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VOGES-PROSKAUER(VP) TEST:- This test is used to assist in the differentiation of Entero- bacteria. Vibrio cholerae, K. pneumoniae and some strains of Enterobacter, ferment sugar, with the formation of ac- etoin which can be detected by chemical methods. v  Principle:- The test organisms are cultured in a glucose phos- phate peptone water for 48 hours. Acetoin formed is converted to diacetyl. It is converted to a pink com- pound by the action of creatine v Requirements:- 1)       Glucose phosphate peptone water 2)       40 g/dl sodium hydroxide  v Procedure:- 1.  Inoculate about 2 ml of sterile  glucose phosphate peptone water with the test organisms. 2.   Incubate at 35°C for 48 hours. 3.  Add small amount (knife point) of creatine powder. 4. Add 3 ml of sodium hydroxidereagent and mix well. 5.  Keep at room temperature (25° ± 5°C) for one hour and observe the color.  v Observations:- 1.       Pink red color : Positive test 2.       No pink red color: Negative test  v Quality control:- Use following microorganisms to confirm the reliability of reagents: Positive control: Klebsiella aerogenes Negative control: E. coli  

OXIDATION FERMENTAION TEST:-  This test is used to differentiate the organisms that oxidize carbohydrates (aerobic utilization) from those organismsthat ferment carbohydrates (anaerobic utilization). Example: Differentiation of Pseudomonas aeruginosa (carbohydrate oxidation) from members of the Enterobacteriaceae (carbohydrate fermentation). v Principle:- The test organisms are inoculated into tryptone or peptone agar medium containing glucose (or mal- tose or sucrose) and the indicator bromothymol blue. The test is carried out in two tubes. The inoculated medium in one tube is sealed with a layer of liquid paraffin to exclude oxygen. Fermentative organisms act on the carbohydrates in both the tubes and the green colored media changes to yellow, due to the formation of acid. Oxidative organisms are able to use carbohydrates only in the open tube and the col- or of the tube changes to yellow.  v Procedure:- 1.  By using heavy inoculum, inoculate the O-F medium to the bottom of the two tubes. 2    Cover one tube (from each carbohydrate pair) with 1 cm layer of sterile paraffin oil. 3    Incubate the tubes at 35°C upto 14 days. Examine the tubes daily. v  Results:-    Open tube                                Paraffin tube                          Interpretation Yellow                                              Green                                          Oxidative organism Yellow                                             Yellow                                          Fermentative organism Green or blue                                 Green                                           No utilization of carbohydrates v Quality control:- Use the following microorganisms to confirm the reliability of reagents: Positive control: Yellow – Green color: Pseudomonas aeruginosa Negative control: Yellow-Yellow color: E. coli

v Principle:- This test is used to detect the enzymatic ability of an organism  to decarboxylase (hydrolyse) an amino acid toform an amine. Hydrolysis of an amino acid results in an alkaline pH change leading to formation of pink color.  formation pink color. vRequirements:- 1.      Glucose non-fermentingorganisms 2.      Glucose fermenting organisms 3.      Decarboxylase broaths (lysine, arginine, ornithine) 4.      Sterile mineral oil 5 Suspensions of suspected organisms grown on 5% sheep blood agar (18-24 hours) in brain- heart infusion broth (BHIB). v Procedure:- A.    Glucose non-fermenting organisms 1.       Prepare a heavy suspension of the organisms in brain-heart infusion broth. 2.       Inoculate each of the three decarbxylase broths and one control broth  (without amino   acid). 3.       Add a 4 mm layer of sterile mineral oil in each tube. 4.       Incubate at 37° up to seven days. 5.       Observe color change.   B.       Glucose fermenting organisms 1.       Inoculate each of the decarboxylase tubes with 1 drop of brain-heart infusion broth   culture. 2.       Add 4 mm layer of sterile mineral oil in each tube. 3.       Incubate at 37° up to seven days. 4.       Observe color change. vObservations: By comparing with the color of the control tube:-   1   Positive:   Purple color (alkaline color change) Lysine:    Klebsiella pneumonia Arginine:     Enterobacteria cloacae Ornithine:    Enterobacteria cloacae  Negative:     No color change or yellow color                             (Due to fermentation of glucose in BHIB).   v  Usefollowing microorganisms to confirm the reliability of reagents: 1   Positive test:    Lysine:      Klebsiella pneumonia Arginine:   Enterobacter cloacae Ornithine:  Enterobacter cloacae Negative test:   Lysine:      Enterobacter cloaeae Arginine:    Klebsiella pneumonia Ornithine:   Klebsiella pneumonia v Note 1         Reference Book Text book of medical laboratory Technology by Dr. Praful B. Godkar 

This test is usedto help in the identification of the organisms which produce the enzyme oxidase. Examples:   Pseudomonas, Neisseria, Vibrio and Pasteurella species. v Principle:- A colony of the test organism is smeared on a filter paper piece, soaked with few drops of oxidase reagent. If the organisms are oxidase producing, the phenylenediamine in the reagent is oxidized to a deep purple color.  Requirements:- Oxidase reagent: 1.0 g/dl tetramethyl p- phenylenediamine dihydrochoride in distilled water (it should be prepared fresh if it appears blue in color.)          2  filter paper strips v Procedure:- 1.           Place a piece of filter paper a clean petri dish. 2.           Add 2 to 3 drop of freshly prepared oxidase reagent. 3.          Smear a colony of the organism on the filter paper by using aglass rod. v Observation:- 1.                Blue purple color : positive test 2.                No blue purple color : negativetest v   Quality control:- Use following microorganisms to confirm the reliability of reagents: Positive control: Pseudomonas aeruginosa Negative control: E. coli

This test is performed mainly to assist in the identification of Enterobacteria.      v Principle:-                 When sulfur-containing amino acids are decomposed by the enterobacteria, hydrogen sulfide is                           produced. It reacts with ferrous ions and the formation of ferric sulfide imparts a black color to the                   medium.     v Requirements:-            1.      TSI medium         v Procedure:- 1.      Stab the butt and streak the slant at 37°C 2.      Observe daily for seven days for blackening caused by H,S.     v    Results:-                   1.           Blackening: Positive test.                      2.           No blackening: Negative test,No HS production.                v Quality control:- Use following microorganisms to confirm the reliability of reagents: Positive control: Staphylococcus aureus Negative control: Escherichia coli