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Objective
The aim of the experiment is to investigate the principles of selective, enriched and differential media on the growth of organism in three types of plates mainly nutrient agar, MacConkey agar plate 1 and MacConkey agar plate 2. Besides, these media are also used to isolate and differentiate selected bacteria to see which media would inhibit or promote their growth in different conditions.
Methods and Materials
Refer to Lab Manual Microbiology 2011 page 63-64
Results
Three plates were used in these experiments, mainly the Nutrient agar plate, MacConkey agar 1 plate and MacConkey agar 2 plate. Using the streak plate technique, a loopful of the mixture is placed onto the nutrient agar plate then the MacConkey agar 1 plate and MacConkey agar 3 plate. The plates were stored at 40C for a day and then incubated at 370C less than 24hours. Then the Gram stain technique was used to determine the microbes if it is gram positive or gram negative. The broth culture provided was Bacillus megaterium, Enterococcus faecalis, Esherichia coli and Proteus vulgaris.
Table 1 : Cellular morphology and the Gram stain of bacterial stains.
Table 2: Various bacterial stains and colony morphology on different media used to incubate the bacterium.
Based on the results obtained in table 1, it can be concluded that of the four bacterium tested for Gram stain, two of the bacterium which is Bacillus megaterium and Enterococcus faecalis were tested gram positive and the arrangement were chain like and pairing respectively. Moreover the other two bacterium which consist of Esherichia coli and Proteus vulgaris both tested Gram negative and both also are observed to be rod- shaped.
All four bacterium were also observed to grow in the nutrient agar plate. But only Bacillus megaterium did grow when placed in the media MacConkey agar plate 1. Besides, both Bacillus megaterium and Enterococcus faecalis did not grow when placed in the media MacConkey agar plate 3.
Discussion.
Nutrient agar is firstly inoculated as all of the four bacterium are able to grow in that media as compared to the MacConkey media 1 and 3. MacConkey 1 and 3 is then inoculated as it contains additives such as dyes which could inhibit the growth of certain bacterium when exposed to it. Therefore in order to prevent this from occurring, the order of inoculation has to be precise so that no contaminants from the MacConkey media would inhibit the growth of other microorganism (Willey et al., 2011)
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The plates are refrigerated then incubated for less than 24hours as when incubation period increases this causes the bacterium cell/ plasma membrane to degrade as incubation period increases. Besides that, the colony forming unit ( CFU ) is also affected by the duration of the incubation period as CFU varies accordingly to the duration of incubation period. Therefore this would apply to the plates as longer incubation time might cause the bacterium growth to decrease or possibly increase drastically. MacConkey agar 1 contains bile salt 2 (oxiod) hence this media is made selective as bile salt 2 supports the growth of enterococci while it inhibits other bile tolerant Gram positive cocci such as staphylococci and non fecal streptococci. On the other hand, MacConkey agar 3 contains bile salt 3 ( oxiod) that would inhibit all Gram positive bacteria such as based on the results which is Bacillus megaterium and Enterococcus faecal (Pattison et al., 2008).
MacConkey agar is also differential as there are two types of bacteria present which is the lactose fermenting bacteria and the non-lactose fermenting bacteria. The lactose fermenting bacteria includes coliforms, Gram positive cocci and rod, and other Gram negative bacteria. Lactose fermenting bacteria could be determined as it appears red or pink as a result of the acidity of the media produced by fermentation of lactose which causes the pH value to drop significantly till the indicator turns to red. These organism are referred to as Lac+ Non-lactose fermenting bacteria or known as Lac – produces beige, translucent or colourless colonies as a result of the ammonia produced by deamination of amino acids in peptone causes the raise of alkalinity in the media hence the indicator would change into the resultant colour (Willey et al., 2011)
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Non-fastidious are organisms produce their own nutrients do not need special agar to grow. Thus, based on this experiment, nutrient agar is a non-selective medium that allows all four organisms to grow which is Bacillus megaterium ,Enterococcus faecalis, Esherichia coli and Proteus vulgaris. As MacConkey agar plate 1 contains bile salt 2, which inhibits the growth of most Gram-positive bacteria except the enterococci. MacConkey agar plate 3 contains bile salt 3 that allows only the Gram negative organism to grow. This can be shown on the table above as when the mixed broth culture is inoculated to the Mac 3 plate, only Esherichia coli and Proteus vulgaris can be seen growing in the media (Vasanthakumari, 2009)
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Conclusion
Based on the experiment, it can be concluded that Bacillus megaterium are rod-shaped and arranged in a chain-like sequence. Besides, Enterococcus faecalis is spherical in shape and usually grouped in pairs. Esherichia coli are rod- shaped and arranged in clusters. Lastly Proteus vulgaris are rod shaped and usually randomly arranged with no particular arrangement. Only the Gram negative bacteria are able to grow on the MacConkey 3 plate . MacConkey agar plate 1 inhibits the growth of most Gram positive bacteria which is why Enterococcus faecalis can be seen growing in the media.
References
Pattison, M., McMullin, P., Bradbury, J.M., (2008). Poultry diseases. 6th edn, pp. 196-197. China:Elsevier Health Sciences.
Vasanthakumari. (2009). Practical Microbiology. pp. 37-38. New Delhi : BI Publications Pvt Ltd.
Willey, J.M., Sherwood, L.M. & Woolverton, C.J., (2011), Prescott’s Microbiology. 8th ed, USA: McGraw-Hill Companies Inc.