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Do a 300 words minimum lab summary. All in one document.

Do a 300 words minimum lab summary. All in one document.
29 Media containing the pH indicator bromthymol blue Simmon’s citrate agar slants A large proportion of disease causing Gram negative bacterial genera belong to the oxidase negative family Enterobacteriaceae (the “enterics”). Many microbiological media/methods exist for the characterization and identification of enteric organisms. The first that we will cover is the differential utilization of citrate (citric acid). Citrate is a carbohydrate which can be utilized as a carbon and energy source by certain enteric genera but not by others. Although the name “enteric” is derived from the fact that these organisms inhabit animal intestines, some enteric genera (see the “aerogenese-group” in the discussion of EMB agar in exercise #10) are found in non-clinical environments as well. These genera are capable of citrate utilization, and include Citrobacter, Enterobacter and Klebsiella. Salmonella is also citrate positive eventhough it is an enteric genus not belonging to the aerogenese-group. Three of our Gram negative non-fermentors (Pseudomonas, Alcaligenes and Acinetobacter) are also citrate positive. Those enteric species found only in animal feces are not capable of citrate utilization. These are referred to as the “coli group” and include the following genera: Escherichia, Shigella, Proteus and our 4th Gram negative non-fermentor, Aeromonas. Simmons citrate agar media contains the pH indicator bromthymol blue, a chemical that is green at neutral pH, and blue at alkaline pH. Organisms capable of citrate utilization will cause the pH of Simmons citrate agar to increase by virtue of the consumption of citric acid (the base NH4OH is generated from NH4H2PO4 during the metabolic process). Any color change from green to blue is considered positive for citrate utilization. To conduct the test, inoculate a Simmons citrate agar slant using your loop. Incubate the slant at 37oC for 24-48hrs. and examine for any color change. SEE IMAGE: Simmons citrate: left negative, right positive. NOTES: * We will only use this test to differentiate Gram negative rods. * Most carbohydrate utilization media will become acidic as the organism produced acid from sugar utilization. Simmons Citrate agar is different. Consumption of the citrate, which is an acid, will result on the media becoming alkaline (basic) resulting in the color change from green to blue. * Any blue color anywhere on the slant constitutes a positive reaction here. O/F Glucose deeps O/F glucose is a semisolid medium incorporating bromthymol blue as a pH indicator, 1 % glucose, and the protein peptone. Here O stands for “oxidative” metabolism of glucose, and F stands for “fermentative” metabolism of glucose. Despite the intended meaning of these terms, the O/F glucose test is actually an oxygen requirement test as it relates to glucose metabolism. That being said, we could take O to mean obligately aerobic, and F to mean facultative. These are the only 2 options as none of our Gram negative rods are aerotolerant anaerobes. As discussed in the citrate lab above, bromthymol blue has a green color at neutral pH. Bromthymol blue turns yellow at an acidic pH. Two O/F glucose tubes are inoculated. One of the tubes is then overlayed with sterile mineral oil which prevents oxygen from diffusing into the medium, and renders this tube anaerobic.  Microbial activity in this tube (the F tube) must be in the form of fermentation. Fermentation carried out by a facultative organism will result in the production of a large amount of acid, causing the F tube to turn yellow. With no oil covering the other tube (the O tube), oxygen will diffuse into the top half of the media allowing aerobic respiration to occur. Carbon dioxide produced from respiration will dissolve in water in the media resulting in the formation of carbonic acid, which will cause the O tube to turn yellow. Facultatives will cause both tubes to turn yellow, which is interpreted as an F result (facultative glucose catabolism). Obligately aerobic organisms will cause the O tube to turn yellow, with no reaction in the F tube. This is interpreted as an O result (aerobic glucose catabolism). We may also see blue coloration, usually at the top of the O tube, due to utilization of the peptone resulting in accumulation of a basic ammonia by-product. The other possible result is “no reaction,” which is no color change in either tube. Results: SEE IMAGES: O/F glucose: F reaction O reaction no reaction  MATERIALS NEEDED:  2 O/F glucose tubes per organism being run sterile mineral oil THE PROCEDURE: Inoculate both tubes of media with a NEEDLE, stabbing through the medium to the bottom as you did for motility tests. Overly 1 tube with ¼ to ½ inch of sterile mineral oil. Incubate both tubes at 37 degrees C for 24-48hrs. Examine tubes for color changes. Expected results: All enterics F (fermenative) Aeromonas sobria F (fermentative), may be weak Pseudomonas aeruginosa O (oxidative) Acinetobacter species O (oxidative) OR no reaction Alcaligenes feacalis no reaction, possible blue in O NOTES: * We will only use this test to differentiate Gram negative rods. * A common mistake on this medium is to apply oil incorrectly. Remember that you should place oil on top of only 1 deep. Place no less than ¼” of oil on the deep.
Do a 300 words minimum lab summary. All in one document.
GRAM NEGATIVE RODS A. Urea hydrolysis Introduction: The urea hydrolysis test (i.e. urease test) indicates the presence of the enzyme urease. Urease decarboxylates urea resulting in the formation of ammonia (NH2) and carbon dioxide. (NH2)2CO + H2O  urease  CO2 + 2NH3 Ammonia is a weak base. Therefore, production of ammonia results in increased pH. As we learned with Mannitol salt agar and carbohydrate fermentation broth, a slightly basic medium containing phenol red pH indicator has a red color. Acidification of these media to near-neutral pH due to acid production from carbohydrate (usually glucose) utilization causes the media to turn from red to yellow. Conversely, ammonia produced in a phenol red-containing medium that begins at a slightly acidic pH will turn from yellow to pink-red. This is the basis of the urea hydrolysis test. As you see below, the urease hydrolysis test can be ran with either agar or broth media, though we will use broth media. The tubes on the right of both images were inoculated with a urease positive organism. The tubes on the left of both images were uninoculated. Inoculation and incubation: Urease positive organisms differ significantly in the amount of urease produced. Difference depends, of course, on species, but also on culture age and general health. For these reasons, a positive reaction could occur in as little as 1hr or as long as 1 week! Inoculate tubes with fresh cells from isolated colonies taken from agar, preferably from a plate. Never use a broth culture as source of inoculum. Leave caps loose as accumulation of CO2 may buffer alkalinity resulting in a false negative reacion. Incubate the inoculated tubes AND an uninoculated control tube at 37OC. Check for a reaction as for 48hrs. If no color change, or very slight change has occurred, place back in the incubator and check again after 72hrs and if necessary, 96hrs. Slow growing organisms, or those that produce small amounts of urease, may take even longer than 96hrs. to give a positive reaction. Beyond 96hrs. a false positive result can occur. However, the control tube will help distinguish between a false and legitimate positive result upon prolonged incubation. Anything from a slight pink color to red is a positive reaction. Expected results: For our purposes, urease test should be used to distinguish Proteus from other enteric Gram negative rods. Clinically, this is important due to the fact that Proteus mirabilis is a rapid hydrolyzer of urea and a common cause of UTIs. Beyond our lab, urease test is used as the presumptive test for the presence of Helicobacter pylori in the stomach of patients exhibiting symptoms indicative of gastric ulcers. Limitations: 1. Do not heat the Urea Agar Slants, as urea decomposes very readily when heated. 2. Urea is light sensitive and can undergo autohydrolysis. Store at 2 to 8C in the dark. 3. Failure to incubate this medium with loose caps may result in false negative results. 4. Significant differences in urease production necessitates continuous monitoring of results.

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