A fixation method for the observation of kinetochores

Abstract

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...

Non-State Armed Groups and the Power to Detain in Non-International Armed Conflict

“…113 This may not answer the first concern specifically, but it is step one: it is the acknowledgement that human rights have to be part of the discussion about the activities and operations of NSAGs. 114 This approach has been strengthened by the emergence of human rights treaty law which expressly addresses itself to NSAGs. 115 Further, there is a consensus that NSAGs that exercise de facto control of territory possess human rights obligations based on the principle of effectiveness and the need to avoid a vacuum in order to maintain the protection of human rights for territories and persons under their control.…”