Abstract
A sample was swabbed from a bike stand in front of George Lynn Cross Hall at the University of Oklahoma and environmentally streaked onto a tryptic soy agar plate. Following incubation at 37C, a distinct colony type was isolated from the TSA plates and sub-cultured to obtain a pure culture. Analysis of the isolate’s morphological characteristics showed that it is a Gram positive, rod-shaped species capable of producing endospores and an extracellular capsule. On the basis of these morphological characteristics, the isolate is similar to members within the genus Bacillus. Further examination of the bacterium through aerotolerance tests (agar deep stab, thioglycollate medium) indicated that it is an obligate aerobe. It grew optimally at 25C and 37C, which is within the optimal temperature range for most Bacillus species, 25C to 40C. In addition, the isolate can perform oxidation but not fermentation, a common characteristic among many Bacillus species.
Introduction
Microbial life can be found almost everywhere: in the air, in foods, on the surfaces inanimate objects, as well as on the human body. Their ability to survive in extreme environments contributes to their ubiquitous nature, and there are very few places in which microbial life has not been found. Microorganisms can live in the hot temperatures of deep-sea hydrothermal vents and in cold temperatures. They can survive in very salty environments such as the Dead Sea, and in very acidic and alkaline environments, such as acid mine drainage and alkaline soils (1).
In addition to demonstrating the ubiquitous nature of bacteria, the goal of this project was to isolate and identify a bacterial species from the environment. Identification and characterization of bacteria is important due to its application in many fields, such as public health, clinical diagnosis, and food safety monitoring (2). Throughout the process of testing and identifying the environmental isolate, important skills for microbiology laboratory were learned and applied, such as aseptic techniques and the proper handling and maintaining of a bacterial culture. Aseptic techniques were necessary in order to maintain a pure bacterial culture because improper aseptic techniques could lead to contamination of the culture. Any contamination would produce unreliable results as the results could have come from any of the organisms. Likewise, maintaining a bacterial culture by sub-culturing was critical identifying the isolate because performing tests on older bacterial cultures can give inconsistent results.
In the laboratory, bacterial species may be identified by their morphological traits and via physiological and biochemical tests. Microorganisms have a wide variety of morphologies, and they also exhibit biochemical diversity in their metabolism and cell structure (2). Thus, using phenotypic techniques to examine a bacterium’s morphological attributes and metabolic activities can provide a useful cue in its identification. Simple tests can be performed to indicate the Gram reaction of a bacterium, colony characteristics, motility, and the presence of spores and capsules, etc. Growth assessments also provide a cue for the identification of a bacterium: how the bacterium grows different oxygen levels, incubation temperatures and pH levels. Furthermore, many bacteria are identified based on their reactions in a series of biochemical tests, such as oxidase, nutrient reduction, fermentation, or utilization of different carbohydrates (2).
Besides the traditional biochemical and physiological methods of identifying bacteria, recent advances in technology provide more cost-effective, rapid alternatives that produce multidimensional data about bacteria. Many of these methods are based on genotypic techniques that are based on profiling an organism’s genetic material. For example, molecular biologic techniques allow bacterial species and strains to be identified by identifying a specific gene or genetic sequence (2). Other modern methods include identification by fatty acid profiling, identification by protein profiles, and genetic fingerprints (DNA assays) (3). The phylogenetic information of the bacterium can then be compared to a database of known organisms to reveal genetic relatedness (2). In some cases of bacterial identification, certain characteristics or test results may be weighted more heavily than others. A given level of similarity in characteristics equates with relatedness. Different strains of the same species may have 90% similarity, and species within the same genus may have 70% similarity. In this case, it should be determined which test results should be chosen and how the tests should be weighted (2).
Besides phenotypic and genotypic examinations, the environment from which a bacterium was isolated can provide cues into its identification. In addition to demonstrating the ubiquitous nature of bacteria, the goal of this project was to isolate and identify a bacterial species from the environment. The environmental sample was swapped from a metal bike stand in front of the George Lynn Cross Hall. Because the bike stand is in a grassy area and surrounded by trees, it is possible that it could have been directly or indirectly contaminated by soil. Many Bacillus species have been isolated from soil or other environments that may have been contaminated with soil (4). Thus, it is possible that the isolate is a member within the Bacillus genus.
Materials and Methods
The environmental isolate sample was swabbed from a bike stand and inoculated onto a tryptic soy agar (TSA) plate. Because environmental samples do not have a high cell density, the EI sample was inoculated using an environmental streak. The plate was incubated at 37°C for approximately 48 hours, following which it was examined for distinct bacterial colonies. The plate contained mostly mold after incubation with the exception of one isolated bacterial colony. The colony was then sub-cultured onto a separate TSA plate in order to obtain a pure culture. Once a pure culture was obtained through subsequently subcultures, the colony morphology was observed. The pure culture was subsequently sub-cultured every week in order in maintain a fresh stock for characterization tests. The characterization tests were performed as per the exercises in Microbiology Laboratory Theory & Application (5), with modifications as shown in Table 1, Table 2, and Table 3.
