  E. faecalis , produced bubbles when the hydrogen peroxide was added to the BHI agar  All organisms, except for  E.  plates, with S. typhimurium and P. 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.  B. subtilis , which was also able to resist solidification following following cooling in an ice bucket. All organisms tested negative for urease, urease, resulting in no color change in the urea broths. The first round of DNase DNase plates were unable to be observed observed due to lack of  indicator added prior to inoculation. For the second attempt with indicator added, only B. only B. subtilis was unable to vulgaris  produced a large decolorized ring and P.  produce a colorless ring around growth in the agar.  P. vulgaris produced and  P. mirabilis  produced a medium sized decolorized ring (see Figure 6). These results are summarized summarized in Table 3.

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

Figure 2: 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

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

Figure 5: 5: BHI agar after addition of H2O2. Notice S. typhimurium bubble formation on bottom compared to E. coli on the top.

Figure 6: 6: 2nd attempt DNase agar plate post inoculation viewed from both sides. sides. Statrting at the top and moving moving clockwise on the bottom up view is B. subtilis, P. mirabilus, and P. vulgaris.

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

Test Performed Gelatin Hydrolysis SIM Deep: Hydrogen Sulfide SIM Deep: Indole SIM Deep: Motility Urea Hydrolysis Catalase DNase

 Escherichia coli

Salmonella typhimurium

 Enterococcus  faecalis

Staphylococcus epidermidis

 Proteus vulgaris

 Bacillus  subtilis

--

--

--

--

--

+

--

+

--

--

--

--

+

--

--

--

--

--

+

+

--

--

--

--

--

--

--

--

--

--

+

++

--

+

+++ ++

+ --

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 Proteus mirabilis

+

 Selective and Differential Media

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

Figure 7: 7: Mannitol salt agar with bottom up and top up views. Inoculated with, starting starting at the top yellow colony moving clockwise, S. aureus, E. coli, and S. typhimurium. Figure 9: 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: 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 10: 10: MacConkey agar with bottom up and top up views. Inoculated with, with, starting from the purple colonies and going clockwise, E. coli, S. aureus, and S. typhimurium.

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

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

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

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

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

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

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

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

Media Mannitol Salt Agar  MacConkey Agar  Phenylethyl

 Escherichia coli

Salmonella typhimurium

 Enterococcus  faecalis

Staphylococcus epidermidis

 Proteus mirabilis

Staphylococcu  s aureus

No Growth

No Growth

No Growth

+ (small yellow)

No Growth

+

+ (Light Pink)

No Growth

No Growth

+ (Light Pink)

No Growth

--

+

+

--

+

+ (Pink w/Dark Centers) +

13

Alcohol Agar  EMB Agar 

Blood Agar 

+ (Metallic Green) + (αhemolysis)

+ (Pink) -(γhemolysis)

-(Colorless/ Light Pink) -(γhemolysis)

-(Colorless)

+ (Pink)

-(Colorless)

-(γ-hemolysis)

+ (αhemolysis)

+ (ßhemolysis)

Gram Positive Bacteria

For the gram positive rods tested, the nutrient gelatin agar was liquefied following cooling in an ice  bucket for only B. only  B. cereus . On blood agar, hemolytic activity through through either a darkening or clearing of the blood agar  was witnessed with C. pseudodiptheriticum and B. subtilis colonies. C. pseudodiptheriticum and B. pseudodiptheriticum,, B. cereus, cereus , and B. 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 ß-hemolytic activity. The non-color  changing organism M. smegmatis was recorded as γ-hemolytic. γ-hemolytic. C. pseudodiptheriticum and B. and B. cereus produced medium sized bubbles when the hydrogen peroxide was added to the BHI agar. Small bubbles where observed for  agar. For the litmus milk test test C. M. smegmatis. smegmatis . No growth of  B.  B. subtilis was observed on the BHI agar.  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 symbols are in Table 6.2. These results are summarized summarized in Table 5. For the gram positive cocci species tested, a yellow color change was observed in all species in the PR  aureus, M. luteus, luteus, and M. roseus (see glucose broth, with the addition of bubbles at the top of the Durham tube for S. for S. aureus, aureus, S. epidermidis, epidermidis , and E. figure 21). For the the PR lactose broths, broths, S. aureus, 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 color change (see figure figure 22). For PR  mannitol broths, no color change was observed for M. for  M. roseus, and E. roseus, S. epidermidis, epidermidis, and S. pyogenes. 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. for  S. aureus, aureus, S. epidermidis, epidermidis , S. pyogenes, pyogenes, M. luteus, luteus, and S. salivarius; salivarius; the latter two producing a larger larger amount of bubbles at a faster faster pace. No growth was observed observed of M. of M. roseus. roseus. On blood agar, hemolytic activity through either a darkening or clearing of the blood agar was witnessed with S. aureus, aureus, M. luteus, luteus, S. salivarius, salivarius, and E. luteus, S. salivarius, salivarius , and E. and E. faecalis colonies. M. luteus, 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 ß-hemolytic activity. The non-color changing organisms, S. epidermidis and S. γ-hemolytic. No growth was observed observed for M. for M. roseus (see Figures Figures 18 and 19). For   pyogenes,  pyogenes, were recorded as γ-hemolytic. mannitol salt agar plates, a yellow color change was observed for S. for S. aureus and E. and E. faecalis. faecalis . S. epidermidis also  produced a yellow color change change but was less significant. significant. No growth was observed observed for S. for S. salivarius. salivarius . For the VJ agar   plates, no growth was observed for M. for  M. roseus, S. epidermidis, and S. salivarius . Both S. pyogenes and E. and E. faecalis

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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 summarized in Table 6.

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

Figure 19: 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.

15

 TABLE 6-2 Litmus Milk Results and Interpretations  TABLE OF RESULTS

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

Result

Interpretation

Pink Color

Acid reaction

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

Acid clot

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

C ur d

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

Symb ol A AC

NC

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

Figure 23: 23: Phenol Red Mannitol Broths. From left to right: M. roseus, S. pyogenes, E. faecalis, S. aureus, S. salivarius , and S. epidermidis.

Figure 22: 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: 24: VJ agar with views of bottom and top. From the top left section going clockwise: E. faecalis, and S. salivarius .

Figure 25: 25: VJ agar with views of  bottom and top. From the top left section going clockwise: M. luteus and M. roseus.

Figure 26: 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: 5: Gram Positive Rod Bacterial Bacterial Tests. Positive and Negative test descriptions descriptions can be found in the last two columns of Table 1. Litmus milk observation symbols can be found in Table 6-2.

Corynebacterium  pseudodiptheriticum

 Bac  Bacil illu luss cere cereus us

Baci Bacill llus us subt subtil ilis is

Mycobacterium  smegmatis

Gelatin Hydrolysis

--

+

--

--

Blood Agar 

+ (α-hemolysis)

+ (ß-hemolysis)

+ (α-hemolysis)

+

+

No Growth

-(γ-hemolysis) + (Very Little)

CA

CKNC (darker than control)

NC

Media/ Test

Catalase Litmus Milk 

17

CNC

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

Media

Staphylococc us aureus

Micrococc us luteus

Micrococcus roseus

Staphylococc us epidermidis

Streptococc us pyogenes

 Enterococcu  s faecalis

Streptococc us salivarius

Phenol Red Glucose

AG

AG (Cloudy)

AG

A

A (Slightly)

A (Clear)

A (Slightly)

A (Clear)

K  (Clear)

Phenol Red Lactose Phenol Red Mannito l Catalase

A

K  (Cloudy)

A

K  (Cloudy)

+ + (βhemolysis)

Blood Agar 

NC (Very Slight  Yellow) NC (Very Slight  Yellow) + -(γhemolysis )

A

K 

-+ (αhemolysis )

++ + (αhemolysis )

NC

A (Clear)

NC

NC (Very Slight  Yellow)

++ + (αhemolysi s)

No Growth

+

No Growth (Contamin ated)

-(γhemolysis)

--

+ (½ Yellow)

--

+

No Growth

No Growth

+ (Little Growth, Pink)

+ (Little Growth, Pink)

No Growth

Mannito l Salt Agar 

+

--

VJ Agar 

+ (Yellow w/ black centers)

+ (Pink with black centers)

