D.Discussion.
To maintain aseptic conditions and avoid contamination by pathogenic microorganisms while carrying out the experiment in the microbiology lab, it was important to work closer to Bunsen burner during inoculation and streaking of bacteria.
First Experiment The S.marcescens bacterial colonies incubated at room temperature appeared red in color while those incubated at 37o C appeared white. The reason for the bacterial colonies to appear as red was due to the production of temperature dependent pigment called prodigiosin (Anandkumar, N., Giri, A., Muthukumaran, G., Pennathur, G., 2004). Prodigiosin is produced around room temperature (Anandkumar, N., Giri, A., Muthukumaran, G., Pennathur, G., 2004). Prodigiosin is synthesized as a secondary metabolite that is not required for bacterial growth (Francia, A., Loren, J., Rius, N., Sole, M., 1997). The enzymes responsible for the synthesis of the pigment will be deactivated at high temperatures and function optimally at room temperature and hence result in red pigment formation. The temperature difference represented the environmental change here. The red-colonies of S.marcescens were temperature dependent. When P.mirabilis plates were observed for colonies, the plate which was added with phenol exhibited swarming, accumulation of similar sized bacteria (Harley, J., Klein, D., Prescott, L., 2002). This occurred as P.mirabilis colonies became non-motile due to the presence phenol increasing their accumulation on the medium. Presence of phenol has made these bacteria as flagella mutants because phenol causes mutations in FLA genes that are responsible for making flagella in these bacteria. So, swarming colonies were observed. The plates that were not supplemented with phenol did not exhibit swarming.
Second Experiment When mutant E.coli were streaked on a McConkey agar plate, after incubation wild type E.coli were observed to have grown on the medium. Mutant E.coli colonies appear white while wild type colonies appear as red (Gilbride, K., Victorio, L., 2006). Mutant E.coli colonies appear white as they cannot ferment lactose sugar while wild type colonies appear red as they can ferment lactose to produce an acid which appears red. Wild-type E.coli consists of lac gene that can give rise to a protein which helps in fermenting lactose. As the resulting colonies appeared red, it indicated that mutant strains back mutated to wild-type. So the bacteria got back their ability to ferment lactose and produce acid. The mutant E.coli strains used here were lac- which means that they lack gene that synthesize the protein which can ferment lactose and hence cannot ferment lactose in the medium. Appearance of red colored colonies after incubation indicates that there was back mutation occurred that can explain the instability of the mutant forms. But wild type red colonies were not observed to have changed to white colony mutants. This could be because of two reasons One could be, due to slow mutation rate around 1108 (Harley, J., Klein, D., Prescott, L., 2002), which means 1 bacterium in 108 can get mutated and lose its ability to ferment lactose. Due to improper streaking large number of bacterial cells may not have grown. The second reason for white mutant strains to appear as red even though they do not produce any acid is because of long incubation time of the plates for about 1 week. Long incubation could have made the medium to lose its ability to differentiate the mutant strain from wild type E.coli and this might have led to incorrect results. From this it can be inferred that genotype change through mutation influenced the E.coli strains to get back their ability to ferment lactose.
Third Experiment Twenty agar plates were examined and occurrence of mutation along the tryptophan biosynthetic pathway was located. A wild type strain of E.coli, EMG23 is called a prototroph that was able to synthesize tryptophan and do not require its presence in the medium for growth. Other E.coli strains were mutant forms called Auxotrophs that had defect in one or more steps along tryptophan biosynthetic pathway. KS463 and EMG4 had mutation points at A, B and C steps. So these could not grow without tryptophan being supplemented in the medium. But when anthranilic acid was added to the medium there was no growth observed which indicates that the enzyme catalyzing step C of the biosynthetic pathway was not produced. So conversion of Anthranilic acid to tryptophan was stopped. No growth was observed also when only casein hydrolysate or indole was added to the medium. This could indicate that the enzymes catalyzing A and B steps were not produced. EMG5 E.coli strain mutated at step B shows that the ability to synthesize indole glycerol phosphate from Anthranilic acid was lost. This inference was made based on the results which showed that when the medium had acid added to it no growth of EMG5 was observed. But growth was observed when indole was supplemented. EMG6 strain of E.coli had mutation at step A, which was inferred from the results which showed that growth took place when all other molecules except casein hydrolysate acting as source of enzymes at all other steps were added to medium. So from this experiment it is concluded that EMG23 was prototrophic and all other strains of E.coli were auxotrophic and could not synthesize tryptophan.
Fourth Experiment Presence of E.coli colonies was seen on one side of the plate where there was thin layer of medium available, which was supposed to have low concentration of streptomycin. As streptomycin is an antibiotic, it inhibits the bacterial growth. But on the other side of the plate where thick layer of medium was available no growth of colonies was observed. The thick layer medium is supposed to have high concentration of streptomycin. So due to its high concentration the absence of E.coli colonies might have resulted due to low rate of mutation in E.coli to get converted to streptomycin resistant strains. As a result streptomycin resistant colonies were not seen on the medium. Probably streaking was not properly performed to get mutants generated. Proper streaking will help in isolation of pure bacterial colonies that may lead to better results and observations.
First Experiment The S.marcescens bacterial colonies incubated at room temperature appeared red in color while those incubated at 37o C appeared white. The reason for the bacterial colonies to appear as red was due to the production of temperature dependent pigment called prodigiosin (Anandkumar, N., Giri, A., Muthukumaran, G., Pennathur, G., 2004). Prodigiosin is produced around room temperature (Anandkumar, N., Giri, A., Muthukumaran, G., Pennathur, G., 2004). Prodigiosin is synthesized as a secondary metabolite that is not required for bacterial growth (Francia, A., Loren, J., Rius, N., Sole, M., 1997). The enzymes responsible for the synthesis of the pigment will be deactivated at high temperatures and function optimally at room temperature and hence result in red pigment formation. The temperature difference represented the environmental change here. The red-colonies of S.marcescens were temperature dependent. When P.mirabilis plates were observed for colonies, the plate which was added with phenol exhibited swarming, accumulation of similar sized bacteria (Harley, J., Klein, D., Prescott, L., 2002). This occurred as P.mirabilis colonies became non-motile due to the presence phenol increasing their accumulation on the medium. Presence of phenol has made these bacteria as flagella mutants because phenol causes mutations in FLA genes that are responsible for making flagella in these bacteria. So, swarming colonies were observed. The plates that were not supplemented with phenol did not exhibit swarming.
Second Experiment When mutant E.coli were streaked on a McConkey agar plate, after incubation wild type E.coli were observed to have grown on the medium. Mutant E.coli colonies appear white while wild type colonies appear as red (Gilbride, K., Victorio, L., 2006). Mutant E.coli colonies appear white as they cannot ferment lactose sugar while wild type colonies appear red as they can ferment lactose to produce an acid which appears red. Wild-type E.coli consists of lac gene that can give rise to a protein which helps in fermenting lactose. As the resulting colonies appeared red, it indicated that mutant strains back mutated to wild-type. So the bacteria got back their ability to ferment lactose and produce acid. The mutant E.coli strains used here were lac- which means that they lack gene that synthesize the protein which can ferment lactose and hence cannot ferment lactose in the medium. Appearance of red colored colonies after incubation indicates that there was back mutation occurred that can explain the instability of the mutant forms. But wild type red colonies were not observed to have changed to white colony mutants. This could be because of two reasons One could be, due to slow mutation rate around 1108 (Harley, J., Klein, D., Prescott, L., 2002), which means 1 bacterium in 108 can get mutated and lose its ability to ferment lactose. Due to improper streaking large number of bacterial cells may not have grown. The second reason for white mutant strains to appear as red even though they do not produce any acid is because of long incubation time of the plates for about 1 week. Long incubation could have made the medium to lose its ability to differentiate the mutant strain from wild type E.coli and this might have led to incorrect results. From this it can be inferred that genotype change through mutation influenced the E.coli strains to get back their ability to ferment lactose.
Third Experiment Twenty agar plates were examined and occurrence of mutation along the tryptophan biosynthetic pathway was located. A wild type strain of E.coli, EMG23 is called a prototroph that was able to synthesize tryptophan and do not require its presence in the medium for growth. Other E.coli strains were mutant forms called Auxotrophs that had defect in one or more steps along tryptophan biosynthetic pathway. KS463 and EMG4 had mutation points at A, B and C steps. So these could not grow without tryptophan being supplemented in the medium. But when anthranilic acid was added to the medium there was no growth observed which indicates that the enzyme catalyzing step C of the biosynthetic pathway was not produced. So conversion of Anthranilic acid to tryptophan was stopped. No growth was observed also when only casein hydrolysate or indole was added to the medium. This could indicate that the enzymes catalyzing A and B steps were not produced. EMG5 E.coli strain mutated at step B shows that the ability to synthesize indole glycerol phosphate from Anthranilic acid was lost. This inference was made based on the results which showed that when the medium had acid added to it no growth of EMG5 was observed. But growth was observed when indole was supplemented. EMG6 strain of E.coli had mutation at step A, which was inferred from the results which showed that growth took place when all other molecules except casein hydrolysate acting as source of enzymes at all other steps were added to medium. So from this experiment it is concluded that EMG23 was prototrophic and all other strains of E.coli were auxotrophic and could not synthesize tryptophan.
Fourth Experiment Presence of E.coli colonies was seen on one side of the plate where there was thin layer of medium available, which was supposed to have low concentration of streptomycin. As streptomycin is an antibiotic, it inhibits the bacterial growth. But on the other side of the plate where thick layer of medium was available no growth of colonies was observed. The thick layer medium is supposed to have high concentration of streptomycin. So due to its high concentration the absence of E.coli colonies might have resulted due to low rate of mutation in E.coli to get converted to streptomycin resistant strains. As a result streptomycin resistant colonies were not seen on the medium. Probably streaking was not properly performed to get mutants generated. Proper streaking will help in isolation of pure bacterial colonies that may lead to better results and observations.
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