An Orange, Gram-Positive Bacterium Isolated from the Environment on Campus (Short but not

concise. What was the bacterium identified as? What was the environment on campus? Better title would be

something like… “Identification of a Soonercoccus bacterial species from the bench at the OU bus stop”. Take

home message what did you find and where did you find it)

ABSTRACT:

An environmental sample was taken from a bench at the South Oval bus stop on the

campus of the University of Oklahoma. Streak plates on TSA agar plates and/or streaks on TSA

agar slants were done during each lab period to ensure that the bacterial culture was pure and

new. Tests were then performed with the environmental isolate in order to determine its specific

characteristics. The colonies were orange, smooth, moist, convex, and circular. These Gram-

positive, rod-shaped, non-motile cells resembled those of the genus Soonercoccus in that it is

also an obligate aerobe, the optimal growth temperature is 20ºC – 35ºC, and the cells are spore

nonproducers, not acid-fast, catalase producers, and orange in color. Also, fermentation with

acid end products occurs (of what sugars?). After various staining tests, this bacteria show that they

are capsule producers, but do not produce spores or inclusion bodies. After certain tests to verify

whether specific enzymes were produced, one can conclude that decarboxylases are present

(which ones?), while oxidase, beta-lactamase, phenylalanine deaminase, urease, and amylase are

not present. Since the enzyme, beta-lactamase, is not produced, the environmental isolate is

susceptible to penicillin and several other antibiotics. Also, after continued tests, the isolate

proved to be unable to metabolize or ferment sugars (which sugars? This sentence contradicts the

previous sentence in which you indicate that “fermentation with acid end products occurs?) and unable to

reduce nitrate. At this point, after determining its specific characteristics, the bacteria correspond

to the genus Soonercoccus. [Specifically, what were the critical tests/results that convinced you that the

purified environmental isolate was in the genus Soonercoccus? Example: Analysis of the environmental isolate

indicated it to be an oxidase negative, catalase positive Gram positive rod that was able to ferment glucose and

lactose but not sucrose, all of which are common characteristics of bacteria within the genus Soonercoccus. In

addition to the biochemical and morphological results that justifies the presumptive identification of the

environmental isolate as a member of the genus Soonercoccus it is well established that Soonercoccus are obligate

aerobes. Based on its growth pattern in fluid thioglycollate broth, the environmental isolate was also considered to

be an obligate aerobe, which further supports its identification as a member of the Soonercoccus genus. Remember

to be brief (<150 words) and concise.]

INTRODUCTION:

The purpose of the Environment Isolate process is to mainly understand and be able to

conduct the aseptic technique. The aseptic technique means to “transfer living microbes from

one place to another without contamination of the culture, the sterile medium, or the

surroundings” (Leboffe and Pierce, 13) [In a science report you do not quote directly from a

source/reference but instead paraphrase (state things in your own words). Furthermore referencing should be done

in the format outlined by the American Society of Microbiology Guidelines]. The aseptic technique is

important because it allows us [personal pronouns like us, I or we should not be used in science/lab reports] to

isolate and maintain a pure bacterial culture from an unknown sample of bacteria in the

environment. In order to obtain pure microbial cultures, it is necessary to make streak plates.

“In a streak plate method of isolation, a bacterial sample is streaked over the surface of a plated

agar medium. During streaking, the cell density decreases, eventually leading to individual cells

being deposited separately on the agar surface” (Leboffe and Pierce, 20) [See above, direct quotes

should not be used and referencing is incorrect]. A specific colony is chosen and the streak plate process

is repeated until a pure colony is obtained. Once a pure colony is obtained, tests can be done to

determine the identification of the bacterium.

However, throughout this entire process, the pure bacterial culture must be maintained by

restreaking the sample during each lab period. By doing this, the culture is kept new and is

capable of presenting accurate results in weekly experiments. Being able to keep the culture

throughout an extended length of time is very important. After each test of the bacterial culture,

accurate identifications of the unknown bacterium can be made. The goal is, by the end of the

semester, to be able to identify to at least the genus of the unknown environmental isolate.

