The purpose of this practical assignment was based on the identification of auxotrophic requirements in bacteria and antibiotic resistance in bacteria, testing of mutagens and effects of Ultraviolet light on mutagens. The experiments were conducted separately under four different objectives. It was found out that methionine, as nutrient is required for bacterial growth. E.coli bacteria were found to be resistant to tetracycline. In addition, cigarette was found to be carcinogenic and exposure to UV light does not kill cell when the duration is below ten minutes.
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Identification of Auxotrophic prerequisites in Bacterial
Materials such as Bunsen burner, minimal media plates, 70% ethanol, auxotrophic E.coli strain, dips, impregnated filter paper discs and pipette use used in the experiment to identify auxotrophic requirements in bacteria. First, the cultured plates were labeled accordingly at the base. With the help of a pipette, a measure of 100µL of E.coli culture was spread at the surface of plates of minimal medium agar. A special technique known as the sterile technique was employed to place all the eight nutrient impregnated discs in agar in an orderly manner as was labeled. The two plates were then inverted and incubated for a period of 24 hours at a temperature of 37ºC. After twenty-four hours, the plated were observed and examined thoroughly and data entered in the table.
In the identification experiment of antibiotic resistance in bacteria, forceps, spreader, Bunsen burner, 70% ethanol, tips, antibiotic discs, pipette, strain of antibiotic resistant E.coli and a plate of nutrient agar materials were used. First, the nutrient agar plate was labeled at the base. A measure of 100µL of E.coli culture was spread at the surface of NA (nutrient agar) plate. The bacteria was then spread using the sterile techniques. The antibiotic-impregnated filter paper discs were placed on the NA plate using the forceps. Antibiotics were placed at suitable distance from one other.. The plate was then inverted and incubated for a period of 24 hours at a temperature of 37ºC. A table was then filled after the duration of the experiment and after examining the plates properly.
In the testing of mutagens experiment, minimal culture plates were labeled accordingly with a permanent marker. TA100 culture of 100µL was applied on the plates’ surface. The bacteria was immediately spread using sterile technique. In the center of one of the plates was placed a filter paper discs before adding 10µL of test solution to the disc. The same procedure was repeated for all the remaining solutions. The plates were inverted and then incubated for a period of 24 hour at a temperature of 37ºC. The observations were then recorded in a table after examining the plates.
In the experiment on the effect of Ultraviolet light on mutagens, the material used include aluminum foil, 70% ethanol, Bunsen burner, UV lamp, five nutrient agar plates, bacteria spreader, four 6cm by 10cm cards and culture of E.coli. First, nutrient agar plates were labeled accordingly followed by spreading of E.coli culture in the plates using sterile technique. A line was drawn on the plate dividing it into two sections; one was to be exposed to ultraviolet light while the other was not to be exposed to UV light. Plates cover was then removed and the 6cm by 10cm card was placed over one of the sections and secured by a tape. It was then exposed to UV light before the plate covers were replaced. The plates were inverted and wrapped with aluminum foil. This was followed by inversion of the plates, which were then incubated for 24 hours at a temperature of 37ºC. The plates were examined after 24 hours and colonies counted and recorded in a table.
The results of the experiment of Identification of Auxotrophic prerequisites in Bacterial are presented in the table below.
Table 1: Responses of auxotrophic bacteria growth.
The results of the identification experiment of antibiotic resistance in bacteria were presented in a table as shown in figure two below.
Table 2: Growth responses of antibiotic resistant bacteria.
The result of testing of mutagens was presented in a table as shown in the figure below.
Table 3: Growth response of TA100.
|Test Solution||Estimated number of colonies|
|Positive control||Able to have colonies|
|Cigarettes||Able to have colonies|
|Negative Control||No colonies|
In the experiment on the effect of Ultraviolet light on mutagens, the colonies’ count on the plates were observed and presented in a table as shown in the figure below.
Table 4: Effect of Ultraviolet light on bacteria visibility.
|Exposure Time||Number of Colonies|
|Irradiated Side||Non-irradiated Side|
|UV lamp 5s||Yes||Yes|
|UV lamp 10s||Yes||Yes|
|UV lamp 20s||Yes||Yes|
|UV lamp 60s||Yes||Yes|
In the experiment of identification of auxotrophic requirements in bacteria, it was established that of the eight nutrients used, methionine nutrient is the one that showed a growth response (Barth, Woodford & Pitt, 1993). Other nutrients such as histidine, arginine, leucine, tryptophan, threonine, thiamine and proline nutrients did not show any growth. This means that of all the eight nutrient methionine is the single auxotrophic requirements in bacteria.
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In the second experiment where the main objective of the experiment was to identify the antibiotic resistance in bacteria, ampicillin, gentamicin, cephradine, tetracycline and chlorampenicol antibiotics were used in the experiment. The antibiotics were tested to find out those or that, which is resistant to bacteria. It was established that growth appeared in the impregnated disc where tetracycline was applied but there was no growth in impregnated disc where all the rest of antibiotic was applied. It is therefore certain that the bacteria strain is resistant to tetracycline antibiotic (Todar, 2008).
In the other experiment of testing of mutagens cigarettes and 2-aminopurine were found to grow colonies while vinger and haridye did not grow colonies. From the experiment, it is true that cigarette introduced mutation into the TA100 strain. Hence, cigarette has a potential of damaging DNA, making it a carcinogen.
In the experiment of finding out the effects of Ultraviolet light on mutagens, E.coli bacteria was exposed UV light for different duration to find out the viability if the UV light on the viability of bacteria. After the bacteria was exposed to UV light for five, ten, twenty and sixty second durations and 10 minutes to sunlight both irradiated and non-irradiated sides were found to have same number of colonies. Hence, the cells of a single celled organism like E.coli bacteria is not destroyed when exposed to a UV light for up to sixty seconds ten minutes for sunlight (Chaudhary & Gupta, 2010). Therefore, organisms need to be exposed to UV light radiation for their DNA to be damaged.
Barth, A., Woodford, N. & Pitt, T. (1993). Complementation of Methionine Auxotrophs of Pseudomous aeruginosa from Cystic Fibrosis. An international journal, 36, 190-195.
Chaudhary, K. & Gupta, S. (2010). Mutagenic effect of UV- light and X-rays on streptomyces nigrifaciens and yield of the antifungal substance, 27(6), 706-707.
Todar, K. (2008). Bacterial resistance to antibiotics. Web.