The process of identifying an organism involves the determination of where an isolate or an organism needs to be placed within a group of organisms that are already known. The process is usually done to conform to a given scheme of classification. Most of the microorganisms that may be found in the environment require to be identified aiming at identifying the different species that may be found in a given environment. The identification enables physicists and health workers to associated different sources with different organisms especially those that cause diseases. Organism classification may also help in the diagnosis of different disease by associating the presence of a given organism with the disease they cause (Fox, 2011).
The process of identifying organisms is mainly conducted through the use of different techniques that have been standardized. The first step in the identification process involves the description of the colony that is made by the organism after being cultured and its cellular structure. Description of the colony morphologically involves observing the organism directly as it grows on the agar. This gives the shape of the organism spherical or rod-shaped, crenated or raised, as well as the pigment of the organism. Some microbiologists have been using visual identification alone in the identification of an organism. This practice has been discouraged due to the fact that only few and experienced people are in a position to identify an organism visually and give positive results (Sandle, 2011). Once a colony has been described, the features are used to differentiate from the other types of organisms that have contrary features. Description of the morphology of the colony thus enables one to further identify the organism. Other methods of identification such as protein analysis, serological methods, and differential staining are then used to provide the specific species in which the organism belongs (Sandle, 2011).
Cellular staining gives crucial information on the content of the cell wall making up the organism together with the shape of the organism. One of the frequently used cellular staining methods is known as Gram staining. The method involves the use of a dye called Gram stain and is done through a four-step technique. The first step involves the addition of the crystal violet stain followed by iodine. Alcohol is then added to decolorize the content and safranins are finally added as a counter stain. When done correctly, a positive result test is indicated by the appearance of blue color since Gram positive organisms are able to hold the crystal violet stain. Those organisms that are Gram negative are not able to hold the crystal violet stain but are able to retain the safranin and thus show a red color (Sandle, 2011).
The Gram reaction works on the basis of the differences that exist in the content of the cell wall of the organism. Those organisms that give a positive result are those that have greater peptidoglycan content and less lipid content compared to the Gram negative organisms. The solvent added works to dissolve the lipid content in the cell wall leading to the leaching out of the crystal violet. In Gram positive organisms, the high content of the peptidoglycan prevents the diffusion of the complex formed by crystal violet and the iodine due to the dehydration of the of the thick walls. In Gram-negative stains, the lack of high peptidoglycan content the lipid layer content is dissolved leading to the leaching out of the crystal violet. The Gram stain, therefore, helps in the differentiation of those organisms that are Gram positive from the Gram negative organism (Sandle, 2011).
Eosin Methylene Blue, which is also referred to as EMB is a selective stain that allows only the Gram positive bacteria organisms to grow. The stain is made up of two different stains methylene blue and eosin in the ratio 1:6 and is used in the formulation of EMB agar. The EMB agar provides a medium where those Gram positive bacteria that are able to ferment lactose or sucrose such as E. coli are distinguished from those that are not able to ferment lactose or sucrose such as the Salmonella. Fermentation of lactose releases acids resulting in reduced pH that encourages the absorption of the dye by the colonies leading to the formation of the purple-black color. Lactose no-fermenters, on the other hand, may result in increased pH, which do not facilitate the absorption of the dye and hence no color formation. A positive result for the test is the presence of nucleated colonies indicated by colonies that have dark centers (ACC, 2013).
The citrate test refers to a test that detects those organisms that have the ability to use citrate as their only source of energy and carbon. Inoculkation of bacteria is done on a media that contains a pH indicator, sodium citrate and inorganic ammonium salts. Metabolism of citrate results in the production of ammonia and sodium bicarbonate leading to a rise in pH level. A positive test is indicated by a change in color from green to blue (Baron, Peterson, & Finegold, 1994).
The other test that is employed by microbiologists in microorganism identification is the catalase test. The test tests for the production of catalase enzyme by the organism using hydrogen peroxide. Presence of catalase enzyme is indicated by the presence of bubbling as a result of the oxygen being produced as the peroxide is degraded (Baron, Peterson, & Finegold, 1994).
