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Microorganisms are ubiquitous in the environment and although majority of them are non-pathogenic, there are some that can cause great damage; regardless of their pathogenic spectrum, organisms need to be identified in order to have a better understanding of their mechanisms. Identification can be done by performing different experiments, beginning with tests that will show broad differences among them leading up to unique characteristics that will only be exhibited by that peculiar organism. Based on their morphology organisms are able to stain differently, such as Gram positive or Gram negative, thus creating a narrower difference for the purposes of its identification. Bacteria that stain Gram positive contain thick peptidoglycan that allows it to retain the Crystal violet dye. On the other hand, Gram negative bacteria are made up of larger pores and thin peptidoglycan, thus allowing the decolorizer to wash off the Crystal violet dye and retaining the counterstain, Safranin (Willey et al. 2008). When performing narrower tests, some organisms' biochemistry will only allow them to perform certain reactions; for example, only some bacteria proteolize certain components such as casein identified through Skim milk medium, others ferment only some sugars, which can be identified by using phenol red broth, or others only hydrolyze certain enzymes, such as urease found by performing urease test broth, among many other tests (Difco/BL, 2010). In addition to these commonly used tests, recent experiments have made advances to the identification of bacteria. Cohan (2006) argues that by employing DNA sequence-based approaches, organisms could easily be classified by the identification of its 16S rRNA sequence, eventually bacteria could be classified based on their protein sequence. The integration of molecular use for bacterial characterization will make bacterial identification more effective. In addition, there are rapid tests that have been manufactured to identify bacteria causing illness in the body. Although there are many bacteria living in the human body that work in conjunction with it, called normal flora; other pathogenic bacteria can work their way in the human body thus causing illnesses. In particular, there are microorganisms living in the pharynx that, as Dr. Maxwell's manual explains, will increase the chances of illnesses only when they are present in larger amounts than normal, or when another pathogenic bacteria invades the system, such as Haemophilus influenza, Streptococcus pneumonia, and Moraxella catarrhalis. The present experiment sought to investigate the amount of bacteria present in the pharynx, and whether these bacteria were hemolytic, as well as identifying organisms based on the knowledge gained about the different tests employed before.
Materials and Methods
To conduct the quantification and characterization of upper respiratory bacteria, a blood agar media plate was first inoculated by swabbing someone's tonsils and following a streak plate technique. The culture was incubated in an anaerobic environment and harvested for 48 hours. A second swab was then performed in which the population density was investigated. A subject's tonsils were swabbed; the swab was then placed in sterile water and allowed to dislodge all the organisms. A series of dilutions ranging from 10-1 to 10-4 were made, however only 10-3 and 10-4 dilutions were used to make a pour plate using nutrient agar. This was then incubated and allowed to grow for a 48 hour period.
To determine the unknown organisms provided, there were two streak plates made for colony isolation from the bacterial broth given, and a dichotomous key to identify the needed tests for identification, seen in figure 1.
Figure 1. Dichotomous key used for the identification of unknown organisms.
The dichotomous key allows for organisms to be identified because it lists all the known characteristics of these and narrows down the options based on the results yielded by the tests. The dichotomous key was built in such a way that it would break down a population in half every time a test was performed. The tests were selected based on their difficulty level and time efficiency. Those tests that require too much time or were too difficult to conduct were excluded. The first test performed was the Gram stain since this allowed the bacterial population to be broken in half. A Gram stain works by making a differential stain based on the bacterial cell's composition as discussed in the introduction. Once the morphology for Gram stained was determined, a series of tests were conducted. The first test after determining the organism was Gram positive was catalase. Organisms such as B. subtilis, S. epidermis, S aureus, C. diptheriae and M. roseus are able to react to catalase tests. Production of catalase results because cells want to prevent formation of hydroxyl radicals by converting hydrogen peroxide to water on oxygen gas as indicated by Dr. Maxwell's manual. This test can be performed by smearing bacterial culture on a slide and adding two drops of hydrogen peroxide. If bubbles immediately appear then the organism is said to be positive for catalase. On the contrary, if the organism does not produce any bubbles it should be examined under the microscope to ensure a false negative is not assumed due to the lack of visibility. If no bubbles appear then the organism is catalase negative. If catalase yielded negative results, an indole test was conducted by using a SIM medium.
