TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is adapted from that detailed in the <em>International Course on Laboratory methods for the Diagnosis of Leptospirosis</em>, by Dr. R.A. Hartskeerl, Dr. H.L. Smits, Mr. H. Korver, Dr. M.G.A. Goris and Dr. W.J. Terpstra from the Leptospirosis Reference Center in Amsterdam, the Netherlands. It is routinely used at UMR PIMIT, St Denis de La Réunion, France, for the isolation of pathogenic <em>Leptospira</em> from wild animals (terrestrial small mammals, bats and dogs). For <em>Leptospira</em> isolation from urine, go directly to step 10.</p>

[BEFORE_START]
<p><em>Equipment: </em></p>
<p>- An incubator at 28 °C.</p>
<p>- A dark field microscope.</p>
<p>- Sterile BenchGuard.</p>
<p>- At least a couple of sterile forceps.</p>
<p>- Sterile filter tips.</p>
<p>- A 100 – 1000 µL micropipette.</p>
<p>- Sterile disposable blades.</p>
<p>- Sterile petri dishes.</p>
<p>- 500 or 1000 mL sterile units of filtration system with a pore size of 22 µm (Stericup<sup>®</sup>, Millipore Express).</p>
<p>- Sterile syringe with a 0.22 µm filter.</p>
<p>- 1.5 – 2.0 mL sterile tubes (hereafter referred as microtubes).</p>
<p>- 5.0 mL sterile culture tubes (Falcon<sup>® </sup>, Round-Bottom, polypropylene and non-pyrogenic).</p>
<p>- A tray for microtubes</p>
<p> </p>
<p><em>Reagents</em>:</p>
<p>- <strong>Three distinct</strong> <strong>media</strong> (i, ii and iii) are used in order to maximize isolation culture success from distinct animal species.</p>
<p> </p>
<p><strong>(i) EMJH + AFAS: </strong>This medium is prepared from <em>Leptospira</em> Medium Base Ellinghausen-McCullough-Johnson-Harris (EMJH) powder (Difco<sup>TM</sup>, Detroit, MI, USA) based on the manufacturer’s recommendations (autoclaving and pH). The resulting autoclaved basal medium is supplemented with Albumin Fatty Acid Supplement (AFAS, purchased at OIE and National Collaborating Centre for Reference and Research on Leptospirosis Academic Medical Center, Department of Medical Microbiology, Amsterdam). Importantly, good quality AFAS is compulsory and in our hands, AFAS purchased at OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Amsterdam, has resulted as of outstanding quality.</p>
<p><strong> </strong></p>
<p><strong>(ii) EMJH + AFAS + RS 1 % + FCS 1 %: </strong>The same as media (i) with addition of rabbit and foetal calf serum (1% each). Both sera should be heated (30min at 56°C) to inactivate serum complement before use.</p>
<p> </p>
<p><strong>(iii) Fletcher semi-solid medium + RS 8 %: </strong>This medium is prepared from Fletcher Medium Base powder (Difco<sup>TM</sup>, Detroit, MI, USA) based on the manufacturer’s recommendations (autoclaving and pH). The resulting autoclaved base medium is supplemented with heat inactivated rabbit serum (8%).</p>
<p> </p>
<p>All media are then supplemented with 5-fluorouracil (5-FU) at a final concentration of 200 µg.mL<sup>-1</sup>. Following the addition of the 5-FU, i and ii media are filtered with the Stericup<sup>®</sup> filtration system. For sub-culture steps, the users have to prepare medium without 5-FU. Dispense 2.5 mL of each media in sterile Falcon tubes.</p>
<p> </p>
<p>- Ethanol 70 %.</p>
<p>- Sterile water (autoclaved).</p>

[GUIDELINES]
<p>All steps should be carried out using sterile forceps. For this, forceps must be cleaned in between each sample by placing them 1 min in a 1.0 mL sterile tube filled with ethanol 70 % and then in a 1.0 mL sterile tube containing sterile water. All microtubes, containing either kidney samples, culture media (for kidney rinsing and <em>Leptospira</em> culture) or water/ethanol for forceps sterilization can be placed in a single tray.</p>

[STEPS]
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