Environmental Isolate

Abstract
In an environment isolation procedure, experiments under categories, such as, morphology, physiology, antibacterial susceptibility, selective media, and biochemical provide results. Both the unknown isolate and members of the Micrococcus genus were shown to be obligate aerobes. By using staining methods, this proved that the organism is gram positive. Morphology, such as, orange pigmentation and coccus shape provide similarities to the Micrococcus genus. Physiological tests were shown to be obligate aerobe, mesophile, neutrophile, and osmotolerant organism. Biochemical reactions such as producing enzymes and fermentation are limited. Micrococcus bacteria are found primarily on mammalian skin and in soil but commonly are isolated from food products and air (Holt, 530). Micrococcus can generally be found anywhere that has been contacted by soil and air. Since the water fountain was outside and exposed to air, the unknown isolate can relate to the Micrococcus environmental characteristics. From this work we conclude that when working this specific unknown isolate it compares to members of the Micrococcus genus.

Introduction
Environment isolation of an organism involves identifying the morphology, types of media, physiological, and biochemical processes. First an organism is examined from the habitat it is taken from. This particular isolate was discovered from a “press” button located on a water fountain. The water fountain was located outside around cold temperatures. This organism possesses unusual abilities to tolerate and combine growth activities living on a metal surface (Jansson). When discovering the microorganism, it usually consists of observing the morphology of the organism based on physical features and gram staining. Different types of media are used to understand how the microorganism can grow. The aerotolerance, movement, and the way it reacts with the environment are determined by utilizing physiological tests. Biochemical reactions of the organism undergoes as a “thumbprint” for its identification (Kaiser). Since each species has its own set of DNA, certain protein enzymes catalyze all the various chemical reactions of which organism is capable to carry out different sets of biochemical reactions (Kaiser). The isolation of this organism will dictate how the type of media, incubation conditions, and metabolic processes the organism can survive in. The research being done to this isolate can give a better understanding what the organism is and how it works. The function of this organism can serve beneficial for developing antibiotics, using the organism to benefit the environment, understanding the importance it has with other organisms, using the organism to treat diseases, and so much more. Based on this information, we hypothesize that the results can identify the organism based on valid references. Therefore, the objective of this study was to identify an organism by its genus species name.
Methods
Morphology
The environmental isolate is obtained and is first observed for its morphological features. The organism is grown on plates that contain the agar medium. According to the colony morphology experiment, the organism is observed by its shape, margin, elevation, texture, and pigment production (Leboffe, 36). The physical descriptions of the isolate are the first step to identifying the organism. To identify if the organism is gram positive or gram negative, a gram stain is performed. Crystal violet, iodine, acid alcohol, and carbolfusion are four chemicals used to distinguish between gram positive and gram negative cells (Leboffe, 108). An acid fast stain is performed for a specific type of species. Nocardia and Mycobacterium contain a waxy-lipid called mycolic acid which makes it difficult for dyes to penetrate the cell wall (Ex 3-8 on D2L, 4). Therefore, these organisms can produce a negative result for a gram stain. By using the kinyoun method, the bacterial sample is stained with carbolfusion, decolorized with acid alcohol, and then counterstained with brilliant green (Ex 3-8 on D2L, 14-17). The capsule stain is a differential stain used to detect cells capable of producing an extracellular capsule (Leboffe, 115). Along with the bacterial organism, Congo red and Maneval’s stain is used to increase the visualization of the halo around the cell. When nutrients are available in high concentrations some bacteria can accumulate and store these nutrients in inclusions (PHB exercise, 1). Black Sudan, citri-solv, and safranin staining are used to show visibility of inclusions if the organism is capable of storing nutrients. (PHB exercise, 15). If environmental conditions become extreme and unbearable to survive, some bacteria produce endospores to insure the survival of the species. Malachite green and safranin are stains used to identify cells that contain spores (Leboffe, 117-119).
Physiological
Bacteria are usually examined for their presence or absence of motility. Inoculating needle is stabbed into semi-solid which then dictates if there is movements present (Ex 5-28, 3). To determine if the organism can survive with or without oxygen is called aerotolerance (Leboffe, 48). Using agar deep stab, fluid thoiglycollate medium and an anerobic jar, will determine if the organism can survive in oxygen (Leboffe, Ex 2-6, 2-7, & 2-8). In exercise Agar deep stab, an inoculating needle is used to stab the agar (Leboffe, 49). Fluid thioglycollate is a medium that is well adapted of strict anaerobes and microaerophiles, but it is also used to confirm if the organism is aerobic or anaerobic by the location in the tube (Leboffe, 48). The GasPak Anaerobic System is a plastic jar in which to create anaerobic, microaerophilic, or CO2 enriched conditions (Leboffe, 50). All incubations were performed at 37oC for 48 hours. The temperature, pH, and osmotic pressure correlate to the growth of the organism. For temperature, the organism is placed in four tryptic soy broth tubes that are grown at 4oC, 24oC, 37oC, and 55oC for 48 hours. Each species is characterized by a minimum, maximum, and optimum temperature known as cardinal temperatures (Leboffe, 54). The organism is placed in five tryptic soy broth tubes at pH2, pH4, pH6, pH8, and pH10 to determine pH growth. Incubations were performed at 37oC for 48 hours. PH tolerance is then used as a means of classification (Leboffe, 56). For determining the osmotic pressure, five tubes will grow under 0%, 2%, 5%, 8%, and 11% salt concentration levels. Osmotic pressure is then characterized by the ability of the solution to pull water toward itself through a semipermeable membrane (Leboffe, 58). Incubations were performed at 37oC for 48 hours.
