Radiation Physics and Chemistry 61 (2001) 619–622 Identification of microorganisms for the analysis of images obtained by neutron radiography J.D.R. Lopes a , V.R. Crispim a, *, C. Lage b a PEN/COPPE, CT/UFRJ, Bloco G, Room 206FIlha da Cid., Universit ! ariaF21945-970, Rio de Janeiro RJ, Brazil b Laboratory Radiobiologia Molecular, IBCCF/CCS/UFRJF21949-900 Rio de Janeiro RJ, Brazil Abstract The main difficulty in identifying infectious microorganisms is the time required to obtain a reliable result, a minimum of 72hours. We propose a reduction to about 5hours through the technique of neutron radiography. Samples containing the bacillus Escherichia coli and the cocci Staphylococcus epidermidis were incubated with B 10 , layered on SSNTD (CR-39) surface and irradiated in the J-9 channel from the Argonauta Reactor (IEN/CNEN) with a flux of thermal neutrons at a rate of 2.2  10 5 n/cm 2 s. Images were observed in an optical microscope after exposure of the plates to chemical development of the latent alpha-tracks. Analysis of the images revealed morphological differences between the species, conferring the technique the perspective to use in microbial diagnosis. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Neutron radiography; CR-39; Microbiology; Bacteria; Boron 1. Introduction After the first results showing detection of micro- organisms by means of the neutron radiography technique (Wacha et al., 2000), efforts were made to identify them. Preliminary assays consisted of the analysis of images obtained with the method for two different bacterial strains: a Gram-negative bacillus Escherichia coli (wild type strain K12-AB1157) and a Gram-positive coccus Staphylococcus epidermidis (wild type strain 2292). The J-9 channel from the irradiator of the Argonauta Reactor was used in the neutron radiography technique, according to operation parameters: a thermal neutron flux of 2.2  10 5 n/cm 2 s, power at 170W, ratio n=g of 3  10 6 ncm @2 mR, L=D ratio of 110, R CD of 20, approximated Maxwellian spectrum (E p ¼ 30meV). First of all, the protocol designed to detect micro- organisms (Wacha et al., 2000) was applied to check if it would be possible to identify them. Whenever necessary, some experimental parameters were modified to achieve better images, like boron concentration and temperature of incubation. The overnight cultures were then incubated with 500 mg/ml B 10 (as sodium borate) and kept at 41C, 251C or 371C for 30min. After incubation with boron, samples were both layered on CR-39 strips or on LB nutrient agar plates to score for the number of viable cells after each treatment. The CR-39 strips were irradiated in the J-9 channel of the Argonauta Reactor for 25 min. The a particles from the 10 B(n,a) 7 Li reaction (Knoll, 1973) strike the CR-39 surface generating latent tracks from 30 to 100 ( A in diameter. To become visible to an optical microscope, the tracks have to be chemically developed in a 5.34N NaOH solution at 901C for 1 h. The resulting images were observed with a conventional optical microscope (Olympus BX-60 M with a 10  ocular and an 100  objective) with 10 3 times magnification. For better identification of the images, the tracks were also analyzed with a phase contrast microscope. *Corresponding author. Fax: +55-21-280-71-44. E-mail address: verginia@lmn.con.ufrj.br (V.R. Crispim). 0969-806X/01/$-see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII:S0969-806X(01)00349-8