A Short History of Histopathology Technique
Michael Titford
Pathology Department, University of South Alabama, Mobile, AL
Abstract
The specialty of histopathology technique dates back to
1838, when Johannes Miiller published his book, On the
Nature and Strz~ctureClzaracteristics of Cancer, the first
book on histopathology. The first compound microscope
had been constructed earlier in 1591 but suffered from se-
vere optical problems. In 1673 Anton van Leeuwenhoek
started the development of simple microscopes with single
lenses but that gave improved magnification and resolution.
The first microtome suitable for sectioning animal tissues
was constructed in 1848, with the popular Cambridge
Rocker (1885), Minot (1886), and sledge microtomes
(1910) manufactured later. Paraffin wax for infiltration and
support during sectioning was introduced during the
mid1800s. Different laboratory chemicals were investigated
for use as fixatives. Formalin, widely used today, was first
used in 1893.
Automated tissue processors replaced hand processing
starting in 1945, and cryostats were first manufactured in
1951. Enzyme histochemistry, electron microscopy, and po-
larizing microscopy all have become diagnostic tools during
the last 50 years. The widespread use of immunohistochem-
istry began in the 1980s has revolutionized cancer diagnosis
and is still under development. This article briefly reviews
the development of histopathology techniques in the United
States and United Kingdom from historical times to present.
(Tlze J Histoteclzrzol 29:99, 2006)
Submitted December 21, 2005; accepted with revisions
March 28, 2006
Key words: histopathology, histotechnology, history
Introduction
Histotechnologists working in the field of histopathology
for many years will have experienced m~lltiplechanges dur-
ing their careers. Within the last 30 years, histotechnologists
have seen the introduction of microcomputer-controlled tis-
sue processors, immunohistochemistry (IHC), in situ hy-
bridization, and laboratory information systems. Cryostats
were introduced in the 1950s, and the introduction of tissue
processors date back to about 1945. There have been many
Address reprint requests to Michael Titford, Pathology Department, Uni-
versity of South Alabama, 2451 Fillingim Street, Mobile, AL 36617.
E-mail: MTitfordOusouthal.edu
The Journal of Histotechnology IVol. 29, No. 2 1June 2006
new techniques introduced since the invention of the mi-
croscope in 1591, which enabled scientists to view tissues
microscopically for the first time. Many of the techniques
used routinely today in histopathology laboratories, such as
IHC, polarizing, and fluorescence microscopy, were devel-
oped in research laboratories. These techniques have revo-
lutionized the field and aided pathologists in making diag-
nosis.
This article attempts to briefly review the evolution in
histopathology techniques over the years, with the emphasis
on histopathology in the United States and the United King-
dom. This writer is indebted to an early book on microtech-
nique, which is still available as a reprint and is recom-
mended for further reading (1). References, some
secondary, are provided for further study.
The birth of histopathology can be credited to Johannes
Miiller (1801-1858). In 1838, Miiller, professor of anatomy,
physiology, and pathology at the University of Berlin pio-
neered the use of the microscope in pathology and published
his book, entitled Oiz tlze Nc~tupaand Strz~ct~ireClzaracter-
istics of Cancer, which was the first book on histopathology
(2).
Microscope Development
The excellent microscopes in use today are a result of
developments in microscopy dating back to 1591 when
Zacharias Jansen made the first compound microscope.
Jansen and his father, spectacle makers in Holland, were
typical of researchers of that period, gifted amateurs with
talent, time, and an interest in what was then a new field of
science. These early compound microscopes suffered from
spherical and chromatic aberration, low magnification, and
could only view specimens with reflected light. The re-
searchers often were more interested in the microscope than
the specimen, but it was a beginning (1,3,4).
Microscopes with better resolution were those made start-
ing in 1673 by Anton van Leeuwenhoek (1632-1723).
These simple microscopes were very rudimentary by to-
day's standards. They had a metal plate with a small hole,
into which was mounted a single small hand ground convex
lens. Items to be studied were permanently glued on a spike
on one side of the plate. The specimen was viewed through
the small lens from the other side. The working distance of
specimen to lens was adjusted with a small screw in the
metal plate (Figure 1).
Each microscope was intended for single use. Leeuwen-
99
 working at the Zeiss factory in Germany, made great ad-
vances in microscopy. He developed the apochrolnatic lens,
substage condensers to gather light, and ilnlnersion lenses
with a resolution of 0.25 micrometers, allowing their use for
microbiology ( 5 ) . Soon, nlany scientists owned microscopes
and, in London, private lessons were available, although no
one thought of themselves as full-time histologists (3). The
modern binocular microscope was developed in 1902. Im-
provements have continued to this day (Figure 2).

Early Fixatives
From early on, researchers sought ways to harden bio-
logical specimens that would enable thin slices to be cut.
Malpighi boiled tissues to solidify and harden them (heat
fixation). Others used common laboratory chemicals, such
as alcohol, acetic acid, chromium trioxide, and potassium
dichromate, to preserve these objects in a life-like state and
harden them for slicing, usually with a scalpel or razor.
Compound fixatives containing two or more components
appeared, with chemicals complernenting one another.
Miiller's fluid appeared in 1860, Zenker's in 1894, and Car-
noy's in 1887. The fixative effects of formalin, now the
most popular fixative, were discovered by Blum in I893 (1).

Microtome Development
Initially, freehand sections were prepared ~isingrazors,
most likely open razors then used for shaving. Later instru-
ments were then designed to hold specimens so thinner
slices could be cut. In another small step of great impor-
tance, the first hand held rnicrotome was introduced by Hill
Figure 1. Leeuwenhoek's ~nicroscopewas invented in 1673 and used a
in 1770 to cut sectioils of twig. Such instruments were cre-
single hand ground lens ( 1 ) . (Image courtesy Science Heritage Ltd.) ated not only to hold a sample, but also to advance it slowly
by use of a screw device so uniformly thin slices could be
cut. The earliest use of lnicrotomes was for botany and were
hoek's microscopes had numerical apertures of about 0.4
and magnified up to approximately x300 and were used to Ocular or eyepiece - -- --- - ---
st~tdynatural history items as well as bacteria, muscle, teeth,
and blood cells. Leeuwenhoek drew detailed sketches of his Draw-tube.----------
observations, which he sent to the Royal Society in London.
