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Living organisms are usually placed into different groups according to their natural relationships. Many features determine the grouping, internal as well as external. It uses information from many branches of biology. There is existence of a vast diversity of genotypes. A variety of microbes inhabit extreme environments like high temperature, pH, pressure, salt concentration and low temperature, pH, nutrient concentration and water availability and conditions having high levels of radiation , harmful heavy metals and toxic compounds (organic solvents).
There are numerous handy applications for classifying unidentified bacteria. For the duration of research, it is of extreme significance to be able to spot unknown bacteria if an assortment is being studied. Trying to classify unknown bacteria can also direct to the unearthing of novel species, for instance, if tests are carried out to establish the identity and the bacterium does not match any preceding solo species tests, it is probable that a novel species has been ascertained. Another very essential application for classifying unidentified bacteria is found in medical laboratories. One of the major tasks of a medical lab is to establish the characteristics of pathogenic bacteria. In numerous cases it is very vital to verify the definite type of bacterium causing ailment so the medical doctor is able to properly take care of the patients.
The makeup of bacteria plays a vital role of what medicines work and which do not. The chemical reactions of the bacteria, in addition, play a massive part in the value of antibiotics. Most antibiotics modify or restrain protein makeup, hinder transcription, hamper translation, have an effect on cell membrane makeup, or rework cell-wall combination. Bacterial opposition is another relevant medical basis for classification. The progression of bacteria has made treatment of ailments much more complicated, which has been augmented by the inappropriate use of antibiotics. Consequently, it is imperative to discern the chemistry, makeup, and resistance of the pathogenic bacteria.
A burn casualty was being treated at a hospital and abruptly caught a severe infection. The symptoms of the patient consisted of ooze of puss from the injuries and difficulty breathing because of a lung infection. The symptoms by themselves were not adequate to classify the bacteria and due to the seriousness of the physical condition of the casualty, it was essential to establish the precise pathogenic bacteria to minimize the quantity of needless antibiotic use, which could be damaging to the casualty. Regrettably, there were not any Analytic Profile Index (API) test strips remaining in the infirmary, which have been used to classify unidentified bacteria since 1986. It was not lawful to dispatch tests between hospitals and new consignments from the contractor were not expected for no less than a week, so a new game plan needed to be implemented immediately to be of assistance in saving the casualtyââ‚¬â„¢s life. The medical laboratory got in touch with a neighboring school and was able to get hold of tests to aid with the detection. The tests studied the capability of fermentation for several sugars, the existence of diverse enzymes, and other biochemical reactions.
There are two ways to classify these microbes that are culture-dependent and culture-independent (molecular). Various methods have been employed for understanding the diversity of microbes in these environments. These organisms have evolved several structural and chemical adaptations, which allow them to survive and grow in extreme environments.
The classic methods to identify bacteria go by the characteristics observed in known strains with predictable biochemical and physical properties under optimal growth conditions. Identification by gene sequencing is more objective, does not require optimal growth or a viable microorganism, and has the added capability of defining taxonomical relationships among bacteria. Identification of the bacteria is possible using the gram stain. In Gram-positive cells, peptidoglcan makes up as much as 90% of the thick, compact cell wall, which is the outermost cell wall structure.
While for Gram negative bacteria the cell walls are more chemically complex, thinner, and less compact. Peptidoglcan makes up only 5-20% of the cell wall, and is not the outermost layer, but lies between the plasma membrane and the outer membrane. This outer membrane is similar to the plasma membrane, but is less permeable and composed of lipopolysaccharides, a harmful substance classified as an endotoxin.
The purpose of the experiment is to identify two unknown bacterial species by gram stain. This gives a difference in color due to the different bacteria present. This will help in determining which bacteria are present in which particular environment and their adaptations to these environments. This will help in finding the effects of these bacteria on humans and other living things. This will also help in finding the cure of the effects of these bacteria.
The materials to identify the unknowns include; broth culture which contains the bacteria t be identified, crystal violet is the primary stain which is used to discolor the bacteria, grams iodine used to bind the bacteria to the slide, 95% ethyl alcohol to discolor the stain, and safranin for the interpretation of the biochemical reaction and is also a counterstain.
