Growth and Survival of Microorganisms in Different Environments - Lab Report Example

Experiment Effect of temperature on bacterial growth Results: Table The growth of different microorganisms after 7 days of incubation at different temperatures. Microorganism Incubation temperature Notes 4-6°C 20-25°C 35°C 55°C Staphylococcus aureus 0 ++ + 0 Micrococcus roseus 0 ++ + 0 Colonies that grew at 20-25°C were darker in colour compared to those at 35°C Pseudomonas fluorescens 0 ++ 0 0 Bacillus stearothermophilus 0 0 0 ++ Was only able to grow at the highest temperature Saccharomyces cerevisiae + +++ ++ 0 Aspergillus niger 0 ++ +++ 0 At ambient temperature, colonies were white with brown colouration at the center. Increased temperature to 35°C produced black colonies Legend: 0 = no growth; + = scant growth; ++ = moderate growth; +++ = profound growth Questions 1. What generalization can be made from the results of this experiment about temperature required for growth of bacteria versus temperature required for growth of yeasts and moulds? Results of the experiment showed that the temperature at which most of the microbial strains (except for B. stearothermophilus) grew ranged from 20-25°C (Table 1). However, at an increased temperature of 35°C, yeast (Saccharomyces cerevisiae) and mould (Aspergillus niger) strains displayed the greater growth compared to the other microbial strains. These results show that yeasts and moulds are more adapted to higher temperatures compared to bacteria, except for some like Bacillus stearothermophilus which grow well only in high temperatures. 2. What effect does the temperature have on culture characteristics? Generally, as temperature increases, growth rate is also increased until a certain temperature limit, where maximum growth rate is achieved. Increasing the temperature beyond the tolerance limit will ultimately kill the bacteria, while low temperatures will inactivate but not kill bacteria. Temperature can also alter the coloration, opacity, and form of the bacterial colonies. Experiment .2: Resistance of bacteria to heat Results: Table.2: The growth of different microorganisms after exposure to 80°C heat. Microorganism Exposure to 80°C (minutes) Notes 0 1 5 15 Escherichia coli +++ +++ ++ ++ Staphylococcus aureus 0 0 +++ +++ (contaminated after 5 minutes exposure) Bacillus subtilis (overnight culture) +++ +++ ++ + The most dense growth was observed at 1 minute exposure B. subtilis (exponentially growing) ++ ++ +++ +++ The most dense growth was observed at 5 minutes exposure Legend: 0 = no growth; + = scant growth; ++ = moderate growth; +++ = profound growth Questions 1. Are any bacterial spores killed when a spore suspension is heated at a high temperature, for example at 80oC for 10 min? Based on the results of the experiment, there were no bacterial spores that were killed by heating the spore suspension at 80°C at different periods from 0 to 15 mins (Table 2). Bacterial spores have different layers, which make them heat tolerant and immune to attacks from other organisms or the effects of chemicals. The results of the experiment show the differences in heat tolerance of different bacterial cultures, and that heating could actually activate the dormant spores. This observation of heat activation is a basis for increasing sterilization time in order to kill and inactivate bacterial spores. 2. Compare the heat resistance of mould and yeast spores to the heat resistance of bacterial spores. Most of the mould and yeast spores are killed in 10-15 minutes of exposure at 60°C. In comparison, most of the bacterial endospores (spores that are found within the bacterial cells) are killed at temperatures that are higher than 100°C. This is due to the presence of layers and structures in the endospores which enable a certain amount of heat resistance. Dry and moist heat will also affect the thermal time to death. Moist heat is more effective in killing bacterial spores. For example the endospores of B. subtilis and B. stearothermophilis are killed under moist heat at 121°C for 1 minute and 12 minutes respectively. 3. How could the isolation of spore-forming bacteria from a soil sample be facilitated? A soil sample contains a mixture of many microorganisms and bacterial species. To separate the non-spore formers and the spore-forming bacteria, water or buffers are added to the soil sample. The mixture is set aside and the soil particles are allowed to settle to the bottom. The upper liquid portion is removed, and heated to 70 - 80°C for 5-10 minutes. All vegetative forms of microbes and yeast and fungal spores will be killed by exposure to these temperatures, which leaves only the spores of sporulating bacteria. These are inoculated to culture media or agar to multiply the bacteria. Experiment .3: Effect of osmotic pressure on microbial growth Table.3: The response of different microorganisms to salt in the media. Microorganism Concentration of Salt (%) 0 5 10 Enterococcus faecalis +++ 0 0 Vibrio natrigens +++ +++ 0 Legend: 0 = no growth; + = scant growth; ++ = moderate growth; +++ = profound growth Table.4: The response of different microorganisms to sucrose in the media. Microorganism Concentration of sucrose(%) NOTES 0 10 25 30 Saccharomyces cerevisiae 0 0 + 0 Growth was observed only at 25% sucrose, with bubble formation. Conditions for the growth are anaerobic, because the growth was at the bottom of the tubes. Spore suspension of Aspergillus niger +++ +++ +++ ++ Growth was observed even at zero sucrose, implying that A. niger could grow even without sucrose. Legend: 0 = no growth; + = scant growth; ++ = moderate growth; +++ = profound growth Questions 1. What other species of bacteria could we have tried in this experiment? Why do they exist in nature? Many bacterial species have the capacity to grow under different environmental conditions. For this experiment we could have tried to use halophilic bacteria, such as Salinibacter ruber which can survive under extremely saline or salty environments (1). We could have also used lactic acid bacteria such as Lactobacillus buchneri that thrive in decomposing plant matter and utilize carbohydrate products in fermentation process (2). These bacteria can be found in nature, and have evolved to survive the environments where they abound. They have developed stress resistance mechanisms, structures, and modified genes and gene expression levels for adaptation to the stress environments. 