MANIPULATION OF BACTERIA
In this experiment that we performed, there were many methods that were used to help us manipulate and identify the bacteria E.coli on a MacConkey agar plate. The first part of the experiment involved the methods of manipulating, identifying and counting the bacteria and the second part was to find out whether the bacteria E.coli was the only type found in the given area by gram staining. E.coli was the chosen bacteria for this type of experiment. It is a gram negative bacterium that will grow rapidly given ‘any culture medium with the necessary energy source, nutrients, pH, and temperature’. Therefore, MacConkey Agar being the medium for its growth will enable us to achieve the experiment outcome. MacConkey Agar only allows the growth of gram negative bacteria as they do not contain bile salts and crystal violets. This was chosen because it groups bacteria on the basis of colour so therefore will make it easier to determine its type. It contains a pH indicator which is its red colour and also a disaccharide called lactose. This makes the area rich enough for the E.coli bacteria to find it easy to adapt to the environment. Some of the methods that were used in part A were streak plating, viable plate counting, spread plating and serial dilutions. Streak plating involves continuous dilution of the bacteria spread over the surface of the Agar plate using the loop. As the bacteria space out, some multiply and form individual colonies over a period of time. When pure coloured strain of colonies are seen, it means that more than one bacterium is involved. However, if one distinct type of colony is seen then only one type of bacterium is being used which is the result that we expected. Viable plate counting which a method is used to determine the number of cells was also used. A solution that doesn’t alter the growth or decrease of the bacteria is diluted into a tube of the sample. The dilution has to be done in many tubes (until the concentration is estimated to have about 1000 cells per ml) in an orderly manner called serial dilution. Serial dilution is when the dilution factor for all the test tubes are the same meaning that you dilute increasingly with the same factor starting from the neat sample. Furthermore, Spread plating is another method that was introduced to us during this experiment. It helps with the viable plate counting method. After the dilution has occurred, a small amount is poured and evenly spread over the surface of a clean agar plate. The number of bacteria can then be counted after the coloured colonies are seen which helps with the calculation of the bacteria in the original sample. In Part B of the same experiment, Gram staining was introduced for the first time. This method was used to check the identity of the purified bacteria. Assumptions are likely to be made that all the bacteria present in the colonies in each plate are the same type, however in some circumstances this is not the case and so gram staining is to verify. Gram staining uses specific coloured dyes in a certain order to wash the surface of a glass slide which has the bacteria smear on it. After the slide is immersed in oil, the type of bacteria will be determined under a microscope, whether its gram negative if purple or gram positive if pink.
One of the aims for this experiment was to familiarize ourselves with the organism E.coli and understand its characteristics after incubation (in a specific temperature) in a MacConkey Agar plate. Another reason for carrying out this experiment was to practice the many techniques involved in this experiment e.g. streak plating was to teach us how bacteria is spread on an Agar plate using a loop and also the way it multiplies after inoculation. I was to become familiar with the equipments and the solutions that were used to enhance my knowledge. Lastly, having a practical experience carrying out these methods was to help me to understand their...
References: for API 20E tests
• Qinn P.J, Carter M.E, Markey B, Carter G.R. Clinical Veterinary Microbiology (1994) Elsevier Limited
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