What is the principle of the citrate test?
Principle of Citrate Test. A citrate test or citrate utilization test is used to determine the ability of an organism to utilize sodium citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source. When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity turning the bromthymol blue indicator from green to blue.
What instrument is used to inoculate in a citrate test?
Inoculate Simmons citrate agar (do not inoculate heavily) by using straight wire from an 18- to 24-hour old colony. Inoculating from a broth culture is not recommended because the inoculum would be too heavy. Incubate at 35 °C for up to seven days. Results
What is the positive organism for citrate utilization test?
Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Hereof, what bacteria are citrate positive? Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms.
What is the pH indicator used in the citrate test?
errors in determining if growth of the test organism did or did not occur. Simmons Citrate Agar contains ammonium dihydrogen phosphate and sodium citrate. The former serves as the sole nitrogen source, whereas the latter is the sole carbon source. The medium also contains the pH indicator bromothymol blue. Microbes able to use the nitrogen and
What does a positive result indicate in the citrate test?
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. Therefore the dye changes from pH of 6.9 to 7.6 (acidic to alkaline) and the green dye indicates the pH going up by its color change to blue.
What bacteria test positive for citrate?
Klebsiella pneumoniae and Proteus mirabilis are examples of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.
What enzyme does the citrate test identify?
Nitrate reduction test is a test that determines the production of an enzyme called nitrate reductase, which results in the reduction of nitrate (NO3).
What is the indicator used in citrate test?
Principle. Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citrate lyase, which breaks down citrate to oxaloacetate and acetate.
What does a citrate test tell you about a bacterial sample?
Principle of Citrate Utilization Test Citrate agar is used to test an organism's ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen.
What bacteria is citrate negative?
Citrate Negative OrganismsEscherichia coli.Shigella spp.Salmonella Typhi.Salmonella Paratyphi A.Morganella morganii.Yersinia enterocolitica.
How do you perform a citrate test?
ProcedureObtain a slant of Simmons Citrate Agar.Using an inoculating needle, stab your assigned organism into the medium, then zigzag the needle back and forth up the surface of the slant.Incubate the tube for at least 48 hours.After the incubation period, examine the slant.
What is the purpose of the enzyme citrate permease?
The function of citrate permease is to import citrate into the cell. Without this enzyme, there can be no utilization of citrate because there is no way to bring it inside the cell.
What test is used to determine if the bacteria utilize aerobic respiration?
Oxidase Test - Virtual Interactive Bacteriology Laboratory. The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. (note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration.
Is citrate test selective or differential?
Subscribe us to receive latest notes. 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.
What test is used for lactose fermentation?
MacConkey agar The selective ingredients are the bile salts and the dye, crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color.
Is Salmonella citrate positive?
When Simmons Citrate agar is inoculated with Salmonella typhimurium , the medium turns royal blue. This is a positive result for the citrate test.
Can E. coli be citrate positive?
Among the E. coli-like isolates from domestic pigeons, pigs, cattle, and horses, 13 (36.1%), 12 (36.3%), 1 (4.1%), and 1 (7.1%), respectively, were found to be citrate positive (Table 1).
Does E. coli use citrate?
Cell Biology Like many organisms, E. coli has a citric acid cycle, and so metabolizes citrate while growing on various substances. It can also grow anaerobically by fermenting citrate.
Is Staphylococcus aureus citrate positive or negative?
Positive (+ve)Biochemical Test and Identification of Staphylococcus aureusBasic CharacteristicsProperties (Staphylococcus aureus)CitratePositive (+ve)CoagulasePositive (+ve)GasNegative (-ve)Gelatin HydrolysisPositive (+ve)41 more rows•Aug 15, 2019
Principle of Citrate Utilization Test
Citrate agar is used to test an organism’s ability to utilize citrate as a source of energy. The medium contains citrate as the sole carbon source...
Media Used in Citrate Utilization Test
CompositionDeionized water = 1,000 mlFinal pH 6.9 +/- 0.2 at 25 degrees C.Preparation 1. Dissolve above salts in deionized water. 2. Adjust pH to 6...
Procedure of Citrate Utilization Test
1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony. 2. Incubate aerobically at 35 to 37C fo...
Quality Control of Citrate Utilization Test
Citrate Positive: Klebsiella pneumoniae ATCC 13883 (growth; blue color)Citrate Negative: Escherichia coli ATCC 25922 (no growth or trace of growth)
What is the principle of citrate utilization?
Principle of Citrate Utilization Test. When an organic acid such as citrate ( remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced which increase the pH of the medium, resulting in the color change. Utilization of exogenous citrate requires the presence of citrate transport proteins ...
What is citrate utilization test?
Citrate utilization test is often used as a part of a battery of tests (IMViC) used to identify gram-negative pathogens of the Enterobacteriaceae family. For instance, test kits such as the API-20E and Enterotube II include citrate utilization medium as one of the diagnostic tests.
What is the iMViC test?
Citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red, VP and Citrate) tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products.
What color is a citrate positive medium?
Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue . The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.
What is the citrate medium used in a citrate utilization test?
In the citrate utilization test, the citrate medium most commonly used is the formula of Simmons. The medium is poured into a tube on a slant. The composition of Simmons citrate agar is as follows: Ingredient. Simmons Citrate Agar (g/L) Ammonium dihydrogen phosphate.
How to avoid false positive reactions?
To avoid false-positive reactions, use a light inoculum to prevent carryover of substances from previous media. Results of the citrate utilization test alone are not sufficient for identification to the species level. References and further reading.
How long to incubate a slant?
Incubate aerobically at 35°C to 37°C for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium. Observe the development of blue color along the slant; denoting alkalinization. * Do not stab the slant, since the test requires an aerobic environment.
What is the medium used to inoculate bacteria?
Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citrase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO 2 ). Production of sodium bicarbonate (NaHCO 3) as well as ammonia (NH 3) from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a change of the medium's color from green to blue.
How to inoculate Simmons citrate agar?
Method. Inoculate Simmons citrate agar (do not inoculate heavily) by using straight wire from an 18- to 24-hour old colon y. Inoculating from a broth culture is not recommended because the inoculum would be too heavy. Incubate at 35 °C for up to seven days.
What is the enzyme that breaks down citrate?
Use of citrate involves the enzyme citrase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO 2 ). Production of sodium bicarbonate (NaHCO 3) as well as ammonia (NH 3) from the use of sodium citrate and ammonium salts results in alkaline pH.
What bacteria are on citrate agar?
Raoultella planticola. Proteus myxofaciens. Two bacteria (both Enterobacteriaceae) on citrate agar, showing a positive result (left side) and a negative result (right side) for the citrate test. Examples:
What does it mean when a test organism has a positive color change?
A colour change in the medium would be observed if the test organism produces acid or alkali during its growth. The usual colour change observed is from green (neutral) to blue (alkaline).
How to inoculate citrate agar tubes?
The citrate agar tubes are inoculated by streaking the surface of the slant. The slant should be streaked back and forth with the loop or the inoculating stick. The cap of the test tubes should be left loosened to ensure adequate aeration. The tubes are then incubated aerobically at 35-37°C for up to 4 days.
What is Simmon's Citrate Agar used for?
Simmon’s Citrate agar is used as the medium to test the ability of an organism to utilize citrate as a sole source of energy. Simmon’s Citrate agar is sold commercially by different vendors in the form of dehydrated powder. However, it can also be prepared in the laboratory if the necessary ingredients are available.
What is the principle of citrate utilization?
Principle of Citrate Utilization Test. Citrate agar is used to test the ability of an organism to utilize citrate as a source of energy. The agar medium contains citrate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. The growth of the organism is indicative of the utilization of citrate as it is an ...
What is citrate test?
Citrate test is used to determine the ability of an organism to utilize citrate as a sole source of energy. A citrate test is performed as a part of the IMViC test to differentiate members of Enterobacteriaceae. The test is also used for the differentiation between fecal coli and members of the aerogenes group on the basis of citrate utilization.
What is the enzyme that breaks down citrate?
The growth of the organism is indicative of the utilization of citrate as it is an intermediate metabolite in the Krebs cycle. The enzyme citrase breaks down citrate into oxaloacetate and acetate, where oxaloacetate is further broken down to form pyruvate and carbon dioxide.
What is a positive slant test?
A positive test is demonstrated by growth with a color change from green to intense blue along the slant. A negative test is demonstrated by no growth and no color change, and the color of the slant remains green. The following table demonstrates the result of Citrate utilization of important medical bacteria: S.N.
What does carbon dioxide do to the alkalinity of a medium?
The release of carbon dioxide induces the metabolism of ammonium salts, causing the formation of ammonia or sodium carbonate, both of which increase the alkalinity of the medium. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.
What is the role of citrate in the fatty acid cycle?
Citrate is the first intermediate of the citric acid cycle and tricarboxylic acid (TCA) cycle. It also plays an important role in fatty acid synthesis which takes place in the cytoplasm. The movement is assisted by two enzymes, citrate-condensing enzyme, which catalyzes the condensation of the acetyl unit with oxaloacetate in the mitochondria, ...
What is citrate test?
The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy. The medium contains citrate as the sole carbon source and inorganic ammonium salts (NH4H2PO4) as the sole source of nitrogen. 1.Dissolve above salts in deionized water.
How long to incubate Simmons citrate agar?
2.Incubate at 35 degree C to 37 degree C for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
What color is a citrate positive medium?
Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue . The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue .
What is the citrate utilization test?
In the citrate utilization test, the citrate medium most commonly used is the formula of Simmons. The medium is poured into a tube on a slant. The composition of Simmons citrate agar is as follows:
How long to incubate Simmons citrate agar?
Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Incubate at 35°C to 37°C for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
Can bacteria grow on Simmons citrate?
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.
What are the components of a sulfate based sulfate based sul
What are its components? 1 Sodium chloride and citrate 2 Dipotassium phosphate 3 Ammonium dihydrogen phosphate 4 Agar 5 Magnesium sulphate 6 Bromothymol blue – It serves as a pH indicator. At neutral pH, the color is green. It turns blue if the pH becomes alkaline and yellow if the pH becomes acidic. 7 Deionized water (6)
What happens when citrate is metabolized?
Once the organism metabolizes citrate it would lead to the breakdown of ammonium salt to ammonia thereby increasing its alkalinity. From a neutral pH, it becomes alkaline making the bromthymol blue indicator present in the medium change its color from green to blue.
What is the enzyme that converts citrate to pyruvate?
An organism that grows on this medium produces an enzyme called citrate permease, which has the ability to convert citrate to pyruvate. The latter will enter the metabolic cycle of the organism so as to produce energy. If citrate is utilized, then expect that there will be a growth of an organism.
How long to autoclave bromothymol blue?
Put about 5 ml into a tube and autoclave at 121 degree Celsius for 15 minutes under 15 psi pressure. Allow to cool in a slanted position.
What happens if you use citrate?
If citrate is utilized, then expect that there will be a growth of an organism. It will then become an intermediate metabolite in the Krebs cycle. (1, 2, 3, and 4) Picture 3: A biochemical test wherein two test tubes represent two distinct results. Picture 4: A Simmon’s citrate agar medium is used for this procedure.
Can citrate grow on slant?
The truth is that some organisms have the ability to grow on citrate but do not produce changes in the slant’s color at all. If this happens, the result is still read as positive. You need to keep in mind though that if the result of the test is equivocal, then it needs to be repeated.
What is the principle of citrate utilization test?
Principle of citrate utilization test. When citrate, an organic acid, is used as the only source of carbon, two things are produced like. Besides, when the ammonium salts are used as the sole nitrogen source , ammonium hydroxide is also produced in the medium. The organism that grows and develops on this medium produces an enzyme called citrate ...
What is the enzyme that breaks citrate into oxaloacetate?
The organism that grows and develops on this medium produces an enzyme called citrate permease (citrate transport protein), which has the ability to break citrate to oxaloacetate and acetate by the enzyme citrate lyase. After that, oxaloacetate is metabolized into pyruvate and CO2.
How long to incubate Simmon's citrate agar?
Do this by touching the tip of a needle to a colony which is 18 to 24 hours old. Incubate at a temperature of 35°C to 37°C for 18 to 24 hours.
What is the IMVIC test?
It is one of the IMVic series groups of tests where IMViC stands for Indole, Methyl red, VP, and citrate test. A citrate utilization test helps detect the organism’s ability to produce citrase enzymes, i.e., citrate lyase. The bacteria which rely on citrate as the sole source of carbon for their energy needs are determined.
Principle of Citrate Utilization Test
Media Composition
Quality Control
- Perform QC on each new lot of media prior to using them. Inspect agar for evidence of freezing, contamination, cracks, dehydration, and bubbles prior to storage and before use. Discard tubes that a...
- Organism
Procedure of Citrate Utilization Test
- Inoculate Simmons citrate agar lightly on the slant* by touching the tip of a needle to a colony that is 18 to 24 hours old.
- Place cap loosely on the tube.
- Incubate aerobically at 35°C to 37°C for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
- Inoculate Simmons citrate agar lightly on the slant* by touching the tip of a needle to a colony that is 18 to 24 hours old.
- Place cap loosely on the tube.
- Incubate aerobically at 35°C to 37°C for 18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
- Observe the development of blue color along the slant; denoting alkalinization.
Results
- Citrate positive: growth will be visible on the slant surface and the medium will be an intense Prussian blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue. Citrate negative: trace or no growth will be visible. No color change will occur; the medium will remain the deep forest green color of th…
Uses of Citrate Utilization Test
- Citrate utilization test is often used as a part of a battery of tests (IMViC) used to identify gram-negative pathogens of the Enterobacteriaceae family. For instance, test kits such as the API-20Eand Enterotube II include citrate utilization medium as one of the diagnostic tests.
Limitations
- Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with a sm...
- Do not inoculate from broth cultures, due to carryover of media.
- To avoid false-positive reactions, use a light inoculum to prevent the carryover of substances from previous media.
- Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with a sm...
- Do not inoculate from broth cultures, due to carryover of media.
- To avoid false-positive reactions, use a light inoculum to prevent the carryover of substances from previous media.
- Results of the citrate utilization test alone are not sufficient for identification up to the species level.