During this entire process of obtaining, testing, and maintaining the unknown

environmental isolate, important techniques in microbiology are expected to be learned. That is,

the aseptic technique, the different tests (especially the stains), and the knowledge of being able

to deduce the identity of the unknown bacterial culture. One of the most important aspects to

learn in this process is to understand and be able to carry out the staining procedures correctly

because these procedures isolate many of the different characteristics of cells that must be known

in order to narrow down the process of identification of the culture. Of course, there are many

other tests, such as different enzyme productions, sugar fermentations, and the effects of

temperature and pH on bacterial growth that are also important and aid in the classification

process [identification not classification process]. The environmental isolate identification process that

relates to what microbiologists do when trying to identify unknown microbial growth is also

comprehended. It was hypothesized that, after many tests and experiments, this environmental

isolate belongs to the genus Soonercoccus. [Thoughts need to be written/expressed with greater clarity (if

you are uncomfortable with or have trouble writing the folks at the OU Writing Center are a great resource!). You

should indicate/discuss the different ways bacteria can be identified (e.g. biochemically, morphologically or by some

of the “more modern techniques” such as…). You should discuss the ubiquity of microorganisms and indicate the

location where you sampled from and discuss in general the types of microorganisms that may be found in this

environment (e.g., over the years numerous different types of bacterial species have been isolated and identified in

soil including spore-forming Bacillus species and many nitrogen-fixing microorganisms). The one thing you do not

want to do in this section is to state what you believe your isolate to be (similar to not giving out the punch line

before telling the joke!), this comes later and is backed with experimental evidence.]

MATERIALS AND METHODS:

The morphological characteristics [isolation and purification was the first thing done not

morphological characteristics, since the first objective was to obtain a pure culture] of the environmental

isolate began by taking a swab sample of a bus stop bench and then making a spread plate [what

kind of streaking procedure was used (i.e., quadrant streak or environmental streak) and why?] of the sample.

Eventually, TSA agar streak plates (with four quadrants) and agar slants were used and then

incubated at 37ºC for each lab period in order to isolate the bacterial culture [you were subculturing

in order to obtain/isolate a pure culture of a single organism]. Once the bacteria were isolated into pure

colonies, the colony color, texture, margin, elevation, and shape were determined. Also, the

gram [Gram should be capitalized since the procedure is named after Christian Gram] stain (using Crystal

Violet, Gram’s iodine, ethanol, and Safranin—gram positive or negative), capsule stain (using

Congo Red and Maneval’s Stain—capsule producer or nonproducer) and endospore stain (using

Malachite Green and Safranin—spore producer or nonproducer were all completed to determine

more specific bacterial morphologies. [What about the acid-fast stain and which acid-fast stain procedure did

you use? Where each of these procedures done exactly as outline in the lab manual and where is the reference for

these procedures?] The physiological characteristics of the environmental isolate were determined by

performing a catalase test (using H2O2 [need to tell the reader what chemical this is] to see if the sample

bubbles) to see if catalase was present, an oxidase test (using a BBL slide to watch for color

changes) to see if cytochrome c oxidase was present, a motility test (using motility test agar) to

see if the bacteria were motile, an aerotolerance test (using various types of agars and broths

[specifically what?]) to see if or to what extent the bacteria could survive in the presence of oxygen,

a temperature test (using nutrient broths incubated at temperatures 4º, 22º, 37º, 50ºC) to find the

temperature(s) that promote optimal bacterial growth, and a pH test (using nutrient broths held at

pH 2, 4, 6, 8,10) to determine the pH(s) for optimal bacterial growth. [What about the test where you

examined the osmotolerance of the EI by growing the organisms at different salt concentrations?] The biochemical characteristics of the environmental isolate were further determined by

performing various tests and exercises, such as an acid-fast stain (ZN or Kinyoun carbolfuchsin

stain, acid-alcohol, and Methylene Blue or Brilliant Green) to see if the bacterium was acid-fast

(presence of mycolic acids in cell walls) or non acid-fast, an antimicrobial susceptibility test

(using Mueller-Hinton agar plates and antibiotic discs of streptomycin, tetracycline, penicillin,

and chloramphenicol) to measure the effectiveness of antibiotics on the bacterium, a beta-

lactamase test (using Cefinase discs) to determine if the bacterium produced beta-lactamase, an

oxidation-fermentation test (using nutrient broths) to see if the bacterium were able to oxidize

and/or ferment specific sugars [which one?], a phenol red broth test (using phenol red broths of

glucose, lactose, mannitose, and sucrose) to see if fermentation of sugars occurred, an MR-VP

[what does this stand for?] (using MR-VP broths and reagents) to determine if acid fermentation

occurred, a nitrate reduction test (using nitrate broths and reagents) to see if nitrate was reduced,

a decarboxylation test (using lysine, arginine, and ornithine broths) to if the decarboxylase

enzyme was produced, a starch hydrolysis test (using starch agar plates and iodine) to see if

amylase was present and if starch was hydrolyzed, and a urease test (using urea broths) to verify

if urease was present and if urea hydrolysis occurred. [It is not clear why certain tests were placed in

certain categories (e.g., acid-fast staining for biochemical characteristics?) No lab manual reference(s) are given.

This needs to be provided in case someone wanted to repeat a specific test and needed to know how to do it or the

materials required. Where all the procedures done as exactly outlined in the lab manual? A better example in this

section would be… “The cell wall composition of the EI was examined by a Gram stain as well as an acid-fast stain

(ref #). The EI was also examined for its capacity to produce endospores by the procedure outlined by Leboffe and

Pierce (ref #) except that the slide was stain with Malachite green under steam for 2 instead of 5 minutes”]. In

general this section needs to be better organized.]

RESULTS:

Table 1 – Morphological Data of Environmental Isolate

Characteristic Observation

Cell morphology Rod-shaped (bacillus)

Gram stain +

Capsule producer +

Spore producer -

Colony color Orange, shiny

Colony texture Moist

Colony margin Smooth, entire

Colony elevation Convex

Colony shape Circular

The environmental isolate was a rod-shaped, encapsulated, gram-positive bacterium. Its colony

morphology included an orange pigment, moist texture, smooth margin, convex elevation, and

circular shape. [Must to refer to the Table you are discussing in the text (e.g., “As shown in Table 1…”).

Specifically how could you tell the EI had a capsule or was not an endospore-former? (e.g., bacteria appeared to be

surrounded by a clear halo after capsule staining). Be a little bit more specific in these observations.]

Table 2 – Physiological Data of Environmental Isolate

Characteristic Observation

Catalase production +

Oxidase production -

Motility -

Aerotolerance Obligate aerobe (what tests determined this?)

Temperature range 22ºC – 37ºC (specify this as optimal growth at)

pH range 6 – 10 (specify this as optimal growth at)

The results of the physiological data are shown above (Table 2). The environmental isolate

produced catalase, but not the cytochrome c oxidase [Consider these more biochemical than physiological

tests]. It did not have motility and was an obligate aerobe [Specifically how could you tell?]. Its

optimal growth temperature was between 22ºC and 37ºC and optimal pH was between 6 and 10. [Based on this data what would you classify the EI as… thermophile? mesophile? acidophile? neutrophile?]

Table 3 – Biochemical Data of Environmental Isolate

Characteristic Observation

Acid-fast (considered morphological test) -

Antibiotic data Susceptible to chloramphenicol, penicillin,

streptomycin, tetracycline

Beta-lactamase data No beta-lactamase production (susceptible to

penicillin)

Oxidation-fermentation results No sugar metabolism (what sugar? Indicate as “-“)

Phenol red results [separate each test based on sugar

with results + or – and G (gas) or NG (no gas)] No fermentation in mannitose and sucrose;

fermentation with acid end products, no gas

produced in glucose and lactose

MR-VP results No mixed acid fermentation; no 2,3-butanediol

fermentation (acetoin not produced)

Nitrate reduction results Does not reduce nitrate

Decarboxylation results [which amino acids?] Decarboxylation; produces specific

decarboxylase enzyme [which ones]

Starch hydrolysis results No amylase present; does not hydrolyze starch

Urease test results No urea hydrolysis; urease absent

The results for the biochemical data are shown above (Table 3). The environmental isolate was

susceptible to at least four different antibiotics and did not produce beta-lactamase, but did

produce the decarboxylase enzyme. It also did not easily metabolize or ferment sugars, but did

ferment acid end products. It did not reduce nitrate. Also, it did not hydrolyze starch or urea. [Not all the biochemical tests that were done are given in the Table (e.g., indole, gelatin hydrolysis, growth on

specific selective media etc…). All test results must be provided and if a test was not performed it must be indicated

and a reason why it was not done provided. Results for many of the biochemical tests can be presented as “+” or “-

“, similar to the way it is done in the Bergey’s Manual. REMEMBER ORGANIZATION IS THE KEY IN THIS

SECTION!]

