FLETCHER'S MEDIA

Cat. no. X67 Fletcher's Media, 2oz. Boston Round Bottle, 10ml 10 bottles/box
Cat. no. U987 Fletcher's Medium Broth Base, Polycarbonate Bottle, 1000ml 1 bottle
Cat. no. SRS125** Rabbit Serum, filtered, sterile, 125ml 1 bottle
**sold separately

INTENDED USE

Hardy Diagnostics Fletcher's Media are recommended for the cultivation of Leptospira spp. Fletcher's Medium Broth Base is recommended for use with sterile rabbit serum.

Cat. no. U987 is not intended to be used for the diagnosis of human disease.

SUMMARY

Leptospirosis is a ubiquitous spirochetal zoonotic disease caused by infection with pathogenic members of the genus Leptospira; Adolf Weil first described leptospirosis in humans in 1816.(3-5,7,10) The spectrum of disease caused by leptospires is wide, ranging from subclinical infection to multi-organ infection associated with high mortality. Syndromes associated with Leptospira infection have been described in the literature as early as the start of the 19th century, and it is believed to have been brought to western Europe in the 18th century by the westward migration of Rattus norvegicus, the common brown rat, originating from Eurasia and now found throughout most of the world. (9) The role of the brown rat as a common source of human infection was uncovered in the early 20th century, and the link between companion animals, such as dogs and livestock, as the source was recognized some years later.(5,7,9,13) Chronically infected animals may remain carriers from years to life and can serve as reservoirs of infection for other animals and humans. Infection usually results from direct or indirect exposure to contaminated urine or leptospiruric animals.(7)

Leptospirosis is usually a biphasic illness and may present as febrile illness, with or without meningitis, or as Weil's syndrome, resulting in hemorrhagic renal failure and jaundice.(6) Acute onset consists of flu-like symptoms persisting for 4 to 7 days, with the second immune phase occurring within a few days. Clinical recognition of leptospirosis is complicated due to multi-organ infection and nonspecific clinical presentation.(6) Two major consistent microbiological sequelae of leptospiral infection are localization and persistence of leptospires in renal tissue and in the human genital tract.

During the first week of illness, direct culture of the organism in blood, milk, or spinal fluid is suggestive of acute leptospirosis. After the first week, fresh urine or, if testing is delayed, urine diluted 1:10 in 1% bovine serum albumin is recommended. However, due to the length of culture, more rapid methods of detection, such as serologic, immunochemical, histological, or PCR, are more helpful for timely treatment of serious infections. The microscopic agglutination test (MAT) is considered the gold standard for identification, but this technique is not well suited for guiding clinical management due to decreased sensitivity with fluctuating titers.(6,13)

In the early 1920s, Fletcher developed an enriched medium for the cultivation of Leptospira from clinical specimens, including urine, blood, and kidney and liver tissues.(2,13) Hardy Diagnostics Fletcher's Media is based on this formulation and contains peptone, beef extract, and rabbit serum, which provide essential amino acids, carbon, and nitrogenous compounds to support bacterial growth. Sodium chloride provides ions necessary for maintaining osmotic equilibrium. Fletcher's Media (Cat. no. X67) contains minimal agar and is a semi-solid medium, making it useful for detecting motile organisms.

FORMULA

Ingredients per liter of deionized water:*

Fletcher's Media (Cat. no. X67)
Sodium Chloride 0.5gm
Peptone 0.3gm
Beef Extract 0.2gm
Rabbit Serum 80.0ml
Agar 1.5gm

Fletcher's Medium Broth Base (Cat. no. U987)
Sodium Chloride 0.5gm
Peptone 0.3gm
Beef Extract 0.2gm

Final pH 7.9 +/- 0.1 at 25°C.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt, store Cat. no. X67 at 2-8°C and Cat. no. U987 at 2-30°C away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

Fletcher's Media (Cat. no. X67)

Fletcher's Medium Broth Base (Cat. no U987)

PROCEDURE

Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. During acute onset within the first week of illness, cerebral spinal fluid (CSF) or blood may be cultured. During subsequent weeks of infection, fresh undiluted or 1:10 diluted in 1% bovine serum album urine specimens stored at 5 to 20°C. for up to a few days are recommended. Consult listed references for more information on appropriate specimen collection and culture.(3-6,8,10,12,13)

For cat. no. U987, add 80ml of filter-sterilized rabbit serum (Cat. no. SRS125) to 1L Fletcher's Medium Broth Base. Mix well.

