Essay: Identification of unknown bacteria

Identification of unknown bacteria is a critical part of microbiology. The purpose of the experiment is for students to learn different characteristics of bacteria, construct a proper dichotomous key, carry out tests to differentiate and narrow down bacteria, and use the results to identify two unknown bacteria. By carrying out this experiment, students learn how to properly identify bacteria, and this knowledge can be useful later in life, whether it is for research purposes or applications in clinical settings.
Two strains of bacteria, one Gram positive and one Gram negative, were provided in a broth tube. Using a sterilized inoculating loop, these bacteria were cultured on four plates: two Tryptic Soy Agar plates, one Mannitol Salt Agar plate, and one Eosin Methylene Blue plate. Following this, the two bacteria were isolated onto separate plates using the isolation streak technique in order to determine which one was Gram positive and which one was Gram negative. Then, using the dichotomous key that was created, various tests were carried out on each of the bacteria to narrow down possibilities and ultimately identify the two different bacteria.
 
Materials and Methods
To begin the experiment, broth tube Unknown #2A containing two unknown bacteria, one Gram positive and one Gram negative, was used as the master stock. Additional subcultures would be created from this master stock. First, a Gram stain was performed on the mixed culture to observe the size, shape and Gram reaction of the organisms. Using a sterilized inoculating loop, the mixed bacterial culture was then streaked onto four plates: three plates, Tryptic Soy Agar (TSA) plate, Mannitol Salt Agar (MSA) plate, and Eosin Methylene Blue (EMB) plate were incubated at 37 degrees Celsius for 1-2 days, and one Tryptic Soy Agar (TSA) plate was kept at room temperature.
After incubating the plates, the resulting bacterial growth on each of the four plates were observed and the morphologies and colors were recorded. One yellow colony and one off-white colony were taken from the 37 degrees Celsius TSA plate, and were streaked for isolation on two separate TSA plates, which were then incubated for 1-2 days. After this incubation period, Gram stains were performed from the two new subcultures to ensure that the two bacteria had been isolated and to distinguish between the Gram positive unknown and the Gram negative unknown. The size, shape, color, and arrangement for both types of bacteria were recorded before moving on with the experiment.
The first test carried out for the Gram positive bacteria was a catalase test, checking for the presence of the enzyme catalase. To perform this test, a sterilized inoculating loop was used to streak a sample of the bacteria onto a glass slide, and a drop of hydrogen peroxide was added to the bacteria. The production of gas bubbles would indicate the presence of catalase, because this would mean that the bacterium was able to convert hydrogen peroxide into water and oxygen. The results of the catalase test were recorded.
After conducting this test, a mannitol fermentation test was carried out. This was done by creating an isolation streak on an MSA plate and incubating the plate at 37 degrees Celsius for 1-2 days. A yellow halo around the bacteria on the MSA plate would indicate that they fermented mannitol, while no change in color in the MSA plate would mean that the bacteria did not ferment mannitol. Observations were recorded for this test. After confirming the results by observing the colony morphology under a microscope and the color on a TSA plate, no other tests were needed to determine the Gram positive unknown bacteria according to the dichotomous key.
The process of identifying the Gram negative bacteria was not as simple, because there were more possible Gram negative bacteria and therefore more biochemical tests needed to be carried out in order to narrow the possibilities down. The Gram negative bacterium was first analyzed by conducting an oxidase test. This was done by using a plastic inoculating loop to place a sample of the bacteria on an oxidase strip. If the strip turned into a purple color, this would indicate an oxidase positive bacteria. If the strip did not change color, this would indicate an absence of cytochrome c oxidase in the bacteria.
Following this test, a gelatin hydrolysis test was carried out to test for the presence of the gelatinase exoenzyme. A gelatinase tube was inoculated with a sample of the bacteria using a sterilized inoculating needle. The tube was then incubated at 37 degrees Celsius for one week before being placed in the refrigerator for approximately 30 minutes. After this process, if the gelatin was liquid, the bacteria contained the gelatinase exoenzyme and was able to hydrolyze, or break down, the gelatin. If the gelatin was solid, the bacterium was gelatinase negative. At this point in the identification process, the identity of the Gram negative unknown was able to be determined using the dichotomous key. Motility, indole, and urease tests were carried out for further confirmation.
The SIM deep for the motility test and urea slant agar for the urease test were inoculated simultaneously. For the motility test, an SIM deep was inoculated with the bacteria by stabbing a sample of the bacteria to the bottom of the tube using an inoculating needle. After incubation at 37 degrees Celsius for 1-2 days, cloudiness observed around the stab would indicate motile bacteria, while a clean stab with no cloudiness would mean the bacterium was immotile.
