Objective: Determine if a solution of 10% formalin would inactivate M. bovis within tissues of infected cattle rendering the tissues safe to be handled at Biosafety level 2 or lower.

Results: A fresh 10% formalin solution rendered M. bovis inactive after incubation in an excess of 10% formalin.

Keywords: Mycobacterium bovis, formalin fixation, inactivation, cattle tissues, biosafety, zinc fixative, cryostat

Mycobacterium bovis (M. bovis) is a zoonotic infectious agent that causes tuberculosis in animals and humans. M. bovis is handled under biosafety level-3 conditions. Historical scientific literature report inactivation and failure of inactivation of Mycobacterium tuberculosis and other mycobacteria by formalin (and similar agents such as embalming fluid). Comparison between the studies is complicated by the fact that the researchers used different solutions that contained formalin, different inactivation/fixation times, and tissues.

With the exception of prions, it has been a widely-held belief that 10% formalin, the most commonly used tissue fixative inactivates microorganisms. The Clinical and Laboratory Standards Institute (CLSI) guidelines state, “Ten percent formalin (3.7% formaldehyde) present in at least ten times the volume of tissues, which has been properly sectioned and adequately permeated, will inactivate all important infectious agents except the agent of Creutzfeldt-Jakob disease (CJD)” \cite{CLSI:2005wh} . Nevertheless, in the literature there is some ambiguity regarding the tuberculocidal properties of formalin in tissues infection with members of the Mycobacterium tuberculosis complex (M. tb complex).

One of the first studies that attempted to culture M. tb from formalin-fixed lung tissue that had been fixed between 72 hours to 9 years in formalin \cite{Kappel_1996}. No growth of M. tb occurred in the 5 cases were examined. A later study investigated 138 cases of tuberculosis in South African miners \cite{Gerston_2004}. Entire lungs were gravity inflated with 10% formalin, immersed in 10% formalin, and stored for 2 weeks to several months. Upon receipt at the lab lungs were immersed in 10L of 10% formalin for 10 days. Viable mycobacteria were cultured from 12 lungs (9%). It should be noted that fixation of the entire lung, as done in this study by Gerston et al. leaves doubts about the degree of fixation of deeper areas of the lung from which samples were obtained.

In a more recent study using experimentally infected guinea pigs a multidrug resistant strain of M. tb resisted inactivation by 10% formalin fixation for 6 months while multidrug susceptible strains were completely inactivated. Pretreatment of tissues for 2 hours in 75% ethanol prior to formalin fixation resulted in inactivation of both resistant and susceptible strains (4).

In each of these examples, the fixation process was not within the scope of typical laboratory process where tissues are routinely removed from formalin after 24 hours and stored in Ethanol to preserve tissue structure and DNA/RNA integrity. Here we report validation of protocols to inactive M. bovis in tissues.