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MISCELLANEOUS STUFF
** Disposal of used diaminobenzidine (DAB) solutions
Question.
How should I dispose of used solutions of 3,3'-diaminobenzidine
(DAB) that have been used for peroxidase histochemistry.
Answer 1.
While DAB itself has not been the subject of in-depth
carcinogenicity studies, it is known to be mutagenic. Further,
all members of the benzidine family that have been tested have
been proved to be carcinogens. In the United States, at least,
all benzidine derivatives are considered carcinogens by the NTP
(National Toxicology Program).
Many people collect the DAB solutions into a bottle containing
5% sodium hypochlorite (which is domestic bleach). After several
hours, the DAB is oxidized to an insoluble polymer.
Chlorine bleach is NOT effective in removing the mutagenic
properties of DAB. While it possibly may break the molecule down
(reaction products are unidentified), introduction of chlorine
into the end products simply produces another mutagenic
chemical. This has been verified by Lunn & Sansone. Using
chlorine bleach is neither chemically sensible nor effective.
Fortunately, most if not all suppliers of DAB have eliminated
this procedure of detoxification from package inserts and
MSDS's.
There are two recommended methods of treatment. The most
commonly used one currently involves potassium permanganate and
sulfuric acid. End products are known to be non-mutagenic. The
second uses horseradish peroxidase to form a solid which is
readily isolated. The fluid remaining is non-mutagenic, but the
precipitate retains its mutagenicity. The only purpose in
performing this method is to reduce the volume of hazardous
waste.
With any commercially available device purporting to detoxify
hazardous chemicals, it is imperative that the user have
documentation from the manufacturer that all reaction products
have been properly tested and found to be non-hazardous. It is
possible that some devices detoxify the liquid and filter out a
hazardous solid. If so, the filter must be handled as a
hazardous waste.
For further information, see:
NTP, 1998. National Toxicology Program Update (January 1998),
Attachment 2. Available on-line at
http://ntp-server.niehs.nih.gov
Lunn & Sansone, 1990. Destruction of hazardous chemicals in the
laboratory. Wiley & Sons (pages 35-41)
Lunn & Sansone, 1991. The safe disposal of diaminobenzidine.
Appl. Occup. Environ. Hyg. 6:49-53.
Dapson & Dapson, 1995. Hazardous materials in the
histopathology laboratory: regulations, risks, handling and
disposal. ANATECH LTD., Battle Creek, MI. (pages 25-27, 109-111
and 162-163)
Richard W. Dapson, Ph.D.
ANATECH LTD.
Battle Creek, MI 49015
(anatech[AT]net-link.net)
Answer 2.
The procedure for acid permanganate oxidation of spent DAB is
as follows. The measurements need not be very accurate.
An acid permanganate solution is made by dissolving
4 g KMnO4 in 100 ml of dilute sulphuric acid (made by
adding 15 ml conc. H2SO4 slowly and carefully to 85
ml of water). This solution is stable. (My experience
is that it's very good at cementing in place the glass
stoppers or screw caps of bottles containing it.)
Add the solution for disposal to an excess of acidified
permanganate and leave overnight (in a fume hood if
the solution contained chloride ions, because these
will end up as chlorine). Next day, neutralize with
sodium hydroxide (carefully; the temperature will
rise) and filter. Leave the filter paper to dry in
the funnel, then put it in a plastic bag for disposal.
If you have a large volume of DAB solution, carefully
add sulphuric acid (150 ml for each litre) and then
dissolve solid potassium permanganate (40 g for each
litre).
Reference: Lunn, G & Sansone, EB (1990). Destruction
of Hazardous Chemicals in the Laboratory. New York:
Wiley Interscience.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)
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** Dilution of concentrated acids: formula etc.
Question.
If I want to make a 1N solution of, for example, hydrochloric
acid how do I convert the liquid, concentrated HCl into a gram
value. The bottle of concentrated HCl says it is a 35-36%
solution.
Answer.
This applies to dilution of all concentrated acids (and also
to strong ammonia (ammonium hydroxide) solutions.
The percentage on the label is weight/weight, not weight/volume,
so you have to take into account the density of the concentrated
acid.
The formula for making one litre of a particular normality, N,
is:
100 X M X N
V = ------------
B X P X D
where V is the volume of concentrated
acid needed, M is is its molecular weight, N is the desired
normality, B is the basicity (1 for most common acids; 2 for
sulphuric; 3 for phosphoric; 1 for ammonia), P is the percentage
by weight in the concentrated acid - the figure on the label,
and D is the density of the conc. acid (specific gravity) in
grams per ml.
No, I didn't work it out myself; it's from Lange's Handbook of
Chemistry.
If the dilution doesn't need to be very precise, you can assume
the following normalities for common concentrated acids:
Hydrochloric (36%) 12N
Nitric (71%) 16N
Sulphuric (96%) 36N (= 18M)
Acetic (99%+) 17.4N
Formic (90%) 23.4N
So to make approximately 0.5N hydrochloric acid, you dilute
the conc. HCl 24 times. To make a litre, you'd measure 42 ml
of the conc. acid (because 1000/24=41.7) and add it to about
800 ml of water. Stir, and make up to a final volume of 1000 ml.
