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The Nucleolus
By definition and etymologg, a nucleolus is a “small nucleus", or any "diminutive
body found within the nucleus of a cell", plant or animal, This general definition is |
qualified in so far as many cells in many widely separated phyla of plant or animal
organization contain particular bodies, morphologically similar , in their nuclei, so
that the term nucleolus has come to be Imrgely restricted to this particular cell organ.
Other types of bodies may be found within the nucleus, having varying degrees of morpho-
logical relationship to 'true' nucleoli: such may be chromosomes, chromocenters, chromatin
net nots, various fixation-coagulation artefacts, intra-nuclear divisbon centers,and perhaps
others, We shall,however, restrict our use of the term nucleolus to bodies which do not
directly and obviously participate in the mechanics of karyokinesis, which typically dis-
appear before or during the early metaphase of the division of the cell, which do not react
positively with the Feulgen chromatin reaction, Various modifications may have to be made,
for which see below, particalarly in the case of protozoid animals, However, the nucleolus
appears so generally and characteristically in the cells ,or rather nuclei, of higher
animals and plants, and similar perhaps more primitive forms appear in the lower animals,
that the nucleolus can be characterized as a definite and specific cell organ. In view
of this obvious fact, it is surprising that the field of nucleolus research has besn soo
neglected, particularly in relation to the immense amount pf work on chromosome cytology.
This paper is a brief resume of the work that has been done on nucleolus, and contains some
suggestions as to future investigation in the area,

Very little of the chemical morphology and physiology is known. Likewise, our conception
of the physico-chemical processes of biological staining is fragmentary; however, the stain
reactions of the nucleoli, and the modifications of these by fixation effects, may give us
& clue to thelr nature and serve as a tool for the investigation of the history and develop-
ment, With respect to fixation reactions, Zirkie has investigated Zea mays root-tips, steining

with Iron-hematoxylin. It should be emphasized with respect to any such studbes that the
Joshua Lederberg pe
‘Nucleocius'

later treatment of the fixed material is of graet importance.Zirkle used elcohol dehydra-
tion and paraffin embedding. (Zirkle, 1928 ,1929a,1931,193%,1934, 1935)

Acetic acié, 5%, fixes the nucleoli so that they do not stain

Formalin and acetic acid, 25% & 5% fix go that some nucleoli in the central region stain
Formiiin, 25% causes stained nucleoli but imperta a totelly different fixation tmage to

the root-tip cells,ie, mitochondria are fixed and stained, Furthermore, many of the

nucleoli are abnormally large, occupying a large proportion of the cell volume (author ,unpub. )

Inasmuch as the Formalin-Acetic combination gives the acid itmage(@issclution of mitochondria,
fixation of the nucleolar reticulum, acetic acid must be the fester penetrant, while the
effect of the formalin is more or less of a mordanting one, To confirm this conclusion, one'
might fix for a short time in acetic acid alone, and then transfer to formalim.The physical
Chemistry of these observations is of course quite cbscure. The author, in Allium cepa ,

has found that after formalin-acetic fixation, some nucleoli in the centrel region are
stained, However,many nuclei which have two or more nucleoli, in this erea,maAy exhibit

one steined,cone unstained nucleolus, This illustrates the erratic manner of the fixatiee,
The author is at ptesent conducting an investig&tion to determine what influence a chang

in the composition of the fixative may have upon the number and distribution of such

nuclei, In the preparations alreddy made, the distribution is entirely random in the

entire central area,

fost other fixative components commonly used, (Chromic acid, Peric acid, osmic oxide-as in
Fleming, Potassium Bichromate, and the heavy metals in combination with bichromates, )fhx
the nucleolus for staining, regardless of their variegated effect on other nuclear ané
cytoplasmic components, This effect applies only to the tron-Hematoxylin stain, For example,
the Iodine-Methyl Violet method (see Johanssen,DA, 1940) which stains chromsomes and
nucleoli very nicely after Bouin's or Navashin's(Belling modification) fails to give a
differential stain to these at all after formalin, Safranin, differentiated with HC} gives
an intermediate image, (Lederberg, unpub. ) After acetic acid, safranin will stain nucleoli
nicely,as Iron-Haemotoxylin will not,

Other acids have been used in a comparison of their effects with that of acetic, (Zirkle,
'34,'34!35) In this respect, in combinetion with fpormealin,the following resukts have been
obtained:

Formic acid, causes no mordanting (staining)of nucleoli,
Acetic Acid, a few nucleoli are stained (v. suprad
Propionic Acid, identical with Acetic

Valeric acid, causes a universal stain of nucleoli

Trichloroacetic acid, fixes nucleoli for stain, butt he image is thet of the formalin

p resent so that this effect may be rresumed as one of slow penetration. Fhe action
is quibe anamélous inasmuch as it is by far the strongest aeid of those mentioned, but

gives nevertheless the basic image in combination with neutral formalin. The butyric

and dicarboxylic acids act on accordance with their fat solubility.

Zirkle('28b, '31) has used certain special fixatives to investigate nucleclar history

in Zea and in Pinus strobus (V infra.)

Nucleoli (true nucleoli, plasmosomes) are often deseribed as being “oxychromatic” or
staining preferentially with acid dyes in contrast with chromosomes, or,more generally,
chrometin, which is'besichromatic', This distinction is not ent#rely valid, and can never
be used as & critical test for distinction between nucleolar and chromatic derivatives.
There is,however, one stain reaction which generally can be used for such a distinction,
namely the Feulgen Nucleal-Farbung. This stain procedure, similar to the Schiff reagent
for the detection of aldehydes in Organic Chemical Analysis consistf#s of the application
of fuchsinnsulfurous acid( i.e., Basic fuchsin which has heen decolorizeé by NaHS Oz )
Joshua “ederberg p. 3
'Nucleolus'

in acid solution) to sections that have been hydrolysed in hot HCL, This hydrolydis pre-
sumably leads to the liberation of aldehydes from the ribose component of the nuclear reticulum
or chromomeme nucleic acids, which reacts with the reagent to form an addition campound which
has a deep purple color, No case has yet been clearly established where nucleoli (plasmosomes)
have reacted positively to the Feulgen stain, There is a posd#iblélity in the case of growing
olcytes.Chromosomes and their derivates (as the interkinetic, nuclear reticulum) ivariably

react positively.