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HiMotility Biochemical Kit for Vibrio parahaemolyticus

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KBM005

Intended use

Biochemical tests for identification of Vibrio parahaemolyticus in accordance with IS specification IS 5887 (Part V).

Kit contents

  1. combination of 12 biochemical tests for the confirmation of V.parahaemolyticus 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.
  2. Technical product insert.
  3. Identification Index.
  4. Baritt reagent A (R029) for VP test
  5. Baritt reagent B (R030) for VP test
  6. Result Interpretation Chart and Result Entry Datasheet.

Material Required but not supplied

  1. 3% H2O2 Solution
  2. McFarland standard
  3. Inoculation loops, pipettes
  4. Enrichment medium / Isolation media

Direction

Preparation of inoculum :

  1. The organism to be identified has to be first isolated and purified.
  2. Isolate the organism to be identified on Nutrient Agar (M001) with or without blood/TCBS Agar (M189).
  3. 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.

Inoculation of the kit :

  1. Open the kit aseptically. Peel off the sealing foil.
  2. Stab inoculate the 1st well. DO NOT INOCULATE THE 2nd WELL.
  3. 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

Temperature of incubation: 35 - 37°C. Duration of incubation: 24-48 hours

Interpretation of results

Interpret results as per the standards given in the identification index. Addition of reagents in well no 3 and 7 should be done at the end of incubation period that is after 18 - 24 hours.

Principle

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 (1,2). 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 (3). KBM005 can be used for screening food samples (3). It can also be used for relevant water samples (1,4). The tests have been designed as per the tests described in IS 5887 Part V. Each KBM005 is a standardized, colorimetric test system based on motility, carbohydrate utilization and other biochemical tests specific for the identification of V. parahaemolyticus. The tests are based on the principle of pH change and substrate utilization. V. parahaemolyticus 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.

Type of specimen

Pure isolate from food & water samples

Specimen collection and handling

Refer direction

Warning and Precautions

Read the label before opening the pack. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Aseptic conditions should be maintained during inoculation and handling of the kits. Reagents should not come in contact with skin, eyes or clothing. Safety guidelines may be referred in individual safety data sheets.

Limitations

  1. Allow the reagents to come to room temperature after removal from the refrigerator.
  2. In case of carbohydrate fermentation test some microorganisms show weak reaction. In this case record the reaction as ± and incubate further upto 48 hours. Orange colour after 48 hours of incubation should be interpreted as a negative reaction.
  3. At times organisms give conflicting result 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 carried out in the laboratory.
  5. Erroneous false negative results may be obtained if the inoculum turbidity is less than 0.1 OD.
  6. Results are more prominent if an enriched culture is used instead of a suspension.
  7. It cannot be used directly for food samples. The microorganisms to be identified have to be first isolated on appropriate isolation media. Only pure cultures should be used.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance

Sterile white opaque strip with 12 wells containing 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.

Quantity of medium

0.8 ml of medium in each well.

Sterility Check

Passes release criteria

Interpretation of results :

Interpret results as per the standards given in the identification index. Addition of reagents in no. 3 and 7 well and should be done at the end of incubation period that is after 18 - 24 hours.

Motility: Well No.1 and 2

Motility is observed as movement of growth from 1st well to 2nd well accompanied with reddening of the medium. 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 2-3 drops of Baritt reagent A (R029) and 1 drop of Baritt reagent B (R030).
  • Positive test is indicated by a development of pinkish red colour in 5 - 10 minutes.
  • No colour change or a copper colour (due to reaction of Reagent A and Reagent B) indicates a negative reaction.

Amino Acid Decarboxylase Test (Arginine, Ornithine, Lysine): Well No. 4, 5, 6

Positive reaction will show color change i.e. Olive green to Purple

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% H2 O2 solution has to be freshly prepared.

H2S Production: Well No. 8

  • Positive reaction will show Black color after incubation period.
  • 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.

Identification Index of V. parahaemolyticus

Sr. No. Test V.parahaemolyticus
1. Motility Motile
2. Motility Motile
3. Voges Proskauer Negative
4. Arginine utilization Negative
5. Ornithine utilization Positive
6. Lysine utilization Positive
7. Catalase Positive
8. H2S Production Negative
9. Mannitol Fermentation Positive
10. Sucrose Fermentation Negative
11. Glucose Fermentation Negative
12. Inositol Fermentation Negative

Result interpretation chart

HiMotility™ Biochemical kit for V. parahaemolyticus (KBM005)

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 - Mannitol fermentation Straw to light pink Dark pink Straw to light pink
10. Sucrose - Sucrose fermentation Straw to light pink Dark pink Straw to light pink
11. Glucose - Glucose fermentation Straw to light pink Dark pink Straw to light pink
12. Inositol - Inositol fermentation Straw to light pink Dark pink Straw to light pink
More Information
Product Name HiMotility Biochemical Kit for Vibrio parahaemolyticus
SKU KBM005
Customized Product Available No
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