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HiMotility Biochemical Kit for Vibrio cholerae

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KBM004

Introduction

Members of the genus Vibrio are defined as Gram-negative rods. They are motile most have a single polar flagellum, when grown in liquid medium. Most produce oxidase and catalase, and ferment glucose without producing gas (1). Three species, V. cholerae, V. parahaemolyticus, and V. vulnificus, are well-documented human pathogens (2,3). V. cholerae, the type species of the genus Vibrio, is the causative agent of cholera outbreaks and epidemics. Indian Standard (IS) defines method for isolation, identification and enumeration of Vibrio cholerae and Vibrio parahaemolyticus. KBM004 can be used for screening food samples and other relevant clinical samples. The tests have been designed as per the tests described in IS 5887 Part V.

Principle

Each KBM004 is a standardized, colorimetric test system based on motility, carbohydrate utilization and other biochemical tests specific for the identification of V. cholerae. The tests are based on the principle of pH change and substrate utilization. V. cholerae .s on incubation exhibit metabolic changes which are indicated by a colour change in the media that can be either interpreted visually or after addition of reagent wherever required.

Kit Contents

  • Each kit contains sufficient material to perform 10 tests.
  • 10 kits of KBM004.
  • Technical product insert
  • Result Interpretation Chart and Result Entry Datasheet
  • Identification Index
  • Baritt reagent A(R029)
  • Baritt reagent B(R030)

Storage and Shelf-life

Store between 2-8°C. Shelf-life is 12 months.

Instructions for use

Preparation of inoculum

  • KBM004 cannot be used directly on clinical or food samples. The organism to be identified has to be first isolated and purified.
  • Isolate the organism to be identified Nutrient Agar with or without blood. Pick up a single isolated colony and inoculate in 5 ml Alkaline Peptone water (M618) and incubate at 35-37°C for 6 to 8 hours until inoculum turbidity ≥1.0 OD at 620nm.

Note: Erroneous false negative result may be obtained if the inoculum turbidity is less than 1.0 OD.

Method of Inoculation :

  • Open the kit aseptically. Peel off the sealing foil.
  • Stab inoculate the 1st well. DO NOT INOCULATE THE 2nd WELL.
  • Inoculate the remaining kit (well no.3-12) by stabbing each individual well (except well no. 2) with a loopful of inoculum. Inoculum should reach the bottom of the wells.

Incubation: Temp. of Incubation: 35-37°C. Duration of Incubation : 24-48 hrs.

Interpretation of results

  • A combination of 12 biochemical tests for the confirmation of V.cholerae based on motility and other biochemical tests. (Kit contains sterile media for motility in first two wells followed by Voges Proskauer, Arginine utilization, Ornithine utilization, Lysine utilization, Catalase, H2S production and 4 different Carbohydrate utilization tests - Mannitol, Sucrose, Glucose and Inositol.

Motility + Nutrient Agar + TTC: Well No.1 and 2

  • Motility: Motility is observed as movement of growth from 1st well to 2nd well accompanied with reddening of the medium.
  • Nutrient Agar+ TTC: Nutrient Agar+ TTC is seen as reddening in the 1st well and wherever the motile organisms have moved in the 2nd well.

Voges-Proskauer's Test: Well No. 3

  • Add 3-4 drops of Barritt reagent A (5% a- Napthol in absolute ethanol, R029) and one drop of Barritt reagent B (40% Potassium hydroxide, R030).
  • On addition of reagent pinkish red color is observed within 10 minutes.
  • No change in colour or a slight copper color (due to reaction of Barritt reagent A and Barritt B) denotes a negative reaction.

Amino Acid Decarboxylase Test: Well No. 4, 5, 6

  • Positive reaction will show color change i.e. Olive green to Purple and negative reaction will show Olive green to yellow or remains unchanged.

Catalase: Well No. 7

  • Scrape a loopful of growth from the surface of the 7th well. Dip the loop in a small clean test tube with 3% H₂O₂
  • Positive catalase test is seen as effervescence coming out from the surface of the loop. No effervescence is observed in case of negative catalase test.

