Systematic Identification of Bacillus Subtilis and Serratia Marcescens Through a Battery of Tests and Plates Introduction

7 July 2016

The purpose of this experiment was to use a systematic battery of tube tests and plates designed to lead to identification of two unknown bacterial species, from the combination of all results. A sample of bacteria was used, labeled “Sample 4”, from which both species was to be obtained, one gram positive and one gram negative. Table 1 is a list of the possible bacteria to be identified; the basic ideas and practice of identification of an unknown sample of bacteria are important for a microbiologist to develop.

Not only is proper procedural practice necessary, the investigator must use critical thinking to solve the puzzle that an unknown bacterial sample represents. The essential idea of bacterial identification and grouping based on testable characteristics is referred to as taxonomy. The types of tests and the efficiency of the identification or taxonomic placement depend upon both the critical reasoning of the microbiologist and a well-designed plan.

Systematic Identification of Bacillus Subtilis and Serratia Marcescens Through a Battery of Tests and Plates Introduction Essay Example

The tests performed and used in the determination of the gram positive bacteria in Sample 4 were the gram stain, esculin hydrolysis, catalase production, and observations from MSA, NA, and blood agar plates. The tests performed and used in the determination of the gram negative bacteria in Sample 4 were the gram stain, TSI slant, citrate utilization, indole production, and observations from EMB, NA, and DNase plates. All tests performed were reasoned to differentiate distinct characteristics of bacteria from the other possibilities, thus identifying the two unknown species in Sample 4.

Table 1: List of possible bacterial species. Bacterial Species Escherichia coli Pseudomonas aeruginosa Proteus mirabilis Shigella flexneri Staphylococcus aureus Bacillus subtilis Enterobacter aerogenes Enterococcus faecalis Serratia marcescens Distinct plate results and observations were expected to be essential to the bacterial determination of both species in Sample 4. During the identification, the bacteria were plated to multiple NA (Nutrient Agar) plates.

Nutrient Agar is a general agar medium containing chemicals essential for growing most culturable bacteria in the lab; it is not considered a selective or differential medium, it simply gives insight into general colony characteristics (Madigan et al, 2012). Each of the two different species of bacteria underwent a gram stain; the gram stain method involves staining and counterstaining a sample of cells, in which the results depend upon the composition and thickness of elements of the cell wall.

Gram negative cells have more chemically complex cell walls, gram positive cells are less complex yet have a thicker layer of a component called peptidoglycan. The difference in results from a Gram stain is because of this distinction in cell wall composition. After treatment with iodine and decolorization with acetone, gram positive bacteria retain the coloration of the first dye used, and gram negative bacteria are counterstained with the color of the second dye (Madigan et al, 2012).

After identification of a bacterial sample as gram positive or negative, the latter may be plated to an EMB plate, the former to an MSA plate. Mannitol Salt Agar (MSA) plates are designed with selective high concentrations of salt, as well as a differential yellow color change which indicates bacteria that ferment mannitol. Eosin Methelyne Blue (EMB) plates are formulated for the encouragement of growth for gram negative bacterial species; EMB plates can also indicate the bacterial fermentation of lactose if the plated colonies are purple after incubation (Levine, 1981).

Two other plates commonly used to identify characteristics of bacteria are the DNase and blood agar plate. A DNase plate is an agar plate used to test for a microorganism that employs the enzyme DNase, which breaks down DNA. A DNase plate contains DNA bound to a dye embedded in the agar, this dye is only colored when bound to the negatively charged DNA particle. If DNA on the plate is broken down by a microorganism, the dye will no longer be bound to it, and thus no longer be colored. A positive result for DNase then, is a clearing zone or “halo” around the bacterial streak on the plate (Menzies, 1977).

A blood agar plate is used to test a gram positive microorganism’s hemolysis activity. Hemolysis is the breakdown of red blood cells, thus an agar plate embedded with red blood cells is used to test for this activity. If red blood cells are broken down, the red color of the agar will disappear and the result is said to be positive beta-hemolysis; if there is no clearing under or around the microorganisms the result is said to be negative gamma-hemolysis (Brown, 1991). Plate results are an important framework on which to rest the slant and tube tests that follow them.

The results of slant and liquid tube tests give indications on less of a spectrum basis than plate results, but can be just as useful when many are compiled together. The bile esculin hydrolysis test is a selective and differential slant used to identify bacteria of the genus Enterococcus. The test contains bile salts to select for the desired bacteria, and differentiates because the hydrolysis of esculin and subsequent combination of the products with iron produces a black color. A positive test for the bile esculin slant is a completely blackened tube (Lindell et al, 1975).

