Introduction
Pseudomonas aeruginosa is a gram-negative bacterium that causes diseases in human beings and animals and can be found in soil, water and external human skin. It is an aerobic, rod-shaped, gram-negative, bacillus with unipolar motility. It is an opportunistic human pathogen affects mainly plants and other organic materials. This is one of the main pathogens of purulent-inflammatory processes, especially in the hospital. It uses a wide specter of biological materials and elements for living.
Main body
According to Liu, P. aeruginosa produces a variety of pigments, including pyocyanin (blue-green), fluorescent, now also known as pyoverdin), fluorescein (yellow-green, and pyorubin (red-brown).P.aeruginosa is initially identified by its pungent smell and its unique appearance (Liu, 2011).
P. aeruginosa is positively identified by its blue color at a temperature of 15 to 30 sec when grown on a nutrient agar.
This microorganism is capable of growing in a jet diesel as a hydrocarbon thus causing microbial corrosion. It generates dark gel-like mats that are sometimes referred to as “algae” (though this is improper) because of their general appearance (Liu 2011).
Although classified as an aerobic micro-organism, P. aeruginosa is viewed by many scientists as a facultative anaerobe because it is well adapted to grow and multiply in conditions that partially aerobic or completely anaerobic. The bacterium achieves optimum anaerobic growth with nitrate as the terminal acceptor. When the nitrate is absent, P.aeruginosa is able to ferment arginine by substrate-level phosphorylation.
Its adaptation to anaerobic or micro-aerobic conditions is essential for certain growth patterns of P. aeruginosa (Ian 1968).
For instance, during lung infection in cystic fibrosis patients in which thick layers of alginate (that normally surround bacterial mucoid cells) can limit the diffusion of oxygen in to the lung cells (Ian 1968).
However, need for an investigative procedure, which can be carried out routinely in the lab for the detection of non-pigmented strains of P. aeruginosa, is obvious. Initial experiments carried out in the laboratory showed that while the cells of P. aeruginosa contain a peroxide-ase and catalase, other gram-negative bacteria similar to P. aeruginosa have similar enzymes which make their use in a practical lab test extremely involving. However, it was discovered that P. aeruginosa contained plenty of cytochrome oxidase which is essential for its identification (Ian 1968).
Data
Cultures of the bacteria used in the initial screening tests were obtained from the microbiology lab and departmental stock culture collection. All cultures were grown on blood agar plates or nutrient, in nutrient broth (4. 5 ml) for 17 to 24 hours at room temperature (24 to 28 C) or 37 depending on the optimal growth condition of the organism.
All cultures were tested biochemically and stains for flagella were obtained on all doubtful strains. During the test, the reagents used were; 1 % a-naphtha in 95 % ethanol and a 1% aqueous solution of p-aminodimethylaniline oxalate. These solutions were freshly prepared weekly because of the auto-oxidation of paminodimethylaniline oxalate, however, they can be used for at least two weeks (Ian 1968).
To detect cytochrome oxidase in broth cultures of the microorganisms, 0. 2 ml of the a-naphtha solution and 0. 3 ml of p-aminodimethylaniline oxalate were added to each tube. The tubes were shaken vigorously to insure homogeneous mixing and the thorough oxygenation of the culture. The appearance of a bluish coloration was indicative of the presence of cytochrome oxidase in the bacterial cells. To find out the presence of the enzyme in microorganisms cultured on agar plates, equal portions of the above reagents are mixed and several drops were allowed to flow freely over isolated colonies (Ian 1968).
The lab investigation on cytochrome oxidase reaction with 57 stock cultures is complied in the table below. Stock cultures were, biochemically, morphologically and systematically labeled eliminating any cases of contamination. Broth cultures of P. aeruginosa gave a positive reaction (blue in color) in a span of 15 to 30 seconds that indicated high concentration of the enzyme (cytochrome oxidase) (Liu 2011).
The table below indicates the laboratory test for identification of P. aeruginosa using cytochrome oxidase.
Micro – organisms
Number of Species or Strains Tested
Cytochrome Oxidase Test (+ positive) or – (negative):
It was noted that broth cultures of A. faecalis gave positive results from 2 to 5 minutes and since the blue color was developed slowly it was easy to differentiate A. faecalis from P. aeruginosa. All the other stocks tested negative for the presence of Cytochrome oxidase.
Colonies of P. aeruginosa on nutrient and blood nutrient agar turns blue after 15 to 30 seconds when exposed to a solution containing equal portions of beta-aminodimethylaniline oxalate and alpha-naphthol, where all other gram-negative bacilli are not affected. Indeed, it is confirmatory possible to indentify and isolate colonies of P. aeruginosa from mixed populations (Liu 2011).
Discussions
From the investigation it was clear that, due to the high concentration of the enzyme (cytochrome oxidase) within the cells of P. aeruginosa it is possible to carry out a laboratory investigation based on the formation of indophenol blue from the oxidation of beta-aminodimethylaniline oxalate in the presence of alpha-naphthol.
This test is recommended especially for nutrient broth cultures of non-pigmented gram-negative bacilli that resemble P. aeruginosa. Similarly, on nutrient or blood agar plates, colonies of P. aeruginosa also tests positively when exposed to an equal compositions of the reagents.
However, in the clinical labs all gram-negative non-lactose fermenters are of importance and therefore, require inoculation into at least the preliminary biochemical media. Since P. aeruginosa is the only known species of Pseudomonas that is pathogenic for man, the test is material- specific when carried out on cultures collected from human specimens (Liu 2011).
Although other strains of Pseudomonas will react positively, it is not certain at this time if the presence of enzyme (cytochrome oxidase) is characteristic of all members of this genus. Until the present day this challenge is being investigated (Liu 2011). Such studies will help researchers and scholar to narrow down their studies to possibilities of negating incidences where some members of this genus would lack cytochrome oxidase.
Conclusion
In conclusion, the process of identification of P. aeruginosa isolated from clinical specimens is based on high concentration of cytochrome oxidase present in the cells of P. aeruginosa. It is important to note other bacilli with similar bio-chemical reactions tests negative for cytochrome oxidase.
References
Liu, D. (2011). Molecular Detection of Human Bacterial Pathogens. New York: CRC Press.
Ian, P. (1968). Identification of Pseudomonas Aeruginosa In The Clinical Laboratory. Upper Saddle River, New Jersey: Prentice Hall.