This study also concluded that 35 ml was the optimal instillation volume for Orbifloxacin delivery of tracer into the lungs. Eyles and colleagues performed instillations of radiolabeled microspheres in either volumes and found that the radioactive microspheres accumulated in the URT when delivered in the lower volume while approximately 50% of the microspheres were delivered to the lungs when administered in the larger volume. Because we could find no studies in which efficiency of pneumonic delivery via intranasal instillation was tested using a bacterial agent, we performed a series of intranasal instillation studies using luminescent FTLVS. Our findings were consistent with those employing dyes or radioactive tracers and confirmed that instillation in small volumes resulted in delivery of FTLVS only to the URT while instillation in larger volumes resulted in delivery of luminescent bacteria to the lungs. The use of anesthesia and the type of anesthesia used during intranasal instillation is another variable that could have a significant impact on its efficiency for delivery of inocula to the lungs. It has been previously shown that delivery of materials to the lungs via this technique is significantly more effective when instillation of mice is performed under anesthesia during the procedure. In fact, one of these studies showed that when intranasal instillation is performed on unanesthetized mice, much of the instilled material was delivered to the gastrointestinal tract suggesting that alert mice tend to swallow a significant portion of the inoculum. In light of these findings, and likely because intranasal instillation is technically much easier to perform on anesthetized mice, the majority of researchers using this technique routinely anesthetize mice prior to the procedure. Therefore, it is surprising that there has only been one other published study that examined the effects of different types of anesthesia on the efficiency of this procedure for delivery of materials to the lower respiratory tract. Because isoflurane and ketamine/xylazine are commonly used for anesthesia in Levobetaxolol hydrochloride rodent-based research, we performed a direct comparison of the efficiency of pneumonic delivery of FTLVS-lux via intranasal instillation under these two types of anesthesia. In light of our general observation that mice anesthetized using parenteral-administered ketamine/xylazine maintain a steadier breathing pattern than mice anesthetized for relatively short periods using inhaled isoflurane, we hypothesized that intranasal instillation would be more efficient for pulmonary delivery of bacteria in mice that had been anesthetized with ketamine/xylazine. To our surprise, delivery of bacteria to the lungs was significantly more efficient when instillation was performed under inhaled anesthesia.