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A paraffin method for serially sectioning a minute object in a known plane.

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Some in\-estigatioiis require that ail object as small as a
protozoan, an egg, an isolated blastomere, an embryo, a i d
similar materials he serially sectioried in a known plane.
Various t e c h i q u e s have been devised to prevent the loss of
a particle SO minute: a ) embedding it iii a small amouiit of
celloidin' or attaching it to a bit of amyloid liver2 or liraiii,'i
all of which are theii reembedded in paraffin; 0 ) embedding it
in paraffin in a gelatin capsule4 or in a glass vial or tube5;
c ) placiiig it in a small depression made in a solid paraffin
block6 ; d ) embedding it in parafiii in a small trough cut into
the bottom of a larger
Other methods have bceii dcveloped t o secure tlic accurate orieiitatioii of such a miiinte
particle, or the exact aligiimeiit of a group of them: a ) attaching objects t o pieces of marked papers or to sheets of gelatin9 or to bits of cleaved Ulva leaf,l" all of' which a r e theii
reemhetlded in paraffin ; 1 ) ) embedding objects together with
* Lee, '21, Microtomist's Vnde-Mecnm, 8th cd., p. 543, ref. t o Entz,
' Ibid.,
11. ,542, ref. t o hlinchin, '1.i.
' Ibiil., p. 266, ref. to IIill, '10.
IhX, 11. 73, ref. to M:iyer, '07; Meres, '12.
' Il>i(l., p. $5, ref. t o Hover, '99; Lauterhorn, '93.
aIl)id., 1'. 75, ref. t o Borycrt, '97; :md p. .X3, note b~ Lee.
' Ild., p. 75, ref. to LefBvre, ' 0 2 and '03.
' Ihid., 11. 81, ref. to I)enne, '02; Knowen, ' 0 0 ; Pattcii, '94; Wootlwortli, ' 0 3 .
' Ibid., p. 81, rcf. to E'ivld and Martin, '94; Lee ant1 YIaycr, '10.
"' Yatsu, '03, J o u r n . Rpplird Mic. and Lab. Mcthods, w l . 6, no. 1 2 , 1). 2669.
H E N l t P J. F R Y
a piece of blackened 11~~1've
fiber as a marker' * ; c ) arrangiiig
objects iii a mixture, such a s clove oil and celloidin, which is
then hardened and reernbcdded in paraffin.12
The method hereafter described avoids the various complications of those methods just mentioned and combines their
advantages. It is simple and quick, as embedding is clone
in paraffin only without previous attachment to any other
substance. There is no loss of an object, as all operations
a r c visible under a dissecting microscope. Accurate orientation in any plane is easily obtained without using anything
except the paraffin, a small embedding dish, and a glass
needle. Objects rangiiig in size from about 10 up to several
(*entimetersa r e easily handled.
When a tiny object is run up arid embedded singly, all fluids
slioultl be filtered. Particles of decayed organisms, sediment,
or dust a r e a s large a s the object uiider treatment and great
care is required to prevent their accumulation. Their presciice elidangers the loss of the object arid causes delay in
separating it out from them.
A device is illustratecl (fig. 1,a)which rapidly filters water
in large quantities, eliminating such particles, so that experiments can be carried on in water free from all sediment.
It is a glass cylinder about 12 cm. high by 4 cm. wide, which
is rather tightly packed with cotton. The water enters the
filter through an intake tube ( a ) in tlie cork a t the top. A
second tube of smaller bore acts as a pressure outlet ( b ) .
Due to tlie latter's smaller size, it maintains some pressure
which aids filtration and yet prevents bursting of the apparatus. The lower ends of both the intake tube and the
pressure outlet have flanges which prevent them from being
forced out of the cork. The cork itself ( G ) is prevented from
cxpulsion by a ring of cement placed above it. The lower
'' let^, '21, Microtonlist's Trade Mecnm, 8th ed., p. 82, ref. to Wilsoii. '00
p. 81, rrf. t o E r i t L , '09; €Ioffm:ciin, '99 and '01; Jordan, '99; Peter,
'$19; Samtrr, '97.