Others include a Bunsen burner used to heat the broth and inoculating loop and needle for separating the bacteria from each other. A staining tray and immersion oil, which helps in viewing the bacteria through a microscope, are also necessary. Lens paperââ‚¬â„¢s use is to check the change in color. The others are bibulous paper, microscope for viewing the bacteria and glassware-marking pencil to mark the changes and differences as observed.
A Trypticase soy agar broth subculture of the unknown bacteria was prepared and refrigerated following incubation. This culture was to be used if there was suspected contamination of the test culture during the identification procedure. A gram- stained smear of the original unknown culture was prepared and the smear examined to record the observation.
Examination of all the biochemical test cultures and their observations and results recorded. Because most bacteria have one of these two types of cell walls, this difference can be used as a feature that can be identified using the Gram stain. The Gram stain is a differential stain that uses two dyes to differentiate between the two basic bacterial cell wall types. A bacterial smear must be heat fixed to microscope slide and a smear of bacteria suspended in a small amount of water on a slide.
Drying the sample is by using heat, which kills the bacteria and attaches the sample to the slide. The slide was flooded with crystal violet, which is the primary stain, which was then rinsed off after a minute with water. The slide was then flooded with iodine, which is a mordant, that binds with the crystal violet and unable to exit the Gram-positive peptidoglycan cell wall. Water was used to rinse the slide. After a minute, it was flooded with acetone alcohol, which is a decolorizor that will remove the stain from gram-negative cells.
The slide was again rinsed with water after about 10 ââ‚¬" 15 seconds, which may remove the stains from the gram-positive cells as well. The slide was then flooded with safranin which is a counterstain and rinsed with water after a minute.
The slide was then dried gently which is then viewed under oil immersion with a bright field compound microscope. The Gram-positive cells will appear purple while the gram-negative cells will be pink having retained the counterstain after the primary stain was removed by the decolorizor. For Gram positive bacteria the broth contains the cocci, micrococcus spp., staphylococcus spp., and streptococcus spp., which was passed through the catalase which gave negative results which means that there is presence of enterococci and streptococcus spp. This was passed through bile esculin, which gave a positive result thus the presence of E. feacalis and S. bovis. This was passed through a 6.5% sodium chloride broth and there was no growth hence the presence of S. bovis in the culture.
For the Gram negatve bacteria which contains bacilli, citrobacter spp., entrobacter spp., Escherichia spp., klebsiella spp., proteus spp., and pseudomonas spp., was passed through lactose which gave a negative result which shows the presence of proteus spp., and pseudomonal spp.. This was then passed through glucose, which gave a positive response for the presence of proteus spp. This was passed through indole, which gave a positive response hence the presence of P. rettgeri, and P. vulgaris, which was passed through H2S production. This gave a negative response hence the presence of P. rettgeri.
If the discolorizer was to be left too long then the Gram-positive cells would have been discolored and therefore the exact results would not have been found adequately by the gram stain test. Therefore extra caution was to be taken for the decolorizer not to stay for long approximately 10-15 seconds. Other tests could be used to determine the unknown bacteria for example by gene sequencing which give accurate results.
These bacteria are usually found in nasal passages, skin and mucous membranes, which cause a wide range of infections as well as food poisoning, and toxic shock syndrome hence they have to be identified to prevent or cure these infections. The unknowns could be identified to be P. rettgeri for Gram negative and S. bovis for Gram-positive test. This shows that physiological tests and stains can identify human pathogens.
The tests used all through the test may be rather old-fashioned, but still have applications in the globe today. Students of microbiology and laboratories employ the tests and can be used in medical labs, if desirable. An improved method for identifying unfamiliar bacteria is to employ API testing strips. The API test is a much more proficient since it is much faster and all tests are done on a solitary narrow piece that can fit in the hand. The employment of the API tests is of vast significance in the medical turf too. The suitable classification of infectious bacteria assists doctors to establish the right antibiotic treatment, which saves countless lives.