No Growth

Gram Negative Rods

For the gram negative rod bacteria tested, in PR glucose broths all, except for  P.  P. vulgaris which created no color change, caused a yellow color change indicative of acidic products being produced. Of these yellow color  marcescens , caused a gas bubble to form in the top of the changing organisms it was observed that all, except for S. for  S. marcescens, Durham tube. For PR lactose lactose broths, E. broths, E. aerogenes, P. vulgaris, S. typhimurium, P. mirabilis, and S. marcescens caused a deeper red color change.  K. pneumoniae, and E. coli produced coli produced acidic products causing a yellow color  change. For PR mannitol broths, all organisms organisms tested caused a yellow color change from acidic products being  produced, except P. except  P. vulgaris and P. 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 mirabilis  producing high amount of bubbles and S. marcescens and S. typhimurium producing 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. of  S. marcescens and P. 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 colonies where observed observed in K. in K. pneumoniae and E. and E. coli. in P. vulgaris. coli . No growth was observed in P. vulgaris . For endo agar plates, colorless colonies of S. of  S. marcescens, marcescens , P. mirabilus, 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. in K. pneumoniae and E. and E. coli. in P. vulgaris (see coli . No growth was observed in P. Figures 27 and 28). For Simmon’s citrate agar, no growth was observed in P. in  P. vulgaris, vulgaris , E. coli, coli , P. mirabilus , and S. marcescens. marcescens .  E aerogenes, 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. for S. typhimurium and P. and P. mirabilus . Dark Pink Pink colonies colonies  E. coli, coli , K. pneumoniae , and E. aerogenes . No growth was observed for  P.  P. vulgaris and S. were observed for  E. and E. aerogenes. marcescens. marcescens. For the TSI slant agars, observations were 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. 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. for  S. typhimurium (see Figure 30). Reaction with with the Kovac’s reagent in the SIM agar deeps yielded a pink ring in only E. only E. coli. beyond the inoculated stab in coli . Growth beyond coli , S. typhimurium, typhimurium , P. mirabilus, mirabilus , and E. aerogenes . the SIM agar deeps was observed in E. in E. coli, and E. aerogenes. In the urea broths, only the P. 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. and E. aerogenes which remained yellow yellow (see Figure 32). In the set of MR-VP tubes tubes  P. vulgaris and E. with 1.2 mL α-Naphthol Reagent and 0.5 mL 40% KOH added, a red color was only observed in E. in E. aerogenes and S. marcescens cultures (see Figure Figure 33). These results are summarized summarized in Table 7.

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

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

19

 TABLE 6-6 TSI Results and Interpretations  TABLE OF RESULTS Result  Yellow slant/ yellow butt

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

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

Interpretation

Sym bol

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/N C

Black precipitate in agar

Sulfur reduction

H2S

Cracks in or lifting of agar

Gas production

G

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

Figure 32: 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 .

20

Figure 33: 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 .

Negative Enterobacteriaceae Enterobacteriaceae Test Results. Results. Positive and Negative test Table 7: 7: Lab 9 - Gram Negative descriptions can be found in the last last two columns of Table 1. Phenol Red Broth symbols can be found in the key of Table Table 2. TSI symbols are in Table 6-6.

Media/ Test

 Enterobacter  aerogenes

 Klebsiella  pneumoniae

 Proteus vulgaris

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) + (pink)

+

++

+

+++

++

+ (metallic)

No Growth No Growth

+ (pink)

+ (metallic)

Endo Agar

+ (pink)

+ (metallic)

No Growth

--

+ (metallic)

-(light pink) -(No Color)

Simmon’s Citrate Agar

+

+

No Growth

+

No Growth

-(light pink) -(No Color) No Growth

MacConkey Agar

+

+

No Growth

--

+

--

No Growth

K/A,G

A/A,G

K/K

2

A/A,G

K/H2S

K/K

Hydrogen Sulfide

--

--

--

+

--

+

--

Indole

--

--

--

--

+

--

--

Motility

+

--

--

+

+

+

--

Urease

--

--

--

--

--

--

--

+

--

+

+

+ (pink) +

+

+

--

--

--

--

--

+

EMB Agar

TSI Slant

MRVP

Methyl Red VogesProskau er

K/H

21

S,G

 

No Growth

 