MacBride, '14, Text-hook of l':ml)ryology, t o ] . 1, pp. 34, 3 5 .
Koriih:cuser, '16. Science, N.S., vol. 44, no. 1124, pp. 57, .58.
c _ _ _
Pig. 1 A, water filter: ( I , intake; 6 , pressure outlet; c, cork with cement
ring above it; d , cotton; e, bolting-cloth tied over flange of bottom edge. B,
round-bottom vial. C, tip of lip pipette. D, vial with paraffin plug. E, a r t dish
f o r embedding; x shows where a n object is oriented a t ccnter of hottom surfact,.
E’, glass needle f o r orienting. G , untrimmed button, showing olijrct at center
of bottom surface.
eiitl of tlie c>!-liiider has a turiied-out flange, so that a piece
of holtiiig-cloth ( c ) of fiiiest mesh (no. 25, with openings about
“5 1-1 sqiiare) caii he t i 4 over tlie moutli to liold the cottoii
( d ) . ‘1’1ie upper layers of thc cottoii a r e cliaiiged when iiecess x y , for after seclimeiit h a s accumulated beyoiid a certain
point it slows up filtratioii aiicl works down into the filtrate.
'Flit various other fhitls used, such a s fixative, alcoliol,
sylol, aiitl paraffiii, a r e illso carefully filtered in the usual waj-.
?‘lie ob,ject is staiiiecl with aqueous eosiii after fixation, aiitl
agaiii with alcoliolic eosin ill 95 per cent alcohol.’:’ This is
important in a\widiiig loss.
‘I’lie ohjwt is trniisferred with R lip pipette (see l)elo\v)
to filtered fixative tliat has previously been placed iii a vial.
r i
1 lie ohject is kept in sight duriiig tlie various changes of
fluids by lookiiig into the vial through the low-power objective of a disswtiiig microscope. Tlie rial (fig. 1, €3) must
have a round bottom which allows clear illuminatioil of its
iiiteriol.. A flat-bottom vial is very uiisatisfactory, since its
i i i t ci* ii-r-egularly liglitetl, which seriously interferes with
vision. Tlie vial is about 25 mm. Ioiig and it slioiild not l)c
Icss tliaii 10 mm. in diameter, so that tlie coiiteiits caii he secii
tliiwugli lwtli lenses of tlie objective. Such rouiitl-l)ottom
ids a r e not usually cai*ried in stock hy scientific supply
houses aiitl they may h i - e to be ordei.ed specially.
E’imm the time of fixatioii uiitil the object is in xylol it is
iintotichctl. Tlie fluids a r e chaiigcd, not the object. This
avoids loss aiitl possiblc crusliiiig or damage. The fluids are
witliclrami with a lip pipette. Its tip (fig. 1, C ) is made from
glass tnhiiig ahout 12 rnm. in diameter. At one elid this is
t l i ~ a ~ v to
i i a diameter of ahout 5 mm. f o r attaclimeiit to tlic
~*uhhertubiiig that coiiiiects it with the mouth-piece. The
other c w l is drawn to a fine capillai-y, 1)eiit at a sharp angle
so that it caii lie coiivetiieiitlj- inserted clou-ii along tlie iiisiclc~
sui.face of the vial without obstriwtiiig vision. The 1ai.g~)
rnitltlle portioii retaiiis the withdraw11 fluids and prerents tlirii.
K l i i n n l ~ l c l , ’9.7. Z(,it.
>Ilk., Rd. 1 2 , h. 3 7 2 ; ;in11 ’96, Il)iil., 1311. 1.3,
getting into the rubber tubing. The latter slionld be thinwalled, of small diameter, aiicl about 3 feet long, so that
tremors a r e not transferred to the liaiid. The mouth-piece is
R short glass tnbe packed with cottoii which helps to control
suction and prcveiits a flow of saliva into tlie rubber tube.