SIM medium is used to determine whether or not an organism can produce indole, be motile, or produce sulfide since it contains sodium thiosulfate and ferrous ammonium. The reaction of ferrous ammonium sulfate with sodium thiosulfate yields a black color. Casein produces indole, and indole is noticed by adding more reagents after incubation. The test should be conducted after the media has been boiled and cooled down, then it is inoculated with the desired bacteria using a stab technique. The media will then be incubated at 35°C for 18-24 hours under aerobic conditions. Since the media is stabbed, motility can be noticed if bacteria diffuse in the media. If the bacteria only grow where the media was stabbed then the organism is not motile. Also, a black color will be noticed if there is any sodium thiosulfate production. Adding 3-4 drops of Kovacs' reagent will yield a red color if the organism has the capability of producing indole, which will be considered a positive reaction. Organisms such as E. coli are able to produce indole but not sodium thiosulfate and they are motile. S. flexneri yields a negative result for indole, sodium thiosulfate production and motility (Difco/BBL, 2010). On the other hand, if the catalase yielded a positive result then a mannitol test was conducted.
A mannitol salt agar test is used to identify S. epidermis and S. aureus since it contains sodium chloride that does not allow other organisms to grow as well as these. Some bacteria are able to grow due to the peptone and beef extract nutrients it can provide. In addition, Streptococci, and Micrococci could be identified. Staphylococci are able to be distinguished because they have the ability to ferment mannitol. S. epidermis depicts pink colonies when harvested in mannitol salt agar. On the other hand, S. aureus white and S. aureus gold require further testing. To carry out this test, bacteria should be inoculated onto a plate with only a single streak and incubated for 24-48 hours at 35°C under aerobic conditions. S. aureus will grow in small or large colonies depicting yellow zones; other Staphylococci will also depict small and large colonies; however their growth will show a red zone. Streptococci will not grow in this medium and Micrococci will depict large colonies of orange or while colors (Difco/BBL, 2010).If the mannitol salt agar test yielded positive results then the bacterial culture was to be examined on a TSA plate for pigmentation to differentiate between S. aureus gold and S. aureus white. On the contrary, a negative result yielded by the mannitol test would require a casein test to be conducted. Casein is tested by using skim milk media.
Skim milk media is used to either cultivate or differentiate organisms based on their ability to coagulate or proteolize casein, which is a milk protein. This comes in handy when differentiating Corynebacterium diptheriae, Micrococcus roseus and Bacillus subtilis from other bacteria since only B. subtilis is able to degrade proteins and coagulate milk. To conduct the test the media should be heated in boiling water for 2-5 minutes and then cooled to room temperature while the cap is tightened. The tubes should be inoculated with a sterile disposable pipet. If dealing with anaerobic bacteria then sterile mineral oil should be added at the top of the broth. The tubes are then incubated at 35°C for 7 days. The results will vary depending on the type of organism being tested, for example, B. subtilis will be curd and acidic (Difco/BBL, 2010). However, no difference will be noted for C. diptheriae or M. roseus so these two would have to be examined on a TSA plate to differentiate in color changes of pigmentation. C. diptheriae grows in white colonies, while M. roseus grows in light pink colonies.
Organisms determined to be Gran negative were identified by conducting a different set of tests. The Gram negative bacteria assigned for identification were Proteus vulgaris, Escherichia Coli, Enterobacter aerogenes, Citrobacter freundii, Serretia marcesens, Bulkholderia cepecia and Pseudomonas aeruginosa. Following a Gram stain test, a lactose test was conducted using phenol red broth base with carbohydrates. This test is used to differentiate bacteria based on their fermentation ability by producing either gas or acid, or both. Organisms such as E. coli, E. faecalis, P. vulgaris, P. aeruginosa, S, typhemurium, S. aureus and Shigella flexneri can be differentiated with this test. The base medium is generally used as a control since it does not contain any carbohydrates. It employs two components, casein and phenol red. Casein is used to detect carbohydrate fermentation, and phenol red detects acid production. When phenol red is added, the acids will cause a reaction that will change from red to yellow color. To analyze if there is any gas production, the Durham tube should be checked since this collects the gas. To conduct this test the tubes should be inoculated with bacteria and incubated for 48 hours at 35°C loosening the cap. The organisms' oxygen demand should be evaluated prior to incubation. A yellow color indicates the presence of acid and the Durham tube shows the presence of gas (Difco/BBL, 2010). If the lactose test yielded positive results then a citrate test was conducted.