Although he was unable to bring himself to share with So-
ciety members the technical details about his microscopes,
Rack and pinlon for
26 were gifted to the Royal Society when he died (3,4). At ---coarse adjustment.
higher magnifications, the lens of the Leeuwenhoek micro-
scope had to be extremely small, the size of a grain of sand, Trlple nose-plece. -- - -
andextreme patience had to be used to prepare the lens and Micrometer screw for
later view the specimen. It is believed that Leeuwenhoek
Objectives. -- - -- tlne adjustment.
was able to achieve dark field illumination that enabled him
to study bacteria (3). Because no microscopic observations
had previously been described, everything he reported was
new. Ills diaphragm --
and abbe condenser.
The compound microscope was further advanced in 1827
when ~ o s e i hLister, father of the famous surgeon, corrected
the spherical and chrolnatic aberrations and together with
P~llar.
Thomas Hodgkin published descriptions of smooth and
skeletal muscle and the lack of a nucleus in the human red
blood cell. Soon after the publication of this article, com- -Stand.
mercial production of microscopes began in Germany,
France, and Italy. The resolution of these lnicroscopes per-
mitted viewing of items one micrometer apart (1). During
Figure 2. Brass ~nicroscopeswere used for Illany years. A mirror below
the 1830s, Amici and Chevalier developed the achromatic the substage condenser collected light and reflected it up into the micro-
lens (for color correction), which greatly facilitated the use scope. Initially, natural light from a window was used and, later, a tungsten
of the microscope. In the late 1870s, Ernst Abbe, while light (38).

A Short History of Histopathology Technique I Titford

called "cutting engines." Another simple microtome, avail-
able until the 1950s, attached to the bench so a knife could
be passed over the specimen and the specimen raised be-
tween cuts (6). The use of the microtome for animal tissues
did not occur until 1848 with the inicrotome of Topping
(Figure 3) (1).
There was a veritable avalanche of new microtolnes de-
signed between 1843 and 1870. Bracegirdle (1) lists some
20 instri~inents,some just being a minor improvement over
an earlier models, but others showing significant advances
in design. Several other important microtomes were devel-
oped later such as the Cambridge rocker (1885), the Minot
microtome (18861, and the sledge developed by Jung in
1910. The Cambridge rocker deserves spec;al attention.&- Figure 4. The rocker was popular in the United Kingdom, It
veloped by Horace Darwin, the son of Charles Darwin, its was robust, inexpensive, and accurate (33). (Image courtesy Leica Micro-
-
simple design and robust construction advanced the field of
histology in biology and pathology laboratories. Further,
svste~iisInc.)

with itsstationary knife and vertical sectioning of the block

it encouraged the use of serial sections. The Cambridge
rocker cut with a slight arc (Figure 4).
By comparison, the heavier sledge microtome was more
suitable for larger blocks and tougher tissues. In the United
Kingdom, the Cambridge rocker and sledge lnicrotomes
were the most popular microtomes in the last half of the
20th Century. In the United States, the most popular micro-
tomes for routine work in hospital laboratories have been
the Minot type rnicrotomes and the rotary lnicrotome pat-
terned after it. The knife remains stationary on the rotary
type microtome and a flywheel raises and lowers the block
which is advanced at the top of its trajectory (Figures 5 and
6) (1).
Microtomes have continued to improve, although the ba-
sic design remains the same. Modern microtomes are low
maintenance and robust and have the rigidity to cut the 3- or
4-pm sections required in today's histopathology laborato-
ries. Motorized microtomes are available to reduce carpal
tunnel syndrome caused by repetitive motion (Figure 7).
Figure 5. The present-day rotaly inlcrotome is modeled after the Minot
(4iown here, 39).
Embedding Media
The problems involved in cutting thin sections of tissue not enough to permit of thin undistorted tissue
were not completely solved until tissues could be infiltrated sections. Different methods had bee,, tried, and the paraffin
with paraffin wax. The early fixatives hardened tissues but wax method was developed slowly over a period of time by
several researchers. Early methods involved surrounding
tissues with molten paraifin wax that would harden an2
provide some support. Later techniques included dehydrat-
ing and clearing tissues and pIacing them in molten paraffin
wax so the wax would creep into the other layers of the
tissue and then harden. Finally, the method was developed
to infiltrate fixed dehydrated and cleared tissues with mol-
ten paraffin wax, which was hardened, providing support
for sectioning. This method remains in extensive use to this
day.
Who actually was the first to embed in paraffin wax is
unclear. Early researchers did not always describe clearly
the techniques they used, sometimes did not want to share
the information, or did not publish the technique promptly.
It has variously been attributed to Klebs (1869), Born &
Stickler (187 1; 7), Giesbrecht (188 l), and Butschli (188 1; 1).

Histopathology Develops as a Field

Figure 3. Cuthcart's microtome was invented in the 1880s and was stdl The early techniques that evolved to create thin sections
ava~lableIn the 1950s (6). (Image courtesy L e ~ c aMicrosystems Inc.) permitted researchers to study cell structure and develop

The Journal of Histotechnology 1 Vol. 29, No. 2 / June 2006


Figure 6. The sledge microto~neis useful for larger, or tougher specimens.
Figure 7. Many other models of rnicroto~iieswere available 100 years ago.