For the urease test, a urea agar slant was inoculated with bacteria using an inoculating loop by first streaking the loop along the surface of the slant and then stabbing the loop to the bottom of the urea agar slant tube. After incubation at 37 degrees Celsius for 2 days, the slant was refrigerated and then observed. A hot pink color in the urea agar would indicate a urease positive result. This would mean that the bacteria were able to convert urea into ammonia.
On the same day that the results of the motility test were observed, an indole test was carried out using the inoculated SIM deep that was previously used to check for bacterial motility. Four to five drops of Kovacs reagent were added to the tube and mixed gently. After 1 minute, the liquid above the agar was checked for a change in color. The presence of a bright red color would indicate that the bacterium was indole positive, while no change in color, or a yellow color, would indicate that the bacterium was indole negative. An indole positive result would mean that the bacteria contained the enzyme tryptophanase and was able to break down, or hydrolyze, tryptophan into indole. After conducting all of these tests, the identity of the Gram negative unknown bacteria was determined and confirmed.
Results
On October 2nd, 2018, the first lab session that the unknowns were handled, a Gram stain was performed on the mixed culture to observe morphology. Under the microscope, both Gram positive and Gram negative bacteria were present. The Gram positive organisms were coccus-shaped in grape-like clusters. The Gram negative organisms were rod-shaped.
One October 9th, 2018, the bacterial growth on the four plates were observed. The TSA plates at room temperature and at 37 degrees Celsius both had decent amounts of bacterial growth on them. On the room temperature TSA plate, one type of bacterial colony was about 3 millimeters in diameter, round-shaped, yellow, fuzzy, and flat. The other colony on the room temperature TSA plate was about 1.5 millimeters in diameter, circular, and off-white, with slight elevation and a defined edge. On the 37 degrees Celsius TSA plate, one colony was round and yellow, with cloudy edges and smooth elevation. The other colony was round and off-white, with defined edges and smooth elevation. The MSA plate was growth positive, and it appeared to only have one type of bacteria on it. The plate turned yellow in the areas where the bacteria grew, and the colonies were off-white, circular, and smooth, with slight elevation and defined edges. EMB plates select for Gram negative bacteria, and the bacterial colonies that grew on the plate were dark pink dots surrounded by fuzz with no green metallic sheen. During this lab session, the two bacteria were isolated through isolation streaks on two separate TSA plates that were then incubated at 37 degrees Celsius.
On October 23rd, 2018, the isolated Gram positive and Gram negative bacteria on the separate TSA plates were observed. To confirm that the plates were isolated subcultures, Gram stains were carried out again. The Gram positive bacteria were circular and off-white on the plate, and were cocci in grape-like clusters under the microscope. The Gram negative bacteria were yellow and fuzzy on the plate, and were singular rods under the microscope.
Results for Gram positive bacteria tests:
Test
Result
Interpretation
Catalase
The solution produced bubbles, indicating gas production.
+/ The bacteria are Catalase positive, or have the Catalase enzyme, and thus have the ability to convert hydrogen peroxide into water and oxygen.
Mannitol Fermentation
A yellow halo was present around the bacteria on the Mannitol Salt Agar (MSA) plate.
+/ The bacteria are able to ferment mannitol.
 
Results for Gram negative bacteria tests:
Test
Result
Interpretation
Oxidase
The oxidase strip turned dark purple.
+/ The bacteria contain the enzyme oxidase.
Gelatinase
The gelatin solution was solid.
-/ The bacteria do not contain the exoenzyme gelatinase and thus do not have the ability to break down gelatin.
Motility
No cloudiness observed in medium.
-/ The bacteria are not motile.
Indole
Solution was yellow after the addition of Kovacs reagent to the tube. No red color was present.
-/ The bacteria do not contain the tryptophanase enzyme and thus do not have the ability to hydrolyze tryptophan.
Urease
No pink color observed in the solution.
-/ The bacteria do not contain the urease enzyme, and thus cannot convert urea into ammonia.
 
Conclusions
It was concluded that the Gram positive unknown bacterium was Staphylococcus aureus. The catalase test was carried out for the bacteria, and the production of gas showed that they were catalase positive, eliminating Enterococcus faecalis as a possible result. After viewing the bacteria under a microscope, it was evident that they were coccus-shaped, not bacillus-shaped, which eliminated Bacillus subtilis as a possibility. One the MSA plate, the bacteria were surrounded by a yellow halo, indicating that it fermented mannitol, which led to the conclusion that the unknown Gram positive bacteria were Staphylococcus aureus. To further support this conclusion, the bacteria appeared off-white on the TSA plate, which matched the characteristics of Staphylococcus aureus in the Gram positive table.
It was concluded that the Gram negative unknown bacterium was Alcaligenes faecalis. First, the oxidase test was performed on the bacteria, and the bacteria were oxidase positive because the oxidase strip turned dark purple. This ruled out Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, and Serratia marcescens. The gelatinase test came out to be negative, and this led to the conclusion that the bacterium was Alcaligenes faecalis. To confirm this result, indole, urease, and motility tests were carried out. Results showed that the bacteria were indole negative and urease negative, which matched the characteristics of Alcaligenes faecalis. The motility test came out to be negative even though Alcaligenes faecalis is motility positive. This may have been the case because Alcaligenes faecalis is an obligate aerobe, so the bacteria in the SIM deep may have died due to a lack of oxygen, leading to no observable motility under the surface of the SIM deep. Aside from this test result, all of the other tests supported the conclusion that the Gram negative unknown bacterium was Alcaligenes faecalis.
Organismal Reports
Staphylococcus aureus is a Gram positive, coccus-shaped bacterium that forms grape-like clusters. It is a facultative anaerobe, beta-hemolytic, coagulase positive, and produces exotoxins. It is halophilic and can grow at a variety of temperatures.
The genus Staphylococcus has two major species: Staphylococcus aureus and Staphylococcus epidermidis. Unlike Staphylococcus aureus, Staphylococcus epidermidis is non-hemolytic, coagulase negative, and does not produce exotoxins. Staphylococcus is generally found on the skin, especially in the area in and around the nose. More specifically, Staphylococcus aureus can be found on skin and hair and in noses and throats. Twenty-five percent of healthy people have S. aureus on their skin.
Staphylococcus aureus is clinically relevant because it can cause diseases, particularly through opportunistic infections. Diseases caused by the Staphylococcus genus are usually opportunistic infections, because the organisms can only infect the body through infected wounds, boils, and abscesses and cannot penetrate the skin independently. As a result, Staphylococcus organisms usually cause nosocomial, or hospital-acquired, infections, as patients in the hospital are more susceptible to opportunistic infections. Once in the blood, Staphylococcus aureus can move to the joints, heart, and bones, and cause septic arthritis, endocarditis, and osteomyelitis, respectively. In addition, bacteremia, or bacterial infection of the blood, can lead to septic shock. Staphylococcus aureus can cause scalded skin syndrome, toxic shock syndrome, and food poisoning. Organisms of the Staphylococcus genus contain Protein A which binds to antibodies and prevents them from attacking, can produce superantigen antitoxins that confuse host defenses, and have antibiotic resistance to penicillin in addition to many other antibiotics. Thus, although antibiotics are effective, they are limited, so Staphylococcus aureus can potentially be dangerous for the host.
Alcaligenes faecalis is a Gram negative, rod-shaped bacteria. Organisms of the genus Alcaligenes can be found everywhere, including marine waters, freshwaters, sewage, soil, feces of healthy individuals, and hospital environments. They are nonfermentative, and therefore are not capable of using glucose, lactose, sucrose, and most other carbohydrates. Alcaligenes faecalis bacteria are motile, oxidase positive, citrate positive, gelatinase negative, urease negative, hydrogen sulfide negative, indole negative, obligate aerobes.
Alcaligenes faecalis is clinically relevant because although it can be found naturally inhabiting the intestines of humans, it is an opportunistic pathogen and thus is commonly found affecting people with suppressed immune systems. It can cause nosocomial infections, because patients in the hospital have weakened immune systems due to surgeries or illnesses, and cystic fibrosis patients are at a higher risk of being infected. It can cause infections such as peritonitis, meningitis, appendicitis, chronic otitis, and bacteremia, or infection of the bloodstream. Usually, when treating diseases caused by Alcaligenes faecalis, the outcome is positive. However, treatment against these diseases can potentially be very difficult due to Alcaligenes faecalis’ resistance to many types of regularly used antibiotics. In the case of A. faecalis diseases, the antibiotics carbapenems, antipseudomonal penicillins, and trimethoprim, are considered relatively more effective in treating the disease. Evidently, Alcaligenes faecalis is a significant bacterium because it can be found in water or aqueous environments and can cause opportunistic infections that may be difficult to treat using antibiotics.