DISCUSSION

Carbohydrate Metabolism and Fermentation

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

 Microbial Enzymes

Production of the enzyme gelatinase allows those organisms to hydrolyze gelatin for the release of soluble  peptides and amino amino acids. (). This prevents gelatination at temperatures temperatures below below 25ºC. Only B. Only B. subtilis was observed to have this capability. capability. H2S production created a black precipitate when reacting with metals in the SIM agar deeps in only the S. typhimurium culture. This indicates indicates that that S. typhimurium produces typhimurium produces cysteine desulfurase used to  breakdown cysteine and methionine. methionine. (). Tryptophanase presence 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 Tryptophanase hydrolyzes tryptophan into  pyruvate which is in turn used in metabolism. (). Indole reacted with the reagent in E. 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. tested. However, it should be noted that that P. vulgaris is known to produce urease. (). This inconsistency inconsistency is  P. vulgaris that caused it to explained by an unknown error that occurred with the laboratory’s stock culture of  P.  behave in a strange manner. This behavior remained despite repeated 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 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. agar. (). Methyl green was not included included upon first inoculation inoculation of the DNase agar plates. plates. This resulted in no available observations. observations. The test for DNase DNase was re-performed on P. on P. vulgaris, B. subtilis, and P. and P.

22

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

 Selective and Differential Media

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

Gram Positive Bacteria

For the gram positive rods tested, gelatinase activity was observed in only B. only  B. cereus . This differed from  previous lab results (see Table 8) which showed gelatinase activity in both B. both B. cereus and B. and B. subtilis . On blood blood agar, agar, C. pseudodiptheriticum 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 therefore only be used to confirm results of another test. (). All organisms tested had different 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 typical of a litmus milk test. For the gram positive cocci tested, in PR glucose CO2 production was observed in the Durham tubes of  aureus, M. luteus, luteus, and M. roseus broths. This differed from previous the S. aureus, previous lab observations of no gas produced, produced, only acidic products. For PR lactose, previous lab observations observations found all organisms tested to produce acidic  production. This differed differed from the observations observations of M. of M. luteus, luteus, M. roseus, roseus, S. pyogenes, pyogenes, and S. salivarius. 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. respectively. M. roseus, S. epidermidis, and S. pyogenes all were observed to use mannitol mannitol oxidatively. However, prior labs 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 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, 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). (γ-hemolysis). Previous lab observations greatly contrast this result listing S. pyogenes as having had ß-hemolytic ß-hemolytic activity. The blood agar plates where where split inaccurately inaccurately with a permanent marker. marker. Proof of  this possibility is best demonstrated in the culture of M. of  M. roseus. plate, M. roseus colonies were roseus. On this agar plate, overgrown by colonies of M. of M. luteus. luteus. Mannitol salt agar plates held more consistent with with the past and present lab observations. Previous labs observed M. roseus and E. and E. faecalis as having no growth. However, M. roseus was observed to have colorless growth indicating no fermentation 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. for S. aureus and S. epidermidis. 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 was true of previous lab observations. For M. For M. luteus, luteus, no mannitol fermentation but tellurite production was observed to have occurred; this differs from previous labs which observed no growth of M. of  M. luteus. fermentation was observed to have occurred luteus. No fermentation  by the pink colonies in either S. either S. pyogenes or  E. growths were were small. Previous lab observations observations  E. faecalis . These growths recorded S. pyogenes as having no growth and E. and E. faecalis as fermenting mannitol. mannitol. These many variations, 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  P. vulgaris which showed no growth. exception of  P. growth. This was a result of the P. the P. vulgaris stock culture impurity. Much like the past lab observations, all organisms besides P. besides P. vulgaris and S. marcescens produced CO2. Both PR  lactose and PR mannitol shared similar similar observations with the previous lab observations. The only different is E. 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, 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, results, showing a metallic surface on the colonies of  K. appearing pink as in the past lab observations. observations. This indicated indicated much  K. pneumoniae rather than just appearing 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 Differentiation of different enterobacteriaceae 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  K. pneumoniae was observed as having heavy lactose fermentation.  P. vulgaris was respectively. respectively. In endo agar  K. observed as having no growth. P. growth. P. mirabilus and S. marcescens where observed as having growth but no lactose fermentation. All these organisms organisms in the past lab results were were observed as having moderate lactose lactose fermentation. In Simmon’s citrate agar, no growth was observed in P. in P. vulgaris, or S. marcescens. vulgaris , E. coli, coli , P. mirabilus , or S. marcescens . This contrasts with past lab results in that growth but no citrate oxidation was observed for  P. and P.  P. vulgaris, vulgaris , E. coli, coli , and P. mirabilus cultures; and citrate oxidation was observed in S. marcescens. marcescens. The TSI slant slant agar observations observations appear to  be easily variable due to the greater amount of qualities to observe.  E. aerogenes , P. mirabilus, 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 in the agar. In the past lab results, 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 H 2S 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 butt indicate glucose fermentation fermentation and peptone catabolism. catabolism. A red slant and red butt indicate no fermentation and the catabolism of peptone, either either aerobically or anaerobically. 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 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. that P. vulgaris and S. marcescens had no growth. In the past lab results, both both were observed fermenting fermenting lactose. All SIM deep agar test testss matched that of  the past lab results except for  P.  P. vulgaris. vulgaris .  P. vulgaris tested negative for H 2S production, indole production, and motility.  P. vulgaris was observed as positive for these tests in the past lab lab results. Further, P. 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. times. The first time, growth growth occurred and no citrate citrate was oxidized. The second time, no growth growth occurred.  P. vulgaris was tested two times in PR glucose, for catalase, catalase, and in Simmon’s citrate agar. The first time, glucose was fermented, many O2 bubbles were produced by catalase, catalase, and citrate oxidation 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 citrate agar. These inconstancies bring into question not only the purity of the stock  P.  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 it produced pink colonies indicating indicating lactose fermentation 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 broth test. These isolated inconsistencies, inconsistencies, those separate from P. from P. inoculation and/or transferring post-inoculation. post-inoculation. Further  vulgaris, vulgaris, are most likely the result of contamination during inoculation testing should be performed in addition to careful practice of aseptic technique.  Table 8: 8: Past Lab Results for Gram Positive Rod Bacterial Tests Performed. The highlighted observations indicates data that differed with this lab’s observations