This model was based on the sledge-type design (40).
theories about how organs and tissues functioned. Early
studies though, were not organized, and were done more for
curiosity. Fran~oisBichat of France (1771-1802), "The Fa-
ther of Histology," encouraged physicians to perform au-
topsies to correlate symptoms of diseases and-the appear-
ance of tissues (3). Bichat was typical of the medical
researchers in Europe at that time in that they worked in
several fields of science. Bichat cared for patients, per-
formed autopsies, performed physiological experiments,
and wrote extensively. He also determined that disease pro-
cesses could affect parts of organs, but not always the whole
organ (3). Researchers of that period also used macrodis-
section, maceration, and injection studies to determine the
structure and function of tissues. These researchers are to be
especially adinired because they were the first workers in
disciplines in which no other studies had been pel-formed.
They proposed theories and made discoveries about normal
and disease processes in the body.
Of all t h e - ~ u r o ~ e acountries,
n it was in Germany in the
1800s that the specialty of pathology came to fruition. Be-
cause of the organization of medical care and research in
Germany at that time, an atmosphere was created that
prompted the development of research institutes. Physicians
were encouraged to perform autopsies. Karl Rokitansky
(1 804-1878) typifies the best of German researchers of that
period. As chief pathologist at the Vienna General Hospital,
he perfor~nedmore than 30,000 autopsies in his career. He
compared disease symptoms with pathological findings and
steered the field of medicine toward a pathological basis of
disease instead of the signs and symptoms being the disease.
Although others in Europe had previously studied specific
diseases and specific organs, Rokitansky was the first to
classify diseases according to the organs involved (3). Jo-
hannes Miiller, previously mentioned, can probably be
called "The Father of Histopathology." He also fostered an
atmosphere of research and discovery in the hospital labo-
ratory. With encoilragelnent and teaching, Miiller attracted
to his laboratory gifted students such as Rudolph Virchow,
Jacob Henle, Karl Reichert, Karl Kuppfer, and others who
made contributions to histologv in the late 1800s. In 1838
the Schleiden-Schwann theorfhad stated that all plant and
animal tissues were composed of cells (7). The first text-
books of histology in the modern form appeared in the
1850s.
Perhaps the most influential pathologist of all times was
Rudolph Virchow (1821-1902), known as the "Father of
Pathology." Although most remembered for his statement
that all diseases are cell based, he also worked in the fields
of public health, archeology, and government and had time
to start a medical journal, which he edited for 50 years.
Disease processes he investigated and named include leu-
kemia, fibrinogen, embolism, and amyloid. Virchow made
many other contributions. With his brilliant mind, he was
able to take the diverse findings of other scientists and cre-
u
ated working theories that by and large were later proved
correct (3).
Meanwhile, microscopy in England remained n~ostlyin
the hands of nonprofessional scientists. Clubs and societies
were created to study the beauty and structure of natural
history items using l ~ i c r o s c o p yThe
~ Royal Microscopical
Society was formed in 1839 and was more academic (1).
The Quekett Microscopical Society was created in London
in 1868 and also remains active to-this day, catering mostly
to the amateur. This club is named after John Quekett
(1815-1861), who was very active in microscopy and is
credited as the first to decalcify tissue in 1848 (7).
In 1845, Hermann Lebert conclt~dedthat the microscope
could be used to differentiate between malignant and benign
tissue by squeezing fluid from the fresh tissue and examin-
ing the cellular morphology. Lebert reported that the ma-
lignant cells had larger nuclei. However, without suitable
staining methods to demonstrate these cells clearly, not
much attention was made of his early cytological observa-
tions (3).
Development of Staining
Researchers in the newly developing field of microscopy
experimented with naturally occussing dyes such as madder
and indigo, but mainly used carmine. Much of Virchow's
work was performed with cannine. Leeuwenhoek had ear-
lier used saffron from crocus to study muscle. Hematoxylin,
reportedly first successfully used by Wilhelin von Waldeyer
in 1863 was another early histological dye (8). William
Perkin opened up a whole new field of discovery when he
A Short History of Histopathology Technique I Titford
prepared the first synthetic dye mauve from aniline in 1856.
A new indt~stryof dye making evolved, primarily in Ger-
many. These dyes were made mostly for the textile industry
(9). Early histologists used these dyes in their studies. Basic
fuchsine was first manufactured in 1865 and aniline blue in
1868. Schwarz devised the first double stain in 1867, and
differentiation was first used in 1868. Several heinatoxylin
formulas such as Delafield's (1885), Ehrlich's (1886) and
Heidenhain's (1892) appeared in rapid succession (1).
Many other staining methods used in pathology appeared
in the late 1880s. The hematoxylin and eosin method was
introduced in 1875 by Wissowzky (7). Ziehl improved upon
Ehrlich's method for acid fast organisms by adding phenol
to the stain solution in 1883 (1). The Gram stain to dem-
onstrate the two main groups of bacteria appeared in 1884,
Congo red in 1886 and the fat stain Sudan I11 in 1896.
Mallory's phosphotungstic acid hematoxylin was intro-
duced in 1897. Gielnsa improved upon the Ro~nanovsky
stains for blood smears with his method in 1902 (1). Two
more recent noteworthy stains are the periodic acid Schiff
method of McManus (1946; 10) and the alcian blue method Figure 8. The "Arlisan" special stainer represents a new technology in
histopathology and was first available in 1999. (Image courtesy of Dako.)
of Steedman (1950; 11).
