BIO221 BIO 221 Lab Module 8 -- Biochemical Assays Lab Report. Already Graded A.

Lab Module 8 – Biochemical Tests Lab Report Name: After reviewing the Biochemical Tests Introduction and Background document, answer the following questions below. 1. Phenol-Red Fermentation Assays a. What ar ...

Lab Module 8 – Biochemical Tests Lab Report Name: After reviewing the Biochemical Tests Introduction and Background document, answer the following questions below. 1. Phenol-Red Fermentation Assays a. What are two common by-products of sugar fermentation? How is phenol-red broth designed to measure both? Two common byproducts of sugar fermentation are acid and gas (mainly hydrogen and carbon dioxide). Acid production - Acid production is indicated by the color changes of medium from normal red to yellow and will remain red for no acid production. Gas production - During fermentation, any gas produced by the microbes gets trapped in the inverted Durham tube and obvious bubbles appear in the inverted Durham tube. b. What does a pink-red color after incubation indicate about the bacterium of interest? Explain your conclusion. It indicates the production of nitrogenous products (pH > 8.2) c. What does a yellow color after incubation indicate about the bacterium of interest? Explain your conclusion. Phenol red is yellow at the pH < 6.8 indicating the production of acid by the fermentation of carbohydrate. If the organism is able to utilize the carbohydrate, an acid by-product is created, which turns the media yellow. 2. Triple Sugar Iron Agar a. What happens when fermenting bacteria uses up all the glucose present in TSI media? Explain your conclusion. The glucose fermenting bacteria ferment the glucose present in the media and produces acidic byproducts leading to the drop of pH in the media. This pH drops results in the phenol red to change its color from deep red to yellow. During the initial hours, the full media will become yellow due to the fermentation of the whole glucose present in the media, but after that the color of the slant will become red again. This is because the amount of glucose present in the slant is less as compared to butt where more media is present. The low acid production in the slant oxidizes rapidly causing an increase in the pH to alkaline condition. Thus, the color of the slant will become red. Hence, after depletion of the entire glucose the TSI media will see as yellow at the bottom and red color at the slant. But some glucose fermenting bacteria are capable of fermenting lactose or sucrose also, in such cases after depletion of the glucose; they will start fermenting lactose or glucose. b. Why is hydrogen sulfide H2S production observed primality in the butt of the tube? Explain your conclusion. The H2S produced during the reaction and reacts with the ferrous sulfate present in the medium. H2S is a colorless gas and when it reacts with ferrous Sulphate - produces ferrous sulfide. Ferrous sulfide is black and is insoluble in the media leading to form as a black precipitate at the bottom of the tube. c. How is sulfur reduction and glucose fermentation observed in TSI media? Explain your conclusion. Some bacteria use thiosulphate anion present in the medium as a terminal electron acceptor leading to reducing it to sulfide. The H2S gas produces reacts with the ferrous sulphate and produces ferrous sulfide, which will be seen as a black precipitate at the bottom of the tube. Some examples of H2S producing bacteria are Salmonella, Citrobacter etc. The glucose fermenting bacteria, ferment the glucose in the media and produces acidic byproducts causing a pH drop in the media. In acidic condition the color of phenol red will become yellow. During the initial hours of incubation, the full media will become yellow (both slant and butt) due to the fermentation of the glucose, but after that the color of the slant will become red again. This is because the amount of glucose present in the slant is less as compared to butt where more media is present. The low acid production in the slant oxidizes rapidly causing an increase in the pH to alkaline condition. The color of the slant will become red and the observation of TSI slant for glucose fermenting bacteria will be yellow but and red slant. 3. Catalase Test a. What benefit is derived from the actions of catalase for a bacteria? The catalase promotes the breakdown of hydrogen peroxide to water and oxygen and also protects the bacterial cell from the reactive oxygen species that are potentially harmful for the cell. It protects the cell from oxidative damages. b. Why are bubbles produced by catalase-positive organisms after the addition of hydrogen peroxide? The bubbles produce the oxygen that is liberated by the splitting up of the hydrogen peroxide into water and oxygen molecule. The oxygen liberated is pure. The catalase in the cells catalyzes the splitting of the hydrogen peroxide into water and oxygen. c. What is the benefit of using hydrogen peroxide for cleaning out a wound? Explain your answer. The hydrogen peroxide when added to the wounds gets split up and produces the free oxygen gas that bubbles up by removing the debris and they also kill the pathogens by breaking their cell walls. The hydrogen peroxide also acts as a signaling molecule that causes the effector cells to respond and promotes the healing of the wound. 4. SIM Media a. Why is it important to only use a single stab to inoculate SIM medium? It is important to inoculate motility medium with single stab as in the absence false positive might result. A single stab help us to determine if the has move outside the stab, while multiple stabs make us difficult to see if the bacteria were restricted. b. What is the significance of indole? Indole production helps us to identify Enterobacteria. Such bacteria have tryptophanase enzyme that is able to hydrolyse tryptophan and produce indole. Also regulates various aspects of bacterial physiology, including spore formation, plasmid stability, resistance to drugs, biofilm formation, and virulence. C. What organism species are able to produce hydrogen sulfide and therefore produce a black precipitate. Sulfur reduction test is useful to identify enteric organisms for example Salmonella typhimurium. 2 species Salmonella and Proteus spp. 5. MR/VP Assays a. Why is it important for some bacteria to neutralize their acid end products? The stable production of adequate acids required to overcome the phosphate buffer. This results in a pH of below 4.4. If the pH indicator (methyl red) is added to the culture broth and the pH is below 4.4, direct production of a red color. The pH is above 6.0 and the mixed acid fermentation pathway is utilized when MR turns yellow in color. b. Why is an orange color considered to be negative in the methyl-red test? The presence of stable red color on the surface of medium indicates sufficient acid production to lower the pH 4.4 and constitutes a positive test. Other organisms may produce small amounts of acid from the test substrate, which is intermediate orange color (between yellow and red). It is not considered as positive test. Large amounts of acid results in significant decrease in the pH of the medium below pH 4.4. This is visualized by using pH indicator, methyl red. To produce a color change, the test organism should produce large amount of acid from the carbohydrate substrate, which is used. Also some bacteria neutralize acid- by products, taking pH to good level for growth continuation. c. Why does the VP-A and VP-B reagents turn the media red in the VP test? Certain bacteria produce acetoin to acetyl methyl carbonyl chief end product of glucose fermentation. In the presence of alkali and atmospheric oxygen acetoin is oxidized to diacetyl which reacts with alpha naphthol to give red color. 6. Simmons Citrate Agar a. What is the function of citrate and what role does it play in the growth of the bacteria? Organisms growing on Simmons citrate agar are capable of using citrate as the sole carbon source and they can metabolize the ammonium salt in the medium. Use of citrate results in the creation of carbonates and bicarbonates as byproducts. The antimicrobial effect of citric acid by lowering the pH, studies have indicated that the chelating or binding metal ions, the substrate for bacterial growth is diminished in the food, and influencing growth. Sodium citrate showed killing Staphylococcus aureus and Staphylococcus epidermidis at less under neutral pH b. Why does a positive Simmons Citrate Agar result in the slant turning blue? When bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to above pH 7.6. Use of citrate results in the creation of carbonates and bicarbonates as bioproducts. Organisms degrading citrate must also use the ammonium salts, producing ammonia, thus increasing the pH of the medium. The increase in pH then causes color change is the bromthymol blue indicator, turning it blue.