Prepared EMJH medium (left) and Leptospira canicola  
growth as haze or smoke at the top of the test tube (right)
II. Detailed composition by Johnson and Harris
Prepare stock solutions (g / 100 ml distilled water each): NH4Cl, 25g; ZnSO4  * 7H2O 0.4g; CaCl2 * 2H2O 1g, MgCl2 * 6H2O 1g, FeSO4 *
7H
2O, 0.5g, CuS04 * 5H2O 0.3g, glycerol 10g, Tween 80 10g,  thiamine, 0.5g, and vitamin B12 0.02g.

Basal medium : Na
2HPO4 1g, KH2PO4 0.3g, NaCl 1g, NH4Cl 1ml, thiamine 1ml, glycerol 1 ml, and distilled water 997 ml.
Ajust to pH 7.4 and  autoclave at 121 ºC for 20 minutes.

Albumin supplement:
Add 20 g of bovine albumin (BSA / "Fraction V") to 100 ml of distilled water. Add slowly the following stock solutions to the albumin
solution while stirring: CaCl
2 2 ml, MgCl2 2ml,  ZnSO4 2ml; CuS04 0.2 ml; FeSO4 20ml; vitamin B12 2ml, and Tween 80 25ml. Adjust
final pH to 7.4, and the final volume, to 200ml.
Sterilize the albumin supplement by filtration.
Add 1 volume of the albumin supplement to 9 volumes of basal medium.

The rabbit serum medium:
Add 5 ml of L-asparagine stock solution (3g of L-asparagine per 100 ml of distilled water),
and 1 ml of the CaCl2 and MgCl2 stock
solution to 100 ml of pooled rabbit serum. Adjust to pH 7.4 and sterilize by filtration. Incubate the serum at 37
ºC for at least 18 h.
Add 1 volume of the rabbit serum to 9 volumes of the basal medium described for the albumin medium.
QUALITY CONTROL
Leptospira interrogans serotype australis ATCC 23605, growth
Leptospira interrogans serotype canicola ATCC 23470, growth
Leptospira kirschneri  ATCC 23604, growth
COMPOSITION & PREPARATION

I. Commercial formulation
Leptospira Medium Base EMJH
disodium phosphate 1.0g, monopotassium phosphate 0.3g, sodium chloride, 1.0g,
ammonium chloride 0.25g, thiamine 0.005g.
Final pH 7.5 ± 0.2.
Leptospira Enrichment EMJH: a solution of albumin, polysorbate 80 and additional
growth factors for Leptospira.
Autoclave the basal medium at 121 ºC for 15 minutes.
Aseptically add 100 ml Leptospira Enrichment EMJH to the medium at room
temperature. Mix thoroughly.
DESCRIPTION
A special medium containing growth factors for Leptospira cultivation.
EMJH
(Ellinghausen and McCullough, modified by Johnson and Harris)
(c) Costin Stoica
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PROCEDURE
Inoculate tubes with undiluted fresh cultures of Leptospira and incubate at 30 ± 2 ºC for up to 7 days.
Examine tubes for signs of growth (turbidity, haze or a ring of growth) or take a small drop from a few millimeters below the surface,
and examine it with dark-field illumination. Use 400 x magnification.
REFERENCES
1. Russell C. Johnson & Virginia G. Harris. Differentiation of Pathogenic and Saprophytic Leptospires. I. Growth at Low Temperatures.
JOURNAL OF BACTERIOLOGY, July 1967, p. 27-31.
2. Leptospira Medium Base EMJH. Difco product leaflet. https://legacy.bd.com/ds/technicalCenter/inserts/S1368JAA(201107).pdf