• Lactose is a disaccharide of glucose and galactose which is connected by a β-galactoside bond.
• Lactose fermenting bacteria consist of both the permease and beta-galactosidase enzymes that are responsible for acid production during lactose fermentation.
• Permease permits the lactose to enter into the bacterial cell wall.
• Then, beta-galactosidase breaks down the lactose into glucose and galactose.
• But in some organisms, permease is absent and hence appear as late or non-lactose fermenters.
• O-Nitrophenyl-β-D-galactopyranoside (ONPG) resembles to lactose in structure, except that glucose is substituted by orthonitrophenyl group.
• In contrast to lactose, the substrate O-nitrophenyl-beta-D galactopyranoside (ONPG) can penetrate the bacterial cell wall even in absence of permease.
• ONPG test is a very sensitive test for lactose-fermentation.
• In this test, O-Nitrophenyl-β-D-galactopyranoside (ONPG) (artificial in nature) acts as a substrate for beta-galactosidase to ascertain the specific enzyme activity that serves in the identification and differentiation of organisms.
• If the organism consists of beta-galactosidase, then it will hydrolyse ONPG to yield galactose and o-nitrophenol.
• The positive test is confirmed by the yellow colour, which is given by ortho-nitrophenol.
• A positive ONPG test is mainly focused on providing the rapid identification of delayed lactose fermentation.

Requirements:

1. Sodium phosphate buffer, 1 M, pH 7.0
2. O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M
3. Physiologic saline
4. Toluene
5. ONPG broth: Ingredients per 1000 mL
   • Na2HPO4 —————- (9.46 g)
   • phenylalanine ——— (4 g)
   • ONPG ———————– (2 g)
   • KH2PO4 ——————–(0.907 g)
   • pH 8.0
6. ONPG disk: 
   • ONPG Differentiation Disk is made by impregnating controlled concentrations of ONPG onto a 0.25 inch diameter filter paper disk.

Procedure of ONPG test:

• For ONPG disk method:
  • Keep an ONPG disk into a sterile tube.
  • Add 0.2 mL saline to it.
  • Use a loopful of test isolate to heavily inoculate the tube.
  • Incubate at 35-37°C for up to 4 hours.
  • Observe for colour change of the disk.
• For broth method:
  • Keep the test medium to room temperature.
  • Use heavy inoculum from a pure 18-24 hr culture to inoculate the test medium.
  • Incubate aerobically, at 35- 37ºC, with caps loosened.
  • Observe for a yellow colour development at 1 hour.
  • If no colour change is observed in the tube even after 1 hour of incubation, continue incubation for up to 24 hours.

Results interpretations:

• Positive result:
  • The positive test is indicated by the yellow colour of ortho-nitrophenol resulted by the hydrolysis of ONPG.
• Negative result:
  • The negative test is suggested by no change in colour, that means the absence of enzyme.

Limitations of ONPG test:

• Further biochemical, molecular, immunological, or mass spectrometry testing on colonies from pure culture is advised for complete identification.
• Cultures which naturally yield yellow colour cannot be tested in this media.
• Inoculation of test organism should be done only from lactose- containing medium.

O-Nitrophenyl-β-D-Galactopyranoside (ONPG) test: Principle, Procedure and Results

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• L-Pyrrolidonyl Arylamidase (PYR) test is one of the rapid tests employed for the identification of group a beta-hemolytic Streptococci and Enterococci on the basis of activity of the enzyme pyrolidonyl arylamidase.
• It is also termed as pyrrolidonyl aminopeptidase.
•  PYR is a bacterial enzyme
• The test disk is impregnated with L-pyroglutamic acid-β-naphthylamide that is hydrolysed by PYR hence serves as a substrate for the detection of PYR
• Hydrolysis of the L-pyroglutamic acid-β-naphthylamide yields beta-naphthylamine which in combination with the PYR Reagent (p-dimethylamino-cinnamaldehyde)  forms a bright pink to cherry red colour.
• Either broth assay method or rapid disk method is used for the test.

