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HiMotility Biochemical Kit for Shigella

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KBM006

Intended Use

Biochemical tests for identification of Shigella in accordance with IS specification IS 5887 Part VII.

Principle

Shigella is a genus of bacteria that is Gram-negative, facultative anaerobic, non-spore-forming, nonmotile, rod-shaped, and genetically closely related to E.coli. Shigella is one of the leading bacterial causes of diarrhea worldwide. Shigella species are classified into four species, S.flexneri, S.boydii, S.sonnei, S.dysenteriae (1,2). Indian Standard (IS) defines method for isolation, identification and enumeration of Shigella species (3). KBM006 can be used for screening food samples (3). It can also be used for relevant clinical and water samples (1,4). The tests have been designed in accordance with IS specification IS 5887 Part VII (3). Each KBM006 is a standardized, colorimetric test system based on motility, carbohydrate utilization and other biochemical tests specific for the identification of Shigella. The tests are based on the principle of pH change and substrate utilization. Shigellae 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.

Instructions for use

Preparation of inoculum:

KBM006 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 Nutrient Broth 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:

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

Interpretation of results

A combination of 12 biochemical tests for the identification of Shigella species based on motility and other biochemical tests. (Kit contains sterile media for motility in first two wells followed by Indole, Voges Proskauer, Citrate utilization, Malonate utilization, and 7 different Carbohydrate utilization tests - Glucose, lactose, mannitol, sucrose, salicin, dulcitol.

Motility + Nutrient Agar + TTC: 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.

Indole Test: Well No.3

  • Add 1-2 drops of Kovac's reagent (R008). Development of reddish pink colour within 10 seconds indicates positive reaction. Reagent remains pale coloured if the test is negative.

Vogues proskeur Test: Well No.4

  • Add 2-3 drops of Baritt reagent A (R029) and 1-2 drops of Baritt reagent B(R030). Pinkish red colour development within 5-10 minutes indicates a positive test. No change in colour or a slight copper colour (due to reaction of Baritt reagent A with Baritt reagent B) denotes a negative reaction.

Citrate and malonate utilization Test: Well No. 5,6

  • After incubation the medium changes colour to blue indicating positive reaction.

Carbohydrate Fermentation Test: Well No. 7 to Well No. 12

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

Result Interpretation Chart

HiMotility™ Biochemical Kit For Shigella (KBM006)

Well No. Test Medium Initial colour of the medium Positive reaction Negative reaction
1. Motility Nutrient Agar + TTC Yellow Yellow to red in both the wells Red along the stabline
2. Motility
3. Indole Peptone Water + Agar Colourless Reddish pink Colourless
4. Voges Proskauer MRVP medium Yellow Eosin, pink to red colour Yellow
5. Citrate Simmons Citrate Agar Green Blue Green
6. Malonate Malonate Agar Light green Blue Light green
7. Glucose Medium as per IS 5887 (Peptone water Straw to light pink Dark pink Straw to light pink
8. Lactose Straw to light pink Dark pink Straw to light pink
9. Mannitol + Andrade indicator Straw to light pink Dark pink Straw to light pink
10. Sucrose + respective carbohydrate) Straw to light pink Dark pink Straw to light pink
11. Salicin Straw to light pink Dark pink Straw to light pink
12. Dulcitol Straw to light pink Dark pink Straw to light pink

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% H2 O2 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.

Storage and Shelf-life

Store between 2-8°C. Shelf-life is 12 months. Product performance is best if used within stated expiry period.

More Information
Product Name HiMotility Biochemical Kit for Shigella
SKU KBM006
Customized Product Available No
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