Playing Darts and Growth of the Genus Corynebacteria Introduction:
It is a well known fact that bacteria can inhibit and thrive in almost any environment and many bacterial species are pathogenic. Exposure to bacteria is inevitable and in order to reduce the risk of encountering a pathogenic organism, proper measures need to be taken to ensure that we stay healthy. The objective of my project is to isolate and attempt to classify bacteria
included in the Corynebacteria genus. There are a few reasons as to why I chose this particular genus. Previously conducted research has indicated that Corynebacteria is very common in the skin flora of humans and may lead to diphtheria, an upper respiratory illness commonly
described as a highly contagious disease. (Farizo and Strebel 1993) Corynebacteria has also been linked to various urinary tract infections (Nebreda-Mayoral and Muñoz-Bellido 1995).
Another reason I chose this genus of bacteria is because these organisms live on most surfaces and can be spread by simple contact with an object, person, or food item that has been contaminated. For this reason, I chose playing darts for isolation of my unknown because many different individuals usually handle them and they may also come into contact with tables, walls and even floors. Darts are usually stored in a standard room temperature setting (~25°C) and pathogenic bacteria may be alive and thriving in these conditions. Pathogenic bacteria might also be transferred to and from various surfaces including the hands of the people using them.
at a local pub. In order to design an experiment that identifies and isolates Corynebacteria the morphology of this genus must be considered.
Corynebacteria are Gram Positive (G+), rod shaped and do not produce spores. They are
non-motile, non-acid-fast and catalase positive. The rods of Corynebacteria are usually slightly curved and may appear in the form of Chinese letters under the microscope (Martinez and Suarez 1994). Based on this information I have designed a experiment in order to isolate and identify Corynebacteria. The methodology is simple and quite easy to follow and is described below.
This information is also briefly outlined in the attached flow chart.
Experimental Design & Possible Results:
I began my experiment by initially utilizing a Gram-Stain test. Corynebacterium have a cell well composed of peptidoglycan and based on this I expected the sample to retain the dark purple color indicating a positive result. The dark purple color helped me differentiate between Gram negative and Gram positive cells that may be part of the Corynebacteria genus. The next test I conducted was a standard simple stain with methylene blue. This bacterial dye binds to bacterial cells through ionic interactions and creates a contrast between the bacteria and the background. The simple stain will help me to identify the presence of granules and a positive result indicates progression towards isolating and identifying Corynebacteria.
then I can reasonably continue my investigation because this result helps indicate that I am closer to identifying the correct bacteria.
The next step in my approach to isolate Corynebacterium was to perform an acid-fast stain. Acid fast staining helps eliminate other bacterial species that retain the carbolfuchsin dye due to high lipid content because Corynebacterium are non-acid fast.
To ensure that the remaining bacteria are non-motile I used the sulfur-iron-motility test.
The SIM test is an effective way of analyzing if the bacteria are motile and are able to reduce sulfur to hydrogen sulfide. Correct identification of motility will be conjectured by observing growth outside of the inoculation stab line. In order to proceed to the next step the SIM test must return a result indicating that the bacteria are non-motile. In addition, adding Kovac’s reagent after the previous observations are made will demonstrate if the bacteria are capable of producing the enzyme tryptophanase. The presence of this enzyme would indicate that
tryptophan was broken down to indole, pyruvate, and ammonium and a cherry red color in the tube would be observed.
The next step I performed was a catalase test to discover if my isolated bacteria are catalase positive. I observed whether hydrogen peroxide was broken down to water and oxygen.. Since Corynebacteria are catalase positive I expected that the bacteria I was testing to be
Weeks prior to actual experimentation I will collect samples to be analyzed by using sterile swabs to gather bacteria living on the surface of playing darts at a local pub. Once the samples are collected I will need to facilitate bacterial growth in order to obtain a sufficient amount of bacteria. I will be using a spread plate technique to grow colonies on a Trypticase Soy Agar (TSA). I have chosen this particular medium because previously conducted research has indicated that Corynebacterium were able to grow well in this environment. TSA also provides a relatively stable growth medium for other species of bacteria.
Once I have created a bacterial colony for testing I expect the experimental process to progress as the elimination of bacteria continues based on the expected results of
Corynebacteria. The target bacteria are gram positive, have granules, do not produce spores, do
not retain acid-fast stain, are non-motile and catalase positive. If these results are obtained from the prospective tests then the experiment will follow the flow chart and provide an opportunity to distinguish between members of the same genus. The final test Starch Hydrolysis can help differentiate between Corynebacteria kutsceri and Corynebacteria xerosis if either are present. The final test will be conducted based on the high probability that I have isolated the target genus.
tests I perform to indicate presence of Corynebacteria and possibly even other bacterial species that are similar in morphology to this genus.
Conclusion:
In conclusion, my project proposal aims to isolate and identify members of the
Corynebacteria genus, which have been described as possible pathogens. This genus of bacteria
is of particular interest because it has been associated with diphtheria, an upper respiratory disease (Farizo and Strebel 1993) and various urinary tract infections (Nebreda-Mayoral and Muñoz-Bellido 1995). Corynebacteria is easily transferred between surfaces and organisms through simple contact and it’s natural presence in addition to the ability of pathogenic members of this genus to cause illness is particularly interesting. In order to reduce the risk of a bacterial infection proper measures need to be taken including sanitization and proper hand washing methods. Engaging in precautionary measures reduces the risk of exposure to possible pathogenic bacterial organisms.
Literature Cited:
Farizo K., Strebel P. (1993) Respiratory Disease Due to Corynebacterium diphtheriae: Case Report and Review of Guidelines for Management, Investigation, and Control. Clinical Infectious Diseases 16: 59-68
Martinez L., Suarez A. (1995) Phenotypic characteristics of 31 strains of Corynebacterium striatum isolated from clinical samples. Journal of Clinical Microbiology 33:2458-2463 Nebreda-Mayoral T., Muñoz-Bellido J. (1995)Incidence and characteristics of urinary tract