Procedure:

• Broth Method
  • Inoculate PYR broth with 2-5 colonies from overnight (18-24 hours) pure culture.
  • Incubate the tube aerobically at 35-37°C for 4 hours.
  • Add 2-3 drop of PYR reagent and observe for colour change.
  • Observe for the red colour development within 1-2 minutes.
• Disk Method (Rapid)
  • With 10 µl sterile distilled water or deionized water, wet the PYR test disc on the strip.
  • Note: Do not flood the disk.
  • From 18-24 hrs culture, place 5-10 colonies of the tested strain on the surface of the disc with a loop and smear them lightly on it.
  • Incubate the disc for 1-2 minutes at room temperature.
  • Add 1 drop of N, N-dimethylaminocinnamaldehyde after incubation.
  • Observe for red colour development within 1-2 minutes.

Results interpretations:

• Positive test:
  • The positive test is indicated by the appearance of bright pink or cherry red color within 1-2 mins. Examples: Citrobacter, Klebsiella etc.
• Negative test:
  • The negative test is suggested by no any color change or a blue color because of indole reaction. Examples: Streptococcus bovis, S. equinus

Limitations:

• PYR is only for the presumptive identification of group A Streptococci and group D enterococci from other streptococci thus other tests are recommended for complete identification.
• If the disk or filter paper are too moist, a false-negative test can result.
• Few isolates of lactococci and aerococci maybe PYRase positive.
• If reactions are read after 20 seconds, non-specific color reactions may occur.

L-Pyrrolidonyl Arylamidase (PYR) test: Principle, Requirements, Procedure and Results

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• Decarboxylases are a group of enzymes which acts by hydrolyzing an amino acid to form an amine.
• Decarboxylase test is mainly performed to differentiate decarboxylase producing Enterobacteriaceae from other gram-negative rods.
• The decarboxylation of the amino acid yields in an alkaline pH and a change in color of pH indicators bromocresol and cresol red from orange to purple is observed.

Requirements:

1. Media: Brain-heart infusion broth, Blood agar, Decarboxylase broth
2. Test-tubes
3. Incubator
4. Sterile mineral oil

composition of Decarboxylase broth

1. Composition———————————–g/ltr
   • Peptic digest of animal tissue——– (5 g)
   • beef extract ———————————-(5 g)
   • bromocresol purple ———————-(0.1 g)
   • cresol red————————————- (0.005 g)
   • dextrose —————————————(0.5 g)
   • pyridoxal ————————————(0.005 g)
   • amino acid ———————————–(10 g)
2. pH-6.0

Procedure of Decarboxylase test:

1. Glucose-Nonfermenting Organisms:
   • Prepare a suspension (≥McFarland No. 5 turbidity standard) in brain-heart infusion broth from 18-24hr old culture on 5% sheep blood agar.
   • With 4 drops of broth, inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and the control broth (no amino acid).
   • To each tube, add a 4-mm layer of sterile mineral oil.
   • Incubate the cultures at 35°-37°C in ambient air.
   • Examine the tubes at 24, 48, 72, and 96 hours.
2. Glucose-Fermenting Organisms:
   • With 1 drop of an 18- to 24-hour brain-heart infusion broth culture, inoculate each tubes.
   • To each tube, add a 4-mm layer of sterile mineral oil.
   • Incubate the cultures for 4 days at 35°-37°C in ambient air.
   • Observe and examine the tubes at 24, 48, 72, and 96 hours.

Results Interpretations:

• Positive test:
  • It is indicated by the alkaline pH and change in coloration from orange to purple.
• Negative test:
  • It is suggested by no change in colour in both test and control tubes and growth is visible in the control tube.

Decarboxylase test-Principle, Procedure and Results

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• Leucine Amino Peptidase (LAP) test is one of the rapid tests which is employed for the detection of the enzyme leucine amino peptidase.
• The LAP test is generally performed for the preliminary characterization of catalase- negative, gram-positive cocci, particularly non-beta hemolytic cocci.
• Leucine- -naphthalamide impregnated disk acts as a substrate.
• The LAP enzyme is responsible for hydrolysis of substrate that yields -naphthalamine.
• The product on addition to p-methyl (an aminocinnamaldehyde reagent) forms a highly visible red colored Schiff’s base.

