After fixation with ethylene glycol, acetic acid and water, 1:1:1, the kinetochore can be studied with the light microscope. In unstained material, when phase contrast optics is used, the kinetochores stand out as dense granules against the more transparent chromosome bodies. Comparative experiments with different kinds of fixation show that the chromosomes swell more in acetic glycol than in conventional types of fixative, such as acetic alcohol. Although the acetic glycol‐treated chromosomes to some extent contract again, when subjected to various post‐treatments, they remain more transparent than after other fixations. Kinetochores, as well as chromosome arms, show a normal Feulgen reaction for DNA after treatment with acetic glycol. Histones were extracted from the chromosomes during the fixation, however, as indicated by reduced fluorescence with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. To facilitate the separation of histories from DNA, a modified fixative with magnesium salt added was tested. This was very successful for the differential staining with crystal violet of kinetochores in smeared microspores. The conclusions are that the changed density relations within the chromosome after acetic glycol fixation are due to swelling of chromatin and extraction of histones. The kinetochore is more resistant towards swelling and extraction. The experimental material was Tradescantia.

A fixation method using ethylene glycol, acetic acid and water, l:l:l, is described. It permits light microscope studies of the kinetochore, either in unstained material usmg phase contrast optics, or, after post-fixation with a chromic fixative, in permanent preparations stained with crystal violet. Haemanthus, Hyacinthus, SciNa, Tradescantia and Vicia were the test materials. Literature on centromere structure is briefly reviewed. Crystal violet demonstrates compact chromatin selectively and probably stains a complex of histones with chromium. The fixative has a swelling action on the chromosomes and extracts substance from them. The kinetochore is more resistant than the chromosome body to this treatment. This resistance is due to a f m r bonding between the constituent components of the kinetochore. a firmness developed from the evolutionary adaptation of the kinetochore to resist damage by the pulling action of the spindle. The achromatic character of the kinetochore after some fixation methods may be due to the compactness of its structure which prevents penetration of dye molecules. After our fixation the kinetochore can be stained by crystal violet and the Feulgen reaction. The same compactness might also explain the differential staining of kinetochores by mitochondrial techniques. Lena Clapham, Department of Genetics and Plant Breeding. P.O.B. 7003, S-7-50 07 Uppsala 7 , SwedenThe introduction of the band-staining techniques with fluorochromes by CASPERSSON et al. (1968) and with Giemsa by PARDUE and GALL (1970) opened a new era in chromosome cytology. Much attention was now focused on the possibility of staining chromosome regions differentially, and many technical modifications and new related methods were developed for this purpose.However, there also exist some earlier studies on differential staining of special regions in metaphase and anaphase chromosomes. One region that, on several occasions, had been subjected to differential staining is the kinetochore, the minute body or granule to which the traction fibres of the spindle are attached. These observations are reviewed by GEITLW (1938), LEVAN (1946), Scm-DER (1953) and LWKX (1970). There are also some early studies in which heterochromatic regions could be demonstrated in the chromosomes during their contraction phases, for instance LEVAN (1946). The present paper is devoted to a more detailed description of some investigations on a method developed by ~STERGREN (1947) for the differential staining of kinetochores. The cells were fixed in a 1:l:l mixture of water, acetic acid and ethylene glycol or glycerol. This was followed by a postfixation in chrome-acetic formalin and staining with crystal violet. The paper by OSTERGREN and h m s m (1973) is a preliminary report by the present writers on the results described here. A fixation in this acetic glycol mixture also permits an observation of kinetochores in unstained chromosomes by means of phase contrast microscopy (OSTERGREN and ANDERSSON 1973). A phase contrast study on the kinetochores...

The kinetochore of Triturus vulgaris was studied after fixation in acetic ethylene glycol, using phase contrast optics. By this treatment dense granules are differentiated from the rest of the chromosome. That these granules are kinetochores is concluded from the following facts: (1) spindle fibers attach to them, (2) size and appearance fit the newt kinetochore as demonstrated by electron microscopists, (3) in a mitotic chromosome the two granules, one for each chromatid, are located in the primary constriction, (4) at the first meiotic, division the granules are located on one side of the chromosome. In the absence of pulling forces, as in colchicine‐treated meiosis, the kinetochores of bivalents are oriented at random relative to the chromosome body and also at random relative to one another. Thus, bivalent structure does not determine how kinetochores initially become oriented. At meiosis as well as mitosis each chromatid most probably has one kinetochore granule, but at meiosis the two granules appear as one until chromatid pairing lapses at anaphase I. The undivided appearance is probably due to close pairing of the two chromatids within the kinetochore region. Unlike normal chromosomes at metaphase I, the colchicine‐treated chromosomes are constricted at the kinetochore region.