Preparation of biological contaminated samples for chemical analysis

Abstract

FFI has established laboratory facilities in order to receive and analyze samples containing a mixture of chemical, biological and radiological (CBR) agents, so-called “mixed samples”. Detailed procedures are needed to correctly handle a mixed sample, and care must be taken during opening of these samples. FFI is currently optimizing such procedures especially addressing the need to analyze simultaneously the sample for the presented C and B agents as well as reducing the risk of exposure to personnel. To make sure that a potential biological agent is not regarded an infectious, or may contaminate the laboratory, inactivation of such agents is essential before further analysis of the presence of chemical or radiological agents. The aim of this study was to investigate if dichloromethane (DCM) used for extraction of chemical warfare agents may inactivate spores and vegetative cells using Bacillus athrophaeus as a model organism (also known as Bacillus globigii, BG-spores) present in aqua’s and soil samples. We further investigated if additional steps in the extraction procedure were able to remove/inactivate bacteria present in the sample. Growth analysis was used for this investigation (CFU). Our results showed that BG spores were able to survive in DCM after an overnight incubation (21 hour). However, the viability (culturability) of vegetative BG cells was reduced (10.000 times) after a 30 minute incubation period. A strong reduction (almost 100 %) of the growth of BG was obtained by first extracting the sample (soil) in DCM followed by a filtration (0.45 μm membrane filter) of the extraction solution at BG concentration levels < 108 CFU/ml. However, an extraction followed by a sterile filtration (0.22 μm pore size) of the sample (soil and liquid) at concentration levels at 106 and 109 CFU/ ml showed a complete reduction (100 %) of the growth of BG. Care must be taken during filtration as the filter may be overloaded. In samples containing a high start concentrations of BG spores, >109 CFU/ml, bacterial growth (< 0.1 %) was observed in some cases. To overcome this problem serial dilution of the samples using different pore sizes may be performed.

FFI har etablert et laboratorium for håndtering og analyse av prøver for kjemiske (C) og biologiske (B) trusselstoffer, samt radioaktive (R) stoffer. En slik kapasitet krever rutiner og prosedyrer for preparering av prøver for analyse av CBR- midler. Ved mottak av en CBR-prøve er et av de første stegene i prosedyren å analysere prøven for innhold av biologiske trusselstoffer. Denne rapporten beskriver et ledd i en mer omfattende prosedyre for hvordan en CBR-kontaminert prøve kan prepareres for kjemisk analyse med minimal risiko for at analyseutøverne skal bli eksponert for biologiske trusselstoffer. For kjemisk analyse er det nødvendig å benytte organiske løsningsmidler, f. eks. diklormetan (DCM), for ekstraksjon av kjemiske trusselstoffer fra en kontaminert prøve. Våre resultater viser at BG sporer (simulant for Bacillus anthracis som forårsaker miltbrann) overlever i DCM og at analysepersonell kan derfor bli utsatt for en eksponering mot biologiske trusselstoffer. Ved bruk av filtrering (0,22 μm porestørrelse) som et tilleggstrinn i prøveprepareringen etter ekstraksjon med DCM, var det mulig å fjerne 99,99 % av sporene. Vi anbefaler at det videre utføres kjemiske analyser for å teste om bruken av et slikt sterilfilter vil ha noen påvikning på kjemiske trusselstoffer og at det testes ut minimum mengde prøvevolum for å få nok prøvemateriale for ekstraksjon av slike trusselstoffer. 4