CONCLUSIONS:

The results in the morphological (Table 1), physiological (Table 2), and biochemical

(Table 3) data tables show many important characteristics that are needed in order to narrow

down a search for the identification of a bacterium. The first step after obtaining pure colonies

(colonies were orange colored) was to conduct a Gram’s Stain. This staining procedure quickly

separated the possibilities of the unknown culture into two categories: Gram positive and Gram

negative [based on what?]. This environmental isolate was Gram positive, rod-shaped bacteria.

Afterwards, other staining procedures and tests to identify other important characteristics proved

that the isolate was not a spore producer, but a capsule producer. The bacterium was an obligate

aerobe meaning that oxygen was required in its existence, and it grew best at temperatures of

22ºC to 37ºC and at pHs of 6 to 10. It did not produce the beta-lactamase enzyme meaning that

it was susceptible to penicillin and some other antibiotics. It did not ferment any sugars, but was

able to ferment acids. It was unable to hydrolyze starch and urea, unable to reduce nitrate,

nonmotile, unable to produce oxidase, but was able to produce catalase.

Therefore, through these tests, the environmental isolate was able to be identified to the

genus Soonercoccus. “Soonercoccus is a non-motile, non-spore forming, non acid-fast, Gram-

positive bacillus that is capable of growing in a broad pH range (5.5 – 9.5), with an optimum pH

of 7.0” (www.campusbacteriausa.org) [Direct quotes should not be used and the referencing is incorrect].

Thus, at this point, the hypothesis [Was there a hypothesis and if so what was it? Remember based on the

nature of the work certain types of reports may not have a hypothesis] is accepted because the given results

are quite similar. These results are further supported in The Big Book of Bacteriology in

addition that it states that Soonercoccus “colonies may show a yellow-orange pigmentation, there

is little or no acid produced from glucose or other carbohydrates, and it is catalase positive”

(Holt, 574) [Direct quotes should not be used and the referencing is incorrect]. It is also additionally

explained that Soonercoccus is “widely distributed in soil” (Holt, 574) [Direct quotes should not be

used and the referencing is incorrect]. For that reason, it makes sense that this was the proposed genus

for the bacterium because this bacterial sample was taken from a wooden bus stop bench campus

likely contaminated with soil. [This is the section where you must CONVINCE the reader of the presumptive

identification of your EI and JUSTIFY your choice. Walk the reader through the LOGIC of how you decided on

your EI being a member of the genus Soonercoccus. Did every test suggest it to be a Soonercoccus? Did you put

more “weight” on some tests rather than others and why (e.g., bubbles in a catalase test were easy to see so felt

confident that the organism was catalase positive but in the phenol red broth sucrose test, the media turned an orange

color rather than yellow (an obvious positive result) or remained red (an obvious negative result). Where you

debating between Soonercoccus and another genus but one test you felt very confident in made you favor the genus

Soonercoccus? Maybe you think it is Soonercoccus but there is another test that would confirm your identification

that you might like to do (e.g., all members of the Soonercoccus genus are lipase positive so performing a lipid

hydrolysis test would help confirm the identity of the EI). REMEMBER THE GOAL IN THIS SECTION IS TO

CONVINCE THE READER THAT YOU HAVE MADE A LOGICAL PRESUMPTIVE IDENTIFICATION

AND YOU NEED DATA AND REFERENCES TO BACK THAT UP!

LITERATURE CITED:

1. 2007. “Soonercoccus Organism Details.” From the website campusbacteriausa.org.

2. Holt, John G. 1998. The Big Book of Bacteriology. 8th Edition. Almost Academic

Press page 574

3. Leboffe, M. J. and B. E. Pierce. 2010. Microbiology Laboratory Theory and

Application. Third Edition. Morton Publishing Company, Englewood, Colorado.

The reference format is not correct. Please see the references section in the American Society of Microbiology

(ASM) Instruction to Authors Guidelines posted on D2L for how to properly reference your citations in both the

reference section and the text.