vast majority of surgical pathology labs do not use agar techniques;
and, as with so much of routine surgical pathology, agar techniques
are not even addressed by, much less approved by the FDA. Agar
utilization is a common sense and intuitive trick which can
be employed into techniques for the betterment of patient care.
However, some of the ways we use agar are in efforts toward
exactitude and thoroughness, the degree of which is hotly debated
in pathology! [disclaimer]
The American Journal of Dermatopathology
Volume 5 Number 2
TIPS FOR AND MOSTLY BY TECHNICIANS:
2004 update at end of this page, below--
Florence Nygaard and Gusstave
CORRECT ORIENTATION OFSPECIMENS FOR HISOLOGIC PROCESSING:
embedding in agar.
Ervin B. Shaw, M. D.
Johnnie M. Johnson, HT (ASCP)
Connie G. Watson, HT (ASCP)...letter responses to the following paper document the history of agar useage, HERE
"Cell-rich body fluids and exudates, fragments
of tissue, biopsied specimen, and larger surgical specimens of
tissues are routinely prepared for microscopic examination through
several procedures: fixation, dehydration, clearing, infiltration
with a supportive hardening compound such as paraffin wax, embedment
in the hardening material to form blocks, sectioning on the microtome,
mounting of sections on glass slides, and staining with various
dyes for reading of pathology. All of this comes to a series of
options. At a time in the course of disease, the patient presents
himself to a physician; the physician selects the site that seems
best to him for biopsy (or more major surgery); the pathologist
selects from the surgical specimen portions most likely to yield
meaningful diagnostic information; and the histotechnologist, after
his processing of the specimen secandam artem, selects the
best mircotome sections to mount on microscopic slides for interpretation.
As the pathologist inspects a tissue sample grossly, he may desire
a particular orientation of it within the block.
"Proper orientation is most necessary in cases
of biopsies of skin in which all structures and layers must be
correctly seen, even in common instances of very small samples.
This desideratum has in the past been difficult to ensure for many
reasons. Markings with India ink or other stains applied during
gross examination may wash off during the passage of the samples
through the various processing solutions; landmarks noted during
gross examination may no longer be discernible enough for proper
orientation after mechanical processing because of the action of
the chemicals, which may distort the shape of the specimen; and,
finally, pieces of tissue may shift around within the processing
cassette. Incorrect embedding may then result in tangential sectioning,
which may interfere with reading or cause incorrect critical measurements,
such as the thickness of a malignant melanoma. There is a previously
reported [first by Dr. Lund in 1961] but little-used technique
for handling of specimens with which these problems can be overcome.(1-4)
[see our modern method
agar recipe] "Commercial laboratory-grade dry agar is
prepared as a 3% (w/v) aqueous solution in the same way gourmet
cooks prepare gravy. Dry agar is added to a small amount of
cool tap water sufficient to wet the particles thoroughly.
A sufficiently large amount of tap water is brought to a rapid
boil, and the wetted agar is added bit by bit to the boiling
water while stirring (a magnetic stirring bar may be used).
The stirring is kept up for a short time until the solution
appears to be clarified, to a point when no particulate matter
is visible. The solution is then poured into test tubes for
immediate use or for cooling, solidification, and storage.
Large batches of tubes may be prepared and maintained in a
laboratory refrigerator. Thereafter, as needed sufficient quantities
are reliquefied by placing tubes in boiling water, or in a
microwave oven, or by holding the stoppered tubes in a 60oC
heat block at the cutting station. In the latter case, one
must be certain that the test tubes are filled only to the
height of the heat block wells.
"By pouring directly from a tube or by dropper
pipettes, one can apply an agar puddle to a correctly oriented
specimen, allow the agar to cool and solidify, and then process
this 'preliminary embedded' specimen in the usual way.
Several aims are thus achieved, namely:
1. The pathologist is assured of maintenance
of desired orientation of specimens from the gut, skin, margins
of malignant neoplasms, and thin-walled collapsible cysts.
2. Reduction of "crush artifacts" of
delicate tissues (neoplasms, lymph nodes, liver) from excessive
pressure by forceps or heavy tamping during final embedment in
3. Reduction of material costs and working time
of personnel by embedding of several specimens from a given case
in a single block in a coded arrangement.
4. Aggregation of small pieces of tissue into
a single block (bone marrow fragments, endocervical curettings,
cutaneous curettings, cell block) for easy final embedding with
consequent reduction of risk of "floaters" showing
up on slides of other cases.
5. Suspensions of microorganisms in coded blocks
to serve as controls of stains.
"This preliminary embedding technique works best
if attention is given to a number of easily learned technical factors,
1. Use of a flat, cool work surface such as
a metal plate or the metal edge of a cutting station sink for
arrangement of a specimen.
2. Making the agar puddle block at least 3 mm
thick with well-rounded borders to prevent its "popping
out" from the microtome block.
