WeeK 17!!!

Oh my its Week 17 already!!!

Hello friends!! How are you guys doing?? I hope everything is fine especially with the projects. Well, this week Im doing on Quality Assurance. Theres not that much work to be done actually. We carry out QA tests on media and agars used in the laboratory. Here are 2 of the tests that I did for the week,

A. Citrate Test

Objective
· To determine the ability of an organism to utilize citrate as a sole source of carbon for metabolism and growth

Principle
· Certain organisms are able to utilise citrate, an intermediate metabolite in TCA (Kreb’s) cycle, as the sole source of carbon.
· In bacteria, cleavage of citrate involves an enzyme system without coenzyme A involvement
· Bacteria break the conjugate base salt of citrate into organic acids (formic acid and acetic acid) and carbon dioxide.
· An organism that can use citrate as its sole carbon source also uses ammonium salts as its sole nitrogen source.
· Simmon's citrate media contains ammonium salts and a pH indicator, bromthymol blue
· Bacteria extract nitrogen from ammonium salts with production of ammonia leading to alkalinisation of medium which causes the indicator, bromthymol blue to change to an intense blue or a Prussian blue colour when pH is above 7.6 showing a positive result

Materials
· Simmon’s citrate media
· Disposable inoculating loops
· Enterobacter aerogenes W 3/3/90 (positive control)
· Escherichia coli (E.coli) ATCC 25922 (negative control)

Procedure
1. Streak the surface of the Simmon’s citrate slant
2. Incubate at 35⁰C overnight (usually 18hrs)

Left: Negative, Right: Positive Picture taken dddddddddddddddddddddddddddddddfrom: http://www.ams.cmu.ac.th/mt/clinmcrb/CMBwebsite/Price%20list_media.htm

Results and Conclusion
· Positive result (Enterobacter aerogenes): Deep blue colour of agar
· Negative result (E.coli): Absence of growth or no colour change
· Citrate test can be use to differentiate members of the Enterobacteriaceae family and other Gram negative organisms

Possible errors
· Too heavy inoculums may give a false-positive result
· Preformed organic compounds from dying bacteria may release carbon and nitrogen which may result in false positive results

Urease Test

Objective
· To test the ability of an organism to break down urea by the action of the enzyme urease

Principle
· Urea which is often referred to as carbamide, a diamide of carbonic acid
· Due to the presence of amides, urea is readily hydrolyzed by a specific enzyme, urease
· Urease is an important microbial enzyme which is used in the decomposition of organic compounds
· Urease breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water
· Urease is detected by plating bacteria onto an amide containing medium, specifically urea
· Urea will be hydrolyzed into ammonium carbonate as the end product,
· Formation of ammonia alkalinises the medium causing a change in the phenol red indicator
· Urea agar is slanted with an adequate butt portion to allow gradation of positive reactions
· Proteus, Providencia and Morganella species produce urease in large quantities

Materials
· Christensen’s urea agar slant
· Disposable Inoculating loops

Procedure
1. Streak the surface of urea agar slant
2. Incubate aerobically at 35⁰C overnight (usually 18hrs)
Left:Negative, Right: Positive Picture taken from: www.nhlmmcgym.com/culture.htm
Conclusion
· Positive result: urea slant change to an intense pink-red to red-violet colourchange Positive result
· Negative result: no colour change; urea slant remain yellow
· Some organisms like Pseudomonas aeruginosa may produce ammonia by breaking down peptones present in the agar slant à False positive results
· Presence of glucose in the Christensen’s medium prevent organisms from using ammonia produced as its sole source of nitrogen which will lead to inaccurate results
· Organism like Helicobacter pylori are able to hydrolyze urea rapidly, producing a positive reaction within 1 to 2 hours

So thats all! Any questions just ask me aites!! Bye! Take care!!