Alcohol-Mediated Horizontal Spread of Bacillus Spores and Assessing the Recurrent Sterilization Needs of Culture-Handling Tools Contaminated with Hardy Spores

Abstract

The study was taken up to assess the chances of alcohol and instrument mediated horizontal transmission of bacterial spores and to fix the sterilization needs of tools recurrently used in bacteriological or plant tissue culture work in the backdrop of encountering mixtures in purified microbial cultures and the re-emergence of contaminants in ‘bacteria-sanitized’ plant tissue cultures. The microbial inoculating needle after contaminating the loop portion with Bacillus subtilis or B. pumilus spores was flamed over a sprit-lamp or gas-burner directly for 10–40 s, or after dipping the instrument (10–12 cm) in spore-adulterated 90 % ethanol or rectified spirit (96 %). The loop and rod portions were monitored for any residual inoculum by bringing the loop region in contact with nutrient agar, or swabbing the rod portion with moistened cotton-bud and imprinting on agar medium. Direct flaming of loop region for ≥20 s eliminated the hardy spores therein, whereas alcohol-dip and flaming increased the chances of spore survival on the rod portion that were acquired from the contaminated alcohol. Similar results were observed with the set of forceps used in tissue culturing work after dip in spore-containing ethanol. Alcohol once got adulterated with the hardy spores served as a source of bacterial inoculum causing unsuspected lateral spread of contamination. Direct flaming of inoculating loop nichrome or the forceps-tips, which came in contact with the microbial inoculum, preferably over a gas flame proved satisfactory for instrument sterilization. Alternatively, the use of autoclaved micropipette tips for handling bacterial cultures in the petri-dishes or attaching the tips to a pair of sterilized forceps is suggested for handling the cultures in tubes. Exposing the forceps and scalpel in a glass bead sterilizer set at 260 °C for ≥5 min effectively sterilized the tissue culturing tools.