:f /j/X/shs.bbgklv
transcribed Thu Oct 10 17:09:40 EDT 2002



Annual Laboratory Reports of the Biology Society [Stuyvesant High School]

          February 1940 to and through January 1941

                               Histology Section
                               Joshua Lederberg, Leader
                               January 8, 1941

These reports are abstracted from the running notes, too voluminous to copy.

(N.B. - This is almost a thesis! J.L.)
                4500 words

To: Mr. Schur

                                    Fall 1945 - Lederberg
.fi
     The major accomplishments and experiments of the Histology
Group are exemplified in the slides that it has prepared.  Since
the preparation of these slides follows, generally, a basic
preparatory process, it would be well to indicate this process.
In the preparation of tissues for histological study, the tissue
must be killed and hardened in the condition it was when alive,
and preserved against future decay and deterioration.  This is
accomplished by the use of a fixative solution, and the process
is called fixation. The tissue must then be made ready for cutting
very thin slices; in order to accomplish this, the fixed tissue,
after having had its fixative thoroughly washed out, is dehydrated,
the water being replaced by paraffin, through an intermediate
stage of dioxane and a solution of paraffin in dioxane.  The
tissue is now said to have been embedded.  The embedded tissue can
now be sectioned; this is done by a microtome, and which delivers
sections of about 10 to 12 u (1u=.0001 cm = 1/25,000 inch) as
indicated.  These sections are now mounted and attached to a slide,
and the slide, holding a thin section of tissue embedded in paraffin,
is ready to be stained.  To do this, the paraffin is removed by
xylol, the xylol is removed with dioxane and that with water.  After
the proper immersion in the desired staining fluid or fluids, the
slide is washed thoroughly, put through dioxane and xylol and is
finally covered with a drop of Canada Balsam dissolved in xylol, and
a thin cover-glass.  This is for mechanical protection and to make
the section transparent.  The tissue - section has had to be stained
in order to bring out and differentiate the various elements and
cells in the tissue!

     Notes respecting the work of the larger part of the first term
of this last year were lost during revision, so that a good deal of
chronological information is lacking;  however, all information
relative to the preparation of the tissues that is necessary for the
interpretation of the results.  Dates, unless otherwise stated,
refer to the date of primary staining.

     Late in February 1940, a fat body was freshly dissected out of
a male frog, a small fragment, 2x5 mm. placed in 10% biological
formal in water, for 2 or three days, washed one hour in 35%
methyl alcohol, then dehydrated overnight in dioxane, infiltered 5
hours in dioxane - paraffin 3 hours in molten paraffin, and
embedded.  The block was then sectioned at 18u, stained in
Delafield's hematoxylin and in 1/2% eosin in 50% dioxane and mounted
under balsam.  In June, 1940, the slide was borrowed by  Mr.
Weinberger, and thence disappeared in some recess of the Biology
Department.

     From the same frog, a piece liver was dissected out and
subjected to a similar process, with the section at 12u, stained
in Delafield's haematoxylin.  Unfortunately, this section is somewhat
wrinkled.  This was not sectioned or stained until April 16, 1940.

     From the same frog, and fixation, etc, the cartilage and
muscle of the episternum.  Stained after 12u section in Delafield's.
4/16/40.

     From the same frog, and fixation, etc., the spinal cord of the
frog in the cervical region, and, also, the lower brain.  Sectioned
at 10u, stained for 3-4 seconds in Delafield's, washed then restained
in 1% Methyline Blue, differentiated in 99% dioxane, and finally
mounted under balsam.  A very poor photograph from this slide,
effective magnification about 450X, enlarged 2.5X.  The picture,
which is the worst of a series, was taken by M. Fink_.  It
shows the ciliated ependymal epithelium surrounding the central
canal.  A series of these sections were stained at about this time.

     April 27, 1940, a smear of human blood was stained about one
minute in Wright's stain.

     (Part of a more detailed notation follows:)
(Mon., April 1 - "No work was allowed today in 521".

     April 2, 1940 - dissection of a rabbit in 419.  The maxillary
turbinats are curious, unfamiliar structures.  I am not sure whether
the paired set of nerves found just subcutaneously above the rear
maxillary teeth are the superior maxillary or one of the facial -
facial, trigeminal - nerves.  They pass just under the orbit and
enter the brain near the optic nerve.  Also, there is a small white
patch, medially at the roof of the mouth, between the soft and hard
palate.  What is it?  Without even a temporary use of the
mu___bator (which has at the time full of bacteriological tubes) it
seems almost hopeless to consider most types of histological work
in the near future.  Oh, for a freezing microtome!  No dehydration.
No difficult and messy infiltrations, no rehydrations to wrench
a section from the slide.  The block of the frog's central nervous
system is well embedded.  Everything is so disorganized.  Quotation
from my journal, 4/2/40.

     4/3/40  "A cross-section of the frog spinal cord was stained".
Because of examinations on April 11 and 12, there will be no work
this week.  Slide was apparently not clear.  Overstained
differentiated, restained.  More experience in technique required.

     4/4/40 Remained afternoon and evening till 6:50pm with teacher-
in-service course.  Spent one hour staining nerve slides.  Both
xyllene and aniline are unsatisfactory in anesting destaining
in acid alcohol.  Stain was by trial and error until right depth
of stain was revealed.  Eosin carmine stain only confuses the field.

     4/15/40 Till about 2 o'clock , aid Mr. Towme in cleaning Petri
dishes.  Thence, some further microtome of block of frog nervous
system".

     4/16/40 same as. In lecture before biology, technique used in
histology was demonstrated.

     4/17/40  12 slides, all nervous, were stained with methylene
blue, in a dioxane modification of a Nissl Stain.

     4/18/40  till 6:45pm at the In Service Course - see 4/4/40.
All afternoon and evening was spent in assisting and in staining some
more of the unstained sections thus far.  Today I used an 85% dioxane
in water solution to distain, absolute dioxane to anest
differentiation.  The chief difficiulty lay in obtaining sufficient
intra-cellular detail, simultaneously with inter-cellular contrast.
In earlier afternoon fixation of a 10mm chick embryo that had been
kept about 4 hours in salt solution for microscopic examination in
Bouin's.

     4/19/40 to '521' where staining of the last nerve slides was
completed.  One in haemotoxylin and one insulfranin.  The remainder
in Nissl - Methylene Blue.  The Canada Balsam we have must be
quite acid as the haematoglin stained slide has turned reddish
again.

     4/22-4/23/40 Embryo dehydrated at home.  In afternoon infilter.
Later, in evening, at Sid Zaharia's incubator, imbred the embryo on
4/24/40.

     May 1, 1940 Sectioning of embryo, more anterior region. Cephalic
region found not embedded at all.  Upper abdominal sectioned poorly,
much wrinkled.  Some sections, more posterior, flattened out nicely.
About 12 sections were made.

     May 8, 1940.  Stain many such sections, largely methylene blue;
poor wrinkling, staining. (Slides: I/22-25, #11.)

     May 9, 1940. Upon request to Weinberger, I was able to obtain
today a number of frog embryos and larvae (tadpoles) for embrylogy.
All were placed in Bouin's.

     May 13, 1940, Stain all embryo slides thus - Slides II/2,3,4,7)
Prepare whole mount of hydra; unsuccessful attempt to kill a
planaria extended

     May 26, 1940. Section blocks of embryo frog, as begun on
     May 9, 1940. Yolk causes poor sectioning
     May 20, 1940 Stain two slides from 5/10/40. Hematoxylin.
     May 23-24 Biology Exhibit.  Stain a number of slides, chick
embryo and a block of head of snake embryo secured by John Jacquez.
Also stain a section of cat cerebrum secured by him.
     May 23, 1940 Frog blood smear, stained with methylene blue and
eosin.
     May 27, 1940 Some blood smears, stain with Wright's.
     May 28, 1940 Stain a few sections of cat cerebellum - Methylene
Blue, Wright's.

     June 5-6, 1940. Preparation of about 200-250 sections of
stomach, lung, heart, intestine and tongue of frog prepared largely
for Mr. Weinberger's shop course. In month of June, 1940.  A series
of sections of stomach, etc, as above, were stained, a great
deal in assistance to Mr. Weinberger.  Also, a block of kidney was
embedded.

         Second Term 1940 - September to January 1941

School opened Monday, September 9, 1940.

     September 13, 1940 - Blocks A 1-14 were sectioned.  Among them,
a small intestine and kidney of a male frog.  (Slides VI/17,22;
VII/21, etc.)  These had been prepared in June of the previous
term with J. Jacquez, Bouin's fixation.  The sections were not
mounted, but floated in a pan of water till

     September 16, when they were mounted: and, on
     September 17, when the types of slides indicated above were
stained at home with hematoxylin and eosin.

