MICR 351 Lab Study Guide It is important to know and understand the procedure, purpose, materials used, and biochemical reactions for all of these tests.1. Oxidase:- Reagent used: Oxidase o Indicator is Tetramethyl-p-phenylenediamine- Why is the purpose of the oxidase test? o To identify the presence of cytochrome c oxidase- How is the oxidase test performed? o Transfer a bacterial colony to filter paper. Add a few drops of oxidase reagent to and observe for a color change within 20 seconds.- Why should you read the results within 30 seconds?o The reagents used for this test are unstable and may oxidize independently shortly after they become moist.- Be able to recognize a positive and negative reaction. o Positive = color change to blue/purple in 20 secondso Negative = no color change in 20 seconds2. Catalase:- Reagent used: Hydrogen peroxide- Why is the catalase test used?o To identify bacteria that produce catalase.- How is the catalase test performed? o Bacteria are added to a microscope slide and one or two drops of hydrogen peroxide is added onto the bacteria.- Be able to recognize a positive and negative reaction. o Positive = bubbleso Negative = no bubbles3. MAC- MacConkey:- Why is MAC used? o MAC is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to ferment lactose. It also selects against Gram-positive bacteria.- What does the bacteria look like on the media? o Gram-Positive = Poor growth or no growth  Organism is inhibited by crystal violet and/or bileo Gram-Negative = Good growth  Organism is not inhibited by crystal violet or bileo Probable coliform = Pink to red growth with or without bile precipitate  Organism produces acid from lactose fermentationo Noncoliform = Growth is “colorless” (not pink or red)  Organism does not ferment lactose. - What does MAC do? o- Is this test selective or differential? Why?o Both. The bile salts and crystal violet inhibit the growth of Gram-positive bacteria (selects against) so only Gram-negative bacteria will grow (selects for). It is differential because it differentiates lactose versus non-lactose fermenting bacteria. The lactose fermenting bacteria will lower the pH and appear red. The non-lactose fermenting bacteria will have a higher pH and will be colorless.4. Malonate:- Be able to recognize a positive and negative reaction.o Positive = dark blueo Negative = No color change or slightly yellow- Why is this test used? o Originally designed to differentiate between Escherichia, which will not grow in the medium, and Enterobacter. Its use as a differential medium has now broadened to include other members of Enterobacteriaceae.- Determine the chemical reaction that takes place.o Figure 5.34 on page 345- What causes the color change? o Bromothymol blue dye is added to indicate any shift in pH. If an organism cannot utilize malonate but manages to ferment a small amount of glucose, it may turn the medium slightly yellow or produce no color change at all. These results are negative. If the organism utilizes malonate, it will alkalinize the medium and change the indicator from green to deep blue.o Malonate is a competitive inhibitor of succinate dehydrogenase. In combination with thesubsequent buildup of succinate in the cell, it shuts down the citric acid cycle and will killthe organisms unless it can ferment or utilize malonate as its sole remaining carbon source. 5. Simmon’s Citrate:- Be able to recognize a positive and negative reaction.o Positive = blueo Negative = No color change; no growth- Why is this test used? o It tells about the ability of organisms to use citrate as their sole carbon source and perform citrate fermentation. It differentiates members of the Enterobacteriaceae.- Determine the chemical reaction that takes place.o Figure 5.32 on page 339o Citrate-positive bacteria hydrolyze citrate into oxaloacetate and acetate using the enzyme citrate lyase. From there, oxaloacetate is converted to pyruvate, from which a variety of products can be formed depending on the pH of the environment. Some bacterial fermentation pathways can make ATP and reducing power (NADH or NADPH).- What causes the color change? o Bacteria that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia (NH3) and ammonium hydroxide (NH4OH), both of which tend to alkalinize the agar. Bromothymol blue dye (indicator), which is green at pH 6.9 and blue at pH 7.6, is an indicator and as the pH goes up, the medium changes from green toblue. 6. BEA- Bile Esculin Agar:- Be able to recognize a positive and negative reaction.o Positive = Medium is darkened within 48 hours  Presumptive identification as a member of Group D Streptococcus or Enterococcus.o Negative = No darkening of the medium after 48 hours  Presumptive determination as not a member of Group D Streptococcus or Enterococcus.- Why is this test used? o It is used for the isolation and presumptive identification of bile esculin-positive enterococci (typically Enterococcus) and the bovis groups of streptococci from non-Group D streptococci.- Determine the chemical reaction that takes place.o Hydrolysis of Esculin; figure 4.3 page 249 Esculin –Esculinase β-D Glucose + Esculetino Bile esculin test indicator reaction; figure 4.4 page 249 Esuletin + Fe3+  Dark brown precipitate- What is the color change?o When esculin is hydrolyzed in BEA, the resulting esculetin reacts with ferric ions in the medium to produce a dark brown/black precipitate, which darkens the medium surrounding the growth. - Is this test selective or differential? Why?o Both, it is an undefined differential and selective medium. Bile is the selective agent added to separate Group D streptococci from other non-Group D streptococci. Bile esculin agar contains oxgall (bile salts) to inhibit the growth of gram positive organisms other than enterococci and group D streptococci. It differentiates between Enterococcus and Streptococcus.7. Nitrate:After incubation, 5 drops each of Nitrate A and Nitrate B have been added and the tube mixed.If your media does not turn red after the addition of Nitrate A and Nitrate B, a small amount of zinc must be added. - Describe how this test is performed and the reagent used. o Inoculate the nitrate broths and incubate the tubes at 37°C for 24-48 hours. Add 8 dropseach of reagent A and reagent B to each tube. Mix well and let stand for 10 minutes. If it is not