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HiAcinetobacter™ Identification Kit

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KB014

Introduction

KB014 is identification system for Acinetobacter comprising of twenty four biochemical tests (Twelve tests in Part A and Twelve tests in part B). The tests include the IMVIC group of tests, carbohydrate fermentation tests, amino acid hydrolysis tests and other tests. The complete list of organisms that can be identified with this system is given in the identification index provided with the kit.

Principle

KB014 is a standardized, colorimetric identification system utilizing twenty four conventional biochemical tests. The tests are based on the principle of pH change and substrate utilization. On incubation, organisms undergo metabolic changes which are indicated as a colour change in the media that is either visible spontaneously or after addition of a reagent.

Kit Contents

  1. Each kit contains sufficient material to perform 10 tests
  2. 10 strips of KB014
  3. Technical product insert
  4. Result Interpretation Chart and Result Entry Datasheet
  5. Identification Index
  6. Kovac's reagent (R008)
  7. Baritt reagent A (R029)
  8. Baritt reagent B (R030)
  9. TDA reagent (R036)
  10. Alpha-Naphthylamine Solution (R009)
  11. Sulphanilic Acid, 0.8% (R015)

Instructions for use

Note: KB014 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.

Preparation of inoculum

  • Isolate the organism to be identified on a common medium like Nutrient Agar (M001) or Soyabean Casein Digest Agar (M290). Pick up a single isolated colony and inoculate in 5 ml Brain Heart Infusion Broth and incubate at 37°C for 4-6 hours until the inoculum turbidity is 0.1OD at 620nm or 0.5 McFarland standards. Some fastidious organisms may require more than 6 hours of incubation. In this case incubate till the inoculum turbidity reaches 0.1OD at 620nm.
  • Alternatively, prepare the inoculum by picking 1-3 well isolated colonies and make a homogenous suspension in 2-3 ml sterile saline. The density of the suspension should be 0.1OD at 620nm.

Note: Erroneous false negative results may be obtained if the inoculum turbidity is less than 0.1 OD.Results are more prominent if an enriched culture is used instead of suspension.

Inoculation of the strip :

  • Open the kit aseptically.
  • Inoculate each well with 50 µl of the above inoculum by surface inoculation method.
  • Alternatively, the strip can be inoculated by stabbing each individual well with a loopful of inoculum.

Incubation:

  • Temperature of incubation: 35 - 37°C. Duration of incubation: 18 – 24 hours.

Interpretation of results :

Interpret results as per the standards given in the result interpretation chart. Addition of reagents in well no 1, 2, 7 & 10 of part A should be done at the end of incubation period that is after 18 - 24 hours.

PART A:

Well No. 1: Indole test :

  • Add 1-2 drops of Kovac's reagent to well no 1.
  • Positive test is indicated by development of reddish pink colour within 10 seconds.
  • Absence of reddish pink colour development denotes a negative reaction.

Well No. 2: Voges-Proskauer's Test :

Add 2-3 drops of Baritt reagent A and 1-2 drops of Baritt reagent B to well no 2.

Reddish pink colour development within 5 – 10 minutes indicates a positive test.

No change in colour or a slight change in colour (due to reaction of Baritt reagent A with Baritt reagent B) denotes a negative reaction.

Well No. 3: Citrate Test :

  • No reagent to be added.
  • Colour of the medium changes from its original green to blue colour if the test is positive.
  • Medium remains green if the test is negative.

Well No. 4: Lysine Test :

  • No reagent to be added.
  • Colour of the medium changes from its original olive green to purple colour if the test is positive.
  • Medium turns yellow if the test is negative.

Well No. 5: Ornithine Test :

  • No reagent to be added.
  • Colour of the medium changes from its original olive green to purple colour if the test is positive.
  • Medium turns yellow if the test is negative.

Well No. 6: Arginine Test :

  • No reagent to be added.
  • Colour of the medium changes from its original olive green to purple colour if the test is positive.
  • Medium turns yellow if the test is negative.

Well No. 7: Nitrate Reduction Test :

  • Add 1-2 drops of sulphanilic acid and 1-2 drps of Alpha-Naphthylamine Solution to well no 7.
  • Positive test is indicated by development of pinkish red colour.
  • Absence of pinkish red colour development denotes a negative reaction.

