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HiE. coli™ Identification Kit

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KB010

Product Identifier

Product Name HiE.Coli™ Identification Kit
SKU KB010

Directions:

KB010 is a standardized, colorimetric identification system utilizing eight conventional biochemical tests and four carbohydrate utilization tests for identification and differentiation of Escherichia coli using pure isolate. Escherichia coli are Gram negative, lactose fermenting coccobacillary rods which are frequently isolated from clinical specimens and non clinical samples.

Preparation of inoculum :

  1. Isolate the organism to be identified on a common medium like Nutrient Agar (M001) or a differential medium like MacConkey Agar (M082).
  2. Pick up a single isolated colony and inoculate in 5 ml BHI Broth (M210) and incubate at 35- 37°C for 4-6 hours until the inoculum turbidity is 0.1 OD at 620nm or 0.5 McFarland standard. Some organisms may require more than 6 hours of incubation. In this case incubate till the inoculum turbidity reaches 0.1 OD at 620nm.
  3. Alternatively, prepare the inoculum by picking 1-3 well isolated colonies and make a homogenous suspension in 2-3ml sterile saline. The density of the suspension should be 0.1 OD at 620nm.

Inoculation of the kit :

  1. Open the kit aseptically. Peel off the sealing foil.
  2. Inoculate each well with 50 µl 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: 18-24 hours

Interpretation of results :

Interpret results as per the standards given in the identification index. Addition of reagents in well no 1, 2, 4 and 6 should be done at the end of incubation period that is after 18 - 24 hours.

Technical Specification

Kit contents
  1. Each kit contains 5/10/20 kits of KB010, sufficient material to perform 5/10/20 tests. Kit contains sterile media for Methyl Red test, Voges-Proskauer's, Citrate utilization, Indole, Glucuronidase, Nitrate reduction, ONPG, Lysine utilization tests and 4 different carbohydrates utilization tests - Lactose, Glucose, Sucrose, Sorbitol.
  2. Technical product insert.
  3. Result Interpretation Chart and Result Entry Datasheet.
  4. Identification Index.
  5. Methyl Red reagent (I007) for Methyl Red test.
  6. Baritt reagent A (R029) for Voges-Proskauer's test.
  7. Baritt reagent B (R030) for Voges-Proskauer's test.
  8. Kovac's reagent (R008) for Indole test
  9. Sulphanilic acid 0.8% (R015) for Nitrate test
  10. N,N-Dimethyl-1-Napthylamine reagent (R009) for Nitrate test
Material Required but not supplied
  1. McFarland standard
  2. Inoculation loops, pipettes
  3. Enrichment medium / Isolation media
Principle Each KB010 kit is a standardized colorimetric identification system utilizing eight conventional biochemical tests and four carbohydrate utilization tests. The tests are based on the principle of pH change and substrate utilization. On incubation E.coli exhibit metabolic changes indicated by a colour change in the media that is either visible spontaneously or after addition of a reagent.
Type of specimen Pure isolate from clinical specimen and non clinical sample
Specimen collection and handling Refer direction
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

Sterile white opaque strip with 12 wells containing media for MR test, Voges-Proskauer's, Citrate utilization, Indole, Glucuronidase, Nitrate reduction, ONPG, Lysine utilization tests and 4 different carbohydrates utilization tests- Lactose, Glucose, Sucrose, Sorbitol

Quantity of medium

0.8 ml of medium in each well.

Sterility Check

Passes release criteria
Interpretation of results (detailed)

Interpret results as per the standards given in the identification index. Addition of reagents in well no 1, 2, 4 and 6 should be done at the end of incubation period that is after 18 - 24 hours.

Methyl red Test: Well No. 1

  • 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.

Voges-Proskauer's Test : Well No. 2

  • 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.

Citrate Test: Well No. 3

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

Indole Test: Well No. 4

  • Add 2-3 drops of Kovac's reagent (R008)
  • Positive test is indicated by a development of pinkish red ring.
  • No red ring indicates a negative reaction.

Glucuronidase Test: Well No. 5

  • Colourless medium turns bluish green indicating positive reaction.
  • Colourless medium indicates negative reaction.

Nitrate Reduction: Well No. 6

  • 1-2 drops of sulphanilic acid (R015) and 1-2drops of N,N Dimethyl-1- Napthylamine (R009).
  • Development of pinkish red colour indicates positive reaction.
  • Colourless medium indicates negative reaction.

ONPG: Well No. 7

  • Colourless medium changes to yellow colour indicates positive reaction.
  • Colourless medium indicates negative reaction.

Lysine utilization: Well No. 8

  • Colour change from olive green to light to dark purple indicates positive reaction.
  • Yellow colour medium indicates negative reaction.

Carbohydrate fermentation Test : Well No. 9-12

  • 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 after incubation Principle Original colour of the medium Positive reaction Negative reaction
1 Methyl red 1-2 drops of Methyl red indicator Detects acid production Colourless Red Yellowish-orange
2 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
3 Citrate utilization — Detects capability of organism to utilize citrate as a sole carbon source Green Blue Green
4 Indole 1-2 drops of Kovac's reagent Detects deamination of tryptophan Colourless Reddish pink Colourless
5. Glucuronidase — Detects Glucuronidase activity Colourless Bluish green Colourless
6. Nitrate reduction 1-2 drops of sulphanilic acid and 1-2drops of N,N Dimethyl-1- Napthylamine Detects Nitrate reduction Colourless Pinkish red Colourless
7 ONPG — Detects ?-galactosidase activity Colourless Yellow Colourless
8 Lysine utilization — Detects Lysine decarboxylation Olive green to Purple / Light purple Yellow
9 Lactose — Lactose utilization Pinkish Red / Red Yellow Red / Pink
10 Glucose — Glucose utilization Pinkish Red / Red Yellow Red / Pink
11 Sucrose — Sucrose utilization Pinkish Red / Red Yellow Red / Pink
12 Sorbitol — Sorbitol utilization Pinkish Red / Red Yellow Red / Pink
Identification Index of various Escherichia species
Tests Methyl Red Voges Proskauer's Citrate utilization Indole Glucuronidase Nitrate ONPG Lysine Lactose Glucose Sucrose Sorbitol
E. coli + - - + + + + + + + V +
E. coli, inactive + - - + - + + V + + V V
E. fergusonii + - V + - + V + + + - -
E. hermannii + - - - - + + + V + V -
E. vulneris + - - - - + + + V V + -
E. blattae + V - - - + + - + + + +

Note: Based on % strains showing reactions following symbols have been assigned from laboratory results and standard references.+= Positive (more than 90%) = Negative (more than 90%) V = Variable (11-89%)
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).

Safety Information

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.
More Information
Product Name HiE. coli™ Identification Kit
SKU KB010
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