Leptospira Medium Base, Korthof, Modified

Intended Use

Recommended for isolation, cultivation and maintenance of Leptospira species.

Composition

Final pH (at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

1)Preparation of Base: Suspend 3.42 grams in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Distribute in 100 ml amounts in flasks. Sterilize by autoclaving at A 115°C for 15 minutes. Cool to 45-50°C.

2)Preparation of Haemoglobin Solution: To the rabbit blood clot, after removing serum, add equal volume of purified/distilled water. Freeze and thaw repeatedly to haemolyse the corpuscles. Sterilize by Seitz or millipore filtration.

3)Complete Medium: To 100 ml sterile base, add sterile 8 ml inactivated blood serum and 0.8 ml sterile haemoglobin solution. Mix thoroughly. Distribute if desired in 2-3 ml amount in sterile screw capped Bijou bottles / tubes. Test for sterility by incubating at 37°C.

Note: A Corresponds to 10 lbs pressure

Principle And Interpretation

Leptospirosis is an acute febrile disease caused by members of the genus Leptospira (1,2). Direct culture of blood is the most reliable way to detect Leptospira during the first week of illness. After the first week of illness and for several months thereafter, leptospires may be isolated by direct culture of undiluted urine specimens. By autopsy, leptospires may be isolated from kidney and liver tissues as well as from blood and urine. Leptospira Medium Base, Korthof, Modified is formulated as described by Korthof (3,4) for cultivation and maintenance of Leptospira species.

Peptone provide amino acids and other nitrogenous substances to support bacterial growth. Haemoglobin solution and inactivated blood serum provide additional sources of nutrients to the Leptospires. The salts supply essential nutrients for the growth of the organisms. Phosphates form buffering system while sodium chloride maintains osmotic equilibrium and also provides essential ions.

All cultures are incubated at room temperature in the dark for up to 6 weeks. The organisms grow below the surface. Material collected from a few centimeters below the surface of broth cultures should be examined weekly for the presence of growth using a direct wet preparation under dark field illumination. Letpospires will exhibit corkscrew like motility (1). Examine the tubes for growth every 5-7 days. Growth occurs as a ringed area (disc) 1-3 cm below the surface of the medium. The absence of a ringed area of growth doesnt necessarily mean leptospires are not present. Remove a small amount of growth from the disc area and examine microscopically (gram stain is not satisfactory). Microcolonies can be fixed with methanol and stained with Giemsa's stain to show rod forms (3).

Type of specimen

Clinical samples - urine

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (5,6). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

In Vitro diagnostic Use only. 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

1. Successive specimens cultured at least 1 day apart increase the likelihood of positive culture, since Leptospira may be shed sporadically (4).

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
Off-white to yellow homogeneous free flowing powder

Colour and Clarity of prepared medium
Yellowish brown coloured, clear to slightly opalescent solution after addition of serum and haemogloin

Reaction
Reaction of 0.342% w/v aqueous solution at 25°C. pH: 7.2±0.2

pH
7.00-7.40

Cultural Response
Cultural characteristics observed with added inactivated blood serum and sterile haemoglobin solution, after an incubation at 30°C for upto 2-7days.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°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 (5,6).

Reference

1. Forbes B. A., Sahm A. S., and Weissfeld D. F., Bailey & Scotts Diagnostic Microbiology, 10th Ed., 1998, Mosby, Inc., St. Louis, Mo.
2. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
3. Korthof G., 1932, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I. Orig., 125:429.
4. Rechcigl M. Jr. (Ed.), 1978, Handbook Series in Nutrition and Food, Vol. III, CRC Press.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
6. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.