Unknown Bacterial Environmental Isolate Report

The identification of an unknown bacterial environmental isolate through a series of morphological, physiological and differential exercises. The purpose of the environmental isolate report is to learn what is necessary in order to take an unknown environmental isolate (EI) and identify it. This was achieved through a series of exercises that provided information on the morphological, physiological and biochemical traits of the EI which were then compiled and interpreted in order to make a presumptive identification of the EI. The EI sample was taken from a door handle to the George Lynn Cross Hall, which is touched by hundreds of students entering the building every day. The most important exercises performed include gram staining, which
showed gram-positive cocci clusters, catalase test, which was catalase positive, the aerotolerance tests which showed a facultative anaerobe, and the overall colony morphology features of white opaque circular colonies. These features taken together successfully narrow the unknown EI species down to the genus Staphylococcus spp (Bergey). Over the course of the semester, the unknown environmental isolate (EI) was examined, tested and evaluated to acquire a likely identification. The EI sample was taken from the outside door handle of George Lynn Cross Hall, which is a surface that many people touch throughout the day. Because of this, it is expected that one would find bacterial species that are commonly found living and growing on skin and hands. This could include bacterial species that are part of the normal skin fauna or even possibly pathogenic species. This sight was chosen to collect the EI from because bacteria tend to collect on frequently touched surfaces such as a door handle (Hurst 2). This sight is also interesting because commonly touched surfaces such as door handles play a role in indirect contamination (spread from person to person via a surface) (Hurst 2). Therefore many different bacterial species could be found at this site. Throughout the semester many different morphological, physiological and biochemical tests were performed and the results were evaluated. Evaluating the results obtained throughout the course of the semester allowed one to apply the knowledge gained of the procedure for microbial testing and identification and apply it to reach a probable identification of the unknown EI. The exercises completed throughout the semester were performed according to the procedure outlined in the Microbiology Laboratory Theory and Application, 3rd edition, Leboffe and Pierce. The supplemental exercises performed were carried out according to the procedure outlined in the D2L handouts Beta-Lactamase Test and Indole Test 2015, Meysick. All exercises performed are listed below in Table 1 along any modifications made to the procedure outline. Exercises were performed to examine the microbial growth patterns and characteristics possessed by the EI (Table 2). The overall colony morphology showed convex, round colonies with smooth, entire margins that were opaque white in color. The results of Ex 2-6, 2-7, and 2-8 show that the EI is a facultative anaerobe, meaning it can grow in the presence or absence of oxygen. Ex 2-9, 2-10, and 2-11 tested different aspects of environmental limitations and showed the optimal environmental conditions for the EI. These results revealed our EI to be a mesophilic, neutrophilic, osmotolerant organism. Ex 5-28 tested the motility of the EI which showed the EI to be a non-motile organism. Several different bacterial stains were performed on the EI which gave important information as to the different traits and characteristics possessed by the bacterial cells of the EI (Table 3).
One specifically important stain preformed was the Ex 3-7 Gram Stain, which showed the EI to be a gram-positive cocci that appeared in small clusters, in addition the endospore stain revealed this to be a non-endospore forming bacterial species. These results are important because they drastically narrow the possible bacterial species the EI could be. Many differential tests were performed on the EI, specifically differential tests that provide information on cellular respiration (Table 4). These differential exercises test the different biochemical processes that the EI can preform. Ex 5-2 oxidation-fermentation test showed the EI to be capable of oxidation and slow fermentation or fermentation only. Ex 5-21 Triple Sugar Iron Agar test showed the EI can ferment glucose and sucrose and/or lactose but is not a sulfur reducer. Ex 5-4 showed the EI is able to preform 2,3 butanediol fermentation and produce acetoin. Many differential tests that tested the enzymatic properties and activity of the EI (Table 5). The results show the EI to be a catalase positive organism. The EI is also coagulase negative. The Beta-Lactamase test showed the EI is
beta-lactamase. Table 6 shows the tests of antimicrobial susceptibility preformed on the EI and the susceptibility or resistance to the antibiotics tested. Based on the results obtained and analyzed, the environmental isolate that was tested is presumptively of the genus Staphylococcus (Bergey). The exercises which gave the most critical information to aid in this identification include Ex 3-7 Gram Stain, Ex 5-5 Catalase Test, Ex 3-10 Endospore Stain, Ex 5-28 Motility Test, Ex 2-2 Colony Morphology, Ex 2-6 Agar Deep Stabs, Ex 2-7 Fluid Thioglycollate Medium, and Ex 2-8 Anaerobic Jar, Ex 2-9 The Effect of Temperature on Microbial Growth, Ex 2-10 The Effect of pH on Microbial Growth, and Ex 2-11 The Effect of Osmotic Pressure on Microbial Growth.

To begin with, Ex 3-7 Gram Stain results produced a gram positive cocci that appeared in small clusters.
This result was imperative for the determination of the specific species since it is an easy way to begin the task of narrowing down the possible species.Ex 5-5 Catalase Test showed catalase positive results. This is a key characteristic of the Staphylococcus species (Bergey). Ex 2-2 Colony Morphology showed circular white opaque colonies with smooth, entire margins when grown on TSA. This is key in helping to determine whether the EI was a Staphylococcus spp or a Mircroccus spp since Mircroccus spp. commonly show yellow color colonies when grown on TSA and Staphylococcus spp are commonly opaque white colonies when grown on TSA (Bergey). Additionally, Ex 3-10 Endospore Stain showed the EI to be non-endospore forming, which corresponds to the characteristics listed in Bergey s Manual of Determinative Bacteriology under the species Staphylococcus (Bergey 464). Ex 5-28 Motility Test showed the EI to be non-motile, which is also a key characteristic of Staphylococcus species
(Bergey). Furthermore, Ex 2-6 Agar Deep Stabs, Ex 2-7 Fluid Thioglycollate Medium, and Ex 2-8 Anaerobic Jar all produced results which indicate the EI is a facultative anaerobe. These results go along with the characteristics listed in Bergey s Manual of Determinative Bacteriology under the species Staphylococcus which states this species is facultative with respect to oxygen requirement, growing very well anaerobically in the presence of a fermentable carbohydrate but growing even better aerobically (Bergey 464). Ex 2-9 The Effect of Temperature on Microbial Growth, Ex 2-10 The Effect of pH on Microbial Growth, and Ex 2-11 The Effect of Osmotic Pressure on Microbial Growth produced results which indicate respectively that the EI is a mesopile, neutrophile, and osmotolerant. These results correspond to the species Staphylococcus which is commonly found on the skin of humans (Bergey 464). These results also correspond to the location from which the EI was isolated, a door handle to a building on OU campus which is frequently touched. All of these results, when gathered together and interpreted via Bergey s Manual of Determinative Bacteriology lead to the final conclusion that the EI can be identified as genus Staphylococcus. It makes sense that this organism was isolated from the environmental site of the GLCH door handle because many of the Staphylococcus species is commonly found on the skin as part of the normal skin fauna and can could have easily been transferred there when a student used the handle to enter the building.