The focus of the Simmons citrate test is to differentiate those organisms that are Gram negative, such as E. coli, E. aerogenes and Citrobacter freundii among others not used in the present lab, and are able to metabolize citrate as their means of carbon through using ammonium dihydrogen phosphate and sodium citrate. A change in color, from green to blue, will be observed. To do so, slants of media are inoculated for 4 days at 35°C. If the slant appears to have blue growth then the test is said to be positive. However, no color change would indicate a negative reaction. E. aerogenes and C. freundii are expected to be positive, where as E. coli will show no color change (Difco/BBL, 2010). To differentiate these two organisms that tested positive for citrate a Methyl Red and Voges Proskauer (MR/VP) test was conducted.
MR/VP medium can be used to differentiate E. coli from organisms such as Citrobacter freundii, E. aerogenes and Klebsiella pneumoniae. Because E. coli's fermentation of dextrose is very acidic it yields a red color after the reaction with potassium hydroxide. The change in color is due to acetoin's oxidation when there is oxygen. Tubes should be inoculated with desired bacteria and incubated at 35°C for 48 hours. The methyl red indicator should be made with 0.1g of methyl red and 300mL of 95% ethyl alcohol and sufficient water. The Methyl Red test is done by adding 5 drops of the prepared methyl red indicator to the broth. The broth should produce a red color for the organism to be MR positive. To perform the Voges-Proskauer test 15 drops of reagent A and 5 drops from reagent B should be added to a 1mL of broth culture. The sample should be shaken and read every ten minutes for color change. In the Methyl Red test, a positive result produces a red color on the surface, and a yellow color would indicate a negative result. The Voges-Proskauer test produces a red color in the first 5 minutes after addition of reagents.
Those organisms that initially tested negative for lactose following the Gram stain were subjected to a glucose test using the phenol red broth with carbohydrates. As discusses above, phenol is used to differentiate between those organisms that are able to ferment glucose and produce gas, acid or both. If the organisms, such as P. vulgaris and S. marcesens, tested positive, then they were tested with for indole formation using a SIM medium. If organisms, such as P. aeruginosa or B. cepecia, tested negative, they were plated on a TSA plate to see changes in pigment color. P. aeruginosa grows in a TSA plate and depicts a green fluorescent color. On the other hand B. cepecia is able to grow; however, this depicts a white color.
Results yielded by the respiratory organisms experiment using a sample from the tonsils showed that the organisms present in the subject's tonsils were δ hemolytic; therefore, there was no hemolytic activity present. When counting the population density for the normal flora present in the tonsils, both dilutions, 10-3 and 10-4, were too numerous to count (TNTC).
Upon receiving the unknown organisms, they were streaked on a TSA plate to harvest pure cultures at 25 and 37°C. The plate incubated at 37°C showed two types of colonies where some were clumped together. The first colony was round convex and depicted beige colors with entire edges as well as a shiny finish. The second colony was of peculiar shape. It depicted colonies of irregular shape and undulated edges as well as an umbonate elevation almost as if it were filamentous. Compared to the precious colony, it depicted a whiter color with groves of a clearer white. The plate incubated at 25°C only contained similar colonies that were smaller and whiter and shinier than those harvested at 37°C. There were only few colonies that showed a white dull color but were hard to distinguish. Once the organisms were isolated, two TSA plates were streaked to harvest the two different bacteria give. Those colonies that depicted entire edges and convex elevation were referred as Unknown A. The colonies with a unique look that contained irregular shape and undulate edges were referred as unknown B. Next, a Gram stain was performed. Unknown A showed cells of rod shape with pink color and some clumping when viewed at 100X under the microscope; therefore this was determined to be Gram negative bacteria. Unknown B showed rod-like cells of purple color and was also viewed at 100X under the microscope, these were determined to be Gram positive cells. Unknown A was tested for lactose fermentation and gas or acid formation and it yielded gas production and lactose fermentation but no acid, thus the test was positive. Unknown B was tested for catalase by smearing cells on a slide and adding hydrogen peroxide; this yielded a positive result determined by the fizz observed. Next, unknown B was inoculated on a mannitol salt agar and allowed to grow for 48 hours at 37°C, this yielded a negative result. Following the lactose test, unknown A was tested for citrate by inoculating a Simmons citrate tube using freshly cultured bacteria from the broth and incubating it at 37°C. After a 48 hour period, the results showed for the organism to be negative. A third unknown was introduced at this time, unknown C. Its Gram stain showed purple cocci when viewed under the microscope at 100X; thus this was characterized as Gram positive. Next, a catalase test was run as following the dichotomous key, the test was positive; therefore, unknown C was inoculated to a mannitol broth. Mannitol broth was used because it yields better results than mannitol salt agar. The mannitol broth did not have any color changes and so it was indicative of a negative result.
