Scanning electron microscopy of food-poisoning bacterium Bacillus cereus using a variable-pressure SEM

A variable-pressure scanning electron microscopy (VP-SEM) with a cooling stage permits long hours of observation of water-containing specimens in their natural or close to natural state, without the conventional specimen preparations of fixation, dehydration, drying and metal coating. It reduces water vaporization and beam damage by keeping the specimens at a low temperature. We observed Bacillus cereus colonies on nutrient agar, which would shrink significantly if any conventional specimen preparation technique were used. We also studied the growing process of the bacteria on raw and steamed rice using the VP-SEM without conventional preparation techniques. Original specimens were directly mounted onto specimen holders and their backscattered electron images observed under the following conditions: specimen stage temperature, -10 degrees C; specimen chamber vacuum level, 30-70 Pa; and accelerating voltage, 15-20 kV. We recognized that the VP-SEM minimized deformation of the colonies due to shrinkage of the nutrient agar, and successfully imaged the morphology of the colonies and bacteria without a decline in bacteria number, which is apt to occur during fixation and dehydration. Also, the growth process of the bacteria on raw or steamed rice could be observed promptly, since there is no specimen preparation step.