Corynebacterium  pseudodiptheriticum

 Bacillus cereus

--

+

Blood Agar 

-(γ-hemolysis)

+ (ß-hemolysis)

Catalase Litmus Milk 

+ AC,G

+ KR

Media/ Test Gelatin Hydrolysis

 Bacillus  subtilis

Mycobacterium  smegmatis

+ + (αhemolys is) + DK

--(γhemolysis) + K

 

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

Media

Staphylococcus aureus

Micrococcus luteus

Micrococcus roseus

Staphylococcus epidermidis

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

NC

A

A

K

A

A

Phenol Red Glucose Phenol Red Lactose Phenol Red Mannitol Catalase

Streptococcus  Enterococcus  pyogenes  faecalis

+ + + ----+ -+ -(γ(γBlood (β(γ(β(γAgar  hemolysis hemolysis hemolysis) hemolysis) hemolysis) hemolysis) ) ) + -No + -Mannitol No Growth Salt Agar  (Yellow) (Pink) Growth (Yellow) (Pink) + No No -VJ Agar  No Growth No Growth (Black) Growth Growth (Yellow) Table 10: 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

 Proteus vulgaris

Salmonella typhimurium

26

 Escherichia coli

 Proteus mirabilus

Streptococcus  salivarius

--(γ-hemolysis) No Growth No Growth

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)

+ (Pink)

Endo Agar

+ (Pink)

+ (Pink)

+ (Pink)

-(Light Pink)

+ (metallic)

-(light pink) + (Pink)

Simmon’s Citrate Agar

+

+

--

+

--

--

+

MacConkey Agar

+ (Pink)

+ (Pink)

+ (Pink)

-(Colorless)

+ (Pink)

+ (Pink)

A/K, H2S

A/A,G

-(Colorless ) A/K, H2S

TSI Slant

A/A,G

A/A,G

A/A, H2S

+ (Pink)

A/K

Hydrogen Sulfide

--

--

+

+

--

+

--

Indole

--

--

+

--

+

--

--

Motility

+

--

+

+

+

+

--

Urease

--

+

+

--

--

+

--

Methyl Red

--

+

+

+

+

+

+

VogesProskaue r

+

--

--

--

--

--

--

MRVP

27

 

 

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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