As listed in Table 1, the first set of tests performed on the isolate were stains, which were done to observe the isolate’s morphological characteristics. The simple and negative stains allowed for the observation of the shape and arrangement of the isolate. The Gram stain, and the Kinyoun acid-fast stain were performed in order to determine the cell wall composition of the isolate. In the capsule stain, the isolate was tested to determine its ability to produce an extracellular capsule. Finally, the EI’s ability to produce spores under poor nutrient conditions was tested according in the endospore stain.
To examine the growth characteristics of the isolate under different environmental conditions, physiological tests were performed as shown in Table 2. The effects of temperature, pH levels, and osmotic pressures on growth were examined in the same medium, tryptic soy broth. For Ex 2-9 (5), the cardinal temperatures at which the EI can grow were determined by inoculating the organism in tryptic soy broth (TSB) tubes and incubating them at temperatures ranging from 4ºC to 55ºC. Similarly, the EI was tested for growth at different pH levels as per the procedure in Ex 2-10 (5). The isolate’s osmotolerance was determined by inoculating the EI in TSB tubes varying NaCl concentrations as per the procedure in Ex 2-11 (5). In addition to examining the isolate’s growth patterns in different environments, the motility of the isolate was also tested as outlined in Ex 5-28 (5).
As shown in Table 3, biochemical tests were performed in order to make a presumptive identification of the EI based on various characteristics: energy metabolism, utilization of different medium components, decarboxylation and deamination of amino acids, hydrolytic reactions, and susceptibility to different antibiotics. Aerotolerance of the EI was determined with agar deep stabs, fluid thioglycollate medium, and anaerobic jar, as outlined in Ex 2-6, Ex 2-7, and Ex 2-8 (5), respectively. The oxidative and/or fermentative pathways of the EI were examined by the Oxidation Fermentation Test as outlined in Ex 5-2 (5). Further examination of the ability of the EI to ferment different sugars (lactose, sucrose, and glucose) was done with the Phenol Red Broth test. The Methyl Red and Vogues-Proskauer Tests were performed to examine the isolate’s capability of performing mixed-acid fermentation and 2,3-butanediol fermentation.
The ability of the EI to respire was examined through the procedures for catalase, oxidase, nitrate reduction, and citrate tests outlined in Leboffe and Pierce. In the catalase test, the procedure was performed to examine the EI’s ability to produce the enzyme catalase. Similarly, the oxidase test and the nitrate reduction tests were performed to test for the enzymes cytochrome c oxidase and nitrate reductase, respectively. In the citrate test, the EI was examined for its ability to utilize citrate as a single carbon source.
The following tests were performed in order to detect enzymes required for hydrolysis reactions: indole (6) starch hydrolysis, urea hydrolysis, casein hydrolysis, gelatin hydrolysis, DNA hydrolysis, and PYR tests. Decarboxylation tests were to examine the EI’s ability to produce lysine decarboxylase, ornithine decarboxylase, and arginine decarboxylase. The coagulase tube test was used to detect the presence of free or bound coagulase produced by the EI. The EI was also examined for its ability to hydrolyze red blood cells in Ex 5-25 (5). Finally, the susceptibility of the EI to different antimicrobial drugs was examined through the Beta-Lactamase Test (7) and the Antimicrobial Susceptibility Test (Kirby-Bauer Method (5).
Results
Table 4: Results of Morphological Tests
Characteristic
Observation
Colony morphology
Opaque, lobate margin, flat, round, moist
Cell shape and arrangement
rods arranged in chains
Gram Stain
+
Acid-Fast Stain
Capsule Stain
+
Endospore Stain
+
+ indicates a positive test
indicates a negative test
Colonies grown on tryptic soy broth at 37C are opaque, flat, round, moist, with a lobate margin, as shown in Table 4. Observation of the isolate using simple and negative stains revealed that the Environmental Isolate is a rod-shaped bacterium grouped in chains, and Gram staining revealed a Gram positive organism. In the acid-fast stain, the bacterium appeared green because it could not retain the carbolfuschin stain after decolorization, which indicates that it does not possess a waxy cell wall. Nevertheless, the EI is a species capable of forming endospores due to the presence of green spheres in the endospore stain. Similarly, the presence of a transparent halo in the capsule stain indicates that the EI possesses extracellular capsules.
