A new enzymatic method for extracting diatoms from organs of suspected drowning cases using papain: Optimal digestion and first practical application

Abstract

Diatom analysis is very effective for positive diagnosis of water inhalation in drowning. However, conventional strong acid diatom testing is laborious and potentially dangerous. We propose a simple, fast, and safe protocol using inexpensive reagents such as papain, SDS, and 5 N HCl for extracting diatoms from lung, kidney, and liver tissues. First, we determined optimal conditions for papain digestion using porcine tissues. Papain digestion was clearly superior to Proteinase K digestion. Next, for assessing the assay effectiveness in practical cases, the papain digestion protocol was applied to 80 tissue samples from 20 suspected drowning victims. Left and right lung tissues (1 g each) were digested in 15-mL conical centrifuge tubes. Kidney and liver tissues (10 g each) were extracted in 175-mL conical centrifuge bottles. Papain dissolved all organs sufficiently and permitted clear visualization of diatoms, although papain’s solubilization activity was still inferior to strong acid digestion. The proposed enzymatic method requires only a low-speed centrifuge and water bath. Diatoms typically can be extracted from tissue samples within 3–5 h. The cost of protease is reduced some 6-fold by using papain in place of Proteinase K. Thus, the proposed method can be useful as a less-laborious, less-hazardous, and less-costly minimal test when the conventional strong acid digestion method is not performed due to personnel, equipment, budgetary limitation, or environmental and safety considerations.