Theresa A. Fassel

Superior preservation of the staphylococcal glycocalyx with aldehyde-ruthenium red and select lysine salts using extended fixation times.

The utility of lysine‐based aldehyde‐ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococcal glycocalyx was improved by substitution of alternative forms of lysine for the free amino form. Paraformaldehyde‐glutaraldehyde fixatives containing alternative lysines, with or without ruthenium red, were compared at short 20‐minute prefixation times and at extended overnight fixation times. Although inclusion of paraformaldehyde made longer fixation times possible, the length of time for “safe” fixation varied per sample and could not be predicted. All alternative lysine forms permitted fixation of at least 24 hours without sample loss. The L‐lysine monohydrochloride or L‐lysine acetate forms permitted longer fixation times than the L‐lysine free amino form, and they had comparable or better preservation of the staphylococcal glycocalyx. Thus, the usefulness of aldehyde‐lysine‐based fixatives with minor changes has been enhanced. Microsc. Res. Tech. 41:291–297, 1998.

PARAFORMALDEHYDE EFFECT ON RUTHENIUM RED AND LYSINE PRESERVATION AND STAINING OF THE STAPHYLOCOCCAL GLYCOCALYX

The utility of lysine in glutaraldehyde—ruthenium red fixatives for the preservation and/or staining of the fibrous staphylococci glycocalyx was improved by inclusion of paraformaldehyde. Short, 20 min prefixation times for paraformaldehyde‐glutaraldehyde fixatives containing lysine, with or without ruthenium red, were compared to an extended overnight fixation. Samples were often lost in fixatives that did not contain paraformaldehyde at extended fixation times hampering the effective use of these fixatives for clinical or environmental applications. Inclusion of paraformaldehyde in the fixation with lysine permitted longer fixation times as well as stabilized the staphylococcal glycocalyx. Thus, the technical usefulness of fixatives employing lysine was significantly improved. Microsc. Res. Tech. 36:422–427, 1997.

The Use of Dimethylsulfoxide for Fixation of Yeasts for Electron Microscopy

Conventional methods of chemical fixation are often inadequate for preserving yeast ultrastructure. The thick cell wall severely limits penetration of fixatives rendering poor detail of the cell wall, membranes, and overall anatomy. Dimethylsulfoxide (DMSO) enhances penetration of chemicals and has been added to fixatives to improve cell preservation. At high concentrations (5 to 50%), however, it affects ultrastructure unpredictably. We found that adding 0.1% DMSO to fixatives greatly improved retention of yeast ultrastructure. Candida albicans, C. glabrata and Aspergillus fumigatus were fixed for 3 hr in 3% paraformaldehyde, 1% glutaraldehyde, 1 mM MgCl2, 1 mM CaCl2, 0.1% DMSO in 0.1 M sodium cacodylate buffer followed by 1% OsO4, 1% K2Cr2O7, 0.85% NaCl, 0.1% DMSO in the same buffer. Thin epoxy sections were post-stained in uranyl acetate and lead citrate. The multilayered character of the cell wall was distinct and well structured. Addition of ruthenium red or alcian blue to the fixatives further enhanced the outer fibrillar layer. The plasma membrane was contiguous and tightly adjacent to the inner mannoprotein layer of the cell wall. The cytoplasm was well preserved and the overall preservation of the yeast ultrastructure was significantly improved.

Analysis of fluconazole effect on Candida albicans viability during extended incubations

Fluconazole is an azole agent with primarily fungistatic activity in standard in vitro susceptibility tests. However, recent work has demonstrated that this drug can reduce Candida albicans viability during prolonged incubations under non-growing conditions. The present study was undertaken to examine more closely some of the parameters of this killing activity. Fungicidal effects of 1.0 microg ml-1 of fluconazole were found during 7-14-day exposures in each of two media that prevented proliferation, distilled water and metal-depleted RPMI 1640 tissue-culture medium. Fluconazole appeared to be stable after being incubated at 37 degreesC for either 7 or 14 days. Strains of C. albicans resistant to fluconazole in standard short-term growth-inhibition assays were also found to be resistant to fluconazoles effect on viability in prolonged culture, suggesting similar mechanisms of action for these effects. C. albicans yeast cells pre-incubated for 7 days in distilled water were not more sensitive to the drug in short-term susceptibility assays. Although all proliferation of the organisms in distilled water cultures appeared to cease after 3 days, fluconazole added at 7 days still reduced C. albicans viability. Therefore, the drug appeared to kill the non-proliferating organisms directly rather than preventing growth and thereby the emergence of younger organisms that would live longer. Transmission electron microscopy demonstrated damage to the cell wall-cell membrane complex and interior contents of yeast cells incubated in distilled water alone; fluconazole appeared to increase the percentages of cells so affected. In summary, extended-incubation susceptibility tests demonstrated that fluconazole has direct fungicidal activity of non-proliferating C. albicans yeast cells. These results may be relevant to the manner in which this drug promotes clearance of chronic fungal infections.

A Double Fluorescence Staining Protocol to Determine the Cross-Sectional Area of Myofibers Using Image Analysis

A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.

Ruthenium Red Preserves Glycoprotein Peplomers of C-Type Retroviruses for Transmission Electron Microscopy

Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B- and D- type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved.