Quantification and assessment of viability of Pneumocystis carinii organisms by flow cytometry

Abstract

Analysis of drug efficacy in animal models of Pneumocystis cainii pneumonia requires an accurate method of quantification of organisms, as well as a means of assessing viability. Lung homogenates were prepared from a colony of athymic nude F344 rats experiencing a spontaneous outbreak of P. carinii pneumonia. With the fluorescent nucleic acid stain propidium iodide, flow cytometric analysis was able to quantify P. carinii cysts and trophozoites reproducibly. As this stain is excluded by living cells, this method was also used to assess the viability of organisms. Application of this technique to analysis of bronchoalveolar lavage specimens was demonstrated.

Uses of flow cytometry in virology.

“…The viability of Pneumocystis carinii has been detected in athymic nude F344 rat lung homogenates and human BAL specimens by flow cytometric analysis of PI-stained cells (102). Only nonviable organisms take up the PI, making it possible to detect and quantitate nonviable organisms.…”

“…(i) Flow cytometry will be used for rapid detection of virus-infected cells and other organisms in body fluids if monoclonal or polyclonal antibodies that specifically react with cell surface or intracellular viral antigens are available. Examples are cells infected with viruses, such as HTLV-1 and -2 (16), hepatitis B virus (29), HCV (199), human herpesvirus 6 (32, 53), varicella-zoster virus (192), and measles virus (59), in blood and microbes, such as P. carinii (102) and Mycobacterium tuberculosis, in pulmonary lavage fluids. Using flow cytometry for direct detection and possible quantitation of these microbes will save time and expense in the clinical laboratory.…”

Use of an ATP bioluminescent assay to evaluate viability of Pneumocystis carinii from rats

“…In previous studies, fluorogenic stains, such as calcein acetoxymethyl ester-ethidium homodimer (Live/Dead kit; Molecular Probes, Eugene, Oreg. ), and classical vital stains have been reported to be useful for this purpose (21,22,26,41). The advantage of these staining techniques is that they permit a cell-by-cell evaluation of the viability of the total cell population present.…”

Recent advances of flow cytometry in fundamental and applied microbiology

“…Numerous papers dealing with changes in the antigenicity of the parasitized host cell have appeared, but are not considered in this review, which is restricted to microbial cell analysis by FCM. Flow cytometric methods have also been applied to direct analysis of free parasites in body fluids [3,11,32,77,202], biological tissues [1,56,69,80,104], and in vitro cultures [1,2,39,45,49,66,68,69,77,80,131,140,145,156,189]. The technical problems encountered are the same as those mentioned for other microorganisms.…”

“…Recent applications of FCM in various fields of microbiology.Plasmid and chromosomal DNA replication regulation system in E coli[6, 18-20, 26, 28, 29, 81, 174, 175, 199] Genes affecting division pattern and morphology in E coli[93,154] Role and distribution of outer membrane proteins and lipopolysaccharides in Gram-negative bacteria[9, 15,67,101,130,132,133,164,181,214] -Regulation of cell cycle progression in yeast S cerevisiae and S pombe[24, 47, 57,70, 78, 89,114,139,161,163] Physiological role of evolutionarily conserved mammalian genes in yeast[134] -Physiological effectors of yeast cell cycle control [90, 92, 138, 177] Yeast vacuolar and cell wall functions [52, 155, 2111 Heterogeneity of the stationary phase of growth [9, 61, 99, 127, 154, 182] -Cell cycle and DNA contents in protozoa [34, 96, 188] Physiological studies of protists: growth, feeding, reproduction, circadian rythms, etc [87, 110, 180, 197, 212] Differentiation -Change in nuclear DNA content in Physarum polycephalum [51, 102, 203] -Cell surface antigen modification in Dictyostelium discoideum[12,165,204,205] Ecological distribution of marine and fresh water phytoplankton[I 12, 190, 213] -Influence of different factors on cell cycles of marine phytoplankton species[87,190,197] Detection of pathogens in biological fluids: blood, urine, wound exudate, bronchoalveolar lavage, tissue homogenates, etc [56,69,104] Monitoring of toxin or drug actions on pathogenic yeast[31, 40, 42,63, 79, 84, 85,142,153] Drug susceptibility of parasitic protozoa[62,74, 77, 95,156,159,189,194,195] Monitoring of the physiological behaviour of photoautrophic algal cultures in photobioreactor[97] Oscillatory states in bacteria, yeast or photoautrophic algal continuous ...…”