Section I : Introduction and Significance of Identifying Unknown Bacteria
In the beginning of the fall semester of 2018, Microbiology instructor Julie Kelly assigned an unknown bacteria to each individual student. This was designed to be an educational experience as well as provide a myriad of examples of different methodologies that can be used to identify a bacteria in a real life setting. The process of identifying an unknown bacteria is regarded to be one of the most important clinical diagnostic tools in a medical setting, as well as an important step in solving environmental issues. The reason it is so important to identify a bacteria in a medical setting is so that you can prescribe the correct antibiotic to counteract the bacteria and lower the chance of over prescribing the antibiotic. In an environmental setting, it can be equally as important to test for bacteria to see if there is coliform contaminated water in the area or an overgrowth of microbes in shellfish for example.
Since all samples were pure with little chance of contamination due to strict aseptic technique, the unknown bacteria was stained and able to be examined under a microscope which helped narrow down both the individual bacterial shape but also whether the bacteria was Gram(-) or Gram(+), cellular arrangement, presence of endospores and presence of capsules. This step is important because it assists the initial narrowing down of bacteria, and can remove a large quantity of possibilities rather quickly. The reason this was reliable was because the aseptic technique used was near perfected by the time the class began testing their unknowns, and had a good understanding of why it is so important. Proper aseptic technique is also responsible for gathering a pure sample, and a mandatory step if you wish to do so.
Another method that was employed was the use of selective and differential media in tests, which would allow certain species of bacteria to grow while inhibiting others which was incredibly helpful. To help understand what a selective and differential media is, a good majority of the course was spent identifying different ingredients and properties of media that would cause certain environments to be selective and differential for bacterial growth. “A differential media contains specific ingredients or chemicals that help us to visually distinguish which species do or don't carry out a specific biochemical process. A selective media is composed of a specific ingredient to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow.” states instructor Angela Hartsock in her online microbiology lecture. What she is saying is that a differential media is a visual distinction and a selective media is a physical distinction.
Throughout all of these tests however, it should be stated that a positive and negative control was used for every test. The importance of a positive and negative control is as high as the importance of each other step, and is a necessary addition to the experiment. A negative control helps determine if there is contamination in media or if it was prepared improperly, as well as give you an idea of what a negative or null result should look like. A positive control does the opposite however and allows you to see what a positive result is expected to look like, and can also be a diagnostic tool for human error if a positive result is not reached. All of these aspects of the classroom diagnostic methodology were used in reaching a conclusion for this series of experiments, leading up to the discovering of each person's individual unknown. Throughout the rest of this report, the student Jaynah Holst’s unknown #III will be the focus.
Section II : Materials and Methods
All of the tests performed in this class on Unknown #III were done to help determine the species, as the samples were given anonymously. The methodology used in this class was a series of tests used to narrow down and determine our bacteria, but before that can be employed it is important to stress the importance of a sterile environment for bacterial growth. To assure the samples of the unknown remained pure, the use of aseptic technique on all experiments was used to avoid contamination of the source bacteria, steps were taken to assure no bacteria became an aerosol, there was cleaning of the tables after every class with the disinfectant lysol, and all media was prepared and autoclaved by the classroom assistant before each inoculation. Afterwards, each sample was disposed of properly and autoclaved again to assure no samples left the classroom.
It should be stated that all important tests were done according to the provided lab textbook and results shall be attached on table 1 provided. Initial microscopic examination showed the cell shape to be a rod that stained Gram (+). Further pivotal tests used to determine Unknown #III’s genus and species were discovered early on to be very exclusive. On the second day of Unknown III’s experiments, Gram staining experiment (3-6) showed early signs of endospores that was later confirmed by the following endospore staining experiment (3-9). From those two experiments, it was clear that the bacteria was either Bacillus Subtilis or Bacillus Cereus, as those were the only two provided that were capable of producing endospores. From this on, research from the textbook required from the class was used as a diagnostic tool to further narrow down the two. The Gram-Positive Rod chart (Chapter 9, 596 Microbiology Third Edition) allowed further information was used to double check. The most important tests were the endospore stain and mannitol fermentation test. These were the final tests that allowed a decision to be made.
TEST
RESULT FOR UNKNOWN III
PURPOSE
INTERPRETATION
Endospore Stain (3-9)
(+)
To test the resilience of the bacteria via presence of endospores.
Endospores visible on stain.
Mannitol Fermentation (5-2)
(+)
To test whether the bacteria can ferment mannitol.
Mild fermentation, gas and acid produced. Secondary test showed definite fermentation.
Table 1. Two tests that narrowed down the results for Unknown III.
Section III : Results
After the second day it was fairly clear what the results were pointing to, a Gram positive rod with the ability to create endospores. Out of the 16 available bacteria, only two were fitting the profile. B. Cereus and B. Subtilis, two very similar bacteria. Two main tests determined the final result of Unknown III, the aerobic tolerance and the mannitol fermentation. Initially, there was confused on the mannitol fermentation test and it was believed that there was a negative result which lead to the initial prediction stated in the Descriptive chart for Unknown Letter Designation III assignment of Bacillus cereus. The fermentation in the mannitol test was faint, so it was redone on the redo day in order to get a more clear result. After the second test, there was no doubt that the unknown III fermented mannitol, leading to a change in prediction to Bacillus Subtilis. Up until the redo day, it was almost certain that it was B. Cereus but the initial prediction was incorrect, later to be confirmed by both of the previously mentioned test.. Unknown III is an obligate aerobe and can ferment mannitol, while Bacillus Cereus is neither of those.
Reading the results for the other experiments appeared to be redundant as it was quite clear from the beginning it was one of the two, Unknown III was fortunately easy to figure out.
The following chart is a descriptive one for how the bacteria was identified.
Figure 1. The above tests showing the method used to decide the species of the bacteria.
TEST
UNKNOWN III
BACILLUS SUBTILIS
Gram Stain
Positive (+)
Positive (+)
Endospore Stain
Positive (+)
Positive (+)
Motility
Motile (+)
Motile (+)
Cellular morphology
Rod
Rod
pH Tolerance
4-7pH
–
Catalase
Bubbles (+)
Catalase present (+)
Anaerobic Growth
Obligate Aerobe
Obligate Aerobe
VP Test
Alkaline (+)
(+)
Acid from:
-Glucose
-Sucrose
-Mannitol
-Lactose
Fermentation of Glucose (+)
Fermentation of Sucrose (+)
Fermentation of Mannitol(+)
Fermentation of Lactose (-)
Fermentation of Glucose (+)
Fermentation of Sucrose (+)
Fermentation of Mannitol(+)
Fermentation of Lactose (-)
Casein Hydrolysis
Clearing (+)
(+)
Gelatin Hydrolysis
Liquid (+)
(+)
Citrate Utilization
Blue (+)
(+)
Nitrogen Reduction
(+)
(+)
Indole Production
(-)
(-)
TOTAL MATCHES
100%
16/16
Table 2. The tests of Unknown III’s results with test results known to describe Bacillus Subtilis in Bergey’s Manual of Systematic Bacteriology(Krieg, 1984).
Part IV : Conclusion
Bacillus Subtilis is believed to be Unknown III, and is widely regarded to be known as one of the most versatile and hardly bacteria that can withstand a myriad of environments. Originally, it was known to be called Vibrio subtilis and was named by Christian Gottfried Ehrenberg but was renamed in 1872 to its current name, Bacillus Subtilis, sometimes nicknamed “Grass bacillus” due to its common location in soil. This bacteria can be found in all sorts of environments ranging from the intestinal tract of animals to the soil that is commonly walked across, and specializes in being versatile by being able to live in both terrestrial and aquatic environments. Part of the reason that this bacteria has such high survivability is the fact that when stressed, it can go into a dormant, highly resistant form called an endospore until the environment proves more favorable. It also has the ability to form biofilms when in high amounts, which also aids its survivability.
The bacteria is rarely known to be infectious to humans, and there is little data available about it since in a laboratory setting, it is common practice to discard B. Subtilis when detected as it’s considered a harmless contaminant unlike its close relative, Bacillus anthracis (anthrax). When B. Subtilis does infect humans, however, it causes food poisoning and related symptoms such as diarrhea and vomiting. Despite causing food poisoning, B. Subtilis is a common probiotic and is used to treat intestinal flora imbalances. When infecting immunocompromised patients, there can be further complications as there can be with any bacteria. Simple hand washing and general sanitary procedures can prevent infection, as well as food safety and handling procedures.
Although typically considered a lab contamination, there are real life and laboratory applications for B. Subtilis. The bacteria is frequently used in studies regarding DNA replication and RNA polymerase σ factors, as well as studied for their ability to undergo sporulation and create endospores. B. Subtilis is also used frequently in agriculture to promote oxidation and other beneficial soil fertilization from cow dung, where the bacteria is frequently found. B. Subtilis helps us grow better food from the soil used by industry by either outcompeting other bacteria or killing them while also producing antimicrobials, iturins, a specialized type of pore-forming lipopeptides. These iturins from B. Subtilis give off strong antifungals as well by inhibiting spore germination which in turn keeps root systems of plants safer from harmful fungi. As well as antifungals, the antibiotic bacitracin was derived from a member of the B. Subtilis family and has very promising prospects and applications for future usage