Easy methods for fluorescent staining of Neurospora nuclei

Easy methods for fluorescent staining of Neurospora nuclei. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol29/iss1/11 observed. Finally 12 meiotic mutants belonging to class I were obtained. Seven mutants belonging to class II, which produced 30 to 90% of abortive white ascospores irrespective of conidial or protoperithecial parents, were also isolated. Among class I mutants, N2AMI52 and N2AM189, were considered to cause chromosomal nondisjunction and three mutants, N2AN6, N2AM213 and N2AM229, were suggested to cause chromosomal degradation. Among class II mutants, N2AM195 was estimated to cause chromosomal nondisjunction, and N2AM83 was found to cause chromosomal degradation of linkage group V and elevation of recombination frequency in linkage group V. This mutation was mapped near al-3 The frequency of spontaneous mutation to meiatic aberration was approximately 2.7 x 10-5. --National Institute for Basic Biology, 38 Nishigonaka Myodaijicho Okazaki, 444, Japan. Nemeti, F. M., M. Schablik and G. Szabo Experiences with Metzenberg's method for preparing Neurospora DNA. Metzenberg and Baisch (1981 Neurospora Newsl. 28: 20) reported an easy method for preparing high molecular weight DNA from Neurospora crassa RL3-8 A. We confirmed their results: however, in our hands the nucleic acid prepared by Metzenberg's method contained large amounts of RNA and palysaccharides. To reduce contamination in our DNA preparation the original procedure was modifie ed as follows: (a) digestion with RNAse was extended to 3 hours; (b) final precipitation of DNA was performed with 0.54 volume of iso-propanol. Since the product obtained in this way still contained equal amounts of DNA and RNA, as determined by the diphenylamine (Richards 1974 Anal. Biochem. 57: 369) and phloroglucine (Dische and Barenfreund 1957 Biochim. Biophys. Acta 23: 639) methods, respectively, an additional treatment with RNAse was performed. The RNA content of the final product was less than 10%. The DNA prepared either by the method of Metzenberg or according to the above modification contained about equal amounts of DNA and polysaccharides (w/w): the latter was determined with the anthron reagent (Roe 1955 J. Biol. Chem. 212: 335). The molecular weight of DNA was determined by agarose gel electrophoresis (Aaij and Borst I972 Biochim. 'Biophys. Acta 269: 192) with λ-phage DNA used as standard, and was found to be about 49 kilobase pairs (kb). About 5% of the product were fragments of lower molecular weight (between 23 and 49 kb). In conclusion, although Metzenberg's mthod provides an easy way for obtaining high molecular weight DNA, the DNA prepared in that way is contaminated to a significant degree with RNA and polysaccharides. Institute of Biology, University Medical School, H-4012 Debrecen, Hungary.

Based on my experience using an Olympus microscope fitted with epifluorescence accessories, the fluorescent methods are indeed very simple, yet highly reliable for staining Neurospora nuclei in conidia, mycelia and asci.
Nuclear counts are reliable and painless. In addition, because of the specificity of several fluorochromes for DNA, they can serve a diagnostic function that is especially needed in fungi where Feulgen staining is difficult or unsuccessful.
A. DAPI and Hoechst 33258 (available from Calbiochem and Sigma, respectively) are specific to AT-rich regions. DAPI (and perhaps Hoechst 33258) also interacts with polyphosphate and S-adenosylmethionine, resulting sometimes in lower contrast between nucleus and cytoplasm. Stock solution may be prepared at 1 mg/ml in distilled water and can be stored in the refrigerator for several months. 1) Place cells or mycelium in 3:1 ehtanol: acetic acid for at least ten minutes. (Fixation in 1% glutaraldehyde, 4% formalin or 70% ethanol is also satisfactory.) Store in freezer if they are not to be used immediately. 2) Wash cells in water once. 3) Dilute the fluorochrome to 0.1 to 0.5 µg/ml (final concentration) and suspend cells in it for ten minutes at room temperature. 4) Wash cells in water once or twice. 5) Suspend cells in 25% glycerol solution. 6) Place a drop of cell suspension on a clean glass slide, squash under a cover glass and seal as necessary. The slides keep well for a week or more if stored in a refrigerator.