Note: 3% H2O2 solution has to be freshly prepared.

H₂S Production: Well No. 8

  • Positive reaction will show Black color after incubation period. while negative reaction will show no blackening.

Carbohydrate Fermentation Test: Well No. 8 to Well No. 12

  • Color of the medium changes from straw to light pink due to acid production if the test is positive.
  • Medium remains straw in color if the test is negative.

Important points to be taken into consideration while interpreting the result

  1. Allow the reagents to come to room temperature after removal from refrigerator.
  2. In case of Carbohydrate fermentation test some microorganisms show weak reaction. In this case record the reaction as + and incubate further for 24 hours. Straw color after 72 hours of incubation should be interpreted as negative reaction.
  3. At times organisms give contradictory results because of mutation or the media used for isolation, cultivation and maintenance.
  4. The identification index has been compiled from standard references and results of tests obtained in laboratory.

Precaution

Samples and microbial cultures should be considered potentially pathogenic and handled accordingly. Aseptic conditions should be maintained during inoculation and handling of the kits. Reagents should not come in contact with skin, eyes or clothing. 3% H₂O₂ is an extremely caustic solution, so avoid contact with skin. In case it does get on the skin, immediately flood the area with 70% Ethanol and not water, to neutralize the action.

Disposal of used material

After use, kits and the instruments used for isolation and inoculation (pipettes, loops etc.) must be disinfected using a suitable disinfectant and then discarded by incineration or autoclaving in a disposable bag.

Identification Index of V. cholerae

Sr. No. Test Medium V.cholerae
1. Motility Nutrient Agar + TTC Motile
2. Motility
3. Voges Proskauer MRVP medium Positive
4. Arginine utilization Decarboxylase Test Medium Base + 1% Arginine Negative
5. Ornithine utilization Decarboxylase Test Medium Base + 1% Ornithine Positive
6. Lysine utilization Decarboxylase Test Medium Base + 1% Lysine Positive
7. Catalase Nutrient Agar for Oxidase Positive
8. H2S production TSI Agar Negative
9. Mannitol Medium as per IS 5887 Positive
10. Sucrose (Peptone water + Andrade indicator + respective carbohydrate) Positive
11. Glucose Positive
12. Inositol Negative

Result interpretation chart

HiMotility™ Biochemical kit for V. cholerae (KBM 004)

Sr. No. Test Reagents to be added after incubation Principle Initial colour of the medium Positive reaction Negative reaction
1. Motility Motility Light yellow to Yellow Yellow to red in both the wells Red along the stabline
2. Motility
3. Voges Proskauer 1-2 drops of Barritt Reagent A and 1-2 drops of Barritt Reagent B Detects acetoin production Light yellow to Yellow Eosin, pink to red colour Yellow
4. Arginine utilization - Amino acid decarboxylation Olive green to light purple Purple Yellow
5. Ornithine utilization - Amino acid decarboxylation Olive green to light purple Purple Yellow
6. Lysine utilization - Amino acid decarboxylation Olive green to light purple Purple Yellow
7. Catalase - Detects Catalase activity Light yellow to Yellow Bubbles on addition of H2O2 No Bubbles on addition of H2O2
8. H2S production - Detects H₂S production Red Black No blackening
9. Mannitol - Carbohydrate fermentation Straw to light pink Dark pink Straw to light pink
10. Sucrose - Carbohydrate fermentation Straw to light pink Dark pink Straw to light pink
11. Glucose - Carbohydrate fermentation Straw to light pink Dark pink Straw to light pink
12. Inositol - Carbohydrate fermentation Straw to light pink Dark pink Straw to light pink

Result Entry Datasheet

No. Test 1 2 3 4 5 6 7 8 9 10
1. Motility + Nutrient Agar + TTC
2. Voges Proskauer
3. Arginine utilization
4. Ornithine utilization
5. Lysine utilization
6. Catalase
7. H₂S production
8. Mannitol
9. Sucrose
10. Glucose
11. Inositol
More Information
Product Name HiMotility Biochemical Kit for Vibrio cholerae
SKU KBM004
Customized Product Available No
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