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Kohn Two Tube Medium No.2
Used for identification of Enterobacteriaceae on the basis of sucrose and salicin fermentation, motility, hydrogen sulphide and indole production.
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Intended Use
Recommended for identification of Enterobacteriaceae on the basis of sucrose and salicin fermentation, motility, hydrogen sulphide and indole production.
Composition
| Ingredients | g/L |
|---|---|
| Peptone | 10.000 |
| Tryptone | 10.000 |
| Saccharose (Sucrose) | 10.000 |
| Salicin | 10.000 |
| Sodium chloride | 5.000 |
| Sodium thiosulphate | 0.016 |
| Disodium hydrogen phosphate | 0.090 |
| Bromothymol blue | 0.020 |
| Agar | 3.000 |
| Final pH (at 25°C) | 7.4±0.2 |
**Formula adjusted, standardized to suit performance parameters
Directions
Suspend 48.13 grams in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Dispense in tubes and sterilize by autoclaving at A115°C for 15 minutes. Cool the tubed medium in an upright position.
Acorresponds to 10 lbs pressure.
Principle And Interpretation
Russell (1) first introduced Double Sugar Medium, a differentiating medium for Enterobacteriaceae. Kohn (2) later developed a technique employing two tubes of composite media for study of culture reactions, for the identification of Enterobacteriaceae. Gillies (3) further made minor modifications in Kohns media. Kohn Two Tube Medium No.2 is used to study carbohydrate fermentation (Sucrose and Salicin) along with motility, hydrogen sulfide production and indole production.
Type of specimen
Clinical isolates: Faeces
Specimen Collection and Handling
Using a straight wire, inoculate with a single stab to about one-third of the depth of the Kohn Two Tube Medium No. 2. Suspend the two test papers (lead acetate and Kovac's) above the medium by bending and trapping them between the cotton wool plug and the side of the test tube. Incubate at 37°C for 18-24 hours and examine for motility, H2S production, sugar fermentation and indole production. Motility is seen as diffused growth spreading from the line of inoculation. The blackening of the lead acetate paper strip indicates H2S production. Fermentation of sucrose or salicin or both is indicated by the colour change to yellow with bromothymol blue being the pH indicator. Indole formation is indicated by the change in colour of the Kovac's reagent paper to pinkish red. After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic Use. For professional use only. Read the label before opening the container. 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. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Well isolated colonies must be used for identification.
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
Cream to yellow homogeneous free flowing powder
Gelling
Semisolid, comparable with 0.3% Agar gel.
Colour and Clarity of prepared medium
Green coloured, clear to slightly opalescent gel forms in tubes as butts
Reaction
Reaction of 4.81% w/v aqueous solution at 25°C. pH : 7.4±0.2
pH
7.20-7.60
Cultural Response
Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.
| Organism | Motility | Fermentation w/ Sucrose/ Salicin | H2S(with lead acetate strip) | Indole |
|---|---|---|---|---|
| ## Proteus hauseri ATCC 13315 | positive, growth away from stabline causing turbidity | acid & gas production or negative reaction | variable reaction | variable reaction |
| Salmonella Typhimurium ATCC 14028 (00031*) | positive, growth away from stabline causing turbidity | negative reaction | variable reaction | negative reaction |
| Salmonella Typhi ATCC 6539 | positive, growth away from stabline causing turbidity | negative reaction | positive, blackening of the lower portion of the strip | negative reaction |
| Shigella flexneri ATCC 12022 (00126*) | negative, growth along the stabline, surrounding medium remains clear | negative reaction | negative,no blackening | variable |
| Shigella sonnei ATCC 25931 | negative, growth along the stabline, surrounding medium remains clear | negative reaction | negative,no blackening | negative reaction |
| Shigella schmitzi | negative, growth along the stabline, surrounding medium remains clear | negative reaction | negative,no blackening | positive reaction,pink colour at the lower portion of the strip |
Key: (*) Corresponding WDCM numbers.
## Formerly known as Proteus vulgaris
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 15-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.
Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. 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 (4,5).
| Product Name | Kohn Two Tube Medium No.2 |
|---|---|
| SKU | M802 |
| Product Type | Regular |
| Physical Form | Powder |
| Origin | Animal |
| Packaging type | HDPE |
| References | 1.Russell F. F., 1911, J. Med. Res., 25:217.2.Kohn J., 1954, J. Path. Bacteriol., 67(1): 286.3.Gillies R. R., 1956, J. Clin. Pathol., 9(4):368. |
| Customized Product Available | No |
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