Inactivation and Ultrastructure Analysis ofBacillus spp. andClostridium perfringensSpores

Abstract

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

Effect of Different Disinfectants on Bacterial Aerosol Diversity in Poultry Houses

“…Glutaraldehyde exhibits a broad bactericidal spectrum with a highly efficient killing capacity for bacteria and virus. Glutaraldehyde also exhibits a strong effect on the spores generated by Clostridium , which can cause necrotic enteritis, and thus is commonly used for the disinfection of bacterial spores during epidemics ( Miner et al, 1993 ; Rutala et al, 1993 ; Brantner et al, 2014 ).…”

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues

“…Immediately before its use, we used phosphate-buffered saline (PBS), pH 7.4, to dilute 16% paraformaldehyde (Electron Microscopy Sciences, https://www.emsdiasum.com) to 4% paraformaldehyde. We fixed tissues destined for EM with 4% paraformaldehyde and 1% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate (Sigma Aldrich) buffer ( 14 ). This combination is referred to as EM fixative.…”

“…Despite a century of use, formaldehyde inactivation of tissues containing Select Agents has been described in few reports ( 12 , 13 ). Frequently, studies test only a small fraction of tissue for complete inactivation ( 14 ), which carries a risk of concealing low numbers of viable organisms in the remaining sample. The process of fixation, and concurrently that of inactivation, is dependent on variables such as time, pH, temperature, fixative concentration, and tissue size/composition ( 10 , 15 – 17 ).…”

Bacillus spores: a review of their properties and inactivation processing technologies

“…However, heat treatment can reduce nutrient content, modify organoleptic qualities such as fresh, and limit the type of package material available to withstand high processing temperatures. For this reason, alternative processes are being developed using chemicals to minimal changes to the fresh properties of food (Brantne et al, 2014;Kim et al, 1999;Sagripanti and Bonifacino, 1996). The basic mechanism of the sporicidal action of chemical agents is not well known, mainly due to the complex nature of bacterial spores.…”

“…The basic mechanism of the sporicidal action of chemical agents is not well known, mainly due to the complex nature of bacterial spores. The spore surface is hydrophobic, and the complete spore presents several sites at which interaction with chemical agents is possible, such as the inner and outer spore coats, the cortex, spore membranes, and the core (Brantne et al, 2014;Kim et al, 1999;Stragier and Losick, 1996). In addition, the complex structure and composition of spores plays an important role in chemical resistance.…”