Silver nitrate preparations have a long history in histol-
ogy and were used in the mid1880s to study the normal Frozelz Sections
structure of tissues. Thin slices of tissue were immersed in In the frozen section technique used today, the tissue is
dilute silver nitrate, and then the silver was reduced and frozen and the tissue fluids in it freeze and then provide
precipitated in the tissue as a fine black powder. The origi- support for the cutting of sections. Stilling in 1842 is re-
nal von Kossa method utilizing silver nitrate for calciiun puted to be the first person to use frozen sections (I), al-
staining was introduced in 1901 (12). The Masson method though F r a n ~ o i sRaspail (1794-1878) is regarded as the
for argentaffin was published in 1914, with Fontana's modi- "Founder of Histochernistry" and to have used frozen sec-
fication in 1925. Among the early argyrophil methods was tions (15). Raspail performed chemical tests on tissues and
Bielschowsky's method for axon and neurofibrils (1904), published his work in 1825 (15). Better documented is the
from which many other methods were developed including method of Rutherford (1871) which used a saltlice slush to
methods for reticulum, basement membranes, urates, mucin, freeze the tissue for sectioning. Later in 1876, evaporation
and Ptzeut~~ocystis cnrirzii, carcinoid tumors (13), and meth- of ether was used as the method of cooling (Figure 9) (1).
ods for organisms such as spirochetes and legionella (14). Welch was the first to use a frozen section to diagnose
breast cancer during surgery in 1891 (1 6). Carbon dioxide
Automatic Stainers
Automatic slide stainers first appeared in histology labo-
ratories during the 1960s. In the United Kingdom the Shan-
don Colnpany introduced their first automated stainer in
1965 (Sheila Dandy and Helen Tucker, Thermo Electron
Corporation, personal communication, 2005). The Staino-
matic,@ manufactured in the 1970s by Garn Rad Incorpo-
rated in Detroit, was widely used in the United States. In the
mid 1970s, Lipshaw Manufacturing Corporation, also in
Detroit, produced the Trimatic0 and Lipshaw AutoIStain
Center.@ The Trimatic was advertised for use as a stainer,
tissue processor or rapid tissue processor, demonstrating
that the early automatic stainers were tissue processors al-
tered for use as stainers. In 1976 the Shandon Varistain
24-30 became available (Sheila Dandy and Helen Tucker,
Thermo Electron Corporation, personal communication,
2005). Automatic stainers provided reproducible results as
well as saving technologist time. In the late 1990s, auto-
matic special stainers came on the market such as the Ar-
tisan@ from CytoLogix. This instrument is capable of per-
forming a variety of special stains si~nultaneouslythat
require different temperatures for staining (Steven Bogen,
MD, PhD, personal communication, 2005), all with "walk-
away capability." The Artisan utilizes bar code technology Figure 9. 111the late 1800s, evaporating ether was used to cool and freeze
and prepackaged reagents. This instrument is now available specimens for frozen sectioning. In this illustration, a Cuthcart microtome
through Dako (Carpinteria, CA; Figure 8). was used (40).

The Journal of Histotechnology I Vol. 29, No. 2 1June 2006

was introduced to freeze tissues in 1901 (1). This last
method remained in use for a long period and saw extensive
use in surgical pathology laboratories, within memory of
some present-day histotech~~ologists. The freezing micro-
tome was clamped to the countertop. Tissue for frozen-
section diagnosis was rapidly fixed in boiling formalin,
cooled with water, and frozen with bursts of carbon dioxide
gas. Several sections were rapidly cut and the resulting free
floating sections transferred from solution to solution for
rapid hematoxylin and eosin staining and cover slipped for
diagnosis (Figure 10; 17).
During the 1960s freezing devices based on the Peltier
effect were available, which attached to sledge and Cam-
bridge rockers. These cooling devices consisted of a series
of bimetal strips. One side of the strip was cooled with
running water, and when an electrical current passed
through the bimetal strip, the opposite surface froze (17).
The commercial production of cryostats began in the
United Kingdom in 1951 by the Bright Instrument Com-
pany, with assistance from Professor A. G. E. Pearse (Alan
Bright, Bright Instrument Company Ltd., personal commu-
nication, 2005). Soon after the Slee Company (South Lon-
don Electrical Equipment), and the Prestcold Company in
Cowley, Oxfordshire also began production ( 15). These
early models were essentially a Cambridge rocker inside a
refrigerated cabinet with the controls outside. Later models
Figure 10. F ~ e e ~ i nlnicrotomes
g were popular before clyostats Mounted
on a bench, the tlssue was frozen with bu1st5 of carbon dlox~deand mul-
tlple sectlolls rapidly cut (6) (Image courtesy of Lelca Mlcrosysterns )
104
used other type microtomes. Pearse was the first professor
of enzyme histochemistry in the University of London.
Later model cryostats in the United States used microtomes
based on the Minot rotary principle. The Harris Refrigera-
tion Company of Cambridge, Massachusetts was an early
manufacturer of cryostats in the United States in the 1960s
(15).
Tissue Processors
For surgical pathology laboratories, time has always been
a factor in the production of slides for diagnosis and before
automation processing was extended over several days. One
recommended procedure took 26-100 h over several days:
96% Alcohol 6-24 h
Absolute alcohol 6-24 h
Chloroform 6-24 h
Chloroform saturated with paraffin 6-24 h
Paraffin bath, two changes 2-4 h
Embed and cool quickly in water (18).
Copper ovens heated with hot water were used to infiltrate
the tissue in dishes with paraffin wax. Vacuum embedding
and infiltration was first used in 1884 (I). The first auto-
mated tissue processor was made in Germany in 1909 (I),
but it is hard to assign the title "first" in regard to laboratory
equipment. Some items of laboratory equipment were first
made in obscure research laboratories or as a single proto-
type by a manufacturer and not further developed. In mod-
ern times, laboratory equipment may not survive "beta"
testing, the rigorous use of equipment on a trial basis in a
working laboratory. Sales divisions of laboratory equipment
companies discuss instruments "maintaining a market pres-
ence" and that is probably a good guide when discussing the
history of histopathology; what instruments were good
enough at that time to be used on a daily basis in routine
histopathology laboratories (Figure 1 1).
