Objectives: Preparation of microbial samples for conventional scanning electron microscopy (SEM) requires often altering dehydration and fixation techniques. More recently, environmental SEM (ESEM) eliminated the high vacuum requirements by using a differential pumping system in combination with a gaseous secondary electron detector (GSED). As a result, hydrated and insulating samples could be viewed without the need for drying or conductive coatings. In this study, ESEM was tested on its ability to observe directly cellular morphology and distribution of root canal bacteria. If so, ESEM could allow seeing the effects of new therapeutic endodontic strategies on bacteria in situ under environmental conditions. Methods: Sterile dentin discs were inoculated with pure cultures of Enterococcus faecalis (LMG 7937), Streptococcus anginosus (LMG 14502) or Fusobacterium nucleatum (LMG 13131). ESEM observation (Philips XL30 ESEM-FEG) was performed at 5-10 kV and 3-5 torr water vapor at 4°C using a Peltier stage and GSED. Next, samples were fixated, dehydrated and gold-coated prior to observation under conventional high vacuum. Results: The GSED images gave clear and useful views of the specimen's shape and surface, but of lower resolution and noisier than the conventional secondary electron detector images under high vacuum. Bacteria are organized as dense aggregates or have formed a confluent mono-species (bio) film. In addition, bacteria were found invading the dentin tubules. Conclusion: ESEM provides a fast, nondestructive way to observe various pathogens directly in their natural state. In near future the effect on bacterial appearance of endodontic treatment modalities such as photoactivated-desinfection (PAD) and ozone-therapy could be explored using micromanipulators for ESEM. Study supported by Fund Scientific Research OT/02/49.

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