After a giveii fluid is withdrawn with a lip pipette, the next
fluid is added with an ordinary one, but it is important that
this sliall not h a r e heeii used. for other purposes. If otlier
oiypiiisrns ill unfiltered fluids litnvc entered it, both organisms
and (lust particles may possibly have hecome attached to its
iiiiier surface. Tf it is tlicw used f o r running up a single
minute object, sucli particles from the inner surface may become cletaclicd aiid there may be much difficulty in picking out
tlie object of tlie experiment from among them.
It is wise to complete the process of emhetlding immediately,
but if time dcmaiicls that the object be stored temporarily in
80 per ceiit alcoliol certaiii precaiitions must he talieii to prevent its loss. Vials should n o t 1)o plugged with wttoii, for if
they a r e accideiitally turiied upside (lowii, a single minute
object is lost among the cottoii fibers. Furthermore, lint from
tlic cottoii gets into tlic alcohol, from wliicli an ohjcct must
later he scparated out. The vial is filled with 80 per ceiit
alcoliol to witliiu a third of thc top. A looped tliveatl is
lowered into the empty space with its eiicls hanging over tlie
edge at clitlier side (fig. 1, 11). The top of tlie 1-ial is filled
Jvitli paraffin. TT'heii this lias tlioronglily contracted, more
paraffiii is added so a s to f o l m a cap that somewhat overflows the etlgcs of tlie vial. Tliesc plugged rials arc packed
in tiers in a larger eoiitaiiicr, with a thick layer of cottoii
hetween each tier, as well as ahove tlie topmost one. This
not oiily prevents plugs from oeeasioiially floating out, hut
keeps tlic vials of upper tiers from pressing upoii lower ones
a r i d forcing tlie plugs loose so that they may sink to the hottom. If this does happen occasioiially, n o liarm is doiie, as
the rounded 1)ottom of the vial prevents the plug from toncliing tlic object. The plug is removed by gently warmiiig the
upper part of the vial, wliicli melts the edge of the plug so
flint it can be lifted out by the embedded string; or a heated
iiceclle is plunged into tlie plug, aiid it is lifted out after the
iiwclle has cooled.
Tliti dishes used f o r embedding (fig. I, E ) a r e the oblong
powclaiii containers iii which many water c~olorsa r e sold.
'I'liq- can he liought empty at art supply stores. They a w
abut 20 mm. long by 15 mm. wide by 10 mm. deep; they
) ~
tlie bettom. A size about twice as
sliglitly I ~ T Y toward
Iwrgtl is also available for a large object. It is necessary to
t~saminecarefidly the inner surfaccs of the bottoms to sev
that they are perfectly smooth, as some dishes have irrcgularitiw. These dishes call be numbered with a diamond glassmarking pencil, rubbing lead into the marks to make the
numerals more visible.
\\'hen an object is in its filial xylol in a vial, it is trarisferrecl
n-itll ii lip pipette to xylol in such a dish. The followiiig proces of changing paraffin and orienting, like t h e preceding
s t cps, arc done under the low-power objective of a (lissectiiig
niicivscope so that the object is kept in view. Reflected light
is used from ahove, as the dish is opaque, but the eosiii-stained
ol)jwt shows u p cleai*ly against tlie white background.
('liangiiig the paraffin as well as orienting the object are
siml)lifietl if tlic paraffiii does not cool too quickly, hence t h t ~
stage of the microscope is kept warm so that the dish does
iiot (+hill. This is accomplished by a small microscope lami)
o r other heating device placed under the stage. After the
sylol lias beeri removed, several changes of paraffin a r e made
i l l the tlisli. If the withdrawn parafiii begiris to solidify iii
tlic 1)ulh of the lip pipette before it is ejected, this is easily
heated to permit expulsion.
l\'heii the object is in its final paraffin it is carefully pusl~etl
to the exact middle of tlie bottom surface of the dish (fig. 1,
II:, .c) with a fine glass needle. When making the needlc,
after tlie terminal hair is drawn, its tip is quiclilj- toiichetl
to a small fiame, so that a tiny ball is formed 011 the eiid (fig.
1, 3')' Such a rounded tip does not tear ohjects a s does a
sliaqi terminal hair. Thc needle is warmed liefore iise, so
that paraffin does not crystallize about it, which causes the
object to cling to it.
