What is citrate utilization test? citrate utilization test purpose.
A positive result is color change from green to blue and/or growth. This means that citrate is utilized. Bacteria that use citrate converts the ammonium which alkalizes the agar.
Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.
At pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative.
Simmons’ citrate agar is a selective and differential medium that tests for an organism’s ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source. It is used for differentiating gram-negative bacteria on the basis of citrate utilization.
- Dissolve above salts in deionized water.
- Adjust pH to 6.9.
- Add agar and Bromothymol blue.
- Gently heat, with mixing, to boiling until agar is dissolved.
- The medium may be used either as slopes in test tubes or as a plate medium in petri dishes.
E. coli can grow on citrate when no oxygen is present through anaerobic processes. E. coli is able to bring citrate into the cell under these conditions because it has a gene called citT that encodes a transmembrane citrate-succinate antiporter.
Biochemical tests revealed that the isolates were Coagulase positive, Catalase positive, Urease positive, Citrate positive, Methyl-Red positive, Voges-Proskauer negative and negative upon starch hydrolysis.
Citrate utilization requires oxygen and thus screw caps, if used, should be placed loosely on the tube. Incubate at 35oC (+/- 2oC) for 18 to 48 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
Simmons’ citrate agar is used for differentiating gram-negative bacteria on the basis of citrate utilization. It is useful for selecting for organisms that use citrate as its main carbon and energy source.
WHY IS THE IDENTIFICATION OF BACTERIAL UNKNOWNS IMPORTANT? Microbiologists must identify bacterial isolates for several practical reasons: • Medical diagnostics — identifying a pathogen isolated from a patient. Food industry — identifying a microbial contaminant responsible for food spoilage.
The citrate utilization test is selective because only certain bacteria can utilize citrate in place of a fermentable carbohydrate.
Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant. E. coli is citrate negative.
Gram-negative bacteria are classified by the color they turn after a chemical process called Gram staining is used on them. Gram-negative bacteria stain red when this process is used. Other bacteria stain blue.
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic rod, which produces gas from lactose within 48 hours. The presence of some coliforms indicate fecal contamination.
When the VP reagents are added to MR-VP broth that has been inoculated with Escherichia coli , the media turns a copper color. This is a negative result for the VP test. When the VP reagents are added to MR-VP broth that has been inoculated with Enterobacter cloacae , the media turns red. This is a positive VP result.
The exogenous citrate enters the cells through the sodium-dependent transporter SLC13A5 (Costello and Franklin, 2016) and is cleaved in acetyl-CoA and OAA.
E. coli O157 evolved from an enteropathogenic E. coli ancestor of serotype O55:H7, which contained the locus of enterocyte effacement containing the adhesin intimin.
In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway.
Biochemical tests used to confirm S. aureus were coagulase test, catalase test, indole production, methyl red test, Voges-proskauer reaction, urease production, citrate utilization and sugar fermentation .
Basic CharacteristicsProperties (Staphylococcus aureus)CitratePositive (+ve)CoagulasePositive (+ve)GasNegative (-ve)Gelatin HydrolysisPositive (+ve)
The citrate test identifies the use of citrate as a sole carbon source, since there are no other nutrients in this medium. The basic end products (carbonates, bicarbonates, and ammonium hydroxide) will cause the brom thymol blue indicator in the medium to turn from forest green to royal blue.
A positive result is shown by the presence of a red or red-violet color in the surface alcohol layer of the broth. A negative result appears yellow. A variable result can also occur, showing an orange color as a result.
The Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is positive for acetoin, it will turn “brownish-red to pink” (tube on the left in the second picture).
What is the substrate for the Citrate Agar? An enzyme called citrase will catabolize the citrate into oxaloacetic acid and acetic acid.
Simmons Citrate Agar is used as a differential medium for the identification of enteric bacteria by using Citrate as the sole source of carbon and Ammonium as the sole source of nitrogen.
CharacteristicsSalmonella TyphiCapsuleNegative (-ve)CatalasePositive (+ve)CitrateNegative (-ve)FlagellaPositive (+ve)
Traits that can be valuable aids to identification are combinations of cell shape and size, gram stain reaction, acid-fast reaction, and special structures including endospores, granules, and capsules.
What three main approaches can be used by microbiologists to identify microorganisms? –Phenotypic- observing bacterial morphology and staining properties as well as biochemical testing. You just studied 45 terms!
Most microorganisms recognized to date as probiotics are Gram-positive, with Lactobacillus and Bifidobacterium being the main species used as treatments of intestinal dysfunctions (Marco et al. 2006). However, some Gram-negatives are also used as probiotics.
Fourth-generation cephalosporins such as cefepime, extended-spectrum β-lactamase inhibitor penicillins (piperacillin/tazobactam, ticarcillin/clavulanate) and most importantly the carbapenems (imipenem/cilastatin, meropenem, ertapenem) provide important tools in killing Gram-negative infections.
Gram-negative bacteria cause infections including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis in healthcare settings. Gram-negative bacteria are resistant to multiple drugs and are increasingly resistant to most available antibiotics.
Recent findings: The high rate of respiratory infections due to Gram-negative bacteria in late-onset ventilator-associated pneumonia has been repeatedly documented. The predominant pathogens are Pseudomonas aeruginosa and Acinetobacter baumannii.