Microbiology Lab
Abstract
This laboratory report tries to recognize an unknown bacterial species. The Gram stain disclosed bacteria to be both gram-negative and positive and via microscopy it confirmed to be rod and cocci respectively. Now two different bacteria were on the agar. One was a coliform and other a non coliform. The bacteria were exposed to all essential microbiological techniques employed to recognize an unknown bacterial species. The unknown comprised about 1% of the total bacterial flora of humans and even most of the warm-blooded animals. The bacteria are the most extensively studied prokaryotic organism, and an essential species in the fields of microbiology and biotechnology, where it has functioned as the host organism for a large amount of work with recombinant DNA. I appeared to have gram negative rods and gram positive cocci in the agar plate. In the meantime, a varied culture was streaked onto a Nutrient Agar plate, EMB and MacConkey Agar plate, so as to achieve isolation of pure cultures. Hence, the major dichotomous key could be prepared.
On the basis of the preamble dichotomous key, the subsequent step was to recognize the particular name of the anonymous bacterium having a genus of Bacillus as well as cocci. This step was merely depended on the biochemical properties of the bacteria. A series of tests was employed for the detection of these bacteria.
Introduction
Prokaryotes are a very assorted collection of living organisms that demonstrate the vigor of nature in their most unusual form. They belong to the kingdom Monera, therefore they have a distinctive genetic evolution dissimilar from all other kingdoms. For the purposes of laboratory, the Gram stain divides bacteria into three main groups which are gram-positive, acid fast bacteria and gram-negative. The Gram stain is actually dependent on the composition of cell wall of particular bacteria. It can be explained e.g. that cell wall of one bacteria take up the primary stain and retain it, another bacteria cell wall only takes up the secondary stain and lastly the Gram incompatible bacteria that may take up any stain and keep it or show conflicting results. Prokaryotes genetically, are regarded as primordial compared to other organisms for example animal cells as they are short of cellular compartments. They intermingle with nearly all higher living systems existing on the earth in any form such as pathogens or in more proper associations (Dixon, 1994). They are even rejuvenated as one of the most imperative saprophytic if not the most significant saprophytes in survival at present.
We all know bacteria are omnipresent in nature. This indicates that they colonize in all probable ecologic niches acknowledged by man including geothermal vents along with boiling water and steam, and very hostile environment for life. This just demonstrates the versatility of the bacteria. In this experiment, this fact is epitomizes to a large extent. The food that man eats certainly contains bacteria in it. As the plant or animal dies, their decomposition begins instantaneously; therefore a requirement to preserve food occurs. It is said that the human body comprised more bacterial cells as compared to the body cells. Nevertheless, a large number of such bacterial are not detrimental to humans but most of them are opportunistic pathogens. The opportunist pathogens are those that attach the persons who are immune-compromised such as person’s immunity reduces after any organ transplantation; pathogen enters the human body and infects them. Examples comprise E. coli and Proteus bacteria both of which are present in the gut. Various drugs have now developed resistance against many bacteria; the more prominent are Klebsiella and Proteus.
The literature just presented above chiefly explains about bacteria and their atmosphere. Now the main attention is on the bacteria and its influence on human beings. As we know, the genetic growth of the bacteria is different to other life forms, so they may share the essential principles of metabolism for example reproduction and production of energy but they are very dissimilar at the metabolic level (Weaver, Debora. Tart, Rebecca, 1999). In case a person develops septicemia due to any type of bacterial, the medical doctor administers a little dose of drugs that particularly target that specific bacteria causing infection while keeping the cells of the body fully protected and intact hence showing a main dissimilarity
As it was mentioned earlier, bacteria interrelate differently with other life forms. There are several species of bacteria recognized by man with different utilizations or different ailments they bring about. The drugs advised for different diseases are dissimilar though basically have the same outcome of exterminating the disease causing agent. Consequently, there are many tests that have been devised to categorize bacteria and to even discover the particular bacteria. Bacteria colonize different niches across the globe.
There have been a great diversity in the area of Microbiology and different organisms come up with different results with the same biochemical typing procedures (Alexander et. al., 2004) such as, some bacteria show motility while others do not therefore by this characteristic only, one can eradicate a handful of bacteria present in the relevant sample. Likewise, different bacteria illustrate different reaction with the same nutrient, e.g. indole for metabolism. It is of serious significance to always keep in mind that, recognition must always be carried out in pure cultures. Hence, two successful culture plates were used in this experiment to obtain isolated colonies. The purity of these colonies was confirmed by the Gram stain. This method was used owing to its less time consumption as well as least expensiveness.
When contamination is discovered to be present, the whole experiment is performed again; otherwise it will affect the results. Also, a few bacterial species vary closely from other bacteria; and can be easily differentiated from just one test (Weaver, Debora. Tart, Rebecca, 1999). Owing to this thought, one can now make a hypothesis for the experiment performed.
The null hypothesis affirms that some species of bacteria will be recognized depending on its distinctive characteristics via removal and double removal whereas the other hypothesis would affirm that all bacteria are fundamentally the same therefore growing them in dissimilar media will provide with no benefit as they will all produce.
Methodology
The unknown microorganisms were present in a nutrient agar, comprising coliform and noncoliform bacteria. The morphology viewed from the plate under the microscope was circular shape, undulate margin, raised elevation, small in size, smooth texture, dull appearance, pigmentation is cream, optical property was opaque.
Figure 1. Agar plates
The bacteria were streaked on 2 streak plates while 2 slants were also prepared and placed one of each at 370C and the other 2 incubated. The first streaking was done at two different temperatures i.e. 300C and 370C. The growth pattern, color of the slant as well as the texture of the working stock culture were seen. This was also carried out on the pure colonies that had been grown in the T-streak plate. This was performed in order to separate a pure culture from the mixed culture.