Objects a r e transparent in the paraffin, and under the dissecting microscope it is a simple matter t o secure accurate
orientation with respect to the plane of tlie bottom surface of
the dish. I f several objects a r e t o be aligned, they can be
arranged in any desired position and as close together a s is
wislied. They keep their exact position during chilling.
There a r e 110 currents that slightly move them later, which
is the case in a mixture of celloidin and clove oil, where the
objects tend to shift out of position as the mixture is
When orientation is completed, tlie dish is carefully placed
in a shallow vessel of water containing ice. The exact depth
of tlie water is important. It must bc sufficient to come just
up to the top edge of the embedding dish, but not so deep
as to wet the top surface of the paraffin. If the dish is completely immersed, so that water comes into contact with the
paraffin, water is sucked deep into the interior of the cont rac ting button.
Immediately after tlie embetldiiig dish has been set in the
water and before the top of the paraffin has solidified, more
meltetl paraffin is carefully added, without disturbing the
lower levels. As much as possible is applied without causing
it to overflow. This additional paraffin prevents the tlepressioii that otherwise is formed iii the top surface and produces
it more evenly shaped buttoii.
After chilling is completed, the button is forced out of tllc
embedding dish by firmly pressing a heavy blunt needle dow-11
into one corner of the dish. This usually lifts the button, I)ut
if unsuccessful, a similar treatment a t an opposite coriier
completes the operation. It is not riecessary to apply a glycerin solution to the dish. In fact, this is undesirable, since thr.
fluid cwllects in tiny droplets oil the glazed surface of the
porcelain, and if sucli droplets happen to be ]!ear the object
they cause irregularities in tlie paraffin. Buttons a r e homogeneous and smooth iii practically all cases. An occasional
exccption is remelted and the process is repeated.
It is easj- t o locate the embetldctl ohject whell trimming the
(fig. 1, G ) . rt i$, 1~tictn.11 that the ob,ject is at tlie miclcllt.
of tlie hottom sillface, siiicc it was placed there with the
glass iiccdle. F u r t h e i , the orientation with respect to tlic
~ ) l a n cof tlic Imttom surface is also lciiown. This hottom suri ' i i w , wliic~liis smooth a i i d l)roa(l, is easilv tliffcreiitia tetl from
t l i c iiiic~vcm t 011 surface ;iiitl the iiarrow sides aiicl ends. If
thc object is so small a s iiot to be readily visihle to the. eye,
t l i c hnttoii is first trimmed roiiglil? hy hantl t o iiear tlic
niitli*cgion,a r i d tlieii tlie process is finislietl uiicler the tlisscctjlig niicroscope.
Tlie locatioii of the ohject is aidecl by its
~1osiiistain. The tliickiiess of the Imttoii permits trimming in
ii 1I y (1 i rec t i on.
Kibl)on is cut so that there are several iiiclies of 'empty'
i * i I ) h ) i i 011 ~ i t l i cside
~ of tlic poi*tioii wntaiiiing the ohject.
l'iiis rilhoii is placcd 011 a loiig i~arrowpiece of glass to I)e
ii~spectctlunder the dissecting microscope where the 'empty'
poi*tioiisa r c cut away. Again tlic cosin stain aitls t h 11rocess.
This mc~tliotlis of r a l u c ~I t o t only for single olljecats of vario i l s sizes, but it works wc~llalso f o r masses of miiiute objects,
such a s eggs. A s the parafin Iwgins t o cool aiitl R few Crystals
a r c forming arouiitl t h e cwriiers of tlic hottom of tlic dish,
tlicy a r e c>niiglitwith tlic glass needle and movctl to tlic w i i t t b r ,
t h e qygs thus heiiig caiiglit aiitl gathered into a pile in tliri
rriitltlle ; tlich crnhctltliiig is tloiie as aho\rc. Tht. tecliniquc is
uscfnl f o r riii'ious c\-tologic*alpurposes.
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serially, paraffin, sectioning, method, objects, know, plan, minut
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