Well No. 8: Malonate Test :

  • No reagent to be added.
  • Colour of the medium changes from its original light green to blue colour if the test is positive.
  • Medium remains light green if the test is negative.

Well No. 9: Urease Test :

  • No reagent to be added.
  • Colour of the medium changes from its original orangish yellow to pink colour if the test is positive.
  • Medium remains orangish yellow if the test is negative

Well No. 10: Phenylalanine Deamination Test :

  • Add 2-3 drops of TDA reagent to well no 10.
  • Positive test is indicated by development of green colour.
  • Absence of green colour development denotes a negative reaction.

Well No. 11: H2S Production Test :

  • No reagent to be added.
  • Colour of the medium changes from its original orangish yellow to black colour if the test is positive.
  • Medium remains orangish yellow if the test is negative.

Well No. 12: ONPG Test :

  • No reagent to be added.
  • Colour of the medium changes from its original colourless to yellow colour if the test is positive.
  • Medium remains colourless if the test is negative.

Part B:

Well No 1 to12: Carbohydrate fermentation test :

  • No reagent to be added.
  • Colour of the medium changes from its original red to yellow colour due to acid production indicating a positive reaction.
  • Medium remains red in colour if the test is negative.

Biochemical reactions of KB014 PART A

Well No Test Reagents to be added after incubation Principle Original colour of the medium Positive reaction Negative reaction
1 Indole 1-2 drops of Kovac's reagent Detects deamination of tryptophan Colourless Reddish pink Colourless
2 Voges-Proskauer 1-2 drops of Baritt reagent A and 1-2 drops of Barrit reagent B Detects acetoin production Colourless Pinkish red Colourless/Slight copper
3 Citrate utilization - Detects capability of organism to utilize citrate as a sole carbon source. Yellowish green Blue Yellowish green
4 Lysine utilization - Detects Lysine decarboxylation Olive green Purple yellow
5 Ornithine utilization - Detects Lysine decarboxylation Olive green Purple yellow
6 Arginine utilization - Detects Arginine decarboxylation Olive green Purple yellow
7 Nitrate reduction 1-2 drops of sulphanilic acid and 1-2 drops of Alpha-Naphthylamine Solution Detects nitrate reduction Colourless Pinkish red Colourless
8 Malonate - Detects capability of organism to utilize sodium malonate as a sole carbon source Light green Blue Light green
9 Urease - Detects urease activity Orangish yellow Pink Orangish yellow
10 Phenylalanine deamination 2-3 drops of TDA reagent Detects phenylalanine deamination activity Colourless Green Colourless
11 H₂S Production - Detects H₂S Production Orangish yellow Black Orangish yellow
12 ONPG - Detects Beta-galactosidase activity Colourless yellow Colourless

Result interpretation chart

Well No Test Reagents to be added after incubation Principle Original colour of the medium Positive reaction Negative reaction
1 Glucose - Glucose utilization Red Yellow Red/Pink
2 Mannitol - Mannitol utilization Red Yellow Red/Pink
3 Xylose - Xylose utilization Red Yellow Red/Pink
4 Inositol - Inositol utilization Red Yellow Red/Pink
5 Sorbitol - Sorbitol utilization Red Yellow Red/Pink
6 Rhamnose - Rhamnose utilization Red Yellow Red/Pink
7 Sucrose - Sucrose utilization Red Yellow Red/Pink
8 Lactose - Lactose utilization Red Yellow Red/Pink
9 Arabinose - Arabinose utilization Red Yellow Red/Pink
10 Adonitol - Adonitol utilization Red Yellow Red/Pink
11 Raffinose - Raffinose utilization Red Yellow Red/Pink
12 Salicin - Salicin utilization Red Yellow Red/Pink

Important points to be taken into consideration while interpreting the result:

  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 for 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 & maintenance.
  4. The identification index has been compiled from standard references & results of tests carried out in the laboratory.

Precautions:

  • Clinical samples and microbial cultures should be considered potentially pathogenic and handled accordingly.
  • Aseptic conditions should be maintained during inoculation & handling of the strips.
  • Reagents should not come in contact with skin, eyes or clothing.

Disposal of used material :

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

Storage and Shelf-life

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

More Information
Product Name HiAcinetobacter™ Identification Kit
SKU KB014
Customized Product Available No
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