THE

PRESENCE

OF

GLYCOGEN IN J.

IN

THE

RAT

LIVER

FOLLOWING AGENTS

I.V

VITRO

IROCESSING

DECALCIFVING TROTT
of Manitoba, May 31,

R.
University for

Faculty

of Dentistry, Received

Winnipeg, 1961

Canada

publication

In the

earlier mucoperiosteum

attempts of

to jaws

preserve it was

ghycogen found that

imi Two effect content rat, ately fixed There 4.0. viously were reaction. liver, acid sodium of 2. Morse adjusted been A six liver on mately acetic
hours,

METHODs

AND

MATERIALs

the a

methods rat, fixed

bylarge oral

used
was

were acetic
periodicof

inadequate
from

(8). the
Schiff were if the as to jaws lost.

If

the of and

experitiients of decalcifying of rat was

ill

were livers takemi into acetic

performiie(l on the

to

test glycogen strain). old

time

mucoperiosteum

removed

palate
reaction found

solutions (Sprague-Dawley from 4 pieces acid The one pH

in
the

alcohol
acid glycogen

fortnahin,

1. A liver

a 1 miiommth and alcohol of the piece as cut periodic into in to of at three fortnic the 7.5. killed into before pieces heitmg for days. l)lacedl

mimIc and was

was

stained

divided for After (9) stainetl Of one at pH citrate (5), with week carefully 3 acid 48 mm
was

itmmmnedi-

( PAS),
in the decalcified seemed First, staining

amounts epithelium. then this three

formiialin fixative tissue 6 acid

However, gly-cogen Possibilities used may be for the

24 hours. fixation initiiediately and

to the of

be methods glycogen

consider. and second,

processed

described
.

e-

fixation

inadequate,

sections by the placed

They
Schiff of and

if the
acids

glycogen used for

it third, of it The

was
maybe

adeqcmatelv
necessary be present

fixed, mayfor in be

then
removing a minimum the

the

the 0.6, at.

remilaining another according pH 2.5, to rate pH

pieces acid in

trichboracetic formmmlae EDTA and ap)roxifixed in 24 6 hours, the

decalcification
to

it,

or

amount before (1).

glycogen can first he of

tissues

another

dcmotistrated these (9). of h)ossil)ilities This decalcifyimug

histochemically has communication agents

NaOH nmale sliced thick amid alcohol

old

comisidered deals glycogen Previous following: discussed acid equal glycogen that umudergo and parts, in h)eriodhiclittle with

elsewhere the action

in livers.
pertinent Schajowicz the effect formic on bone acid of publications and tuitric acid :20 Cabrini acid, include (6) hy-drochloric sodium citrate the have

formahin seven
was

hours

The groups.
and above. at 1)H

specimiietms The first

following Atiother 7.5 for 7

were group
fixation group

divided
used l)laeed last were wa.s The

into
as processed in group

three
controls as EI)TA was

the
and

histochemical
cartilage. Schiiff positive except They

behaviour
poimut sul)stanc-tS for glycogen,

of
out

days.

placed in formiiic as in the previous cotitrols, a known tive

at.

acid and experiment, periodic passed through experimental

sochitmm citrate, for 7 days. acid Schiff posi(lie

modification,

slide the same

was

always titne as

solutions
slides, and

which
Schiajowicz disodium at pH

is quickly
and

lost
Cabrini

except
also

in
tested

cartilage
the effect

cclls.
of (EDTA),

the

ethylenediaminetetraacetate 7.0 and were on bone, I)efOre on hiistochemical kidney. the same fixed Concerning as tissue before. reactiotis

a slide from in chia.stase

Glycogen

in

bone, the in his for itt

cartilage results study 48 hours

gly-cogen, Loe l0 in (3), formalin

+,
tion,

+ +,
as

each experimental group was digested before staining (4). was estimated in the sections as 0, + + +, and + + + +. No tiote was mmiake
,

of the

densit this

distribution, been considered

AND

or

degree

of polariza(9).

had
REsULTs

previously

in

clecalcifying

EDT; positive
of

1)H mathis The glycogemi fixed

in

DI5CUSsmON

7.2. terial
it to
is

.\lthough was
tiot

periodic
seemi in whether or not. the

acid
cytoplasm

Schiiff

first
in

requirement
tissues is

for that was acctic it must

dcmonstratimmg be adledluatelv that

osteoblasts,

t-lear

thmc author

considlers

the

cells. )rocessitig

It

found
acid

previously

be

glycogemi

for rouititie 699

alcohol

formahiti

700 TABLE
Pre8ervation Times of of Liver Fixation and

J.

R.

TROTT

I
After Methods Different of

reflect
specimens

this. adequate length

Thus
thicker

it appears
than about

desirable 3 mm

not

to have

Glycogen

if complete

Decalcification
Fixation (Acetic Alcohol Formalin) Decaicifying

(7 Days)
Agent (7 days)

and The
much

fixation of fixation
as

difference,

of glycogen is to occur. does not appear to make can be seen from Table I. that even though the glycogen is still retained. (7) suggest that there
the cartilage cells which

It is interesting
Control EDTA Formic acid

to observe

1)H
is

fixative is 4.0, Schajowicz & Cabrini

a capsule around

of the

6 hours 24 hours 48 hours

7 days

+++ +++ ++++

++++

+++ ++ +++
++++

0 0 0
0

allows
subsequent

glycogen

to be retained
decalcification

in the
procedures.

