4.

3 Cultivation

This section provides the salient features of in vitro leptospiral cultivation and pertinent
recipes for successful leptospiral growth using information primarily derived from Faine
et al. (1999) and Zuerner (2005). For highly informative, detailed methodologies and
culture conditions for the laboratory maintenance of Leptospira and in vivo growth of
Leptospira in laboratory animals the reader is directed to the classic book by Faine et al.
(1999) and the Current Protocols in Microbiology articles written by Zuerner (2005) and
Haake (2006). A description of leptospirosis in laboratory animals can also be found in
the chapter by William A. Ellis, this volume.

One of the essential requirements for the preparation of all solutions and media needed
for the cultivation of Leptospira is the use of glass-distilled or doubledeionized water that
has been sterilized by autoclaving (routinely 20 min at 121 °C) and then cooled.
Sterilization is necessary to eliminate saprophytic leptospires which are ubiquitous in
water sources. One should also note that filter-sterilization cannot be used in place of
autoclaving, since saprophytic Leptospira can pass through microbiological filters. The
other critical factor in the successful propagation of pathogenic leptospires is the source
and quality of the serum albumin supplement added to the medium to serve as a fatty
acid detoxicant. The most frequent form of albumin used is bovine serum albumin (BSA).
Analysis of different BSA lots for their suitability for successful leptospiral cultivation,
especially for isolation of leptospires from clinical samples, is essential. Upon
identification of a suitable BSA lot, purchase of a large quantity of that particular lot is
recommended to provide sufficient material for successful cultivation in the future. BSA
can also be delipidated by extraction of the dry powder with chloroform/methanol (2:1,
vol/vol). A 0.22 μm filter, instead of autoclaving, must be used as a final sterilization
step for prepared media that contains albumin, as albumin is heatlabile. Alternatively,
leptospiral growth media may be commercially purchased. All media should be heated
at 56 °C for 30 min to ensure killing of saprophytic leptospires. It should also be noted
that pathogenic Leptospira species constitute Biosafety Level 2 (BSL-2) pathogens and
thus appropriate biosafety guidelines should be followed.
4.3.1 Growth in Liquid Media

The success of leptospiral growth in liquid media depends upon the nature of the seed
inoculum. Well-adapted laboratory strains can be directly passaged as liquid cultures,
using 1–10 % of the volume of the fresh medium as the seed inoculum. Three different
types of media commonly used for liquid culture of Leptospira are described. In all cases
reagents should be added in the specified order. EMJH medium The most frequently used
liquid medium for culturing Leptospira is the Johnson and Harris (Johnson and Harris
1967) modification of Ellinghuasen McCullough medium (Ellinghausen and
McCullough 1965) (EMJH). Rabbit serum can be added to this medium; if serum is to
be added it must be collected from a sero-negative rabbit and heat-inactivated by
incubation for 30 min at 56 °C. EMJH supplemented with albumin is prepared from a
series of stock solutions (all prepared in sterile, distilled water) as outlined below.

Basal Salt Stock Solution (Zuerner 2005):

1 g Na2HPO4

0.3 g KH2PO4

1 g NaCl

1 ml of ammonium chloride stock solution (25 g NH4Cl/100 ml water)

1 ml of thiamine stock solution (0.5 g thiamine/100 ml water)

1 ml of glycerol stock solution (10 ml glycerol/100 ml water)

 Add components to ~800 ml sterile, distilled water with stirring.

 Adjust pH to 7.4 with dilute solutions of NaOH or HCl.
 Bring volume to 1 liter with sterile, distilled water.
 Sterilize by autoclaving.
 Store up to 1 month at 4 °C.

BSA Stock Solution (Zuerner 2005):

Add 10 g bovine serum albumin, fraction V, to 50 ml sterile, distilled water with constant,
slow stirring (avoid foaming). To facilitate dissolving of the BSA, this solution can either
be gently heated to <50 °C or left at 4 °C overnight. Use immediately to prepare the BSA
Supplement (see recipe below).

