AimsTo examine whether bacterial spores are vulnerable to impaction injury during standard spread-plating or to other modes of physical impaction. Methods and ResultsEmploying heat-challenged spores of Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Lysinibacillus, Paenibacillus and Brevibacillus spp. from day-4 to day-10 nutrient agar (NA) plates in 50% ethanol, plating the spore suspension to the extent of just drying the agar surface on fresh NA (50-60s; SP-B) was tested in comparison with the spreader-independent approach of spotting-and-tilt-spreading (SATS), or a brief plating (<10s; SP-A). Spore CFU was significantly reduced with SP-B in different organisms (23-40%) over SATS independent of the spore size. Comparing 4-, 7- and 10-day-old B.pumilus spores, the former two displayed significant CFU reduction in SP-B indicating a spore age-related effect. Continuous plating for 2-5min showed a reduction in spore CFU in all organisms depending on plating duration. CFU reduction effect with SP-B was less manifest on refrigerated plates where no friction was experienced but acute on prewarmed and surface-dried plates. Spreader movement over agar surface subsequent to the exhaustion of free moisture proved highly detrimental to spores. A simulated plating study by plating the spores over a plastic film till drying showed a significant reduction in spore CFU. DAPI staining and glass bead-vortexing studies confirmed spore disruption through physical impaction. ConclusionsBacterial spores are vulnerable to injury during spread-plating or with other forms of physical impaction with variable effects on different genotypes independent of the spore size but altered by spore age. Significance and Impact of the StudyImplications during spore CFU estimations employing spread-plating and during spore surveillance, and the recommendation of SATS as an easier and safer alternative for spore CFU enumeration.