cell

during This

appears unlikely, because free access of fixative to agents should

that work, followed it

if the capsule allows the cell, decalcifying From the and from that
of chelating

TABLE
Preservation Different of Liver Decalcifyin.g
Solution

II
after Solutions
pH

also

have
are

Glycogen

7 Days

in

experiments vious fixation,

access. reported,
appear use

two
pre-

would by the

adequate
agents

Glycogen

for
the

the

decalcification
prerequisites at best even for

of bone all. It
for he

and if glycogen

cartilage,
is to

are
be

necessary

Control 5% Trichloracetic
Formic
(Itiorse,

++++ Acid & Sodium Citrate

demonstrated

is doubtful
the used EDTA

if Loe
at a

(3)
of pH

0.6 2.5 7.5

0 0 +++

used glycogen, of 7.2

the

fixative though

demonstration

Acid

1945)

EDTA.

decalcification.
SUMMARY

gave the However,

tissues From that

most satisfactory longer fixation

are I, to be where Table the

results might be
size of

in rat desirable
specinien

liver. for
was

It quately

has

been retained

shown in rat

that livers

glycogen after

can fixation

be

adewith

(lecalcified.

acetic 48 hours
clisocliutn

alcohol

formalin

for
followed

6 hours,
by

24

hours, with

or 7 clays, 7.5. Formic

acid fixation.

treatment citrate
the

controlled,
to have any lost glycogemu solution acid-sodium

the

length
great during the

of fixation
bearing seven on days he all

did the
seen

not appear amount of

in the EDTA formic

ethyhenecliaminctetraacetate

adjusted

to

pH

acid-sodium
solutions remove

or
glycogen

5%

trichloracetic following

(Figs.

1, 2, 3). citrate

It will
removes

that the

glycogen
control from the size However, the of

Acknowledgments: possible Council by of a grant Catuacla from and!

This the the

work National author much M.

was

made

from
(Fig.

the
4).

tissue
Similar

as effectively
results (Table was tiot for were II),

as a diastase
oi)tained

is

Research grateful to of Medicine, the

first experiment the there specimtn was a

where
the

for
the

their
technical

support.
advice of of

He
of Pathology,

is also
Miss

indebted Melville
d)f

controlled. glycogen (Figs.

tendency

in

the

Department University done technician, by-

Faculty

section
Deeper

to he unevenly areas show the tissue All surface; sections

FIG. liver, liver, liver,

distributed

less the shown

gly-cogemi
3+ figure

5, 6, 7). thatu those

does not

Manitoba.
D. typing

The Hart,
by

technical N.R.C. Miss V.

work laboratory
Rees.

was

Mrs. an(l

near

stained

by

periodic

acid

2. Rat Fto. 3. Rat Ftc;. 4. Rat sever dirtys. Ftu. 5. Rat

FiG.

1. Control rat liver, fixed seven fixed six hours in acetic alcohol fixed sevemi days in acetic alcohol fixed seven days iii acetic alcohol

Schiff day-s

method, in acetic

photographed at 250X. alcohol formalimi

formalin, formalin, formalin,

left sevemi day-s in EI)TA solution. left. seven clays in E1.)TA soluition. left in formic acid and sodium citrate houmrs, twenty-four controlled. thetu placed hours, Notice
in

liver, fixed iii acetic alcohol formalimi for twenty--foumr acid for sevemi clays. FIG. 6. A section of rat liver, fixed in acetic alcohol formalin for El)TA solution for seven days. The size of the specimen wa.s not acetic
of glycogen. 7. Another glycogen. illumstration of the same section as Figure

5(

trichlorplaced peripheral in

then the

density FmG. scattered

6, taken

deeper

in the

section,

showing

702 1tFFhh(hNClA I.
KCGLER,

J.

It.

TROTT

of

teeth

amid

hones

for

sectioning

itt

l)arathin.

relation
st rat 1959. iort.

J. H. ANt) 1LKIN5ON, h)et weetm tot a! glycogemi

ammd its hiistochetmiical

W.

J.

(.

J. Dent.
6. ScttAJowt(Z,

Res.

24: 143-153,

1945.
L. The effect amid emmzymnes of i)one amid

comitemit

myocardimttmi

.1. Histoehem.
H. I).

(ytochem. Blakiston,
fortuiat ion.

of ox detnomm 7: 398-402, and

of acids ott the

cartilage.
7. 1955. Setts,jowtcz, agents
TROTT,

F. AND CABRmNI, H. decalcifyi tmg solut ions) histochemical behavioumr J. Histochem. (ytochem.
F. as
AND CABRINI,

3:

122-129,

2.

LIm,l.mm,

Hmstopatlmologic

Technic

Practical
3. 4.

Histoehemistrtj.

New
Odun
t.

York, 1954 (P 35. Los, H. Bomme tissue

histological

and

.1 eta

Scand.

17: 311-427.

1959.

8.

5.

MUMANIs, J. F. A. AND MOWRY, H. W. Staining .llethods, Histological and 11 istochemical. Paul B. Hoeber Imic., New York, 1960, (p. 126). Momts, A. Formic acid-sodium citrate (Iccalcificat iomm amid butt yl alcohol dehydrat iomm

decalcifiers. Stain Technol. J. H. An imivestigation conmtemmt of the gingivae. 234-241, 1957. 9. TR0TT, J. H. An evaluation
comnmiuotily used of ghycogen. 703-710, 1961. for the

L. Chelatimmg histochemicmul 31: 129-133, 1956. into the ghycogemm Dent. Practit. 7: of methods amid stainimig

R.

fixation

J.

Histochem.

(ytochem.

9: