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.