Selective Media
Different types of media are designed to enhance the isolation procedure by inhibiting or encouraging growth of some organisms (Leboffe, 129).When bacteria are grown in mannitol salt agar, sodium chloride uses its high concentration to dehydrate and kill most bacteria (Leboffe, 137). Staphylococcus aureus is identified if growth is present. MacConkey agar is used to isolate and differentiate members of the Enterobactericeae based on the ability to ferment lactose (Leboffe, 139). Eosin methylene blue agar is used to isolate fecal coliforms (Leboffe, 142). Organism is inoculated into medium and determined if it is gram negative and positive for coliform. Hektoen Enteric agar is used to isolate and distinguish between Salmonella and Shigella (Ex 4-7 on D2L, 27). The agar is inoculated and used to determine the growth rate. Blood agar is used to differentiate bacteria based on an organism’s beta, alpha, and gamma hemolytic characteristics (Ex 5-25 on D2L, 2). All incubations were performed at 37oC for 48 hours.
Antibacterial Susceptibility
Antibacterial susceptibility testing can be used to help identify an organism or simply select an appropriate antibacterial agent to be used to fight it (Leboffe, 214). The bacitracin, novobiocin, and optochin susceptibility tests are shown to test a bacterial sample for susceptibility or resistance from bacitracin. A beta-lacamase test is used on a dryslide to determine if the Beta-lactam ring of drug inactivation (Beta-Lactamase exercise on D2L, 11). For the antimicrobial susceptibility test, the antimicrobial drugs Penicillun, Ciprofloxin, Chloramphenicol, and Trimethoprim are used to test for susceptibility and resistance of bacterium. Bacteria can have different patterns of antimicrobial susceptibility depending on the antibiotic used (Ex 7-3 on D2L, 3). All incubations were performed at 37oC for 48 hours.
Biochemical
An organism can be identified by its chemical properties it possesses. To determine if the organism can ferment carbohydrates, several tests are performed to differentiate the bacteria. The oxidation-fermentation test is used to identify the bacteria that can oxidize or ferment sugars (Leboffe, 155). Glucose, lactose, and sucrose are added to phenol broth to determine fermentation. Phenol red broth is used to differentiate members of Enterobacteriaceae and to distinguish them from other gram-negative rods by inoculating each tube and determining growth rate (Leboffe, 158). Methyl red and Voges-Proskauer is a broth medium that tests what organisms can ferment mixed acids and ferment glucose (Leboffe, 161). There are tests that identify an organism as an aerobic or an anaerobic respirer generally are designed to detect specific products of (or constituent enzymes used in) the reduction of the final electron acceptor (Leboffe, 165). In the catalase test, organisms are identified for positive or negative catalase enzyme. A slide test is used instead of slant test. After the addition of hydrogen peroxide, bubbles present indicate a catalase enzyme (Leboffe 166). Oxidase test is used to identify organisms with the enzyme cytochrome c oxidase. Organism is transferred onto a dryslide and is determined for color change indicating that there is a presence of cytochrome c oxidase enzyme (Leboffe, 170). The nitrate test is used to identify which organism can or cannot reduce nitrate (Leboffe, 173). Nitrate broth is obtained, inoculated, and incubated to determine presence of gas. Utilization media are highly defined formulations designed to differentiate organisms based on their ability to grow when an essential nutrient is strictly limited (Leboffe, 175). The citrate test is used to identify which organism uses citrate as its carbon source (Leboffe, 175). After inoculation and incubation, change of color in the slant tubes will dictate the results. Decarboxylation media is used to identify Enterobacteriacae by using lysine, ornithine, and arginine broths (Leboffe, 179). Tubes are inoculated and sterile mineral oil is placed on the top of tubes that are meant for anaerobic growth. The indole test determines whether an organism has tryptophanase based on the presence of indole in the culture media after bacterial growth (Indole Exercise, 8). This exercise is performed to identify if the organism has the tryptophanase enzyme. Triple Sugar Iron Agar is a rich medium designed to differentiate bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction (Leboffe, 206). After inoculation and incubation, color changes and gas production are analyzed. The purpose of the coagulase test is to determine if the coagulase enzyme is present in S. aureus and to differentiate from other organisms (Leboffe, 222). Only the tube test was performed. Tubes are inoculated and incubated then viewed for presence of gas. Reactions that use water to split complex molecules are called hydrolysis reactions (Leboffe, 184). Starch hydrolysis test is used to identify organisms that contain alpha-amylase enzyme which can break down starch (Leboffe, 184). Some organisms produce the enzyme urease which hydrolyzes urea (Leboffe, 187). The urea hydrolysis test will only be tested for 24 hour period. Broth will be tested instead of slants. After inoculation and incubation, color is observed. Casein hydrolysis test is used to identify organisms that produce the enzyme casease which can hydrolyze milk (Leboffe, 190). Gelatin hydrolysis test is used to identify organisms that produce gelatinases enzyme which can hydrolyze gelatin (Leboffe, 192). If gelatin is liquid after inoculation and incubation, the organism is determined if gelatinase enzyme present. There are organisms that produce the deoxyribonuclease (DNase) enzyme which can hydrolyze DNA. The DNA hydrolysis test is used to differentiate between different species (Leboffe, 194). After plate is inoculated and incubated, clearing is observed to confirm presence of DNase enzyme. All samples were incubated at 37oC for 48 hours.