The urease test is used to test for the presence of Helicobacter pylori. The test works on the basis of the bacteria having the ability to produce the urease enzyme. The enzyme works to catalyses the breakdown of urea to carbon dioxide and ammonia, which increases the pH level of the medium. A positive test is indicated by the conversion of the yellow color to red (Baron, Peterson, & Finegold, 1994).
PR (phenol red) fermentation test is a test that is used to determine the ability of an organism to digest a given sugar. The test is done by using a pH indicator (phenol red) to detect any change in pH through color change (yellow at pH of 6.8 and below and fuchsia at pH 7.4 and above). Change in color indicates the results are positive while no color change indicates that the results are negative. The gelatin test, on the other hand, is used to test those bacteria that can use gelatin as a source of energy. A positive test is indicated by the medium failing to solidify after it is refrigerated and a negative result is indicated by solidification after refrigeration.
Pseudomonas aeruginosa disease is tested using an ankle-brachial index or ABI. The test compares the blood pressure that is in the ankle with that one in the arm to indicate the efficiency of blood flow in the limbs. A normal result for ABI ranges from 1.0 and above with a range of between 0.9 and 1.30. A Doppler ultrasound may also be used detect the disease and show whether a given vessel is blocked. The Doppler ultrasound results also indicate the severity of the disease. Other tests include the treadmill test, arteriogram, blood tests, and magnetic resonance angiogram (NIH, 2011).
Pseudomonas aeruginosa refers to a common bacterium that may lead to disease development when in animals including humans. The bacterium is found in different habitats including the soil, water, and several man-made environments. The organism has the capability of living in normal conditions as well as in the hypoxic areas enabling it to colonize numerous environments both natural and artificial (NIH, 2011).
The organism makes use of a wide range of food sources with its versatility enabling it to infect tissues that have a reduced immunity and those that are damaged. When such colonization takes place in essential organs such as the kidneys, urinary tract, and lungs, there are high chances that the incident may cause death. The bacteria may also be seen medical equipments such as the catheters leading to cross-infections. The pseudomonas aeruginosa bacterium is an opportunistic and nosocomial organism infecting mostly the immunocompromised people. It has been reported to the major cause of infections that occur in the outer ear and burn injuries (NIH, 2011).
The isolation of P. aeruginosa is usually done from those sites that are not sterile such as the sputum and mouth swabs where the colonization mainly takes place but not infection. Since their isolation from these areas is not an indication of infection, it is necessary that a physician be consulted as most of this colonization may require no treatment. Isolation of P. aeruginosa is from sterile sites such as the blood, deep collections or bone is an indication of infection and treatment is recommended. Naturally, P. aeruginosa show resistance to a huge number of antibiotics and may show additional resistance especially when treatment fails through porin modification. Some of the antibiotics that have been successfully been used to eradicate P. aeruginosa include quinolones, monobactams, antipseudomonal, and cephalosporins.The administration of these antibiotics usually occurs through injections. This has resulted in a number of hospitals use of some antibiotics is highly regulated to avoid development of resistance (NIH, 2011).
ACC. (2013). Eosin Methylene Blue Agar. Retrieved April 25, 2014, from Austin Community College: http://www.austincc.edu/microbugz/eosin_methylene_blue_agar.php
Baron, E. J., Peterson, L. R., & Finegold, S. M. (1994). Bailey and Scott's diagnostic microbiology. Missouri: Mosby.
Fox, A. (2011). Culture and Identification of Infectious Agents. Retrieved April 24, 2014, from http://pathmicro.med.sc.edu/fox/culture.htm
NIH. (2011). What Is Peripheral Arterial Disease? Retrieved April 25, 2014, from National Institutes of Health: https://www.nhlbi.nih.gov/health/health-topics/topics/pad/#
Pommerville, J. C. (2010). Alcamo's Laboratory Fundamentals of Microbiology. London: Jones & Bartlett Publishers.
Sandle, T. (2011). Identifying Bacteria - Introducing the Gram Stain. Retrieved April 24, 2014, from http://digitaljournal.com/blog/14180