The pure culture that was obtained tested negative and positive on the Gram-stain. The culture took up the initial stain and appeared red as well as purple. This validates that the unidentified bacteria used in this experiment must in some way or is expected to interact with humans. This elucidates its most favorable growth temperature. Thus it would be of fundamental importance to watch asepsis within the lab and therefore disinfection of that place is of supreme importance since the human skin has several resident bacteria.
Figure 2. Microscopic picture
The most favorable growth condition (temperature) was then found out. The pure culture grew best when incubated at 370C. The colonies were isolated at 370C then streaked was performed on colony one and colony 2 on a EMB and MAC plate as shown below.
Figure 3. EMB agar
Figure 4. EMB agar
Figure 5. MAC agar
Figure 6. MAC agar
The next step was the making of a dichotomous key on the basis of the characteristics obtained from the experimentation. Microscopy was then carried out to disclose the shape of the particular bacteria. The gram-negative bacteria from the pure culture were bacillus as they emerged as rod shaped while positive ones from the pure culture emerged as cocci. The size of the bacteria was also found out. (Stanier,1986).
The next step was biochemical typing. Here, the bacteria were come into contact with different biochemical tests that facilitated recognizing the species of the bacterium.
All of the following tests were performed to detect the unknown bacteria after the EMB or MAC plates.
- Glucose
- Lactose fermentation test
- Manitol test
- Indole test
Results
The bacteria were initially exposed to Gram stain so as to view the morphology as well as physiological properties. The bacteria stained negative and positive both for the Gram stain. Examination under the microscope demonstrated that the bacteria were Bacillus and cocci.
Figure 7. Microscopic picture showing gram positive and gram negative bacteria
The results from EMB and MAC plate and re isolation of two colonies and restreaking of plates were:
Figure 8. Agar plate
Figure 9. MAC
Figure 10. Colony 1 and 2 on agar
Figure 11. EMB agar
Figure 12. EMB agar
The result from glucose test was:
Figure 13. Glucose test
Figure 14. Glucose test
The results of Lactose Fermentation were:
Figure 15. Lactose Fermentation
Figure 16. Lactose Fermentation
Chart #1
This is the first dichotomous key
Key: P = Polymorphic. Reference: Gideon and Bergey’s Manual
In the above chart, bacteria are separated into two big groups i.e. gram negative and gram positive. It is central to note that the following tests were determined by the controlling tests specifically the Gram stain and microscopy that was carried out.
Chart #2
The given section of the results is the comprehensive dichotomous key (Chart #2).
- Gram Stain +
- + - +
Bacillus subtilis B.cereus S. epidermidis S. aureus
- +
-
+
Proteus vulgaris
+ -
Escherichea coli
Shigella flexneri
Legionella pneumophila
+ _
+ _
Salmonella typhimurium Pseudomonas aeruginosa
Shigella flexneri Klebsiella pneumonia Enterobacter aerogenes
_ +
Shigella flexneri Pseudomonas aeruginosa
Alcaligenes faecalis
_ +
Pseudomonas aeruginosa Alcaligenes faecalis
Light green Of-white, grayish gray pink
Blue green Colorless translucent
Shiny glistening
Shiny to greenish
Key
-: negative
+: positive
Discussion
The results above has not disclosed about the unknown bacteria. The Gram stain and microscopy have not made known the genus of the unknown bacteria via observation of morphological characteristics. The Gram stain had not decreased down the list of supposed bacteria. The most favorable growth temperature however additionally removed other bacteria unable to interact with human beings. Streaked colony 1 and colony 2 on an EMB and MAC plate were observed. Different colors were detected. Colony 1 showed gram positive, non coliform bacteria which were for sure S. Aureus while other colony 2 was gram negative, coliform. Neon green and shiny and metallic colors were the prominent one on EMB. Escherichea coli, Klebsiella pneumonia and Enterobacter aerogenes bacteria appeared positive for lactose fermentation. Glucose test was positive for S.aureus. Indole test was also performed which but results have not been studied yet.
Conclusion
Based on the results from the tests, the unknown bacteria were still needed to be confirmed. However, certain bacteria are identified and separated. One now has enough evidence to believe the null hypothesis which affirmed that some species of bacteria would be recognized on the basis of their unique features through removal and reject the substitute that affirmed that all bacteria are essentially the same therefore growing them in dissimilar media would give no benefit as they will all grow eventually.
References
Alexander, S.K., Strete. D, and Niles, M.J. (2004) “Laboratory Exercises in Organismal and Molecular Microbiology”. Boston; McGraw Hill higher Education, p123-158.
Bergey, Berge'ys Manual of Systematic Bacteriology. 2. Baltimore, Philadelphia, Hong Kong, London, Munich,Sydney,Tokyo: A Waverly Company, 1986. 965-1599. Print.
Kramer, J.M., Gilbert, R.J., Bacillus cereus. In: Doyle, M.P. (Ed.), “Foodborne Bacterial Pathogens. Marcel Dekker,” New York, pp. 22-70. 1989.
Lodish; Harvey with Berk, Matsudaira, Kaiser, Krieger, Scott, Zipursky and Darnell , “Molecular Cell Biology,” 5th edn, W.H. Freeman and Company,2004.
Richard L. Hellmich et al, “Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen,” 2001, Pubmed Central, The National Academy of Sciences. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC59744/ retrieved on 4th April 2013.
Stanier, R. and others. “The Microbial World.” 1986
Strunk Jr, W., White, E. B.,. “The Elements of Style, third ed. Macmillan,” New York. 1979
Weaver, Debora. Tart, Rebecca. “Laboratory Exercises in General Microbiology. 2nd. New York: The McGraw-Hill Companies,” 1999. 1-130. Print.
