Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.
Campylobacter pylori is a newly recognized microaerophilic, gram-negative spiral bacterium. The role of this organism in the pathogenesis of human gastritis and ulceration is currently being widely investigated. Published reports of the ultrastructure of C. pylori exhibit poor preservation of subcellular detail indicating that certain conventional fixation procedures may not be appropriate for optimal results. Consequently, we utilized freeze substitution and conventional methods to examine the influence of fixation technique on the preservation of ultrastructural detail.C. pylori strain NCTC 11638 was grown for 24 h in liquid media. Conventional fixation was performed with (1) the Ryter-Kellenberger procedure, (2) to 3% glutaraldehyde with secondary fixation in 2% OsO4 (3) combinations of formaldehyde (F) and glutaraldehyde (G) ranging from 10% F/0.5% G to 2% F/1% G with secondary fixation in 2% OsO4 and (4) fixation at 0°C. For all fixatives, pH was 7.2-7.4; osmolarity varied between 300 and 2000 mOsM depending on the fixative.
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