Reported Cryptococcus neoformans heat-killing protocols show incomplete killing

Abstract Heat killing is commonly employed to kill microorganisms, including Cryptococcus neoformans (C. neoformans), particularly in immunological studies and vaccine development. To assess the reliability of existing methods, we systematically reviewed 50 publications and identified 23 distinct protocols differing in temperature, duration, suspension concentration, and sample volume. Using two virulent strains, H99 and B3501, we experimentally tested these conditions and found that only 9 protocols achieved complete (100%) killing, whereas 14 resulted in partial killing. Consistent with this, only 17 publications (34%) reported complete killing, and just 20 studies described plating suspensions for validation, raising concerns that incomplete killing may have gone undetected. Further analysis revealed that suspension volume and fungal concentration critically influenced outcomes. These findings highlight substantial variability and insufficient validation across reported protocols, which may undermine reproducibility and biosafety. Our study provides evidence-based guidance for selecting effective heat-killing conditions, thereby supporting safer and more reliable use of heat-killed C. neoformans in experimental research.