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HiListeria™ Identification Kit

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KB012A
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Intended use

KB012A is a combination of 12 tests for identification of Listeria species from clinical specimen and non clinical samples. It can also be used for validating known laboratory strains.

Kit contents

  1. Each kit contains 5/10/20 kits of KB012A, sufficient material to perform 5/10/20 tests. (Kit contains sterile media for Catalase, Nitrate reduction, Esculin, Voges Proskauer's, Methyl red, and 7 different carbohydrates utilization tests - Xylose, Lactose, Glucose, α-Methyl-D-Mannoside, Rhamnose, Sucrose, Mannitol .
  2. Sulphanilic acid 0.8%(R015) for Nitrate test
  3. N, N-Dimethyl-1-Napthylamine reagent (R009) for Nitrate test
  4. Baritt reagent A (R029) for VP test
  5. Baritt reagent B (R030) for VP test
  6. Methyl Red reagent (I007) for MR test
  7. Technical product insert
  8. Result Interpretation Chart and Result Entry Datasheet Identification Index

Material Required but not supplied

  1. McFarland standard
  2. Inoculation loops, pipettes
  3. Enrichment medium / Isolation media

Direction

Preparation of inoculum :

  • KB012A 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 on either PALCAM Agar (M1064) or Tryptone Soya Agar (M290). Pick up a single isolated colony and inoculate in 5 ml BHI Broth (M210) and incubate at 35-37°C for 6 to 8 hours until inoculum turbidity is 1.0 OD at 620nm.

Inoculation of the kit :

  1. Open the kit aseptically. Peel off the sealing foil.
  2. Inoculate each well with 50µl of the above inoculum by surface inoculation method.
  3. Alternatively, the kit can also be inoculated by stabbing each individual well with a loopful of inoculum.

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 Result Interpretation Chart. Addition of reagents in well no 1, 2, 4, 5 should be done at the end of incubation period that is after 24 to 48 hours.

Principle

Each KB012A is a standardized, colorimetric test system based on motility, carbohydrate utilization and other biochemical tests specific for the identification of Listeria species. The tests are based on the principle of pH change and substrate utilization. Listeria species 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 clinical specimen and non clinical sample

Specimen collection and handling

Refer direction

Warning and Precautions

In Vitro diagnostic Use. For professional use only. 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 clinical 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 clinical specimens. 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

Kit contains sterile media for Catalase, Nitrate reduction, Esculin, Voges Proskauer's, Methyl red, and 7 different carbohydrates utilization tests - Xylose, Lactose, Glucose, α-Methyl-D-Mannoside, Rhamnose, Sucrose, Mannitol

Quantity of medium

0.8 ml of medium in each well.

Sterility Check

Passes release criteria

Interpretation of results :

Interpret results as per the result interpretation chart. Addition of reagents in well no 1, 2, 4, 5 should be done at the end of incubation period that is after 24 to 48 hours.

1.Catalase Test: Well No. 1

  • Scrape a loopful of growth from the surface of the 3rd 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.

2.Nitrate Reduction: Well No. 2

  • Add 1-2 drops of Sulphanilic acid (R015) and 1-2 drops of N,N-Dimethyl-1-Napthylamine Reagent (R009).
  • Immediate development of pinkish red colour on addition of reagent indicates positive reaction.
  • No change in colour indicates negative reaction.

3.Esculin Hydrolysis : Well No.3

  • Positive test is indicated by a colour change to black colour.
  • Green or no colour change indicates a negative reaction.

4.Voges-Proskauer's Test : Well No. 4

  • Add 1-2 drops of Barritt reagent A (R029) and 1 - 2 drops of Barritt 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.

5.Methyl red Test: Well No. 5

  • Add 2-3 drops of Methyl red indicator (I007).
  • Positive test is indicated by a development of red colour.
  • Yellowish orange colour indicates a negative reaction.

6.Carbohydarte Fermentation Test : Well No. 6 to Well No 12

  • Positive test is indicated by a colour change to yellow colour.
  • Red or no colour change indicates a negative reaction.

Result interpretation chart

No. Test Reagents to be added in wells Principle Original colour of the medium Positive reactio Negative reaction
1 Catalase 3% H₂O₂ solution Detects Catalase activity Colourless Effervescence coming out from the loop No Effervescence seen
2 Nitrate Reduction 1-2 drops ofsulphanilic acid and 1-2 drops of N, N-Dimethyl-1-Naphylamine Detects Nitrate reduction Colourless Pinkish Red Colourless
3 Esculin Hydrolysis Detects Esculin hydrolysis Cream Black Cream
4 Voges Proskauer's 1-2 drops of Baritt reagent A and 1-2 drops of Baritt reagent B Detects acetoin production Colourless/ Light yellow Pinkish red Colourless/ slight copper
5 Methyl red 1-2 drops of Methyl red reagent Detects acid production Colourless Red Yellowish-orange
6 Xylose — Xylose utilization Pinkish Red / Red Yellow Red / Pink
7 Lactose — Lactose utilization Pinkish Red / Red Yellow Red / Pink
8 Glucose — Glucose utilization Pinkish Red / Red Yellow Red / Pink
9 α-Methyl-D-Mannoside — α-Methyl-D-Mannoside utilization Pinkish Red / Red Yellow Red / Pink
10 Rhamnose — Rhamnose utilization Pinkish Red / Red Yellow Red / Pink
11 Sucrose — Sucrose utilization Pinkish Red / Red Yellow Red / Pink
12 Mannitol — Mannitol utilization Pinkish Red / Red Yellow Red / Pink

Identification Index of various Listeria species

Test Catalase Nitrate Reduction Esculin Hydrolysis Voges Proskauer's Methyl red Xylose Lactose Glucose α-Methyl-D-Mannosid Rhamnose Sucrose Mannitol
Listeria grayi + + + + + + + V +
Listeria monocytogenes + + + + V + + + V
Listeria innocua + + + + + + + + V
Listeria seeligeri + NR + + + + NR + V
Listeria ivannovii Sub sp. ivannovii + + + + + + + V
Listeria ivannovii Sub sp. londoniesis + + + + + + + V
Listeria welshimeri + NR + + + + NR + + V +

Note: Based on % strains showing reactions following symbols have been assigned from laboratory results and standard references.
+ = Positive (more than 90%) - = Negative NR = Not Reported v = Variable reaction

Storage and Shelf Life

On receipt store between 2-8°C. Use before expiry date on the label. Product performance is best if used within stated expiry period.

Disposal

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 disposal bag. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (2,3).

More Information
Product Name HiListeria™ Identification Kit
SKU KB012A
Customized Product Available No
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