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Experimentation with Mycobacterium tuberculosis requires biosafety level 3 (BSL-3) containment because of the potential for human infection. General safety considerations for working with M. tuberculosis are described by B. W. Allen (1). Removal of M. tuberculosis suspensions from BSL-3 containment should be done only if there is certainty that all bacteria are killed. When preparing samples for electron microscopy (EM), the use of low concentrations of glutaraldehyde alone is believed to be less damaging to the M. tuberculosis envelope structures (7). M. tuberculosis cultures are fixed in a final concentration of 5% formalin before absorbance determination at 600 nm. M. tuberculosis is susceptible to phenolic disinfectants (6), and a commercial product such as Vesphine IIse can be used to sterilize M. tuberculosis samples in the BSL-3 biosafety cabinet. The efficacy of these methods for killing M. tuberculosis samples has not been reported, and it is unknown if bacterial clumping interferes with the activity of these chemicals. For these reasons, we evaluated the killing efficacy of several mixtures using M. tuberculosis Erdman, a common BSL-3 laboratory strain. M. tuberculosis Erdman was grown either as a pellicle or as a liquid suspension before fixation for 1 h. We believe the 1-h fixation period is optimal for safe and efficient sample processing. Our results suggest caution in assuming sterilization of M. tuberculosis samples after incubation in EM fixative and certain laboratory disinfectant solutions.

(The data in this paper are from a thesis to be submitted by J. Reid Schwebach in partial fulfillment of the requirements for a Ph.D. from the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.)

Growth and preparation of M. tuberculosis. All work with M. tuberculosis was conducted in a BSL-3 safety cabinet at the Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, N.Y. A frozen vial of M. tuberculosis Erdman obtained from the laboratory of Barry Bloom was thawed and suspended in 25 ml of 7H9 Middlebrook medium (Difco, Detroit, Mich.) containing 0.5% glycerol (Sigma, St. Louis, Mo.) and 0.05% Tween 80 (Sigma). A 25-ml starter culture was grown in a 490-cm² roller bottle (Corning 430195; Corning, N.Y.) rotated at 1.25 rpm in a 5% CO₂ incubator at 37°C until the absorbance at 600 nm reached 0.3 (approximately 0.6 to 1.7 × 10⁸ CFU per ml). One or 3 ml of this culture was then added to 50 ml of 7H9 medium either with 0.05% Tween 80 (7H9-T medium) or without Tween 80 (7H9 medium), respectively, and was grown in the same manner as the starter culture for 7 days. Entire cultures were then harvested by centrifugation at 2,000 × g for 8 min in a Sorvall RTH-750 swinging bucket rotor using a Sorvall RT7 centrifuge (Kendro Lab Products, Newtown, Conn.). The absorbance of the 7H9-T culture at this time was 1.1 at 600 nm. Each bacterial pellet was then washed twice in phosphate-buffered saline (PBS) (0.137 M NaCl, 0.003 M sodium phosphate, pH 7.4) before suspension in 15 ml of PBS. Bacteria grown in 7H9 medium were then sonicated for 15 s to disperse clumps using a Branson Sonifier 250 sonicator (Danbury, Conn.) with a cuphorn sonicator (Branson 102 Converter) set at 60% peak output constant-duty cycle prior to antimicrobial fixation.

Fixation of M. tuberculosis and counting of CFU. Fixatives contained EM-grade glutaraldehyde (Polysciences, Inc., Warrington, Pa.), paraformaldehyde (Electron Microscopy Sciences, Fort Washington, Pa.), Vesphine IIse (Calgon Vestal Vesphine IIse [EPA Reg. No. 1043-87] containing o-phenylphenol [9.09%], p-tertiary amylphenol [7.66%], and inert ingredients [83.25%]; Steris Corp., Calgon Vestal Division, St. Louis, Mo.) in tap water at a standard concentration (40 ml of Vesphine IIse in 1 gal of H₂O) or 10% formalin buffered in PBS (Biochemical Sciences, Swedesboro, N.J.). The final concentration of the fixatives in each solution after the addition of bacilli is indicated in Tables 1 and 2. The 0.5, 1.0, and 2.5% glutaraldehyde and the 2% paraformaldehyde solutions contained 100 mM HEPES (Gibco, Grand Island, N.Y.), while the solutions with both glutaraldehyde and paraformaldehyde contained 100 mM trihydrate sodium cacodylate at pH 7.4 (Electron Microscopy Sciences). All fixatives were prepared fresh and diluted with PBS. As an additional test, bacteria were incubated in PBS containing 1 or 10 mM sodium azide (Sigma). One milliliter of M. tuberculosis suspension in PBS was added to 1 ml of fixative. Based on the absorbance of the 7H9-T cultures at 600 nm, we estimate that 7 to 18 × 10⁸ bacteria were fixed in samples containing 7H9-T M. tuberculosis, a similar number of organisms as would be useful for scanning an EM sample preparation.