3. Beveling by scalpel of the edges of the agar
block also helps prevent "popping out."
4. Allowing the agar block, at first clear,
to harden to slight opacity before placing the block into the
processing cassette. This helps to prevent rounding of desirable
flatness of the bottom [down] surface during processing.
5. Seeing to it that agar blocks are not so
thick that they do not dehydrate evenly during processing and
thereby cause an irregular rather than flat bottom surface to
6. Taking care that bubbles do not form during
pouring or pipetting in the agar block because bubbles tend to
retain clearing agents and interfere with the smoothness of sections
7. Blotting, if necessary, of fixative, mucus,
or other sources of moisture before covering with the agar solution.
8. Permitting the agar to solidify completely
because fresh tissue is otherwise more difficult than is fixed
tissue to hold firmly in a block.
9. Remembering that agar blocks resist drying
in room air for about half an hour and therefore batches [of "grossed" cases]
must be collected and placed in processing cassettes at proper
10. Slipping and lifting agar blocks from the
work surface by sliding a scalpel blade under them and sliding
them off the blade into processing cassettes.
11. Remembering that thin specimens standing
on edge tend to flop over when poured agar touches them, and
avoiding the error by placing a very small starter drop at edges
12. Use of slices ["walls"] of solid
agar to retain thin curettings or other fragments difficult to
orient in perpendicular positions while melted solutions of agar
13. Making markers of agar colored black by
India ink [or other colors] that may be sliced and show up well
on finished slides when used as dividers, or shaped to code margins
in tissue blocks.
"The same technique is useful in processing some
types of frozen sections. It is especially useful in processing
frozen sections taken in Mohs surgery in which both proper orientation
and marking of margins are critical. Such use of agar is very helpful
in handling small specimens that ordinarily would be difficult
to place at particular planes for frozen sectioning. Proper orientation
of fresh tissue for freezing in order to do immunohistological
and enzyme studies is also feasible. Agar freezes on dry ice and
thaws without distortion. In cryologic applications, as much excess
agar as possible should be trimmed away so as to avoid thick differentials
of texture between the solidified embedding compound, ice-hard
agar, and frozen tissue.
- Ruby Boldorac, HT(ASCP), Creative Imagination section, "Formalin-Agar for Embedding Small Tissue Specimens, Laboratory Medicine 10(12):766, Dec. 1979, [not found online & text transferred to this website, HERE].
- Lund, H.Z., Baker, L.L., and Anthony, J; "Preliminary Embedding of Small Biopsies in Agar: A Summary of 15 Years Experience With the Technic", Archives of Dermatology 111:1963-1964, 1975 (now JAMA Dermatology beginning Jan. 2013).
- Lund HZ, Forest WW, Harris JR, Brown EH, "Preliminary embedding in agar-agar, Its Value in the Processing of Multiple Separately Identified Specimens, and in the Orientation of Small Biopsy Specimens", Am. J. Clin. Path. 36(6): 562-564, 1961 [does not show on-line except that Google Scholar directs to Technical Bulletin of the Registry of Medical Technologists, 31:192-194, 1961, (HERE) which does not show the text, Nov. 2016].
- Shackelford RI and Jones JL, "An Embedding Medium for Permanet Sections of Exudative Material and Fragments of Tissue Removed for Biopsy", Am. J. Clin. Path. 32(4): 397-398, 1959 [does not show on-line except that Google Scholar directs to Technical Bulletin of the Registry of Medical Technologists, 29:155-156,1959, (HERE) which does not show the text, Nov. 2016].
Write for reprints to: Ervin B. Shaw, M. D. The Lexington County
Hospital, 2720 Sunset Boulevard, West Columbia, South Carolina
29169. [none available as of 25 Nov. 2001, so, I posted it, above, on-line; however, the article can be purchased through that journal's web site as of, at least, 6/29/2013 AND HERE are free on line letters to the editor & responses subsequent to publication.]
- Using an absorbent paper/cloth towel, blot fixative away
from tissues before applying agar to make the agar-tissue button
(a "watery" button is weak)
- Permanent blades that need re-sharpening vs. disposable histology razor knife blades: beveling of edges of agar pre-embedded button is less advantageous (wasted effort) with ultra-sharp blades.
- Once remelted for use, non-fresh agar (in heat
block more than a few days) tends to molecularly degrade (I think)
so that the dehydration phase of tissue/agar processing will
sometimes result in collapse of the agar button into a plastic-like
wafer which is hard to microtome. So, we discard any unused liquid
- Foreign fibers from such as the prostate "blot papers" will
stay attached to the core fragments and interfere with optimal
- there are reports that an agar-gelatin mix performs better in allowing microtomb sections to spread optimally on the waterbath HERE.
agar tips] We've continually used
agar pre-embedding, in large volume, since 1977; and I would
never practice pathology without it!
(posted 9 February 2002; latest update 29 June 2013)