B. Olivomycin (available from Calbiochem) is structurally related to mithramycin with specificity for G-C.
Stock solution is prepared as 1 m g / m l 0.067 M Sorenson's phosphate buffer, pH 7.0, and stored in the refrigerator for a year or more. Place cells in ethanol: acetic acid (3:l) for at least ten minutes and store in the refrigerator if they are not to be used immediately. (Glutaraldehyde, ethanol or formalin may also be used for fixation.) Rinse cells in the phosphate buffer. Stain cells for five to ten minutes in 100 µg olivomycin per ml buffer to which magnesium chloride is added (25 mM). Follow other steps as given for DAPI above. The slides may be stored in the refrigerator for several months without loss of fluorescence intensity. According to the literature, mithramycin (

Equipment
For most observations, I recommend an epifluorescence microscope (also called reflected-or incident-light fluorescence microscope) employing a super-pressure mercury lamp. A mercury light source is very versatile because of its wide-range emission spectrum, including near-UV. The near-UV is essential when employing DAPI or Hoechst 33258. Olivomycin, acriflavin or auramin-0 do not require the near-UV emission, and can be used with either a mercury or a quartz-halogen light source. In addition to the light source, one or more sets of specifically matched excitation filter/dichroic mirror/barrier filter combinations are needed to deliver the radiation of desired wavelength to the specimen and to transmit the resulting fluorescence to the observer.
In an epifluorescence microscope, the light source is located at shoulder level. Radiation form the lamp travels a horizontal path through a 'narrow pass' excitation filter. A dichroic mirror placed at a 45" angle to the horizontal path reflects the radiation downwards so that it goes vertically through an objective to the specimen. The fluorochrome-DNA complex in the specimen absorbs this radiation and in turn, emits light of longer wavelength, which travels up through the same objective and through the dichroic mirror directly to the observer. (The dichroic mirror is opaque to shorter wavelengths but transparent to longer wavelengths.) The transmission peak of the excitation filter should match as closely as possible the absorption maximum of the fluorochrome-DNA complex (365 nm with DAPI or Hoechst 33258, or about 425 nm for olivomycin). The dichroic mirror selected in each case should be opaque for excitation wavelength but transparent to the fluorescence emitted by the specimen (470 nm with DAPI or Hoechst 33258, and about 540 nm with olivomycin). Acriflavin or auramin-O fluorescence may be examined with the same filter/mirror combination as for olivomycin. A barrier filter placed between the dichroic mirror and the eyepiece improves contrast by absorbing shorter wavelengths. It is chosen to have a cut-off point somewhere above that of the dichroic mirror but just below the emission maximum of the fluorochrome-DNA complex.
Since the epifluorescence method used the microscope objective as its own condenser, the objective must be capable of transmitting the desired wavelengths to the specimen. Fluorite objectives such as Zeiss Neofluors, Olympus UV-FL, Nikon UV-F, and Leitz FL, among others, are most useful for DAPI or Hoechst 33258. These objectives are also better than achromats for bright-field microscopy. The most expensive planapochromats (with their many lens elements) may not transmit the near-UV wavelengths necessary for DAPI or Hoechst 33258. Excitation of olivomycin, acriflavin and auramin-0, when complexed with DNA, is in the blue range requiring a separate filter/mirror combination as compared to DAPI. In this mode, normal bright-field as well as fluorite objectives can be used.
Most existing research microscopes can be adapted for work with various DNA-specific fluorochromes by fitting them with UV, violet, blue or green excitation accessories. Cost varies from $3,000 to over $10,000 for accessories alone, depending on microscope make. New Olympus or Nikon research microscopes equipped for epifluorescence work appear to be as good for out' purposes as are the much more expensive Leitz, Zeiss or Reichert units.

Observation
I routinely observe cells at 400x or higher magnification. High magnification objectives have the advantage of producing a brighter image because of their larger numerical aperture. After staining with DAPI or Hoechst 33258, nuclei as well as DNA-containing cytoplasmic organelles fluoresce bluish whereas olivomycin-, acriflavin-, or auramin-O-stained nuclei fluoresce golden yellow against a generally dark background. Both auramin-0 and acriflavin are very good for staining Neurospora for nuclear counts as well as for quantitative DNA measurements. I use acriflavin because of its brighter fluorescence and because the staining solution need not be prepared fresh every time.
Fluorescent images fade as a result of intense excitation radiation; fading is more rapid when high magnification objectives are used. DAPI and Hoechst 33258 are relatively stable and allow observations for five to ten minutes on each microscope field. Olivomycin (or mithramycin) fluorescence fades dramatically within a few seconds, especially in the fresh preparations. Despite the initial rapid fading in these fresh specimens, residual fluorescence is adequate to allow reliable counting of nuclei and observation of major nuclear features. After storage for a week or more at 4°C, the olivomycin-stained specimens show remarkable resistance to fading and observations may be made for up to ten minutes on each microscope field. Acriflavinor auramin-O-stained specimens fade less rapidly; observations may be made for five minutes or longer, even on fresh specimens.