     September 18, 1940 - the group prepared 105 slides for the
Bio Dept consisting of different colored threads variously mounted.
Study of stomach and kidney.

     September 19, 1940 - Examination of I/8.  Note possible
granulation or ciliation of cells of frog marrow at 430X.

     September 20, 1940 - Examination at 970X of many slides,
particularly I/8.

     September 23,1940 - A lecture meeting in the Histology Group,
dealing with the use of the microscope, demonstrating the oil-
immersion microscope, and lecturing on the structure of the
elementary structures.  Also, preliminary examination of prepared
blood smears at 970X.  There was also a discussion of Havell's
Theory on Blood Coagulation.  

     September 25, 1940 A lecture on the general structure of the 
cell and the theories of histological fixation and staining.  Then,
the mechanism of the microtome, and general basic histology
technique.

     continued 9/25/40 Some sections were taken of tissues the same
as previously.

     9/27/40 Staining Technique introductory.  Group members stained
sections previously sectioned.

     9/30/40 Wright Stain Technique on blood smears; also examination
thereof.  General group work.

     October 2, 1940 Various stains applied to blood smears to
determine optimal staining.  All were fixed in absolute Methy alcohol
after the smear dried.  Then dried again.  Then stained, washed and
dried.  Stains used were picric acid, methyl violet, picric acid
followed by Methylene Blue; light Green. Carbol-Fuchsin; methylene
blue and alcoholic gentian violet.  Nuclei of leucocytes seem
best stained by methyl and gentian violet.  These also stain the
hemoglobin of the erythocytes.

     October 9, 1940.  Fixation - Theory reviewed; Entire
histological technique demonstrated.  All Group work.

     October 14, 1940.  Blood Histology for entire group.
Demonstrate and explain slides made previously thereof.  Also group
examination on Wright staining technique.  Preparations 1,2,3,4,5,
6,7 and 02 outlined on attached sheet were performed by the entire
group.  Slide I/8 on frog marrow was shown to demonstrate o1
Materials for o3 were not available.  Results were highly successful,
but detailed notes were taken more by the numbers of the group.

     For two weeks,we tried to get a rabbit at a usable time, and
were unsuccessful until October 30, 1940.  Also, Jewish holidays
entirely disrupted a plan schedule: 10/3,4; 10/17,18; 10/24,25;
also, work was not allowed on 10/2/40.

     October 23 - Histology Theory review for Group.  Also, theory
on connective tissue.

     October 28 - Lecture from 10/23/40 continued; examination
of slides showing cartilage, fibrous, areolar and elastic tissue.
No rabbit was available.

     October 30 - A rabbit was finally gotten so that preparations
1,2 and 3,4 and 6 on Sheet 2 were attempted, with reasonable
success, except in the case of 3; there was no sunlight; (?)
development did not proceed; 11 was available from external sources.
Cartilage was studied indirectly from organ slides - rat embryo,
late chick embryo, larynx.  In preparation 1 and 2 a peculiar
type of striated fibre, resembling algae, was seen, still
unidentified.  Van Gieson's had to be prepared at the time, but
not hastily.

     November 1 - Arista....[Society]

     November 4 - Lecture and slides on cartilage, bone and bone
development, with 11 of Sheet 2.  Slides on rat and chick embryo,
long ago prepared, were most helpful.  A number of stomach slides
were restained with Van Gieson's, Methylene Blue-Acid Fuchsin and
Wright's stain.

     November 8, 1940 Stain sections of _______ with hematoxylin
eosin. Cursory study.

     November 13, 1940 Due to general lack of knowledge of anatomy
in group, a rabbit and a frog were rapidly dissected with explanation
11/14,15 - Midterms.

     November 18, 1940 Lecture on muscle structure (summary due to
lack of facilities) and also of the alimentary canal in general.

     11/20/40 Lecture on structure of pharynx, esophogus and stomach
with examination of slides and discussion.

     - in afternoon, at home, fix from a far from fresh frog, in
10% formalin, the liver, spleen, pylorus region of stomach, tongue,
lung, kidney, oviduct, sciatic (spinal) nerve, episternum, fundal
and cardial regions of stomach.  This is to be known as sirus B.  
Fixation proceeded to Monday, 11/25/40.  Tissues whose sections do
not appear in following accounts were discarded because of obvious
decay before fixation.