Procedure:

1. Place a LAP disk in a sterile petri dish, and leave the disk to warm to room temperature.
2. Lowly dampen the LAP disk either with reagent grade water or with a little sterile distilled water.
3. Use a wooden applicator stick to rub a small amount of several colonies of an 18 to 24 hour pure culture onto a small area of the LAP disk.
4. Incubate at room temperature for 5 minutes.
5. Add 1 drop of cinnamaldehyde reagent after the incubation period, and read within one minute.

Results interpretation:

• Positive result:
  • The positive test is indicated by the development of a red/pink color.
• Negative result:
  • The negative test is suggested by no change or slight yellow color.

Limitations:

• The confirmation of test organism to be gram-positive coccus and catalase negative is mandatory for the LAP test.
• There might be chances of false negative if inadequate columns are taken.

Leucine Amino Peptidase (LAP) test

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• Microdase test is also termed as modified oxidase test.
• It is one of the rapid tests employed for the differentiation between Staphylococcus and Micrococcus which are Gram positive cocci having catalase enzyme.
• The test is based on the detection of oxidase enzyme.
• Filter paper disks impregnated with tetramethyl-p-phenylenediamine in dimethyl sulfoxide (DMSO) is used in order to detect the oxidase enzyme.
• 1% (w/v) tetramethyl-p-phenylenediamine in certified grade dimethyl sulfoxide is the preparation for modified oxidase reagent.
• DMSO serves for the permeability of cells to the reagent along with providing solubility and stability against auto-oxidation.
• When exposed to atmospheric oxygen, the oxidase enzyme reacts with the oxidase reagent and cytochrome C to yield the coloured compound, indophenol indicated as blue or purplish blue coloration on the disc after the bacterial colony is introduced on the disc.

Requirements:

1. Media: Blood agar
2. Oxidase Disc
3. Filter paper disks impregnated with tetramethyl-p-phenylenediamine dihydrochloride in DMSO.
4. Incubator
5. Forceps
6. Petri dish
7. Glass slide
8. Fresh pure colonies of test organisms

Procedure:

1. Use forceps to place the disk in an empty petri dish or on a clean glass slide.
2. Use a wooden applicator stick and rub a small amount of various colonies of an 18- to 24-hour pure culture grown on blood agar onto a small area of the microdase disk.
   Note: Do not rehydrate the disk before use.
3. Incubate at room temperature for 2 minutes.
4. Observe for a blue colour development.

Results interpretations:

• Positive result:
  • The positive test is suggested by the development of blue to purple colour within the 2 mins.
• Negative result:
  • The negative test is indicated by no any change in color.

Limitations:

• Neither too young nor too old cultures are used as they might yield inaccurate results.
• Microdase test is recommended only for the Gram-positive, catalase positive cocci.
• Microdase is limited only for the routine testing of oxidase activity of Staphylococcus and Micrococcus.

Microdase (Modified Oxidase) test: Principle, Procedure and Results interpretation

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• After E. coli, Staphylococcus saprophyticus is the second most causative organism for the Urinary tract infections (UTIs) in sexually active young women.
• Laboratory identification of Staphylococcus saprophyticus is performed on the basis of haemolysis, coagulase and resistance to novobiocin.
• Thus, after isolation of coagulase negative staphylococcus, laboratory should further identify the isolate and find out if the isolate is sensitive to novobiocin or not.
• Novobiocin is the antibiotic produced by the actinomycete Streptomyces nivens.
• Sensitivity to novobiocin is detected by placing a novobiocin impregnated paper disk on a agar plate seeded with the organism under identification.
• If the bacteria are sensitive to novobiocin, the formation of visible zone of inhibition  is seen around the disk.
• The zone of inhibition represents the area where the growth of organism was inhibited by the antibiotic concentration.
• No zone of inhibition suggests the resistance of organism to the antibiotic.