Identifying unknown organisms can be done by grouping their characteristics while separating the given population in half. There are many easy tests that are commonly used for identification purposes and each of these biochemical tests works in such a way that allows for some organisms to be selected, while others are only differentiated. Additionally, when there is a notion of what the bacteria causing illness may be, there are rapid tests that can be performed to identify it. The first experiment sought to determine whether or not normal flora in the tonsils was hemolytic. Based on the results, it was determined that the bacteria present in the subject's tonsils went through gamma hemolysis; hence the bacteria were not hemolytic since it did not depict any clearing around the colonies. Clear zones are observed when the red blood cells are completely destructed, and as Dr. Maxwell's manual explains, this is referred to as β hemolysis. When bacteria is able to breakdown red blood cells while partially destroying hemoglobin, then a green halo will be noted around the colonies; the cells are said to have had α hemolysis. By employing different tests, the second experiment sought to identify three unknown bacteria. To identify the given unknowns, a series of tests that followed a dichotomous key were performed. Figure 2 shows the tests that were employed to identify these three unknown organisms.
Figure 2. Tests performed to identify unknown organism following the dichotomous key. Tests highlighted in yellow belong to unknown A. Those highlighted in orange belong to unknown B. Lastly, unknown C followed the same test as unknown B; however, different results were obtained.
Unknown A was identified as Gram negative, positive for lactose fermentation and negative for citrate metabolism. Based on these results, it was determined that unknown A was E. coli. This bacterium belongs to the Enterobacteriaceae family, is non-motile and its cells are straight rods living in the intestines of humans or other warm blooded animals whose optimum temperature is around 37°C (Willey et al. 2008). It is not able to metabolize citrate to make carbon; however, it is a lactose fermenter. Those strands that are pathogenic can cause infections such as urinary track infections usually transmitted through a fecal contact or severe diarrheas transmitted through the ingestion of contaminated foods or water (Hawley, 2000). E. coli has been used to prevent chronic colitis as well as murine acute colitis using the Niddlr1917 strain (Kamada et al. 2005). Unknown B stained Gram positive, tested positive for catalase and was negative for mannitol. Based on these findings and the peculiar colonial morphology, it was determined that unknown B was B. subtilis; therefore no further testing was conducted. B. subtilis is a gram positive bacterium belonging to the Bacillus genus, able to produce catalase enzymes. Its shape is straight rods and is capable of forming endospores that allow it to survive in harsh environments. It is also considered a chemoorganotroph and a mesophilic bacterium. B. subtilis has been used to investigate quorum sensing, cell division, gene regulation, etc. (Willey et al. 2008). Following, unknown C's cells depicted a purple color under the microscope therefore it was determined to be Gram positive. It was able to react to catalase and was negative for the fermentation of mannitol. Based on these results unknown C was identified as S. epidermis. S. epidermis is a Gram positive bacterium, non hemolytic, usually found on the skin. Recently, it has been determined to be opportunistic pathogen since it infects medical devices that are implanted in patients and is responsible for endocarditis. It is cocci shaped and belongs to the Staphylococci genus (Willey et al. 2008).
As it has been observed, an unknown bacterium can be identified by performing different tests such as the ones above because they provide useful information and a clear answer to what the characteristics of the unknown are. Bacteria react differently depending on the nutrients they are provided or their biochemistry. For example, when one restricts bacteria from some nutrients they are likely to die. This is the method used by several biochemical tests; they inhibit bacterial growth while promoting growth for other organisms with different properties. Other methods only show whether or not the bacteria is motile; perhaps this could be the determining factor when differentiating bacteria. Newer methods work by employing quick tests that allow bacteria to be identified fairly quickly when the existing information points to that direction. By understanding bacteria's physiology and biochemistry identification of unknowns is possible as it has been supported by this experiment.