Culturing Leptospira interrogans serovar Icterohaemorrhagiae can be notoriously finicky, especially when it comes to media supplements. Based on your description, a few key points may help troubleshoot the issue:

1. FD066 Himedia Supplement Specificity

FD066 is typically formulated for EMJH medium, which supports Leptospira growth. However, it's optimized more for saprophytic strains like L. biflexa. Pathogenic strains such as L. interrogans often require additional enrichment, particularly BSA (Bovine Serum Albumin) and long-chain fatty acids like Tween 80 or oleic acid.

• Check the FD066 datasheet—some batches may lack sufficient BSA or fatty acid content.

• If BSA is absent or minimal, supplementing with 1% BSA (fraction V) can significantly improve growth.

2. Locally Made vs. Commercial Supplements

Your observation that Gila Labs and homemade supplements work better suggests:

• Better adaptation to local strain requirements.

• Possibly fresher or more bioavailable components (e.g., fatty acids, albumin).

• Commercial supplements may suffer from batch variability or storage degradation.

3. Batch-to-Batch Variation

The fact that your first homemade batch worked but subsequent ones didn’t could point to:

• Contamination or pH drift during preparation.

• Oxidation of fatty acids or degradation of BSA if not stored properly.

• Autoclaving issues—some components (like albumin) should be filter-sterilized, not autoclaved.

✅ Suggested Troubleshooting Steps:

• Prepare EMJH base and add BSA (1%) and Tween 80 (0.1%) freshly.

• Use filter sterilization for sensitive components.

• Incubate cultures at 28–30°C in dark conditions, and monitor for up to 4 weeks.

• Consider serial dilution inoculation to reduce inhibitory effects from rich media or toxic metabolites.

FD066 may not be sufficient alone for pathogenic Leptospira—especially serovar Icterohaemorrhagiae. Supplementing with BSA and ensuring proper media preparation protocols should help. Also, keep a close eye on batch consistency and sterility.

Trishant Dinda, Feel free to share your exact media prep steps—I’d be happy to help refine them further.

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