Requirements:

1. Isolated colonies of an aerobic, catalase positive, coagulase negative gram positive cocci.
2. Mueller Hinton agar
3. Blood agar/ Tryptic Soy agar
4. Incubator
5. Novobiocin disk
6. Distilled water
7. Sliding calipers/ metric ruler

Procedure of Novobiocin susceptibility test

1. The test isolate taken is of 18-72 hours and in pure culture.
2. Prepare a suspension of the test isolate in tryptic soy broth equal to a McFarland 0.5 standard or equivalent.
3. Dip a sterile swab into the suspension and rotate it against the side of the tube above the fluid level in order to remove excess inoculum.
4. Inoculate a blood agar or Mueller Hinton agar plate by streaking the expressed swab over the entire agar surface and repeat in 2 planes.
5. Allow the agar surface to dry for exactly15 minutes before applying a Novobiocin Disk.
6. Prepare a lawn of growth over the entire plate by use of sterile swab, swabbing over the entire plate in 3 directions and around the edge of the plate.
7. Using alcohol-dipped and flamed forceps, aseptically apply a novobiocin antibiotic disc to the surface of each inoculated plate.
8. Use sterile forceps to gently press the discs down to make sure that they adhere to the agar surface.
9. Incubate plate aerobically for 18 to 24 hours at 35 to 37°C.
10. Use sliding calipers or a metric ruler to measure the diameter of the zone of inhibition.

Results interpretation:

• Positive result:
  • The positive test is indicated by the zone of inhibition greater than 16mm suggesting the sensitivity of the organism.
• Negative result:
  • Negative test is indicated by the zone of inhibition less than 16mm which suggests the novobiocin resistance of the organism.

Limitations:

• Only isolated colonies of aerobic, catalase positive, coagulase negative gram positive cocci are to be tested.
• Biochemical, immunological tests are further recommended for the complete identification of the organism.
• Novobiocin disks can mislead the results if the test is performed for isolates other than urinary specimens.

Novobiocin susceptibility test: Principle, Procedure and Results interpretations

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• Acetate agar is employed to test an organism’s ability to utilize acetate. The medium consists of sodium acetate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth of organisms suggests the positive test for acetate utilization. During the metabolism of acetate by the bacteria , the ammonium salts are broken down to ammonia, which elevates alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to blue. This medium is usually used for differentiating Shigella spp from Escherichia coli. Shigella spp are not able to metabolize actetate whereas approximately 94% Escherichia coli utilize acetate.

Requirements:

1. Media: Composition of Sodium acetate agar
   • Ingredients                                         gm/l
   • sodium chloride …………………………………….5.0 g
   • magnesium sulfate ………………………………..0.1 g
   • ammonium phosphate, monobasic …….1.0 g
   • potassium phosphate, dibasic …………….1.0 g
   • sodium acetate …………………………………….2.0 g
   • agar ………………………..………………………….20.0 g
   • bromothymol blue ……………………………. 0.08g
2. Sterile inoculating loops or sticks
3. Sterile pipette
4.  Incubator at 35°C
5.  Sterile saline

Procedure of Acetate utilization test:

•  Using an 18- to 24-h culture from a non-inhibitory culture plate, prepare a turbid saline suspension.
• Inoculate the slant with 1 drop of the suspension.
•  Alternatively, streak the slant back and forth with a light inoculum picked from the centre of a well-isolated colony.
• Place cap loosely on tube.
•  Incubate aerobically at 35 to 37°C for up to 5 days for Enterobacteriaceae; incubate at 30􏰀C for nonfermenting, gram-negative rods for up to 7 days.
• Observe a colour change from green to blue along the slant.

Results interpretations:

• Positive test:
  • It is indicated by the growth of organisms and conversion of colour from green to intense blue along the slant.
• Negative test:
  • No colour change indicates no growth and is suggestive of negative test.

Limitations:

• Stab on slant should be avoided as the test requires aerobic environment.
• Inoculation from broth cultures is avoided due to carry over of media.
• Light inoculum is preferred to avoid false positive results.

Acetate utilization test: Principle, media composition, Procedure and Results

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• Bacitracin is the antibiotic produced by Bacillus licheniformis and this drug interferes the formation of peptidoglycan, a major component of bacterial cell wall.  Bacitracin blocks bactoprenol from transporting NAM and NAG sugars across the cell membrane thereby halting further synthesis of peptidoglycan. Bacitracin test is usually performed for the presumptive identification and differentiation of beta-hemolytic group A streptococci (Streptococcus pyogenes– susceptible) from other beta-hemolytic streptococci. This test detects whether the organism is susceptible to bacitracin or not. It is also used to distinguish Staphylococci species (resistant) from micrococci (susceptible).
• A disk infused with a small amount of bacitracin (0.04 units) is placed on an agar plate, permitting the antibiotic to diffuse into the medium and halt the growth of sensitive organisms. Following incubation, the inoculated plates are analysed for zones of inhibition surrounding the disks.
• If there is a zone around the edge of the disk where the organism has not grown, the organism is sensitive to the bacitracin in the disk whereas if the organism grows up to the edge of the disk, it is resistant to the bacitracin infusing the disk. 

Requirements:

1. Test microorganism
2. Blood agar (trypticase soy agar + 5% sheep blood)
3. Inoculating loops
4. Incubator
5. Disk impregnated with bacitracin

Procedure of Bacitracin test:

1. Use a sterile inoculating loop, select two or three suspect colonies of a pure culture.
2. Streak the isolated colonies onto a blood agar.
3. Using sterile forceps, place a bacitracin disk in the first quadrant (area of dense growth).
4. Gently tap the disk to make sure appropriate contact with the agar surface.
5. Incubate the plate for 18 to 24 hours at 35^oC in ambient air.
6. Observe zone of inhibition around disk.

Results interpretations:

• Positive test:
  • If any zone of inhibition greater than 10mm is seen then the microorganism is susceptible to the antimicrobial compound infused in the disk and is suggestive of positive test.
  • eg. Beta-hemolytic streptococci (Streptococcus pyogenes), Micrococcus
• Negative test:
  • If no zone of inhibition is seen then the microorganism is resistant to the antimicrobial compound impregnated in the disk and it is indicative of negative test.
  • eg. Streptococcus agalactiae, Staphylococcus

Limitations:

• An expired out blood agar plate should not be used because it decreases diffusion of the bacitracin yielding a false-negative result.
• Different concentrations of bacitracin results discrete zone sizes, thus differential disk (0.04 units) is used in comparison to sensitivity disks (10 units).
• When testing isolates, a light inoculum may result in false zone of inhibition. So it is important that an inoculum resulting in confluent growth be used.
• Different biochemical and/or serological tests such as latex agglutination are recommended  on colonies from pure culture for complete identification.

Bacitracin test: Principle, Requirements, Procedure, Results interpretations, Limitations

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The bile solubility test is employed  to distinguish Streptococcus pneumoniae from alpha-haemolytic Streptococcus spp. The basis of bile solubility test is the lysis of pneumococcal cells when bile salts especially sodium desoxycholate and sodium taurocholate is applied to the colony under particular conditions of time and temperature, whereas other streptococci do not. An amidase is an intracellular autolytic enzyme possessed by pneumococcus that is responsible for the rapid autolysis of the organism, when cultivated on artificial medium. The bile salts lowers the surface tension of the medium and cause cell membrane disruption. The working mechanism of the test is not clearly known; however, one theory states the bile salts accelerates lysis of pneumococcal cells by activating the autolytic enzyme.

Requirements:

1. Inoculating Loops
2. Test tubes or slide
3. Pipettes
4. Sodium desoxycholate
5. 0.85% NaCl
6. Broth culture medium

Procedure of Bile solubility test:

A. Test tube method:

1. Dispense 0.5 ml of sterile saline or suitable broth into a small test tube.
2. Prepare a heavy suspension of the organism in normal saline.
3. Shake or vortex to form a uniform suspension of the organism
4. Divide the suspension into two tubes, one labelled “TEST” and the other labelled “CONTROL.”
5. Dispense 5 drops of bile reagent into the tube marked “TEST.”
6. Add 5 drops of saline to the tube marked “CONTROL.”
7. Gently mix each tube.
8. Incubate the tubes for 3 hr at 35°C, checking hourly for clearing, or examine each tube by Gram stain or methylene blue wet mount for lysis of cells at 15 min.

B. Direct plate method:

1. Place a drop of bile spot reagent near a suspected 18 to 24-h colony; gently roll the drop over several representative colonies by tilting the plate. Take care not to dislodge the colonies.
   NOTE: Make sure not to touch the agar surface with the tip of the dropper of bile reagent.
2. Keep the plate right side up and incubate at 35°C for 15 to 30 min or until the drop has evaporated.
3. Placing the plate on a heat block is a substitute for use of an incubator.
4. Observe for flattening of the colony.
5. Make sure the colony did not simply float away.