The results of the physiological tests, as shown in Table 5, shows that the EI is a nonmotile, obligate aerobe that grows well under the following conditions: 25ºC and 37ºC, pH 6, and 2% NaCl. Colonies grew well under aerobic conditions, while growth was inhibited under anaerobic conditions. In the agar deep stab test, the bacterium grew only at the top of the stab line, where the oxygen concentration is the highest. Similarly, there was only growth observed near the top of the fluid thioglycollate medium broth, indicating that it cannot grow in the absence of oxygen. In addition, the EI grew only in the aerobic plate in the anaerobic jar test. Based on the growth patterns under different environmental conditions, the isolate is classified as a mesophilic and neutrophilic species that can survive only under low salt concentrations.
Table 6 shows the results of the biochemical tests. The isolate can obtain its energy by means of aerobic respiration but not fermentation. In the Oxidation-Fermentation test, a yellow color change was produced only under both aerobic conditions, indicating that the EI can oxidize glucose to produce acidic products. In addition to glucose, the EI can also utilize lactose and sucrose, and this conclusion is based on the fact that the color of the phenol red broth changed to yellow in all three tests. Although the EI does perform fermentation of these three carbohydrates, it appears that this bacterium cannot perform mixed acid fermentation nor 2,3-butanediol fermentation due to the lack of color change in Methyl Red and Vogues-Proskauer tests.
It appears that the isolate lacks some respiration enzymes, specifically catalase, oxidase, and nitrate reductase. The addition of hydrogen peroxide to the bacterial smear in the catalase test did not result in bubble production, which shows that the isolate is catalase negative. In the oxidase test, there was no cytochrome c oxidase present to reduce the chromogenic reducing agent tetramethyl-p-phenylenediamine, as indicated by the DrySlide window remaining a white color. In the nitrate reduction test, it was concluded that nitrate was still present in the broth when the media became red upon the addition of zinc powder. While the isolate cannot reduce nitrate, it can utilize citrate as a single carbon source, as indicated by the color change to blue in the citrate test.
The results of the hydrolysis tests show that the isolate possesses some enzymes required for hydrolytic reactions. Extracellular enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been cleaved. In the blood agar test, the lack of change in the test medium around the colonies showed that this bacterium does not have a hemolytic effect on red blood cells. In addition, the isolate does not possess the enzyme coagulase.
The last set of biochemical tests were tests for antimicrobial sensitivity. From the beta-lactamase test, it appears that the EI does not possess a beta-lactamase because the bacterial smear on the nitrocefin slide was yellow. These results are further supported by the Kirby-Bauer test, which showed that the EI is susceptible to penicillin, a beta-lactam antibiotic, in addition to streptomycin, ciprofloxacin, and trimethoprim.
Discussion/Conclusions
Bacteria can be identified in the laboratory through their morphological and physiological traits and biochemical assessments. Many bacteria are identified and classified based on their phenotypic characteristics, growth characteristics, and their reactions in various biochemical tests. In this exercise, the initial tests performed on the isolate were the stains listed in Table 1. Results showed that the isolate is a Gram positive, rod-shaped species with the ability to produce an extracellular capsule and endospores. These characteristics suggest that the isolate could be a Bacillus or a Clostridium species because species belong to these two genera are usually Gram positive, endospore-forming bacilli (4).
Following the initial stain tests, physiological tests and biochemical were performed to produce a broader identification profile of the isolate. In the aerotolerance tests, it was determined that the isolate is an obligate aerobe. Because Clostridium and Bacillus species differ in their aerotolerance, the aerotolerance of the isolate is a key factor in concluding which genus the isolate belongs to. While the morphological results may have suggested that the isolate could belong to the genus Clostridium, most Clostridium species are obligately anaerobic (8). Most Bacillus species, however, are aerobes or facultative anaerobes (4). Hence, the isolate appears to belong to the genus Bacillus and not Clostridium. In addition to similarities in oxygen requirement, the optimal growth temperature of the isolate is also similar to those of members within the Bacillus genus. The majority of established Bacillus species are mesophiles, with optimal temperatures between 25C and 40C. Analysis of the isolate’s growth patterns under different temperatures revealed that the optima were 25C and 37ºC, which are included within this range.
From the results of the biochemical tests, it appears that the carbohydrate catabolism of the isolate shows similarities to those of Bacillus species. Many members within the Bacillus genus produce small amounts of acid via oxidation rather than fermentation of carbohydrates, which corresponds with the results of the Oxidation-Fermentation test. Acidic products were produced only in the presence oxygen, which indicates that this bacterial isolate is capable of glucose oxidation but not fermentation. With regards to other biochemical tests, previously identified Bacillus species exhibit varying characteristics. For example, catalase is produced by most species, but some species have shown different reactions depending on the strain. There are also variations between different species in hydrolysis, citrate utilization, nitrate reduction, and Voges-Proskauer tests (4).
Despite the wide variety of physiological and biochemical abilities that Bacillus species exhibit, the morphological characteristics, optimal temperature requirements, and carbohydrate utilization of the isolate allows for the presumptive identification of it as a member within the Bacillus genus. In addition, many Bacillus species have been isolated from soil (4), which concurs with the location from which the isolate was isolated.