Hoechst 33258 Staining for Detecting Mycoplasma Contamination in Cell Cultures: a Method for Reducing Fluorescence Photobleaching

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Note from the Biological Stain Commission: Certification of Wright Stain Solution

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Fluorescence fading and stabilization in cytofluorometry

Summary The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as 33258 Hoechst and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO₂ and cresylviolet-SO₂, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromoleculedye complexes generally induce fluorescence stabilization.     

Photometric analysis of antifading reagents for immunofluorescence with laser and conventional illumination sources

Bleaching of stained objects is a major problem in immunofluorescence. The prevention of fluorescence fading would allow longer observation times, photographic documentation, fluorometry, and pattern recognition. Fluorescein kinetics and fluorescence intensities (FI) of fluorescein isothiocyanate (FITC) conjugate-stained Sephadex beads were studied with previously described "antibleaching" reagents using an argon laser as the excitation light source. Eight antibleaching reagents were tested (sodium azide (NaN3), sodium iodide (NaI), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), 1,4-di-azobicyclo-(2,2,2)-octane (DABCO), p-phenylenediamine (PPD), n-propylgallate, and sodium dithionite (Na2S2O4]. Sodium azide and sodium iodide were found to increase FI. This was likewise found with mercury arc illumination and hence they may prove useful for routine immunofluorescence tests. PPD was found to accumulate on the surface of the beads and to disturb immunofluorescence by autofluorescence. The value of any of the other reagents in immunofluorescence is questionable.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.     

Non-aqueous permanent mounting for immunofluorescence microscopy

It is generally assumed that an aqueous mounting medium is necessary for the preservation of immunofluorescent-labelled microscopical preparations and polyvinyl alcohol-based solutions (e.g. Mowiol) being the most frequently used mounting media; however, both the quality and intensity of the fluorescence signal in most immunolabelled preparations after aqueous mounting slowly diminish with time, and finally, samples become unsuitable for examination. In the present work, we describe a very simple and rapid non-aqueous mounting procedure for cultured cells and tissue sections, which preserves the fluorescent signal in an excellent way after immunodetection or use of other specific labelling methods. It is based on the current histological protocol in which, after fluorescence labelling, preparations are dehydrated in ethanol, cleared in xylene and mounted in DePeX. Using this non-aqueous mounting medium, the fluorescent signal remains high and stable, allowing a suitable and permanent preservation of labelled and counterstained microscopical preparations.     

Influence of light and mounting medium on the fading of Feulgen stain.

Serial microspectrophotometric estimations of the absorbance of Feulgen-stained interphase nuclei of human fibroblasts were made on slides mounted in a variety of mounting media and stored in light or in darkness. All preparations showed some fading, at rates which varied with different mountants, were greater in the light than in the dark, and were greater in the monochromatic illumination of the microspectrophotometer under working conditions than in normal laboratory light conditions. Fading was minimised by using XAM improved white neutral mounting medium (G.T. Gurr), storing slides in darkenss, and by reading samples as quickly as possible.     

Fluorimetry of bovine myotendon junction by fibre-optics and microscopy of intact and sectioned tissues

Summary The autofluorescence of tendon, epimysium and endomysium at the myotendon junction of the deep digital flexor in the bovine forelimb was measured with a fluorescence microscope and with a bifurcated light guide composed of quartz optical fibres. Data were adjusted for spectral variation in the radiance of the halogen illuminator used to standardize the photometer. Samples of myotendon junction were examined intact, in slices several millimetres thick and after being frozen in liquid nitrogen and sectioned at 20 µm. Sections were examined with and without a mounting medium and with and without immersion oil objectives. Type I collagen fibres were identified by their scarcity of branching, relatively large size and yellow staining with silver. Type III collagen fibres were identified by their extensive branching, small size and black staining with silver. Purified Types I and III collagen were also examined. Type I collagen fibres had a strong fluorescence emission peak between 410 and 450 nm and a shoulder at 510 nm. For the strong peak, results obtained by fibre-optics were positively biased relative to those obtained by microscopy. Type III collagen reticular fibres lacked a strong emission peak at 410 to 450 nm. Although their overall fluorescence was weaker than that of Type I collagen fibres, Type III collagen fibres had similar or slightly stronger emissions around 510 nm. The Type I emission spectrum of collagen fibres was converted to a spectrum similar to the Type III spectrum by conditions that caused the fading of fluorescence (storage as dry or mounted sections and exposure of sections to UV light). It is suggested that, with fibre-optic fluorimetry of intact tissues, Type I collagen fibres may emit a pre-fading spectrum while Type III collagen fibres may emit a post-fading spectrum, and that the preservation of Type I and the fading of Type III collagen is a consequence of the surface to volume ratio of their fibres.     

Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy.

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM(1)); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM(n) = EM(1) * A ((n-1)). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.     

Fluorescent C bands of human chromosomes with 33 258 Hoechst stain

Summary Air-dried preparations of human metaphase chromosomes normally exhibit a Q band fluorescence pattern with 33 258 Hoechst stain while the C band regions of 1, 9, 16, and most acrocentric short arms appear dull. If these stained and mounted slides are stored at room temperature in the dark for several days, a spontaneous change from C-negative to C-positive bands sometimes occurs. We postulate that the pH of the buffered saline mounting medium during storage of the slides causes the C band shift since the results can be duplicated experimentally by lowering the pH of the mounting buffer from 5.5 to 4.0.     

Combined Bright Field and Fluorescence Staining of Paraffin Sections for Studying the Fertilization Process in Plants

PAS-toluidine blue O—aniline blue staining of paraffin sections allows study of histological and cytological detail while retaining aniline blue induced fluorescence in all “callose sites”. Because most autofluorescence is eliminated by the PAS-toluidine blue prestaining, the detail and contrast of the fluorescence image is superior to slides stained in aniline blue alone. Slides are stained by the PAS reaction, 0.03% toluidine blue O, alkaline 0.005% aniline blue, and mounted directly in aqueous mounting medium.     

Influence of mounting media on the fading of basic aniline dyes in epoxy embedded tissues.

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. The lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.     

Influence of Mounting Media on the Fading of Basic Aniline Dyes in Epoxy Embedded Tissues

A simple cytophotometric technique is used to quantitate stain fading of basic aniline dye-stained epoxy-embedded tissues mounted in six different commonly used mountants. Significant fading was detected with all six mountants, although rates varied. the lowest rate of fading was observed with immersion oil and the highest rate of fading with Canada balsam. No significant differences in fading rates of four synthetic mounting preparations were observed.     

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy

 Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best. Accepted: 1 April 1996     

A new technique for retarding fading of fluorescence: DPX-BME

The antioxidant beta-mercaptoethanol (BME) used in conjunction with the permanent mountant DPX (DPX-BME) retarded fluorescent fading of mithramycin, acridine orange and Hoechst 33258 stained chicken erythrocytes, each to a varying degree. The initial fluorescence of all dyes examined was more intense with DPX-BME than with DPX alone. Specimens mounted in DPX-BME showed strong fluorescence and excellent morphology; if kept in the dark, they could be stored indefinitely without deterioration. Retarding fading of fluorescence with DPX-BME faciliated quantitation of DNA using fluorescence cytophotometry.     

A New Technique for Retarding Fading of Fluorescence: Dpx-Bme

The antioxidant β-mercaptoethanol (BME) used in conjunction with the permanent mountant DPX (DPX-BME) retarded fluorescent fading of mithramycin, acridine orange and Hoechst 33258 stained chicken erythrocytes, each to a varying degree. The initial fluorescence of all dyes examined was more intense with DPX-BME than with DPX alone. Specimens mounted in DPX-BME showed strong fluorescence and excellent morphology; if kept in the dark, they could be stored indefinitely without deterioration. Retarding fading of fluorescence with DPX-BME faciliated quantitation of DNA using fluorescence cytophotometery.     

Feulgen stain stability in relation to three mounting media and exposure to light

Fading of Feulgen-stained rat liver nuclei was studied with sections mounted in Eukitt, Clearmount, and Permount, each under conditions of light and dark storage. Repeated microdensitometric measurements were made on selected nuclei over a 160 day post-staining period. In all three media the stain faded significantly when exposed to daily laboratory lighting, but faded least in Eukitt. Slides stored in darkness faded less than those exposed to light but, again, Eukitt-mounted preparations showed the least fading. Eukitt's low refractive index, however, imparts a high refractility to the cytoplasm which may be undesirable for scanning microdensitometry, and its apparently long stabilizing period may be disadvantageous if care is not taken to measure slides at comparable times during the period following mounting.     

Chromatin fluorescence after carmine staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.