C. Direct slide blood culture test:

1. Add 1 drop of blood culture broth to 1 drop of bile reagent on a glass slide and allow to dry.
2. As a control, add 1 drop of broth blood culture to 1 drop of water and allow to dry.
3. Gram stain and examine for cocci.

Results interpretation:

Test tube method:

• Positive result: It is indicated by clearer or loss of turbidity in suspension in contrast to control which is turbid. Eg. Streptococcus pneumoniae
• Negative result: It is denoted by the turbidity of suspension. eg. Streptococcus mitis

Direct plate method:

• Positive result: It is demonstrated by the flattening of colony or disintegration of colony within 30 mins, leaving an area of alpha hemolysis where the colonies were located.
• Negative result: is indicated by the intact colony or no change in integrity of the colony within 30 min.

Direct slide blood culture method:

• Positive result: The complete lysis of all the cocci in smear is suggestive of positive result.
• Negative result: Intact colony of cocci in both control and test is indicator of negative test.

Limitations:

• Even if the lysis doesn’t take place, the organism can still be S. pneumoniae . Thus colonies resembling S. pneumoniae but bile insoluble should be further processed to other biochemical tests for identification.
• The bile solubility test is not valid with old cultures that have autolysed.
• While the test is performed using the plate method, proper care should be taken not to  dislodge the colony being tested, thus yielding false-positive results. If the direct plate method is difficult to interpret, the test should be repeated using the tube or slide method.

Bile solubility test: Principle, Procedure, and Result

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Hippurate hydrolysis test is performed for the identification of Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Streptococcus agalactiae. The identification is carried out by detecting the ability of the organism to hydrolyse sodium hippurate to benzoic acid and glycine by the action of the enzyme hippuricase. Hippurate is the glycine conjugate of benzoic acid. In 48hr long classical method, the ability of bacteria spp to hydrolyse hippurate was tested by using ferric chloride indicator. Ferric chloride detected benzoic acid produced during hydrolysis. A 2 hr rapid technique has been developed which uses ninhydrin as indicator for the detection of glycine. Ninhydrin reacts with glycine forming a deep blue or purple colour.

Requirements:

1. Sterile wooden sticks or inoculating loops
2. Incubator at 35°C
3. Test tubes
4. Distilled water
5. Ninhydrin
6. Ferric chloride

Procedure of Hippurate hydrolysis test:

A. Classical Method:

1. First prepare sterile Sodium Hippurate broth and inoculate with test organism.
2. Incubate at 35°C for 8-24 hours
3. Centrifuge the broth and remove the precipitate.
4. Add Ferric chloride reagent in the supernatant.
5. Observe for the precipitate and if the precipitate still remains after 10 minutes, it indicates presence of benzoic acid. Thus the test is positive for hippurate hydrolysis

B.  Ninhydrin Method:

1. Prepare a hippurate tube
2. Add 0.2 ml of distilled  water at a pH of 6.8 to 7.2 to reconstitute lyophilized tube test reagent.
3.  Add 2 drops of distilled water to an empty tube for disk or tablet tests.
4.  Defrost one 0.4 ml tube per test for prepared reagent.
5.  In the tube, make a heavy suspension  from an 18 to 24 hr culture. Use care not to pick up agar, which contains protein.
6. For disk or tablet tests, add reagent after inoculation of the tube with the culture.
7. Incubate the tube for 2 h at 35 to 37°C.
8. After the 2 hr incubation period, add 2 drops of the ninhydrin solution to the hippurate reagent-organism mixture.
9. Add an additional 2 drops if test has 0.4 ml of hippurate.
10. Re-incubate at 35 to 37°C for 30 min. Observe the tubes at 10-min intervals for the appearance of a deep blue colour, which is a positive test. The colour change will usually appear within 10 to 15 min after the ninhydrin indicator solution has been added.

Results interpretations:

• Positive result: The appearance of a deep blue colour within 30min is suggestive of positive reaction.
• Negative result: A faint purple colour or no change in colour indicates negative test.

Limitations:

• Other biochemical tests are recommended to confirm the identification.
• A small percentage of C. jejuni organisms are hippurate negative and must be identified by other methods.
• There are high chances of false positive results if incubation with ninhydrin exceeds 30min.

Hippurate hydrolysis test: Principle, Procedure, Result interpretations and Limitations

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