Results
Table 1: Morphology characteristics of the unknown organism Morphology | Gram Stain | Acid-fast stain | Capsule stain | PHB inclusion body stain | Endospore stain | Orange/smooth and circular in shape. Slightly raised and entire | + | + | + | ND | ND |
(+) = Positive, (-) = Negative, (ND) = Not Determined
According to table 1, morphology is used to describe the physical characteristics of the microorganism. The pigmentation of the organism was a bright orange. It had a raised elevation and circular in form. The margins are entire. With the organism being gram positive, this indicates that there is a thick layer of peptidoglycan (Ex 3-7 on D2L, 3). The acid-fast stain had a slight positive result. This indicates that the cell wall is surrounded by wax lipids. For the positive capsule stain, the organism contains a capsule that protects, adhere, and prevent phagocytosis (Ex 3-9 on D2L, 2). PHB inclusion test was unable to be performed due to lack of time. The endospore stain results were skewed by the fact that there were not enough samples visible.
Table 2: Physiological characteristics of unknown organism Motility | pH growth | Temperature growth | Osmotic growth | Agar deep stab | Fluid Thioglycollate | Anaerobic Jar | ND | 6-10 pH | 24-37oC | 0-5% | Obligate aerobe | Obligate aerobe | Obligate aerobe |
(ND)= Not Determined
Motility results were not determined due to lack of time and materials. According to table 2, pH growth is in the range of 6-10pH, temperature is in the range of 24-37oC, and osmotic growth of NaCl is in the range of 0-5%. Since the unknown microorganism has a growth of pH level 6-10, this indicates that it is a neutrophile. Neutrophiles grow well at a neutral pH (Ex 2-10 on D2L, 16). The temperature range of 24-37oC indicates that the organism is a mesophile. Mesophiles optimal temperature for growth is approximately normal human body temperature (Ex 2-9 on D2L, 7). With an osmotic pressure between 0-5%, this indicates that this is a possible osmotolerant bacterium which will grow over a wide range of salinities (Leboffe, 58). According to table 2, experiments Agar deep stab, Fluid Thioglycollate, and Anaerobic jar result in a strict aerobe. The organism can only grow in conditions with oxygen present.

