Pre-Lab Questions1. What is the difference between chemically defined and chemically complex media? Give either a clinical or environmental research example for which each media type would be the most appropriate choice for culturing microorganisms.Chemically defined media contains ingredients in known chemical composition while complex media contains unknown ingredients. Chemically defined media provide only the required nutrients in defined concentration while complex media provides varied growth factors in excessive concentration. Defined media is used in culture of in vitro cells while complex media is used in growth of fastidious pathogenic bacteria and viruses. Glucose broth is an example of defined media and nutrient broth is an example of complex media.2. Why is differential media typically inoculated with isolated colonies that have been previously cultured on general growth media?Differential media are growth media that allow the growth of certain bacteria so that they can be innoculated. They help to distinguish between closely related organisms. The difference can be visually distinguished by certain biochemical reactions that may be caused by one species but not by the other.Innoculation of differential media is done to obtain reproducibility of quantitation results. When bacteria grow on general growth media there is no way to distinguish the different species or groups. When innoculated on the differential media, they can be isolated on the basis of the biochemical reaction they might or might not cause.3. Use a textbook or a reputable internet source (such as www.cdc.gov) to research and describe a scenario in a lab or clinical setting in which a selective and/or differential test would be necessary.Experiment 1: Bioprospecting for Starch Degrading BacteriaData Tables Table 3: Starch Agar and Gram Iodine Results Sample Growth on Starch Agar? (Yes or No) Clear area around colonies? Do these bacteria contain amylase? A yes yes yes B yes yes yes C yes yes yes D NO NO NO Post-Lab Questions1. Why is cow manure used as a potential source of starch-degrading bacteria? (If you are not familiar with the process of digestion in cows, use a reputable internet source to inform your answer.)2. What are some other potential sources of starch-degrading bacteria?Cow dung have a fecal composition that contains lower amounts of protein and a higher percentage of starch. cows fibrous diet dictates a lower need for amylase in the small intestine. The microorganisms found in the cattle manure will harvest the starch as their main source of food instead of protein. so, the cow dung acts as a potential source of starch degrading bacteria.3. What component makes starch agar selective for starch-degrading bacteria?Starch degrading bacteria are most important for industries such as food, fermentation, textile and paperstarch-degrading bacteria in pure culture from soil using a starch laden agar mediumThe two bacterial strains Bacillus amyloliquefaciens and Bacillus licheniformisCow manureMillions of enzymes, each with a specific role, are required in nature to break down compounds during the decomposition process (and make compounds in catabolic processes). By purifying these enzymes and producing them on a large scale, they can be used for a vast range of applications in the service of mankind. Humans have used bacteria and the enzymes they produce for thousands of years (e.g. cheese making). Common exploitation of microorganisms for decomposition of substances include use of bacteria in sewage treatment plants and in composting. On a more experimental basis microbes have been used to decompose contamination from oil spills and other hazardous contaminants in ground water (bioremediation). Almost all enzymes used in these applications originate from microorganisms found in the soil4. Why were each of the following steps performed in this experiment?a. Serial dilution: Serial dilution: It is normally done if the chemical concentration is too high than the desired composition. It is used to diminish the thick culture to a more usable concentration.b. Growth on the nutrient agar plates: Growth on the nutrient agar plates: Growth on the nutrient agar plates is carried out for isolation and purification of cultures. This is used to produce the bacterial lawns needed for the tests. When the growth appears on the agar we are able to perform our startch degrading bacterial experiments. Therefore, this is used for isolation and purification of culture ultimately.c. Streak on the starch agar plates: Streak on the starch agar plates: Streak on the starch agar plates is a qualitative isolation method in order to isolate and sample individual bacterial colonies.Experiment 2: Selection and Differentiation of Body Inhabiting Gram-Positive BacteriaData TablesTable 4: Experiment 2 MSA Growth Observations Surface Tested Growth (Yes or No) – Nutrient Agar Growth (Yes or No) – MSA Agar MSA color around colonies (Red or Yellow) Other Observations SKIN YES NO NONE NONE NOSE YES YES YELLOW CLOUDY CONTROL NO NO NONE NONE TABLE NO NO NONE NONE Post-Lab Questions1. What chemical in MSA confers selectivity? How?Mannitol Salt Agar (MSA) is both selective and differential for gram-positive bacteria, containing ahigh (7.5%) salt (sodium chloride)concentration, which makes it selective for Gram positive bacteria since this level of salt is inhibitory to most other bacteria., and mannitol as the carbohydrate source for fermentation, which makes it differential. It encourages the growth of a group of certain bacteria while inhibiting the growth of others. The high concentration of salt (7.5%) selects for members of the genus Staphylococcus, since they can tolerate high saline levels. Organisms from other genera may grow, but they typically grow very weakly.2. What chemical makes MSA differential? How?sugar mannitol and the pH indicator phenol red3. Why are the nutrient agar plates used in this experiment?Luria-Bertani broth or lysogeny broth as well as nutrient agar are both used to promote growth. Neither are selective or differential for any bacteria but provide a medium for as many types microbes as possible. LB has many protein sources like tryptone, casein or yeast extract, glucose, vitamins, minerals and trace elements and salt. Nutrient agar contains peptone, year extract and salt.4. Was there any growth on you MSA plates? Did any of the colonies change the color of the MSA? What does this tell you about the bacteria taken from each area of your body?Mannitol salt agar is both selective and differentiated medium. It has high concentration of salt, 75% of grow to halophilic.Experiment 3: Selection and Differentiation of Body Inhabiting Gram-Negative Bacteria from Liquid SamplesData TablesTable 5: MacConkey Agar Results Sample Growth? (Yes or No) Colony Color (Clear or Red) Analysis Water from the fridge Yes red Experiment 3 Post-Lab Questions1. What types of bacteria are inhibited on MacConkey agar? What ingredient(s) in MacConkey agar selects against those bacteria?due to crystal violet and bile salt Gram positive bacteria are inhibited on MacConkey agar plates2. What ingredient(s) makes MacConkey agar differential?the presence of crystal violet and bile salt inhibits the growth of Gram positive bacteria and select Gram negative bacteria.3. Using a textbook and a reputable online source (such as the CDC), research and describe some potentially pathogenic members of the intestinal bacteria family Enterobacteriaceae. Which pathogenic species are lactose fermenters that will grow on MacConkey agar?4. Use a reputable internet source to research and describe some potentially pathogenic intestinal bacteria that do not ferment lactose that will grow on MacConkey agar.Non-Lactose fermenting bacteria such as Salmonella, Proteus species, and Shigella cannot utilize lactose and will use peptone instead and will grow on MacConkey agar. This forms ammonia, which raises the pH of the agar, and leads to the formation of white/colorless colonies.5. Use a reputable internet source to research and describe what variations of MacConkey agar can be used to detect other species of bacteria.6. How would you verify that the colonies that grew on a MacConkey agar plate were Gram-negative?MacConkey’s (MAC) medium selectively allows the growth of gram negative bacteria. Gram positive bacteria cannot grow in MacConkey’s medium due to the presence of bile salts and crystal violet. Gram negative bacteria are characterized by their inability to retain crystal violet stain, which is used to differentiate gram positive and gram negative bacteria.7. Look up the formula for MacConkey agar either in a microbiology textbook or online. Is this a chemically defined or a chemically complex media? Why is that important?A chemically defined media is a media in which all of its chemical constituents are known and we also know the ratio in which we use them whereas a complex media is the type of media where the constituents are plant, animal and fungi extracts but we don’t know the proportion in which they are mixed and it is also not uniformly made each time.Since Mac conkey agar has a defined composition in which we know the amount of each constituent we need, it is a chemically defined media.Mac conkey media is important because it is also a selective and differential media that selectively allows only gram negative bacteria to grow.