THE THEGRY OF FIXATION

In this article, all data for which no reference is given is derived from
the previously unpublished work of the author.

The major portion of the research herein described was performed at the
American Institute Science Iaboratorey, 310 Fifth Avenue, New York City. Some of tf
the final phases were done at the Histology laboratory, Zoology Department, at the
Columbia University. The author wishes to express his thanke to the American Insti-
tute of the City of New York, and to Columbia University for having provided the
facilities and the materials for this work.

Fixation is a process which converts a biological entity in the form of
living tissue to a dead mase of proteins,fats,cellulose,minerals,etc,, with the
fond hope that the morphology of this dead mass corresponds fairly closely to that
of the living material, This presumptive hope is valid in those cases where it has
been possible to compare the morphology of fixed material with that of the living.
This comparison can only rarely be mide at all, and then only with extreme difficuw
ty, for available methods of microscopic and phiycco-chemical analysis are so crude
that close studies can not ordinarily be made without the rapidly ensunng death of
the living materia}.

Chemically considered, fixation is mostly an irreversible coagulation and
denaturation of proteinaceous material, To the biologists' regret, the organic
chemists have really established very little about the chemical changes that con-
stitute coagulation. Indeed,wtil a much clearer picture than we now have is made
available on these chemical precesses, most discussion on the chemistry of fixation,
and therefore its theoretical basis, is worse than academic,I have,however,attemptt
to stumble in the direction of a few vague hypotheses on the basis of such meagre
information as is now available, However it might be sald that the work I have dorm
has raised far more questions than it has settled, The only theoretical basis on
which any chemical-fixation work can be analyzed is on that of the reactiona of
proteins, .
Zirkle (1928, et.seq.,) has probably done the most important recent work
on fixation. He has shown (1928, '29) that the fixation imeges, which are the ep-
peerances exhibited by stained,mounted sections fixed in a particular amnner, vary
according to the pH of the fixative solution when a whole series of metallic chro-
mates are used for fixatives, When the fixative vH is below a point thet ia ap-
proximately 4.4, a so-called acid image is produced. In this case,the cytoplasm of
the cell (Zea root-tips, Iron-hemetoxylin stain were used) 2s Cisrunted to give
ea stringy or reticular appearance to the truly homogeneous living cytonlesm in
which distinct vacuoles are embedded. In contrast with the false victure thet ts
given to the cytoplasm, the components of the nucleus amd Lite cerivatives is pro-
bedly the truest thet can be obtained. Chromosomes spindle fibers,nucleoli,and the
nuclear reticulum are fixed in such a way that subsequent staining gives high con-
trast and that study of these elements is alded;furthermore,there is ea fairly aceu-
rate preservtaion of these elements,as shown by comparison with living meterial.A
controversy still exists over the spindle fibers,which is not auite settled.In the
cytoplasm,however, mitochondria wre dissolved out.

On the other hand,a basiv fixetion,where the pH is above lh, gives the
following image: Cytoplasm is accurately fixed as an homogeneous substretum in
which vacuocles,with distinct bounding membranes, and mitochondria, are disnersed.,
In the nucleus however, the chromatic reticulum has disanpeered, and is renlaced
by a homogeneous mass, ,vhich @irkle éefdines ac fired tkergclyymh. In contrast to 4
Fixed reticulum,which frequently shrinks from the nusleclus to leeve ean erty slec
or "helo', the keryloymnh is al in direct contact with the nucleolus, Thromosomes
ere only faintly rarer end then only in metarhese tletes, Provheses ord
telocvheses are iIndistineuishable,

This cone nootion of weld and basic fixetion oon be extended to other ?
and mixtures, Chart IT is 2 summary of Zirkée's d&ta on 2 larse vertety of chericels,

de

CHART ONE |

Fixative Nucl Fix Nytonl Fix Mitochonéria Mucleoli Note
Chromates, ph hh or less A A h steinee. tyre acid
iCrg07, oH WG z& A A ust - gee (1)
tearboxyl acids (2) A A A et
Cxealic aciff A A P?? st
Dicarboxyl & Formalin B B P st
Alivhatic aciés (3) A A A unst
Trichloroacetic ac B B P st
Aliphatic ac % Formalin (4) BA BA PA st see kb
u salts, acids (3) A A A Bt
Formic acid & formelin A A A unst
Aldehydes (5) A A P at
Aldehrdes & pleric ac 4a A fh > st,
Fommlin & pieric 22 B B ° et
Amines & Chromic ec at 5.9 3 B P st 6
Formamide " " " A A A st

i, This is an anamolous case, The nucleoli were fixed es very lerge irreculer er bodies, No
explanshior is aveilable.

 

2, Acetic,Pronionic, Malonic,Succinic,Gluteric,malic,tarterte ectds
%, Acetic,Pro plonio ,Butyric, Vale eric ,Glycollic ,Glyceric Gluconic,lectic and Formic acids
4, Except formbs,ectde Wh, Acetic arid & Formelfohvde sxmordents only central nucleolt

Fe Forme léehrde ,Ac seta ldehyde , Propional,Chloral Peorcent Pormeléchriac

Lng
6. Bthylene dlanine,tthyl amine, Diethylamine ,Tricthrlenine Mrcthrlarine "rimethylanine

a Orne
2 -
Prridine, Dit sca: manine