In 1945, Edwin Weiskopf, founder of the Technicon Cor-
poration in New York, started production of tissue proces-
sors that became popular in the United States (19). In these
early processors, the tissues were carried from solution to
solution in a basket. Only the paraffin solutions were
heated. These models had agitation but no heated solutions
or vacuum infiltration. The flat round baskets contained
compartments into which the specimen was placed together
with an identifying label. In the United Kingdom, the Shan-
don-Elliot Company began manufacture of its tissue pro-
cessor which became popular in that country in 195 1 (Sheila
Dandy and Helen Tucker, Thermo Electron Corporation,
personal comn~unication,2005), and a similar model was
produced by Hendrey Relays Ltd. These instruments (irrev-
erently called "dunk and dip") were programmed with a disc
and timer mechanism where a peg resting against the disc
edge fell into a notch that activated the instrument. Short
cycles were available. Although these machines advanced
tissue processing, there were problems with these instru-
ments including baskets hanging up overnight and tissues
drying out, baskets breaking loose allowing tissue intermin-
gling, and organic solvent fumes escaping into the labora-
tory' Later lnodels of TechnicOll processors in
Vacuum infiltration, and processing solutions heated with
mineral oil to better facilitate the exchange of solutions
A Short History of Histopathology Technique / Titford
Figure 11. Before tissue processors, tissues were infiltrated and processed
over several days. Wax infiltration was performed in dishes on top of
copper ovens (41). (Image courtesy of Lexis Nexis Butterworth.)
during fixation, processing and infiltration with wax (Fig-
ures 12 and 13).
During 1979, two co~npaniesstarted marketing fluid
transfer tissue processors, which used stationary tissue pro-
cessing chambers into which the cassettes were placed.
These new instruments were the Histo~naticO(Fisher Sci-
entific Company, Pittsburgh, PA), and the Tissue Tek I11
Figure 12. An early British tissue processor fro111 the late 1940s.
The Journal of Histotechnology IVol. 29, No. 2 1 June 2006
Figure 13. "Ultra" tissue processor@ from Technicon heated processi~ig
solutio~isand wax. Vac~luluand agitation was applied during processing.
vacuum infiltration processor@ (Miles Company, now
Sakura Finetek USA, Torrance, CA: Sunny Hong, Sakura
Finetek USA, Inc., personal communication, 2005; 20). In
these instruments during processing, the solutions including
molten paraffin wax, are pumped into the chamber. Cas-
settes are exposed to agitation, heat, vacuum and pressure
during processing. After processing a "purge" cycle cleans
the chamber. Microprocessors are used to program instru-
ments. This technology is still widely used (Figure 14).
In 2004 a new development occurred when a continuous
throughput tissue processor was marketed. The Tissue Tek
XpressO (Sakura Finetek USA) uses thin tissue slices, spe-
cialized cassettes, microwave technology and propriety pro-
cessing solutions to process up to 120 specimens each hour.
Development of Other Histology Procedures
Although the development of tissue processors, cryostats,
and staining methods represent major milestones in the de-
velopment of histopathology technique, there have been a
variety of other develop~nentsthrough the years which have
improved the quality and turn around time of histopathology
cases.
Early histotechnologists determined that optimum paraf-
fin hardness improved sectioning and generally, thinner sec-
tions were possible with harder wax. In the 1950s, paraffin
waxes were available of different melting points to facilitate

Figure 14. "Histomatic"@ (Fisher Scientific) was the first o f the fluid
transfer processors.
sectioning. These waxes often contained contaminants and
had to be filtered before use. Other methods to control hard-
ness of waxes included the addition of compounds such as
ceresin, beeswax and bayberry wax. In the 1960s plastic
polymers were added to control hardness and improve sec-
tioning. Cooling the blocks further hardens the wax and
irnproves sectioning. This method also remains in use to this
day.
Slides, Coverslips, and Mountants
In the early days of microscopy, researchers mounted
several specimens on "sliders" for passing across the mi-
croscope stage. In 1839 The Microscopical Society of Lon-
don decided on the size of 1 inch by 3 inches for the mi-
croscope slides in their collection which became the
standard around the World. (I).
Early microscopists used cover slips of the mineral mica
to cover their preparations while viewing specimens under
the microscope. These preparations were often aqueous
mounted or cleared specimens and the mica reduced evapo-
ration of the fluid. In 1840 the Chance Glass Company of
Birmingham, England started prod~rctionof glass cover
slips (1). In 1985, Hacker Instruments and Industries Inc
(Winnsboro, SC) introduced the RCM 3650 automatic glass
coverslipper, t h e first widely used instrument of its kind
(Elfi Hacker, Hacker Instruments & Industries Inc., personal
communication, 2005). Automatic coverslippers not only
save time and use less lnountant but also reduce employee
exposure to xylene.
The mountant used almost exclusively in the early days
of histopathology technique was Canada Balsam, a natural
resin extracted from the Canadian fir tree, Abies bnlsmnea.
First used in 1835 (I), Canada balsam had many of the
attributes desired in mountants today. The balsam dried at a
refractive index (RI) of 1.53 (compared with the RI of glass
of IS), adhered well, was miscible with xylene and dried
fairly quickly with little or no shrinkage. Some batches of
Canada balsam did discolor to a light brown color and if
acidic, balsam causes fading of basic dyes and may bleach
out Prussian blue deposits. Synthetic mountants became
popular in the 1960s.
Aqueous mountants are used for techniques where or-
ganic solvents and clearing agents will destroy or change
cellular components under investigation. Early formulations
used glycerin and gelatin, while other formulas included
gum arabic, sugars and buffering salts.
Other Laboratory Ware and Consumables
The Coplin jar was invented by W.M. Coplin in 1897 and
remains in use to this day (1). Early histotechnologists used
paper molds for embedding or Leuckhart's type molds. Par-
affin blocks were then mounted onto wooden blocks or a
microtome stage for sectioning. In 1890, Gaskell introduced
the flotation bath (1). Another important consumable was
developed in 1958, when Dr. James B. McCormick (a mem-
ber of the National Society of Histotechnology) founded
Lab-Tek Plastics and introduced embedding rings to histo-
technology. These rings could be labeled and fit securely
into the microtome. The ring system was used in conjunc-
tion with metal cassettes and molds. Later in 1968 after
Miles acquired Lab-Tek Plastics, another invention of Dr.
