Veterinary Epidemiology and Zoonosis

Culture and Identification of Leptospira

DIAGNOSIS OF LEPTOSIROSIS BY CULTURE AND IDENTIFICATION

• Two types of media are used for culturing of Leptospira:
  • Rabbit serum based Fletcher’s medium and Stuart’s medium, and
  • Non-serum based Kortho’s medium and EMJH medium.

Preparation of Ellinghausen McCullough Johnson Harris (EMJH) basal medium

• Liquid media for MAT and semisolid media for maintenance of Leptospira.

EMJH liquid medium

• EMJH base medium : 2.3g
• Distilled water : 900mL
• Adjust pH to 7.5 using 1N NaOH. It should be sterilized by autoclaving.
• Check the medium for sterility by incubating at 37°C for about 48 hours and then store at 4°C until use.

EMJH semisolid medium

• Add 0.2% bacteriological agar to the liquid medium before autoclaving.
• Adjust pH to 7.5 using 1N NaOH. It should be sterilized by autoclaving.
• Check the medium for sterility by incubating at 37°C for about 48 hours and then store at 4°C until use.

EMJH albumin enrichment

• The following stock solutions should be prepared in 100mL sterile triple distilled water separately. Ferrous sulphate solution should be prepared freshly.

Calcium chloride 2H2O	1.50g
Magnesium chloride 6H2O	1.50g
Zinc sulphate 7H2O	0.40g
Ferrous sulphate 7H2O	0.50g
Tween 80	10.00g
Vitamin B12	0.02g

• Prepare the enrichment supplement by adding 10g of bovine serum albumin in 100mL of distilled water. The following quantities of stock solutions should be added slowly to the albumin solution.

Calcium chloride	1.0mL
Magnesium chloride	1.0mL
Zinc sulphate	1.0mL
Ferrous sulphate	10.0mL
Tween	12.5mL
Vitamin	1.0mL

• Adjust the pH of the albumin solution to 7.5 using 1N NaOH and make the final volume 100mL with distilled water and then sterilize by filtration.

Preparation of EMJH enriched media

• EMJH albumin enrichment is added to basal medium at 10% level.

EMJH basal medium	90mL
EMJH albumin enrichment	10mL

Culturing of leptospira

• Aliquot 5mL of EMJH enriched media in to sterile culture tubes (10mL capacity) with screw cap.
• Incubate the tubes at 37°C for 48 hours to check sterility.
• Inoculate the suspected specimen into the medium to about 1 to 10% level.
• Incubate at 35°C for seven days.
• Observe for growth of leptospira by DFM.

Figure: Leptospira culture in EMJH medium

Interpretation of result

• Leptospires are microaerophic. Growth of leptospira indicates the presence of Dinger’s ring just below the surface of the culture media.
• Leptospires are seen as spiral-shaped with hooked ends against dark background under DFM, measure about 6 to 20 microns long by 0.1 microns in diameter. They are motile with screwing movement and often form different shapes (secondary structures) like ‘S’ or ‘V’ or ‘U’ or ‘L’ or ‘C’ or ‘hair-pin shape’.

Precaution

• Care must be taken while handling live leptospira cultures. Material used for culturing leptospires should be disposed after autoclaving.

Note

• Culture should be carried out once in every 10 days in fresh liquid medium. Culture should be carried out in fresh semisolid medium once in every two months.