A triple sugar iron (TSI) slant is used to identify sugar fermentation in a microorganism; it contains a red pH-sensitive dye that will turn yellow under acidic conditions, such as contact with the acidic byproducts of sugar fermentation. The three sugars, sucrose, lactose and glucose, are present in specific concentrations, 1%, 1%, and . 1% respectively (Hajna, 1945). The combination of the color change results and the location in the tube of the changes allows for a multitude of varying results.

The TSI slant is a useful launch point for an investigation of this type, because the varying results can give a solid idea of what direction the remainder of the tests must take. A citrate utilization test is used to determine if an organism uses citrate as its only source of carbon, a positive result will change the dye in the slant from green to blue due to the byproducts changing the pH in the tube (Kiska et al, 2002). An indole production test is used to indicate if an organism can degrade the chemical tryptophan into indole and other products.

After incubation, the tube is tested for indole production by the addition of Kovac’s reagent; the reagents indicates a positive result if it is colored red in the tube, negative if it is not red (Watanabe et al, 1972). A catalase test is a slightly different category of test than plates or slants; it used to determine if a microorganism uses the enzyme catalase. If, upon placement of a drop of hydrogen peroxide on a bacterial colony, bubbles are produced, the bacteria has broken down the H2O2 into water and oxygen.

The oxygen production is responsible for the bubbles, thus bubbling is said to be a positive test for the enzyme catalase (Keilin et al, 1938). The systematic inventory of the results obtained from all of the tests allowed the gram negative and gram positive species of bacteria in Sample 4 to be determined as Serratia marcescens and Bacillus subtulis, respectively. Procedure: The bacterial tube labeled “Sample 4” was obtained, the bacteria inside were streaked for isolation to an NA plate and incubated overnight at 37° C.

Distinguishing by isolated colony color and morphology on the NA plate, a gram stain was performed on one of each of the two distinct colony types. The gram negative bacteria was both plated to an EMB plate and streaked to a new NA plate for isolation. The gram positive was both plated to an MSA plate and also streaked to a new NA plate for isolation. The plates were incubated overnight at 37° C. Colonies isolated on the gram positive NA plate were used to inoculate all of the following tests. A bile esculin slant was streaked along the surface with a loop.

The slant was incubated overnight at 37° C and the results were recorded. A blood agar hemolysis activity plate was streaked for isolation, and then incubated overnight at 37° C, the results were recorded. Finally, a catalase test was performed by directly placing a drop of hydrogen peroxide on a colony of the NA plate and the results were recorded immediately. Colonies isolated on the gram negative NA plate were used to inoculate all of the following tests. A TSI slant was first stabbed through to the bottom, and then streaked along the surface with a loop. The surface of a citrate slant was streaked in a zigzag pattern.

A tube of broth containing tryptophan was inoculated with a loop full of bacteria for the indole test. After incubation, two drops of Kovac’s reagent was added to this tube and the color of the drops was recorded. All of the tests above were incubated for 24 hours, with the exception of the citrate test which incubated for 48 hours at 37° C, the results were subsequently recorded. A DNase plate was marked down the center; each half was designated to one of the two species, and a single straight streak of a colony from the respective NA plates was set onto the agar.

The DNase plate was incubated at 37°C for 24 hours, results were observed and recorded. As all results were recorded, they were compared to previously collected data to decide what still needed to be tested to come to a bacterial determination. Results: The following results are summed up in Table 2. The initial NA plate gave two distinct colony morphologies and colors: large fuzz-edged off-white colonies, and small, pink smooth colonies. The gram stain for the off-white colonies returned purple long bacillus bacteria; the gram stain for the pink colonies showed many small pink bacillus.

The MSA plate showed a yellow color change with medium sized colonies. The EMB plate inspection gave small smooth pink colonies. The bile esculin slant showed no color change. The blood agar plate also showed no change in color and no clearing zone around the bacterial colonies. Upon placement of hydrogen peroxide on the colonies for the catalase test, bubbles were observed immediately. A yellow butt and red slant was the observed result from the TSI slant. The off-white colony side of the DNAse plate showed no clearing, the other half had pink smooth colonies with a halo clearing zone surrounding the streak.

Upon addition of Kovac’s reagent to the indole test, the reagent was a yellow-orange color. The citrate slant resulted in a color change from green to blue of the entire slant. Table 2: Summary of results obtained from both species in Sample 4. TestOff-white Colony ResultsPink Colony Results NA Platelarge, fuzz-edged colonies, off-white colorsmall, smooth colonies, pink color EMB PlateN/Asmall, smooth pink colonies MSA Platesmall colonies, yellowing of agarN/A Bile Esculin Slantno color change, yellowN/A Blood Agar Plateno change, no clearingN/A Indole TubeN/Aorange-yellow reagent drops

Citrate TubeN/Ablue color change TSI SlantN/Ayellow butt, red slant Catalase TestN/ABubbles Dnase Plateno clearing zone observedbright pink colonies, obvious clearing zone Discussion: A compilation of all results led to the determination of both species of bacteria present in Sample 4. The purple color, indicating a thick peptidoglycan layer in the cell wall, of the gram stain for the off-white colonies implied the presence of gram positive bacteria. The pink coloration, indicating a thin peptidoglycan layer in the cell wall, of the gram stain for the pink colonies was indicative of gram negative bacteria.

Based on the following results and reasoning, the gram negative species in Sample 4 was determined to be Serratia mercescans. First examined in the determination were the TSI slant results. The yellow butt and red slant of the test imply the bacteria fermented only one of the three sugars present in the agar: glucose. This is understood when the concentrations of each sugar is examined; if either lactose or sucrose had been fermented, the entire tube would have changed color, if no sugars were fermented, the tube would have remained the same red as before.

The significance of the butt of the tube changing color and not the slant indicated fermentation of only glucose, as there is not enough glucose in the slant to turn the whole tube yellow. These results are supported by research suggesting S. marcescens ferments glucose, but does not ferment either lactose or sucrose (Wilfert et al, 1970). The DNase plate was essential to the identification because of the distinct result; the obvious clearing zone around the bacteria indicated DNase activity, and the pink colonies on the plate were definitive for that test. S.

marcescens is proven to test positive for DNase activity 92. 4% of the time, as well as show consistently pink colored colonies (Wilfert et al, 1970). The citrate utilization test was positive because the dye in the tube changed color from green to blue, denoting an alkaline pH change resulting from the breakdown of citrate by the bacteria. The result of the indole production test was negative, because the added Kovac’s reagent was not colored red. S. marcescens has been proven to test positive 100% of the time for citrate utilization, and test negative 99% of the time for indole production (Wilfert et al, 1970).

The NA and EMB plates further solidified the identification of the gram negative bacteria as S. marcescens. The species has consistently smooth, pink colonies on nutrient agar, as well as on EMB. The sample was also consistent in testing negative for the fermentation of lactose because it did not result in purple colonies on the EMB plate, the color remained unchanged. Overall, the distinctive compilation of the test and plate results in comparison to known characteristics of all possible bacterial unknowns for the gram negative bacteria allowed for determination as S.

marcescens; possibly most notable: the constant pink coloration of the colonies. The gram positive species in Sample 4 was determined to be Bacillus subtilis based upon the observed results and observations, as well as detailed research information describing typical B. subtilis characteristics. A very powerful early indication of B. subtilis came from information attainable from the gram stain, cell morphology. Every cell observed on the slide was bacillus or rod shaped; of the three possible gram positive bacteria (B. subtilis, S. aureus, and S. faecalis) only B. subtilis is morphologically bacillus, the others are coccus.

The result of the catalase test was clearly positive, the immediate production of bubbles indicated a bacteria able to break hydrogen peroxide into water and oxygen. B. subtilis has been shown to test positive for the enzyme catalase; the species is even occasionally used as a model organism in laboratory catalase research (Bol et al, 1991). The response of the bacteria to the esculin hydrolysis test indicates a negative result. No black color change indicated that the esculin in the slant was not broken down, and there were no byproducts to bind to iron and turn the tube black.

In an aside to the initial morphology result, the esculin hydrolysis and catalase tests indicate the gram positive unknown is not S. faecalis, a species known to show color change for esculin hydrolysis and negative for the catalase enzyme (Zervos et al, 1987). The blood agar showed no change in the agar, indicating gamma-hemolysis. B. subtilis has not been known to typically lyse blood cells, the model laboratory organism S. aureus has been known to exhibit this characteristic (Fairweather et al, 1983). This final test, in conjunction with the gram stain morphology result, discounts the possibility the unknown species is S.

aureus, leaving the final determination of the gram positive unknown in Sample 4 as B. subtilis. In the era of phylogeny, the determination of a bacterial species’ place in the tree of life by way of nucleotide base pair sequencing, taxonomic studies such as this have taken a back seat in true bacterial identification. Phylogeny, however, requires much more time and analysis than the determination of bacteria by testing for certain characteristics; this difference is worth the sacrifice in exact results in situations where an unknown bacteria has caused infection or contamination.

Thus, batteries of tests and plates are used commonly in clinical situations where the medical response may need to be immediately determined to help an individual, or prevent an outbreak. Typically, the same strategy of testing by design is used by those in the medical field to launch an investigation into an unknown pathogen or contaminant. The investigator must combine all their knowledge and skill to come to an accurate conclusion, making this type of experiment an essential tool for microbiologists.

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