McCormick's was introduced: the cassette system widely
used today where the tissue is processed in the labeled cas-
sette and the cassette later becomes an integral part of the
paraffin block (20). The plastic cassette fits snugly into a
metal mold or disposable plastic mould. Quick release
clamps on microtomes facilitate rapid exchange of blocks
while sectioning. Development of embedding rings and cas-
settes led to the production of cabinets to file these blocks.
In the mid1980s, cassettes of different colors were manu-
factured, enabling histotechnologists to color code their
work. Another item of laboratory equipment that has con-
tributed to histopathology technique is the forceps warmers
that removed the need for a Bunsen burner in the histology
laboratory. These in turn were replaced by embedding cen-
ters that combine a reservoir for molten wax, a cooling plate
to harden blocks and a holding tank for cassettes waiting to
be embedded. The introduction of disposable microtome
knives in 1978 by the Feather Company was an important
advance (7). Previously, steel knives were used for routine
microtomy. These knives required frequent honing and
stropping. Small foci of calcium in the tissue or a metal
staple left in the tissue would create a nick in the knife-edge,
which would have to be honed out, or unsightly scores
would appear in sections cut at that spot. Disposable knives,
while possibly not so sharp and stable as steel knives, are far
more usef~rlbecause of their availability and temporary na-
ture. They are also used in cryostats (Figures 15 and 16).
Alternative Embedding Media
Other embedding media were devised at different times.
Celloidin was first used in 1877 (1). Celloidin, and its less-
viscous partner low-viscosity nitrocellulose (i.e., LVN), is
prepared from the nitration of cotton and was used exten-
sively in the past for preparation of large sections of friable
material or bone. An advantage of celloidin was that the
processing was performed at room temperature and did not
involve the shrinkage associated with paraffin embedded
tissues. The disadvantages of celloidin are that thin sections
are not possible and the celloidin sections have to be
handled as "free-floating" sections. In addition, celloidin is
explosive. Processing takes approximately one week. Cel-
loidin is still used infrequently today in microanatomical
A Short History of Histopathology Technique / Titford

Figure 15 Left to right: early histotechnologists embedded in Leuckhart's
molds, two brass "Ls" used with a metal plate. In 1958, enlbedding rings
were introduced. Tissues were processed in small round cassettes and
embedded using a plastic ring. In 1968, the early model of the present day
cassette was introduced with a metal lid, followed by the all-plastic cas-
sette.
Figure 16. Early embedding center. Operations were at different levels.
Cassettes were kept stored in a heated wax pot until embedded.
studies or situations in which the shrinkage during routine
paraffin processing alters the relationship of tissues. Speci-
mens benefiting from celloidin sectioning include eyes,
bone and brain.
Ester wax, developed by Steedman in 1947, was recom-
mended for the sectioning of hard tissues, insects, and cor-
tical bone. Ester wax good adhesion with specimens and
thin sections with minimal shrinkage were possible. Xylene
could be avoided during processing (17).
Plastic Sections (Glycol Methacrylate)
For a few years in the mid-1980s, glycol methacrylate
(GMA) became a popular new embedding medium for the
production of thin (one micrometer) biopsy sections. In this
technique, small samples of biopsy tissues are fixed, dehy-
drated to ethanol, infiltrated with GMA, a catalyst is added
and the block polymerized at room temperature. Using a
glass knife prepared from fractured glass or a tungsten car-
bide knife and heavy duty microtomes, 1-pm sections are
possible. The resulting sections gave excellent clarity and
lacked the shrinkage and artifacts associated with formalin
fixation followed by paraffin processing. This method was
especially beneficial for biopsies such as liver, kidney, and
bone marrow. In kidney biopsies, the glomerular basement
membrane is more easily observed. Small bone biopsies
also could be sectioned without decalcification. Many light
The Journal of Histotechnology I Vol. 29, No. 2 / June 2006
microscopy staining methods are not adaptable to GMA
sections, and the technique is labor intensive and long; as a
result, interest waned. GMA is still used in academic and
research environments.
Enzyme Histochemistry
In 1939, a new field of enzyme histochemistry was cre-
ated when, working independently, Gomori and Takamatsu
developed methods for demonstrating the enzyme alkaline
phosphatase in frozen sections (7). Soon, using specific sub-
strates, a wide variety of enzymes were demonstrable in
frozen sections. The use of enzyme histochemistry has less-
ened in histopathology with the arrival of other diagnostic
methods such as IHC but is still used to diagnose muscle
biopsies in neuromuscular disorders, acetylcholinesterase in
Hirschsprung's disease, some enzyme deficiency disorders
and other uncommon uses. A subset of enzyme histochem-
istry methods are used on bone marrow biopsies and aspi-
rate smears in conjunction with flow cytometry to diagnose
leukemia. Most of these methods require unfixed bone mar-
row aspirate smears. (Enzyme methods are used to visualize
reactions in IHC, but the initial step in method in immuno-
logical.)
Electron Microscopy
Ernst Ruska and a team in Germany developed the first
electron microscope in the late 1920s, and Siemens built the
first commercial electron microscope in 1939. In the United
States, RCA started production in 1941 (21). Described
briefly, the use of electrons as a "light" source with their
shorter wavelength gives improved resolution and higher
magnifications. Using light microsco~yand oil immersion
lenses, resolution of 0.2 p m or 2000 A is possible at xlOOO
magnificatjon. Using electrons as the light source, resolu-
tion of 2 A is theoretically possible. In reality, because of
the thickness of sections used in EM, the low magnificatiolls
used, and imperfections in e!t electron microscopes them-
selves, resolution of 10-20 A is more usual. Most electron
microscopy performed in histopathology is in the x3000 to
x 10,000 range.
Found primarily in medical centers and university set-
tings, electron microscopy came into use in the late 1950s
and early 1960s and they were widespread by the 1970s.
Although it was used initially to study the ultrastructure of
biological samples, researchers interests soon turned to dis-
eased tissues and it was determined that diagnosis of some
difficult cases could be made using electron microscopy.
Electron microscopy is also a good example of a new in-
strument being devised and then ancillary equipment being
developed later to use the instrument to its full potential.
The first electron microscope developed by Ruska basically
just focused the electron beam. Later, researchers placed
samples in the electron path and examined the shadow ef-
fect created by passing the beam through the sample. Still
later, ultramicrotomes were devised to cut ultra-thin sec-
tions and specialized plastics used to provide the extra sup-
port required. Only then could biological samples be exam-
ined. Originally, fractured glass knives were used to prepare
the ultra-thin sections. Now, diamond knives are commonly
used. IHC removed much of the need for electron micros-
copy in surgical pathology, but it still has uses in renal and
muscle pathology and in other areas in addition to research.
Polarizing Microscopy
Polarizing microscopy has wide applications in clinical
laboratories. In the polarizing microscope technique, when
two polarizing lenses are rotated so that their axes are per-
pendicular to one another, the background appears dark.
Birefringent substances between these two lenses appear
bright against a dark background. Birefringent materials en-
countered in histology include collagen fibers, bone, striated
muscle, hair and cholesterol. Amyloid deposits in thicker
(8-10 bm) sections stained with Congo red are also bire-
fringent.
Another use of polarizing microscopy in histopathology
is to differentiate between the uric acid crystals of gout and
calcium pyrophosphate. For this, a third filter called a first-
order red plate is used by the researcher to determine wheth-
er the crystals have positive or negative birefringence. Most
modern high-end microscopes used in diagnostic surgical
pathology have polarizing attachments.
Fluorescence Microscopy
In 1941 Coons, Creech, and Jones demonstrated that a
flourochrome-tagged antibody could be used to label anti-
gens in a frozen section (22). When viewed with a fluores-
cence microscope, the visible light created demonstrates the
site of the antigen. Using a later technique with fluorescein
isothiocyanate (FITC), the method is still used today to
demonstrate autoimmune deposits in renal and skin biopsies
and other tissues. Modern day fli~orescencemicroscopes use
an epi-illuminated system where ultra violet light from a
mercury vapor lamp is directed down onto the section
through the objective, and visible light of longer wavelength
returns up through the objective and to the oculars. These
techniques work best of frozen sections. Some disease pro-
cesses exhibit natural tluorescence, for example porphyria
will fluoresce in paraffin sections with correct filtration.
Autofluorescent substances include elastic fibers, collagen,
vitamin A and lipfuscin (23). Various fluorochrornes are
available that will stain some tissue structures and organ-
isms. These fluorochromes may be exited by blue or green
light, or by ultraviolet. Very ilsef~11is the auramine-
rhodamine method for acid fast bacilli. A disadvantage of
fluorescence methods is the temporary nature of the slides
and the expensive tluorescence microscopes.
Immunohistochemistry (IHC)
IHC is another area of histopathology using immunologi-
cal procedures and as used in histopathology laboratories
today usually means the preparation of permanent formalin
fixed paraffin embedded sections in which an antigen-
antibody method has been used to demonstrate a protein in
the section. An important event occurred in the develop-
ment of this procedure in 1966 when researchers first used
3,-3'diaminobenzidine (DAB) to create a stable colored
compo~uldat the site of the protein in the tissue. Later, in
1970, Sternberger et al. developed the peroxidase antiper-
oxidase method (i.e., PAP) that inserted a secondary anti-
body that attaches to the primary antibody and a peroxidase
antiperoxidase complex, which is visualized with DAB or
other compounds (22,24). These methods increased sensi-
tivity 100- to 1000-fold over earlier methods. A still further
improvement to the aforementioned method occurred in
1974, when Taylor and Burns developed a working method
for IHC suitable for formalin-fixed, paraffin-ernbedded tis-
sues. This had multiple benefits. IHC could be performed on
the same blocks as routine histology hematoxylin and eosin
and special stains because paraffin blocks retained their im-
munoreactivity over time. In addition, the morphology of
paraffin embedded tissues was better than with the frozen
sections used in immunofl~~orescence procedures. In addi-
tion, retrospective studies c o ~ ~ be
l d performed.
A variety of different procedures are now available that
increase specificity and sensitivity. Masking of antigenic
sites by formalin fixation is reversed by different techniques
collectively called "Antigen retrieval" (24,25).
IHC methods have revolutionized cancer diagnosis in the
histology laboratory. Becausesome epitopes of antigens are
not easily demonstrated, several complimentary antibodies
may be used for each type tumor. IHC became available to
routine histopathology laboratories in the United States in
the early 1980s, when kits for specific antigens became
available from Dako Corporation (Carpinteria, CA) and Im-
mulok Inc (later, Immulok Division, Ortho Diagnostic Sys-
tems, Raritan, NJ). The first antibodies in wide use were for
prostate specific antigen (PSA), glial fibrillary acidic pro-
tein (GFAP), human chorionic gonadotropin (HCG), and
some imn~~~noglobulins. Initially, the commercially avail-
able antibodies were polyclonal, developed in laboratory
animals, most commonly rabbits. Later, more specific
monoclonal antibodies were produced, grown in hybridoma
cultures. Soon after, universal kits became available, where
the primary antibody was "plugged in" to the universal kit.
The first widely used IHC stainer, the Code-on@ from
Fisher Scientific (Pittsburgh, PA), used capillary action to
automate IHC staining. The most recent IHC stainers avail-
able use a spray on mechanism. Automated IHC stainers
provide reproducibility, red~lceerrors, and save technologist
timer. The method remains a very powerful diagnostic tool.
Because not all tumors express identical proteins, algo-
rithms have been developed that use panels of antibodies to
demonstrate general lines of differentiation of tumors or a
battery of antibodies may be used to achieve the same effect
(24).
Laboratory Information Systems (LIS)
LIS began arriving in clinical laboratories in the mid-
1970s but workable surgical pathology packages were not
available until later. LIS for surgical pathology laboratories
provide word-processing features, patient histories, printed
slide labels, billing, and other features. Demographic data
(patient name, sex, race, medical record number, date of
admission, etc) provide useful background information re-
lating to histopathology specimens. Diagnoses are coded
and can be retrieved for research or quality control pur-
poses. Newer LIS systems also provide bar coding for speci-
mens, cassettes, and slides and further reduce essors.
Books and Literature
An account of the history of histopathology technique
would not be complete without a mention of the textbooks
that characterize the techniques used. There has been no
shortage of such books. An early book devoted to technique
was by Arthur Bolles Lee, The Microto~nist'sVcide-Mec~lm
(1885; 26), which described general microscopy techniques
for biology. Early pathology texts, such as in John Warren's
A Short History of Histopathology Technique / Titford
Surgiccrl Patlzology arzd Therr~l)eirtics(1895; 27) and Aldred
Warthins's Practical Pathology for Stildetzfs nrzd Plzysicicirzs
(1897; 28), included sections on histology technique, either
at the end of a chapter dealing with a particular organ sys-
tem, or at the end of the book. As the field of histopathology
technique grew, textbooks became available devoted solely
to histopathology technique. In the United States, Mallory
and Wright first published Pntlzological Techrziqile (1901;
29), which went into several editions, and in the United
Kingdom Harry Carlton published Histopatlzologicnl Tech-
nique (1926; 30), which also went to several editions. Lillie
published his excellent reference book, Histo~~ntlzologic
Technic (1948; 31), and later collaborated with Fullmer on
a forrrth edition. In the United Kingdom the late Charles
Culling (later an eminent member of The National Society
of Histotechnology) published H~zrzrlDookc!f'Histol7atho/og-
icnl Techr~iques(1957; 32), and R.A.B. Drury and E. A.
Wallington honored Carleton by rewriting his book and en-
titling it, Carletorz's Histological Teclzrziq~ie(1967; 33). The
enzyme histochemistry period was served by Histoclzernis-
tr-y-Tlzeor-etical a i ~ dApplied by A. G. E. Pearse (1953)
which in later editions went to three volumes (15).
Theor-y arzd Practice of Hi.stoteckrzologj (second edition;
1980), by Dezna Sheehan and Barbara Hrapchak (12), was
the first of several excellent texts to appear in the last 25
years, accompanied by Tlzeoly arzcl Practice of Histological
Teclzlziques edited by John Bancroft and Alan Stevens (sev-
eral editions, 1977; 34) that provided up-to-date detailed
procedures with references, principles of the technique, and
tips on the method, More recently John Kiernan's Histn-
logical arzd Histoclzerizicrrl Metl7ocls- Theory arzd Practice
(three editions; 35) gives a more academic approach to his-
topathology techniques with fewer of the older traditional
staining methods. Numerous other fine textbooks also are
available or fill a special need in enzyme histochemistry,
IHC or research. Modern texts also give fill1 citations for
their methods.
Finally laboratory accreditation agencies and professional
organizations, including the National Society of Histotech-
nology and The American Society of Clinical Pathologists,
have published books, booklets and other training aids spe-
cializing in histopathology technique, safety, and manage-
ment. In the United Kingdom, the Royal Microscopical So-
ciety publishes a series of handbooks relating to histology
techniques (36).
References
The Present State of Histopathology Technique
A multitude of changes have occurred in histopathology
technique since the days of Leeuwenhoek and Miiller, and
the now-quickened rate of change promises to continue. In
the United States, much of this is driven by the high cost of 2. Johannes Miiller (1801-1858) Anatomist, physiologist, pa-
medical care and the desire of medical ins~~rance companies
to get the maximum amount of treatment of the lowest cost,
which translates to more inforormation and rapid turnaround
times. Also, in the United States and in Western Europe,
new regulatory requirements, waste disposal requirements,
and safety issues are enco~~raging companies to make avail-
able less hazardous fixatives on which old techniques will
work, as well as the newer IHC methods (37). To speed up
procedures, microwave processors are now used in some
laboratories for processing and staining. New instrunlents
such as the Sakura Finetek Xpress,O which was disc~~ssed
The Journal of Histotechnology IVol. 29, No. 2 1 June 2006
earlier in this review, processes thin slices of tissue rapidly
using proprietary chemicals. "Core" histology laboratories
are being created to maximize work output as large histol-
ogy laboratories work more efficiently. The realization that
some cancers involve one or more gene abnormalities has
resulted in increased interest in gene expression profiling
and the use of tissue microarrays. These studies have re-
sulted in the development of diagnostic histological sub-
types of tumors that may affect patient management and the
likelihood of tumor metastasis, although at this time such
techniques are performed in specialized laboratories.
Strangely, however, the standard embedding procedure for
histopathology still uses paraffin embedding, which seems
un~~sually out of date for the present age. One wonders if a
water-soluble embedding medium with the characteristics
of paraffin wax is possible. Such a medium would reduce
shrinkage, speed up turnaround times, and enhance IHC.
Conclusion
This article has reviewed the history of histopathology
technique from the construction of the earliest microscopes
in the 1500 and 1600s to the present time. The earliest
practitioners were hobbyists and amateurs. The first micro-
tome was developed in the 1700s. Early researchers used
common laboratory chemicals to preserve and fix the tissues
with embedding in paraffin wax following in the late 1800s.
About the same time the aniline dye industry was created
making available many dyes suitable for staining tissues.
Automated tissue processors became common starting in
1945. More recently, electron microscopy, fluorescence mi-
croscopy, polarizing microscopy, and IHC have been
adapted for diagnostic work in histopathology. The devel-
opment of new techniques for histopathology and the re-
finement of older techniques promise to continue as the
medical com~~lunity strives to extract the maximum amount
of diagnostic information from patient tissues.
Acknowledgments
Adrian Hoff provided excellent photographic assistance
and prepared old illustrations for publication. The staff of
the University of South Alabama Charles M. Baugh Bio-
medical Library gave unlimited access to the William Gard-
ner collection of historical pathology books. John Kiernan
reviewed the manuscript and recommended changes.
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