     11/25 - series B transformed to 50% methyl alcohol for washing 
Aid to Mr. Towme.

     Tues. 11/26 B series transferred to dioxane for dehydration
overnight.

     11/27 placed in dioxane paraffin for infiltration; later in
afternoon, into molten paraffin over two nights, when, on
  
     11/29 the series, or a large part of it, was embedded and
blocked off.

     December 2, 1940 Section blocks embedded 11/29/40 and mount
on slides.  Fix narcissus root tips in: Carnoy's 10% formal,
Zinker's, Bouin.

     December 3, 1940 Some sections were stained//7% Cr03, Flemming's
achromatic from blocks sectioned 12/3. The stomach was found to
be somewhat autodigested with a little detail remaining,
however (hem,- eosin).  Fixed root tips from yesterday washed in
70% Methy alcohol, with Li C03 or iodine where indicated.  Stained
were stomach, kidney and oviduct.  Cursory examination.

     December 4, 1940 root-tips in dioxane; overnight, for
dehydration.  Stain Frog Liver, series B, hem-eosin; Frog spinal
cord, Series A with Methylene Blue.  Section more stomach (lung,
kidney and spleen (B.) Oviduct was much ripped and wrinkled.

     December 5, 1940; root-tips in dioxane - paraffin, then paraffin
for infiltration; Buy slide labels.  Bring classification to date,
clean, relabel slides.

     December 6, 1940 Embed root tips; Help Mr. Towme.

     December 9: Group members stain stomach, liver, kidney cells
their technique still imperfect, but fair.

     December 11 - help Mr. Towme.

     December 12 - Section and stain root-tip (no. 4 - fixed with
Flemming's a chromatic - dichromate-chromic acid-acetic acid
fixative.  Results inconclusive.  Also section Spleen B. Close
group reexamination of slides on rat, chick and snake embryo
respectively.

     12/13 Examine stained, mounted sections of Spleen B - with
hem-eosin. "Sinuses and blood vessels are very characteristic, but
well defined.  Malpighian bodies are not at first recognzied.
Capsule transmits few _?__. Dense pulp.  Striated venous
walls.  Also, stain smear of blood of turtle.  Apparently, type of
amphibia.  Rearrangement and house-cleaning of group property.

      12/16/40. Aid Mr. Towme - preparation of glass holders with
?_ were for bacteriology.

     12/17/40 Duodenum. Ureter peritoneum, liver, and esophogus -
trachea were taken from a rabbit and all fixed in Bouin's.
Subsequently washed through to 70% Methyl Alcohol, wherein they
now repose.

     12/18/40 Dissection and fixation in Bouin's of skin, peritoneum,
liver, appendix, spleen, duodenum-pyloris, stomach, duodenum
pancreas, fat body (sub cutan), kidney, lung, heart, esophogus and
trachea of a male white mosue.  Subsequent washing; now in 70% Methyl
alcohol.  Fix in 10% formal, subsequent washing - the brain and
high spinal cord.

     12/19/40 "subsequent operations" of yesterday

     12/20/40 Prepare slide of Paramecia caudatum, hematoxylin
stain.  Fix in Schaudim's, wash through iodine alcohol, water.
Stain, dehydrate in dioxane, clear in xylol, mount in xylol-halsam.
Stain not too precise or clear.  Also, stain stomach, pyloris B,
in hem-eosin.

     Take inventory

                   Christmas vacation

     January 2, 1941 Group buys a quantity of chemicals with personal
funds.

     1/3/41 Fri. Stain a section of Liver B with Iron-Hematoxylin.
Mordant_ in 4% Ferric Alum, 25 min; Wash one minute in tap water;
stain 5 mins in 1/2% hematoxylin aq; differentiate to suit with
Ferric alum, wash, dehydrate, etc.

     Stain some wet blood smears fixed in absolute MeOH.  Proceed
as above for iron-hematoxylin.

     Stain a number of specimens from a protozoa_ culture marked
Spirostomum.  Form resembles Blepharisma.  Iron-hematoxylin.
(see 12/20/40 for technique)

     1/6/41 Restain Spirostonum from 1/3 to satisfy.  Mount in
xylol-damar.

     1/9/41 Section again; a number of blocks in series A,B,C (root
tip).
     (Date.)