2. Is the absence of growth, as observed under some conditions of this experiment, due to microbiocidal or microbiostatic effect? How could this be proved experimentally? Microbiocidal refers to the capacity of the compound to kill the microbes completely while microbiostatic compounds disrupt the growth of microbes. By looking at the results of the experiment, it is difficult to conclude if the absence of growth was due to either microbiocidal or microbiostatic effect. To prove the effect, one can do a sub-culture on regular nutrient media, if there is growth then the treatment resulted in microbiostatic effects. However if there is no growth from the sub-culture, then the original treatment resulted in killing all bacterial cells and was therefore microbiocidal in effect. 3. Considering the outcome of inoculating S. cerevisiae and A. niger on varying concentrations of sucrose, what is the industrial significance of this observation. The growth of S. cerevisiae and A. niger on sucrose are significant because it shows the means by which these microorganisms can be utilized to produce other products from carbohydrate sources. Results for S. cerevisiae do not show this; probably due to some experimental error (Table 4). A. niger, which grows well on sugar, is employed in the industrial synthesis of citric acids, gluconic acid, and many enzymes for producing certain products from sugar substrates. Yeast has many uses in the food industry such as its fermentation of carbohydrates produces carbon dioxide production, which is utilized in producing bread, wine, and beer. 4. Having previously investigated the effect of temperature on S. cerevisiae and A. niger, what process could be employed to control their growth in an industrial setting. In an industrial setting, the growth of yeast and Aspergillus can result in the production of contaminants in the final product. Therefore, all the procedures carried out must be done carefully to reduce unwanted growth of the microorganisms which can be toxic and result in food poisoning. All the materials for food preparation or product preparation should be sterilized by boiling or autoclaving them for a minimum time and temperature. All the raw materials used have to be thoroughly washed, and if juice is to be prepared, this should be boiled for a sufficient period of time. Experiment .4: Bacterial variation due to environmental change Results Describe the pigmentation of S. marcescens after incubation at both temperatures. Figure 1. Agar slants of S. marcescens after incubation at room temperature (left) and at 37 °C. Bright red pigment production peaked at room temperature and was lost at higher temperature. Describe the Gram-stain morphology of S. marcescens incubated at the two temperatures. Figure 2. Gram stain morphologies of S. marcescens were similar when incubated at room temperature and 37°C. Gram stain revealed that S. marcescens is a Gram negative (because the cells were stained red) short rod. Table .5: Growth of P. vulgaris in media supplemented with phenol. Microorganism Phenol concentration (%) Notes 0 0.1 1 Proteus vulgaris +++ + 0 The growth in the first plate with 0% phenol was in all the plate. The growth in the second plate with 0.1% phenol was very limited and surrounded with phenol. Legend: 0 = no growth; + = scant growth; ++ = moderate growth; +++ = profound growth Figure 4. Growth of P. vulgaris on nutrient agar with (Being highly motile P. vulgaris swarms across the plate from its point of inoculation. Phenol prevents this phenomenon from happening. Figure 5. Hanging drop technique shows that P. vulgaris is highly motile. The growth of the culture is dispersing out of the stab. Figure 6: Reddish colour of P. vulgaris colonies grown in a phenol-supplemented media after incubation for 7 days. Questions 1. What was the phenotypic change in the cells of P. vulgaris grown in the presence of phenol? Proteus vulgaris is a highly motile bacterium, and literally “swarms” across the plate. Phenol has an anti-microbial effect, therefore the growth of P. vulgaris is negatively affected (3), (4). Without phenol, the bacterium grew rapidly all over the plate (Table 5), and a thin film is produced above the bacterial colonies. Distinct concentric zones and colour of the colonies were observed. In plates with phenol, the motility and growth of P. vulgaris was inhibited (Table 5). This effect of phenol on bacterial growth has been reported elsewhere (5). 2. What two other activities of the cells of P. vulgaris were correlated with the phenotypic change observed? Did the Phenotypic changes observed in your exercise affect virtually all cells in the culture? P. vulgaris also degrades urea to ammonia, which can increase the pH of the medium. In the presence of phenol red for the overnight one , the colour of the medium changed from yellow to red or dark pink. With time, this colour could get darker. However, incubating the cells at 37°C lightens the colour to light pink, which could be due to the effect of temperature on the rate of urea degradation to ammonia, resulting in lower pH and the lighter colour. It was observed that colonies formed at 37°C were less dense and exhibited limited growth. References: 1. Salinibacter ruber gen. nov., sp. nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Anotn, J, et al. 2002, International Journal of Systematic and Evolutionary Microbiology, Vol. 52, pp. 485-491. 2. Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchneri, with or without homofermentative lactic acid bacteria. Driehuis, F, Oude Elferink, S and Van Wikselaar, P. 2001, Grass and Forage Science, Vol. 56, pp. 330-343. 3. Effect of phenol molecular structure on bacterial transformation rate constants in pond and river samples. Paris, D Wolfe. N, Steen, W and Baughman, G. 1983, Applied and Environmental Microbiology, pp. 45(3):1153-1155. 4. Antiseptics and disinfectants: activity, action and resistance. McDonnell, G and Russell, D. 1999, Clinical Microbiology Reviews, pp. 12(1):147-179. 5. Phenol resistant bacteria from soil:identification-characterization and genetical studies. Ajaz, M, et al. 2004, Pakistan Journal of Botany, pp. 36(2):415-424. Read More