Method of Use:

1. Prior to inoculation, the medium should be brought to room temperature.

2. Inoculate the medium with one or two drops of blood or urine and distribute throughout the medium. Leptospires are more likely to be isolated from blood during the first week of illness. Thereafter, they are more likely to be isolated from urine. Both undiluted and 1:10 dilute urine in 1% bovine serum albumin should be cultured. NOTE: 5-fluorouracil can be added to the medium to culture heavily contaminated urine specimens.(6)

3. Leptospira may also be cultured from liver and kidney tissue. Aseptically macerate tissue specimens and inoculate using 1:1, 1:10, and 1:100 dilutions for optimal results. Consult appropriate references for detailed information about the processing and inoculation of tissues and other specimens.(3,6,10,13)

4. Incubate cultures at 29 +/- 1°C. for 7 to 10 days. Examine cultures by quality dark-field microscopy every 1 to 2 weeks using a 100 watt light source for optimal detection. NOTE: The organism may take up to 16 to 26 weeks to detect in culture, depending upon the serovar and number of cells present in the initial sample.(13)

INTERPRETATION OF RESULTS

Examine tubes for growth every 5 to 7 days. Growth occurs as a ringed-area (Dinger's ring) or disk 1-3cm below the surface of the medium.(8) The lack of ringed growth does not necessarily denote the absence of leptospires in the culture. Remove a small amount of growth from the disk area and examine microscopically by dark-field, Giemsa (Cat. no. 591A16) or fluorescent stain. Gram staining is not satisfactory. Cells can be fixed with methanol (Cat. no. VMT032) and stained with Giemsa stain to show long thin tightly-coiled spirochetes up to 20um in length.(12)

LIMITATIONS

Fletcher's Medium is non-selective and heavily contaminated specimens may overgrow leptospires, preventing successful direct cultural isolation.(9)

Cat. no. U987 is a base only, and the addition of filter-sterilized rabbit serum is required.

Successive specimens cultured at least 1 day apart increase the likelihood of positive culture, since Leptospira may be shed sporadically.(9)

Pathogenic Leptospira are generally 0.1um in diameter and 10-20um in length, aerobic, tightly-coiled spiral-shaped bacteria, with distinct pointed hook-like ends, and axial, periplasmic flagellae; CO2 is essential to trigger growth of the bacterium, though the organism is aerobic.(8,9) Gentle agitation of cultures, such as an orbital agitator or nutating mixer (Cat. no. 520GS), may increase yield.

Some strains may require a medium containing pyruvate for optimal growth or grow best when incubated at the host body temperature. The most widely used medium currently in use for culture of Leptospira is Ellinghausen-McCullough-Johnson-Harris (EMJH) medium which contains fatty [oleic] acids and bovine serum albumin.(6)

Both fresh, undiluted urine and 10-fold diluted urine specimens in 1% bovine serum albumin should be cultured, since undiluted urine may contain metabolites that can inhibit the growth of Leptospira.(6,13)

Leptospires do not stain well with aniline dyes and silver-staining methods lack sensitivity and specificity for the organism, though these techniques may be a useful adjunct for histopatholgical diagnosis.(13) In addition, due to the small width of leptospires, contrast-enhancing techniques such as dark-field are recommended for optical microscopes.

Complete identification of isolates may be required to determine if the organism is pathogenic or saprophytic and the etiologic agent of disease.(9)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, Giemsa stain (Cat. no. 591A16), methanol (Cat. no. VMT032), other culture media or supplements (Cat. no. SRS125), incinerators, and incubators, microscopes, nutating mixers (Cat. no. 520GS), etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Hardy Diagnostics tests Fletcher's Media and Fletcher's Medium Broth Base for sterility, pH, and fill volume only.

USER QUALITY CONTROL

Physical Appearance

Fletcher's Media (Cat. no. X67) should appear slightly opalescent and light amber in color.

Fletcher's Medum Broth Base (Cat. no. U987) should appear clear and colorless with no precipitate or debris.

REFERENCES

1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Fletcher, W. 1928. Recent work on leptospirosis, tsutsugamushi disease and tropical typhus in the Federated Malay States. Trans. Roy. Soc. Trop. Med. Hyg.; 21:265-287.

3. Tille, P.M., etal. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

4. Gochenour, W.S. 1953. Laboratory Diagnosis of Leptospiral Infections. New York Acad. of Med. Bull.

5. Hirschberg, N., L. Maddry, M. Hines. 1956. Laboratory Services in the Diagnosis of Leptospirosis. Am. J. of Pub. Health.; 46:45-53.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. Klatskin, G. 1955. Leptospirosis. Yale J. of Bio. and Med.; 27:243-266.

8. Leptospirosis Information Center. Culturing leptospires in the laboratory. [accessed 12/15/2011]. http://www.leptospirosis.org/topic.php?t=28

9. Levett, P.N. 2001. Leptospirosis. Clin. Microbio. Rev.; 14(2):296-326.

10. Jorgensen et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

11. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

12. Weinman. 1981. In Balows and Hausler (ed.), Diagnostic Procedures for Bacterial, Mycotic and Parasitic Infections, 6th ed. American Public Health Association, Washington, D.C.

13. World Organization for Animal Health (OIE). 2008. Leptospirosis in The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Terrestrial Manual). Paris, France. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.09_LEPTO.pdf


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