BSA Supplement (Zuerner 2005):

To 50 ml BSA Stock Solution (see recipe above) add the following stock solutions with
constant stirring:

1 ml of calcium chloride stock solution (1 g CaCl2 · 2H2O/100 ml water)

1 ml of magnesium chloride stock solution (1 g MgCl2 · 2H2O/100 ml water)

1 ml of zinc sulfate stock solution (0.4 g ZnSO4 · 7H2O/100 ml water)

0.1 ml of copper sulfate stock solution (0.3 g CuSO4 · 5H2O/100 ml water)

10 ml of ferrous sulfate stock solution (0.5 g FeSO4 · 7H2O/100 ml water)

1 ml of vitamin B12 stock solution (0.02 g/100 ml water)

12.5 ml of Tween 80 stock solution (10 ml/100 ml water)

 Adjust pH to 7.4 using approximately 0.4 ml of 2 N NaOH.

 Bring the final volume to 100 ml with sterile, distilled water.
 Sterilize by filtration through a 0.22 μm filter.
 Store indefinitely at −20 °C.

Complete EMJH Medium (Zuerner 2005):

Start with 100 ml of the BSA Supplement (see recipe above) and add the following
components using aseptic technique. To further reduce the incidence of contaminants,
which is particularly important during primary isolation of pathogenic Leptospira, add
10 ml of 5-fluorouracil stock solution (1 g/100 ml water) to 890 ml Basal Salt Stock
Solution (see recipe above). If rabbit serum is added, reduce the volume of Basal Salt
Stock Solution to maintain a final volume of 1 liter. Store up to 1 month at room
temperature.

Modified Complete EMJH Medium for growth of Leptospira at 37 °C (Ellis and

Thiermann 1986; Bolin unpublished):

As mentioned previously, optimal growth of Leptospira in vitro is accomplished at a

temperature range of 28–30 °C, although Leptospira can be successfully grown at 37 °C
using a version of the Complete EMJH Medium that is modified as outlined below:

 Best growth is achieved by preparing all solutions using commercially purchased

sterile, distilled water (e.g. Gibco #15230-204). Sterile, double glass distilled
water prepared in-house may also be suitable.
 To the BSA Supplement add 0.1 ml of manganese sulfate stock solution (0.3 g
MnSO4 · H2O/100 ml water) in place of the 0.1 ml of copper sulfate stock
solution.
 To 100 ml of the BSA Supplement, add 1 g of lactalbumin hydrolysate, 100 µl of
10 mg/ml superoxide dismutase and 0.04 g sodium pyruvate. Allow the solids to
dissolve without swirling the bottle.
 Vacuum-filter the Complete EMJH Medium using a 1 liter, 0.22 µm filter
apparatus.
 The observed growth rate for L. interrogans serovar Copenhageni is enhanced in
this modified version of the Complete EMJH Medium. Final densities of ~109
bacteria/ml are achieved within 3 days at 37 °C using a seed inoculum of 2 % of
the final volume.
 It has been our observation that the modified version of the Complete EMJH
Medium has a shorter half-life than the conventional Complete EMJH Medium.
If sustained maximal leptospiral growth is desired, the modified medium version
must be prepared at approximately 2-week intervals and stored at 4 °C.
Stuart’s medium (Zuerner 2005) This medium was originally formulated by Stuart
(1946) and subsequently modified by Faine (1994). It is rich in rabbit serum, which
fosters leptospiral growth, but also has a propensity to precipitate phosphates present in
the medium. Such precipitates can obscure viewing of bacteria upon darkfield analysis
of cultures.

1.93 g NaCl

0.34 g NH4Cl

0.19 g MgCl2 6H2O

0.13 g L-asparagine

0.67 g Na2HPO4

0.087 g KH2PO4

 Dissolve components in distilled water to make a final volume of 1 liter.

 Adjust pH to 7.5 with dilute solutions of NaOH or HCl.
 Sterilize by autoclaving for 20 min at 121 °C, cool.
 Store for up to 1 year at room temperature.
 Prior to use, aseptically add sterile rabbit serum to 10 % final concentration.

Korthof’s medium (Faine et al. 1999; Korthof 1932) This is another commonly used
serum-containing medium that, similar to Stuart’s medium, enhances leptospiral growth,
but can complicate darkfield viewing of bacteria if phosphate precipitates form.

