In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain

A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h.     

Rhodamine 123 as a vital stain for mitochondria of plant cells

Abstract. Rhodamine 123 (Rh 123), a relatively new mitochondrial marker, little used in the study of plant cells, was tested on excized leaves of Elodea canadensis Michx. and on suspension-cultured cells of Ranunculus serbicus Vis. In both preparations, the dye accumulated rapidly and selectively in the mitochondria whose number, morphology and cell distribution could be easily observed. In the presence of Rh 123, cytoplasmic movements could also be perceived and the spatial arrangement of the mitochondria with respect to that of the auto-fluorescent chloroplasts was studied in connection with a normal or altered cytoskeletal framework. The specific uptake of Rh 123 by the organdies seemed to be potential-dependent since it was influenced by cations, ionophores and inhibitors of electron transport. Short exposures to the stain were practically non-toxic, whereas prolonged treatments (6–20 h) provoked specific alterations in structure of the mitochondria. The data reported here indicate that Rh 123 may be an excellent vital stain to study the morphology, function and dynamics of the mitochondria in living plant cells.     

Harmine as a substitute for 33258 Hoechst in the FPG technique

Summary We studied the effectiveness of harmine as a substitute for 33258 Hoechst in the fluorescence-plus-Giemsa technique, using Allium cepa chromosomes after 5-bromo-2-deoxyuridine (BrdU) incorporation. Harmine showed a photosensitizing capacity which was somewhat higher than 33258 Hoechst and used half of the time established for the usual treatment.     

Harmine as a substitute for 33258 Hoechst in the FPG technique

We studied the effectiveness of harmine as a substitute for 33258 Hoechst in the fluorescence-plus-Giemsa technique, using Allium cepa chromosomes after 5-bromo-2'-deoxyuridine (BrdU) incorporation. Harmine showed a photosensitizing capacity which was somewhat higher than 33258 Hoechst and used half of the time established for the usual treatment.     

Dimeric bisbenzimidazole Hoechst 33258-related dyes as novel AT-specific DNA-binding fluorochromes for human and plant cytogenetics

Ten new fluorescent dyes have been tested as possible tools for human and plant chromosome banding; eight of the tested dyes were dimer derivatives of bisbenzimidazole (Hoechst 33248). The dimer compounds differed from each other by the length of the linker connecting benzimidazole moieties and by the structure of the moieties themselves. Four compounds selected for the study, namely, DB(7), DB(8), DB(17), and DB(18), upon UV excitation (365 nm) exhibited a bright blue fluorescence in nuclear heterochromatic granules, but at longer excitation wavelengths (blue or green) did not show any fluorescence in nucleus or cytoplasm. All these compounds were shown to produce a high-quality distinct banding in human and plant chromosomes, comparable to that obtained after standard DAPI staining. Further study was carried out so that to evaluate contrast of the band borders, fluorescence intensity, as well as a photobleaching rate for different dyes. As a result, we selected DB(17) as a fluorochrome of choice for chromosome banding. Spectral characteristics of this compound allow employing it in combination with FISH analysis. Investigation of linum karyotypes using DB(17) has demonstrated applicability of this dye for banding of very short plant chromosomes. The known higher stability of dimer DNA-binding fluorophors, as compared to that of monomer ones, suggests the possibility of using DB(17) for banding and identification of flow-sorted chromosomes.     

Bolton-Hunter reagent as a vital stain for developing systems

The Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy, 5-[¹²⁵I]iodophenyl)propionate, was used as a vital stain for developing amphibian and tunicate embryos and for isolated cells (human erythrocytes and cultured chick limb mesenchymal cells). We found that the Bolton-Hunter reagent can be used on living cells at room temperature with techniques that are quite similar to the techniques routinely used to label isolated macromolecules in vitro. At concentrations of vital stain that were sufficient to label intracellular proteins in intact-cells, labeled cells underwent normal developmental sequences. Under these conditions, vital staining with the Bolton-Hunter reagent disproportionately labeled exterior proteins, and it seems likely that the Bolton-Hunter reagent is an especially good vital stain for cell surface and cell membrane proteins. The Bolton-Hunter stain is covalently bound, is not reutilizable, and appears not to disrupt natural physiological and developmental processes. Thus, we used the Bolton-Hunter reagent to follow the natural life spans of proteins in vivo and we were able to distinguish particularly long-lived proteins in Xenopus embryos.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Multiple binding modes for Hoechst 33258 to DNA

Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.     

An investigation of hydroethidine as a fluorescent vital stain for prokaryotes

Abstract Hydroethidine (HE), manufactured by the chemical reduction of ethidium bromide (EB), was found to be decomposed to a bright red-fluorescent product by a range of microorganisms. Therefore, HE seemed to have potential as a fluorescent vital stain for microorganisms. However, inhibited or killed bacterial cultures still fluoresced red. Short periods (2–4 min) of excitation of sterile solutions of HE with a UV light yielded an orange/red product. Thus, HE seems to be decomposed biologically by a number of microorganisms but also abiotically by UV light-mediated process to red fluorescent material. These observations suggest that the oxidation of HE has only limited potential for assessing microbial activity.     

Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle

A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco ( N. tabacum cv. Xanthi) leaf protoplasts.
In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome. The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2 n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2 n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2 n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

Summary We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.