Table 3: Selective media characteristics of the unknown organism Mannitol Salts Agar | MacConkey Agar | EMB Agar | Hektoen Enteric Agar | Blood Agar | Inhibited by high salt concentration/not a staphylococcus organism | No growth/gram positive/inhibited by crystal violet and bile salts | No growth/gram positive/inhibited by Eosin Y and methylene blue dye | No growth/gram positive/inhibited by bile salts | Beta-hemolysis |

According to table 3, this organism is highly inhibited by high salt concentrations. Since most of these mediums contain higher salt, it is difficult for the bacteria to survive. MacConkey Agar, EMB Agar, and Hektoen Enteric Agar prove that gram positive bacteria are inhibited by bile salts. MacConkey Agar and EMB Agar are selective for gram-negative organism and both contain indicators to differentiate lactose fermenters and nonfermenters (Leboffe, 139). Mannitol provides the substrate for fermentation and makes the medium differential (Leboffe, 137). Accordingly, this organism does not ferment mannitol. Blood agar is used to detect hemolytic ability of gram-positive cocci species (Leboffe, 217). The organism mostly represented a beta-hemolysis clearing by indication that plate had very large clearing around the organism.
Table 4: Antibacterial Susceptibility characteristics of the unknown organism Characteristics | Bacitracin Test | Beta-Lactamase Test | Antimicrobial Susceptibility Test | | Bacitracin Sensitive | ND | Penicillin, Ciprofloxin, Chloramphenicol, and Trimethoprim sensitive |
(ND)= Not Determined
According to table 4, this microorganism is bacitracin, penicillin, ciprofloxin, chloramphenicol, and trimethoprim sensitive. The disc-diffusion method is used as an antibacterial susceptibility test (Leboffe 214). This bacterial organism is highly susceptible to many antibacterial agents.
Table 5: Biochemical characteristics of the unknown organism Characteristics | Oxidation-Fermentation Test | Phenol Red Broth Test | Methyl Red & Voges Proskauer Tests | | No sugar metabolism | Cannot ferment glucose, lactose, and sucrose. Makes alkaline products | Unable to produce mixed acid fermentation and acetoin |

Fermentation converts carbohydrates to pyruvate but uses it to produces one or more acids (Leboffe, 155). According to table 5, this organism cannot undergo fermentation.
Table 6: Biochemical characteristics of the unknown organism continued… Characteristics | Catalase Test | Oxidase Test | Nitrate Reduction Test | Citrate Test | | - | - | - | - |
(-) = negative
The catalase test is used to identify organisms that produce the enzyme catalase which is most commonly to the differentiate members of the catalase-positive Micrococcaceae (Leboffe, 166). Result may be incorrect by not having the organism lively present. According to table 6, catalase, oxidase, nitrate-reduction, and citrate tests all showed a negative result of enzyme production.
Table 7: Biochemical characteristics of the unknown organism continued… Characteristics | Decarboxylation Test | Indole Test | Triple Sugar Iron Agar Test | Coagulase Test | | + | (-) for tryptophase enzyme | No fermentation/peptone catabolized aerobically with alkaline products | - |
(+) = Positive, (-) = Negative
Table 8: Biochemical characteristics of the unknown organism continued… Characteristics | Starch Hydrolysis | Urea Hydrolysis | Casein Hydrolysis | Gelatin Hydrolysis | DNA Hydrolysis | | (-) for alpha-anaylase enzyme | (-) for urease enzyme | (-) for casease enzyme | (-) for gelatinase enzyme | (-) for DNase enzyme |

Other than the decarboxylation test, table 7 and 8 produce negative results. Decarboxylation tests are designed to differentiate members of gram-negative rods (Leboffe, 179). The results most likely are incorrect because the microorganism is gram-positive cocci. All other tests in table 7 and 8 prove negative production of enzymes.
Conclusions
Both the unknown isolate and members of the Micrococcus genus were shown to be obligate aerobes. Micrococcus bacteria contain cells that are spherical and irregular clusters (Holt, 530). Micrococci are strict aerobes and are gram positive bacteria (Holt, 530). Both unknown isolate and micrococcus genus produce little or no acid from carbohydrates (Holt, 530). Since the unknown isolate can grow in osmotic pressures between 0-5%, this can compare to micrococci bacteria that are halotolerant which grow at 5% NaCl (Holt, 530). By performing selective media tests, the unknown isolate can only grow in simple media conditions. For a Micrococcus bacterium, they usually grow on simple media (Holt, 530). The unknown isolate performed negative tests for catalase and cytochromes. The Micrococcus bacteria are positive for both tests (Holt, 530). There could be a possibility of undetermined problems that occurred during the experiment that caused negative results. The unknown isolate was discovered on a cold winter day and found on a water fountain. Micrococcus bacteria are found primarily on mammalian skin and in soil but commonly are isolated from food products and air (Holt, 530). Micrococcus bacteria can generally be found anywhere that has been contacted by soil and air. Since the water fountain was outside and exposed to air, the unknown isolate can relate to the Micrococcus environmental characteristics. According to tests run on a water fountain, Micrococcus bacteria provided resistance to cold temperatures and metal contact (Geldreich, 109-110). Micrococcus bacteria can possess unusual abilities to tolerate and to use very toxic organic molecules as carbon sources, and combines these activities with tolerance to metals (Jansson). This can compare to the unknown isolate that was discovered under these conditions. Since Micrococcus bacteria are nonsporing, the bacteria can still survive in lower temperatures by undergoing a type of dormant phase. (Holt, 530). Acid production from sucrose, lactose, mannitol, and some other carbohydrates originally reported was not observed in any strains studied under Micrococcus bacteria (Kocur, 291). In comparison, this applies to the unknown isolate when experiments were performed. Micrococcus bacteria are susceptible to penicillin, streptomycin, chlorophenicol, tetracycline, novobiocin, neomycin, and erythromycin (Kocur, 290). As performed in the antimicrobial susceptibility tests, the unknown isolate was susceptible to similar antibiotics compared to Micrococcus bacteria. By gathering all results from the performed experiments, there was enough information to identify the unknown organism. Therefore, the isolate can be presumptively identified as Micrococcus.

References 1. Geldreich E. E. 1996. Microbial quality of water supply in distribution systems. Lewis Publishers.109-111. http://books.google.com/books?id=wP7m7zE2IEC&pg=PA111&lpg=PA111&dq=micrococcus+on+water+fountain&source=bl&ots=hD6xEwwPE&sig=loKGJN76DsomnxNBrQsw78XD674&hl=en&ei=s4y7TfX9K5TBtge446zCBQ&sa=X&oi=book_result&ct=result&resnum=1&ved=0CBkQ6AEwAA#v=onepage&q&f=false 2. Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., and Williams S. T. 2000. Bergey’s Manual of Determinative Bacteriology. Lippincott Williams and Wilkins. 9: 528-537. 3. Jansson J. 2008. Micrococcus luteus. http://genome.jgi-psf.org/miclu/miclu.home.html 4. Kaiser G. E. 2010. Identification of bacteria through biochemical testing. http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/lab8.html 5. Kocur M., Schleifer K. H, Kloos W. E. 1975. Taxonomic Status of Micrococcus nishinomiyaensis. International Journal of Systematic Bacteriology. 25: 1-4. 6. http://ijs.sgmjournals.org/cgi/reprint/25/3/290.pdf. 7. Leboffe M. J. and Pierce B. E. 2010. Microbiology Laboratory Theory and Application. Morton Publishing. 3: 33-268. 8. Leboffe M. J. and Pierce B. E. 2010. Microbiology Laboratory Theory and Application on D2L. 3: Ex: 2-10, Ex: 2-6, Ex 2-7, Ex: 2-8, Ex: 2-9, Ex: 3-3, Ex: 3-7, Ex: 3-9, Supplemental Exercise PHB, Ex: 4-7, Ex: 5-25, Ex: 5-28, Ex: 7-3, Supplemental Exercise Beta-Lactamase.

References: 1. Geldreich E. E. 1996. Microbial quality of water supply in distribution systems. Lewis Publishers.109-111. http://books.google.com/books?id=wP7m7zE2IEC&pg=PA111&lpg=PA111&dq=micrococcus+on+water+fountain&source=bl&ots=hD6xEwwPE&sig=loKGJN76DsomnxNBrQsw78XD674&hl=en&ei=s4y7TfX9K5TBtge446zCBQ&sa=X&oi=book_result&ct=result&resnum=1&ved=0CBkQ6AEwAA#v=onepage&q&f=false 2. Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., and Williams S. T. 2000. Bergey’s Manual of Determinative Bacteriology. Lippincott Williams and Wilkins. 9: 528-537. 3. Jansson J. 2008. Micrococcus luteus. http://genome.jgi-psf.org/miclu/miclu.home.html 4. Kaiser G. E. 2010. Identification of bacteria through biochemical testing. http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/lab8.html 5. Kocur M., Schleifer K. H, Kloos W. E. 1975. Taxonomic Status of Micrococcus nishinomiyaensis. International Journal of Systematic Bacteriology. 25: 1-4. 6. http://ijs.sgmjournals.org/cgi/reprint/25/3/290.pdf. 7. Leboffe M. J. and Pierce B. E. 2010. Microbiology Laboratory Theory and Application. Morton Publishing. 3: 33-268. 8. Leboffe M. J. and Pierce B. E. 2010. Microbiology Laboratory Theory and Application on D2L. 3: Ex: 2-10, Ex: 2-6, Ex 2-7, Ex: 2-8, Ex: 2-9, Ex: 3-3, Ex: 3-7, Ex: 3-9, Supplemental Exercise PHB, Ex: 4-7, Ex: 5-25, Ex: 5-28, Ex: 7-3, Supplemental Exercise Beta-Lactamase.