0.8 g peptone
1.4 g NaCl
0.02 g NaHCO3
0.04 g KCl
0.04 g CaCl2
0.24 g KH2PO4
0.88 g Na2HPO4

 Dissolve components, one at a time, in distilled water to make a final volume of

1 liter.
 Steam the solution at 100 °C for 20 min (boiling can be used in place of steaming).
Cool overnight at 4 °C.
 Filter off the resulting precipitate through Whatman No. 1 filter paper.
 Dispense into working aliquots.
 Sterilize by autoclaving for 20 min at 121 °C, cool.
 Aseptically add sterile rabbit serum to 10 % final concentration.
 The final pH of the medium should be 7.2–7.6.

Isolation of Leptospira from contaminated samples The simplest way to eliminate

contamination from Leptospira cultures is to filter the culture using a 0.22 µm sterile
filter; leptospires will pass through and the filtrate can be incubated and subcultured
(Faine et al. 1999). Alternatively, successful isolation of Leptospira from samples
contaminated with common clinical and environmental microorganisms has been
achieved using a combination of the antimicrobial agents sulfamethoxazole (40 µg/ml),
trimethoprim (20 µg/ml), amphotericin B (5 µg/ml), fosfomycin (400 µg/ml), and 5-
fluorouracil (100 µg/ml) (Chakraborty et al. 2011). Leptospires may also be recovered
from contaminated cultures by animal inoculation. The culture is injected
intraperitoneally and blood is taken aseptically under anesthesia after 10–30 min.
Hamsters, mice, and guinea-pigs have been used.

4.3.2 Growth in Semisolid Media

Pathogenic Leptospira, and particularly leptospires recently isolated from clinical

samples, grow well in a semisolid medium where they form a dense zone of growth
referred to as a Dinger’s disk (Lawrence 1951). To facilitate growth in a semisolid
medium, aseptically transfer ~100–250 µl of culture to a tube containing 6–8 ml of
semisolid medium (see recipe below). Transfer the Dinger’s disk to a tube containing
fresh semisolid medium when a disk forms that has visible density. The timing of transfer
depends upon the leptospiral strain being grown, with some strains requiring weekly
transfer and others taking up to 6 months to attain sufficient growth to allow transfer.

Semisolid EMJH medium (Zuerner 2005):

 Add 1.5 g agar to 900 ml of Basal Salt Stock Solution (see recipe above).
 Sterilize by autoclaving for 20 min at 121 °C.
 When the Basal Salt Stock Solution/agar mixture has cooled to ~50 °C add 100
ml BSA Supplement (see recipe above) per liter.
 Store in 1 liter bottles for up to 1 year at room temperature.

4.3.3 Growth on Solid Media

Growth of isolated colonies of Leptospira on solid media is often difficult and, for some
fastidious pathogenic leptospiral strains, unachievable. To isolate individual leptospiral
colonies, streak cultures or spread limiting dilutions on solid EMJH medium (see recipe
below). Minimize potential contamination by working in a sterile fashion, sealing the
plates with Parafilm, and placing the Parfilmed plates in a sealed plastic bag. Invert the
plates, incubate, and monitor for growth at weekly intervals. The time to development of
isolated colonies depends on the strain, with some exhibiting growth within 10 days of
plating and others taking up to 6 weeks to arise. Colonies appear embedded just below
the agar surface, are white in color and typically have a diameter of 1–2 and 2–3 mm for
pathogenic and saprophytic leptospires, respectively. Isolation of colonies is achieved by
gently aspirating the colony into the tip of a sterile filter tip or Pasteur pipette, followed
by introduction of the colony into semisolid or liquid medium and incubation until
growth appears.

Solid EMJH Medium (Zuerner 2005):

 Add 8 g agar to 900 ml Basal Salt Stock Solution (see recipe above).
 Sterilize by autoclaving for 20 min at 121 °C.
 When the Basal Salt Stock Solution/agar mixture has cooled to ~50 °C add 100
ml BSA Supplement (see recipe above) per liter.
 Pour into individual Petri plates with ~40 ml/plate, preferably in a biological
safety cabinet to decrease the chances of plate contamination.
 Store solidified plates in the original Petri plate bag, inverted, at 4 °C.