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INTRODUCTION:
Pathologists or microbiologists play a very vital role in the management and diagnosis of any disease. They get immense help by performing certain series of tests which aids them reaching to a conclusion. One such test is called as gram staining which is used widely as the most primitive test for the identification of various bacteria
According to Beveridge, the gram stain essentially delineates bacteria into two broad categories. It does that by giving color to these bacteria and bacteria that attains purple or violet color are referred to as gram positive bacteria while, on the contrary, bacteria that attain red color due to decolorization are referred to as gram negative. The cells or bacteria that are gram positive have an impermeable and broad wall that prevents from getting decolorized. This wall is composed of polymers and peptidoglycans. In gram negative bacteria, the cell wall is lipid rich and thin and is susceptible to disruption by the decolorization. Although gram staining is the most fundament and basic testing modality that is used for the diagnosis and identification of bacteria, it has its downsides too. This test is subjective which means it is highly operator dependent and its result can vary according to the expertise of the performer. According the Rutgers University New Jersey, inadequate decolorization during gram staining is the most common cause of poor results.
Similarly, based on the gram staining capability of bacteria cant alone demarcate various bacteria and therefore, need for further testing is warranted to pinpoint the specie and variety of the bacteria. According to Baron, many bacteria are diagnosed based on their biochemical properties and their behavior in a particular biochemical reaction. Some tests that are used in a routine are oxidase, amino acid degradative enzymes, carbohydrate fermentation and reduction of nitrates. More specific tests that are pathognomic for a designated genus like coagulase test for staphylococcus.
The gram staining was done to create the basic difference. The specimen is supposed to take up the crystal violet dye, when heat fixed to it. Slides are then treated with a mordant that aids in fixing the dye or stain. Once fixed, the slide is washed with 95% alcohol and then contrast dyeing occurs with safranin. The prepared slides are supposed to be viewed under oil immersion lens.
A dichotomous key was formed to basically categorize the organism based on their gram staining. The gram staining that was done sorted the bacteria into two broad categories and from there, further specific and focused testing and evaluation was done. Both, gram positive and gram negative were then split into two categories based on the shape of the organism; rods and cocci. These dichotomous key was used and applied on both, group A and group B. Group A showed positive test for catalase but a negative lactose fermentation test. While on the other hand, group B was a gram negative bacteria which showed positive lactose fermentation test. Rest of the tests like indole test, urease test and gelatinase test were negative.
Catalase test is used to determine whether an organism possess the catalase enzyme which converts hydrogen peroxide into water and oxygen. Most aerobic and facultative anaerobes give a positive catalase test. Indole test assess whether the organism possess tryptophanase enzyme which cleaves tryptophan to indole. When indole accumulates in the cell, it is detected using kovac’s reagent. In lactose fermentation, bacteria starts producing glucose in the absence of oxygen. It basically includes glycolysis and some other additional reactions. A McConkey nutrient agar is used to test for lactose fermentation which contains a fermentable sugar, peptone and a pH indicator. In gelatin hydrolysis, presence of gelatinase enzyme is assessed in bacteria which aids them in breaking down gelatin into its constituents. A specific medium called nutrient gelatin deeps are used to test for gelatinase and liquefaction represents a positive result. Lastly, bacteria that are capable of producing urease and breaking urea into ammonia and carbon dioxide are assessed using urease test. A pink slant observed signifies a positive urease test.
RESULTS:
As mentioned in the methods section, there were two groups of bacteria that were supposed to be identified. These two groups were subjected to undergo gram stainin first followed by metabolic tests. The results of this laboratory practical are, therefore, categorized below:
Unknown Group A:
Gram Staining:
It was a gram positive staining bacterium.
Metabolic Test:
The catalase test splits hydrogen peroxide into oxygen and water producing gas bubbles. Therefore, organism in group A is catalase positive but it doesn’t ferment lactose because it didn’t change to yellow color confirming a negative lactose fermentation test.
Unknown Group B:
Gram Staining:
It was a gram negative staining bacterium.
Metabolic Test:
The organisms in group B dint convert into liquid after performing gelatinase test and thus giving a negative gelatinase test. Since these organisms produced yellow color upon performing lactose fermentation test, it is safe to say that these organisms are lactose fermenters. In indole test, appearance of pink color suggests a positive test but these organisms didn’t produce pink color giving a negative indole test. Similarly, urease producing bacteria produce pink color while performing urease test which means a positive test but organisms in group B produced yellow color suggesting a negative urease test.
CONCLUSION:
In group A, the bacterium is a catalase positive which means it belongs to any of the following; staphylococci or micrococci family. A negative lactose fermentation means that the organism belongs to the micrococci family ab=nd most likely the organism is micrococcus luteus. A positive urease, negative coagulase and bacitracin susceptibility will confirm the identification.
In group B, the bacteria is a lactose fermenter which means it is either E.Coli, Enterobacter or Klebsiella. While a negative indole test means this organism is either Enterobacter or Klebsiella because E.Coli gives a positive indole test. In urease test, differentiation between Enterobacter and Klebsiella can be made because Klebsiella is an urease positive organism while Enterobacter is urease negative. So, based on the observations tabulated above, it is safe to conclude that the organism in group B is Enterobacter.
Works Cited
Baron, Samuel. Medical Microbiology. New York: Churchill Livingstone, 1996. Print.
Beveridge, TJ. "Use Of The Gram Stain In Microbiology". Biotechnic & Histochemistry 76.3 (2001): 111-118. Web.
Rutgers, The State University of New Jersey. N.p., 2016. "GRAM STAIN INFO". Web. 3 Apr. 2016.
