Bacteria are microscopic single celled organisms which are said to be the smallest yet most ancient forms of cellular life. Bacterial cells are prokaryotic and wide spread. Complex behaviors often associated with the organisms have prompted scientists around the world to keenly monitor the characteristics of various types of bacteria in an attempt to control them (Havey and Cling 42). This field of research has in the past gained popularity especially in the health and medical sectors.
Materials and Methods
Microscopic and scientific technology has been vital in the study of bacteria and other microscopic organisms. This has been achieved through the invention of various test methods that are known to produce results which are specific to a given class of bacteria (Hwang and Ederer 51). Results obtained from the various tests can then be applied to a specific type of bacteria. Most of these tests involve staining to identify the various features of the microscopic bacteria being observed under a microscope.
Organisms/Colonies Morphology Test
A clean and uncontaminated petri dish is removed from a plastic bag and placed on a level surface (Greenwood and Pickett 45). This was done carefully to avoid touching the inside of the petri dish or expose it before the experiment. A 1ml pipette was then filled with water and emptied into the bacteria medium bottle. The medium bottle was inverted twice to mix water with the medium. The mixture of water and medium was then poured into the petri dish and swirled to completely cover the surface. The petri dish was then covered. The dish was then incubated for 48 hours under room temperature. The contents were then observed.
Gram Stain Test
A sample of the unknown bacteria was picked from a colony based on the colour. A drop of purple crystal violet dye was then placed on an uncontaminated slide. A colony from the petri dish was then transferred on to the cover slip and immediately inverted onto the crystal violet dye. Alcohol was then used to wash the slide thus washing the dye out of the cell surface. A counter stain containing pink dye was then used to stain the cells and the cells observed.
A drop of Hydrogen Peroxide was placed on a clean slide. A colony of the unknown bacteria was identified from the contents in the petri dish. A colony was then placed on the cover slip and immediately inverted onto the hydrogen peroxide.
Slide Coagulase Test
Two separate portions of concentrated homogenous suspensions were prepared on a slide (Greenwood and Pickett 45). Three loopfuls of citrated plasma at room temperature were placed next to one of the solutions. The suspension was then carefully mixed with the plasma and left for about 10 seconds. The two solutions were then compared after the 10 seconds.
A strip of a filter paper was impregnated with 1% Tetramethyl-p-phenylenediamine dihydrochloride solution. Forceps were then used to place the filter paper onto a glass slide. A glass rod was then used to pick a colony from a freshly grown culture. The colony was then placed onto the impregnated strip of the filter paper. Forceps were then used to touch the impregnated strip of the filter paper onto the colony. The colour of the colony was then observed.
Tube Coagulation Test
A volume of 24 hour culture was prepared. The volume was then placed in a sterile test tube containing an equal volume of sterile oxalated human plasma and the solution swirled to mix (Havey and Cling 42). The solution was then incubated for 4 hours and was regularly checked at intervals of 30 minutes. The solution was then left for 24 hours.
Acid and Mannitol
0.5 millilitres of 50% sulphuric acid was added into two millilitres of a medium containing the unknown bacteria and then observations made.
A sample of the bacteria colony was inoculated and incubated for 48 hours at a temperature of 37 degrees centigrade. 8 to 10 drops of naphthol, 5 percent ethanol and 8 to 10 drops of 40% potassium hydroxide containing 0.3% creatine were added into a 1 millilitre quantity of the culture and shaken well.
A culture medium was heavily inoculated in a medium bottle. The solution was then incubated for 24 hours at 37 degrees centigrade and observed (Havey and Cling 42). The solution was then incubated for seven days upon negative results.
Deoxyribonuclease (Danse) Test
The unknown bacteria were spot inoculated on the surface of the agar with heavy growth. The bacteria were then incubated for around 18 to 24 hours at around 37 degrees centigrade.
Resistance and susceptibility
Novobiocin and Polymyxin antibiotic solutions were placed in two separate test tubes. Colonies of the unknown bacteria were then introduced into the solutions and observations made.
The cells observed in a microscope were circular in shape. This signified that the bacteria were morphologically cocci. Upon completion of the gram stain test, the bacteria were observed to still possess the purple colour, the colour of the primary stain. This signified that the unidentified bacteria were Gram positive cocci. Bubbles were also observed at the end of the Catalase test signifying the production of oxygen.
The bacteria were thus classified as Catalase positive. At the end of the slide Coagulase test, a uniform suspension was observed signifying that the unidentified bacteria were Slide Coagulase negative. Similar results were obtained in the tube Coagulase test with no coagulation noted in the test tube. This indicated that the bacteria were tube Coagulase negative. The Oxidase test did not signify any colour changes implying that the unknown bacteria were Oxidase negative.
There was no formation of crystal precipitation in the solution in the acid/ Mannitol test making the bacteria acid/Mannitol negative. The absence of colour change during the acetone production test signified that the bacteria did not have any neutral products. There was no colour change noted at the end of the Urease test. This indicated that the bacteria do not produce urea which would have otherwise been indicated by a colour change to bright pink.
A clear zone was observed to surround the colony at the end of the Deoxyribonuclease test. This indicated that the bacteria were Deoxyribonuclease negative. Introduction of Novobiocin and Polymyxin antibiotic solutions produced uncertain observations and the effect of the antibiotics could not be identified as a result of malfunctioning antibiotic disk. Based on these observations, it is clear that the bacteria in question were Streptococcus haemolyticus.
Though the tests were carried out effectively, the microscopic size of the bacteria has continued to be a challenge in the classification of bacteria. Care must also be taken to prevent the individuals carrying out the test from being infected (Greenwood and Pickett 45). The abundance of bacteria in the atmosphere and in the environment may end up contaminating cultures, a situation that would give wrong results. Observation of bacteria under extremely high magnification powers can help in the classification of the organisms.
Greenwood, Girolamo, and Johnson Pickett. Clinical Microbiology, New York: CBS Interactive Network, 2004. Print.
Havey, Cohn, and Johnson Cling. Microbiology, New York: Henry and Holt, 2010. Print.
Hwang, Padmanabh, and Nahavira Ederer. Clinical Microbiology, Harvard: Harvard University Press, 2005. Print.