Remember to pour the acid slowly into the water, especially
sulphuric acid, which generates a lot of heat when mixed with
water.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)
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** Disposal of waste from "special stains."
Question.
How should I safely dispose of the waste chemicals
generated in a variety of special staining porcedures?
There is no consensus here, especially about the use of
"copious running water" for dilution. A sample of the
opinions stated in replies to the HistoNet listserver
in the Summer of 1998 follows.
Answer 1.
Identify the substances that are dangerous in quite small
amounts, such as mercuric chloride or sodium cacodylate,
and follow your institution's guidelines for collection
and disposal. Most substances used in special stains (dyes,
acetic acid etc) can be flushed down the sink with plenty
of running water.
John A. Kiernan
London, Canada.
Answer 2.
There are disposal practices that are forbidden for "Industrial"
users that are allowed for "Educational" users.
The last time (some years ago) I took a Hazardous Waste Disposal
course, I found out that Industry has strict regulations on
e.g. Osmium tetroxide disposal, but it was *recommended* that
university labs dump it down the sink. This was allowed,
as long as the Os concentration didn't exceed some specified level
at the sewage treatment plant. Storing the Os for disposal (even
using corn oil and kitty litter) was more likely to result in
legal troubles because of laws on how waste must be stored, for
how long, and whether at a "local" site (your lab) or a central
collection site, etc.
Hazardous waste laws change frequently.
Philip Oshel
(oshel[AT]terracom.net)
Answer 3.
Here is a brief synopsis of advice appropriate for the
USA, and to a great extent, Canada. Further details can
be found in our book, Hazardous Chemicals in the
Histopathology Laboratory, 3rd ed.
First and foremost, never mix different wastes together
unless directed to do so by a licensed waste hauler, or
until you have determined that it is safe and proper to
do so. Why? You could easily create something far more
hazardous. You might be mixing a low-hazard solution
that could go down the drain with a high-hazard solution
that could only be hauled away; that creates a far
larger volume of high-hazard material that you have to
pay to get rid of. A good example would be mixing
mercury waste from B-5 or de-Zenkerization with a
trichrome solution. Remember, too, that alcoholic waste
is burnable and thus less expensive to haul away than
aqueous waste. Don't dilute alcoholic waste with a lot
of aqueous waste, or you will be billed at the aqueous
price.
Second, ALWAYS contact your local wastewater authority
for advice. In many cases, they can assist in
determining disposal procedures, particularly in those
communities with proactive outreach programs. Have
information ready for them: type of waste (flammable,
toxic, etc.), components (don't say Mallory's trichrome,
rather list the ingredients), volume and how often.
Include MSDS's. Every community has its own unique set
of limits for certain chemicals. Chromium, silver and
mercury are stringently regulated, so keep those wastes
separate from others.
Third, use common sense. Stain waste that does not
contain heavy metals, and is of small volume (few
hundred ml) is so insignificant that in most sewer
districts it can be trickled down the drain. NEVER pour
waste down the drain if silver, chromium or mercury is
present. This includes rinses following those solutions
in the staining program.
Do not pour waste down the drain all at once. Trickle it
from a small carboy outfitted with bottom spigot. Never
use "copious amounts of water" to flush waste; it is
against EPA regulations anywhere in the United States.
Finally, use what others are doing as a guide only.
They may or may not have opted for legitimate means of
disposal, and even then, their constraints or lack
thereof almost certainly will not pertain to you unless
you are in the same community.
Richard W. Dapson
ANATECH LTD
Battle Creek, MI 49015
(anatech[AT]net-link.net)
Answer 4.
I have to ask why using copius amounts of water is bad
when disposing of waste. I can understand arguments
about wasting water, but that would preclude putting
solutions down the drain in the first place. So, if
you are allowed to put something down the drain, I
would think the volume would be beneficial for
dilution.
Tim Morken,
Centers for Disease Control
Atlanta, GA 30333, USA
(timcdc[AT]hotmail.com)
Back to Table of Contents
** Magnification of a photomicrograph
Question.
I'm trying to find the calculation used to determine the
magnification of a photomicrograph. I know you have to take into
consideration several things besides the objective.
Can someone help?
Answer 1.
There are a couple of "gotchas" in figuring magnification. You
need the magnification of the objective multiplied by the
magnification of the ocular. However, and here is where you
need to do some double checking, be sure the ocular in the path
to the camera is the magnification you use. On some
microscope/camera combinations, a different magnification is
used for the camera ocular.
Then there is the matter of whether the microscope has a "tube
lens." If the microscope you used is not one of the newest
infinity corrected types, then there is most likely a
magnification lens BETWEEN the objective and the ocular. These
generally fall into the magnification range of 1.5 x, which
again would have to be multiplied with the other two
magnifications. On some microscopes, the tube lens magnification
is marked on a surface betwen the objectives and the oculars,
but on others, theres is no external marking. In that case, you
will need an original manual for the scope. To complicate
matters even further, many camera connect to the microscope
trinocular tube with a reduction tube. So the magnification the
camera sees is the combination of the various lenses used,
divided by the reduction tube. The reduction tubes commonly fall
into the range of 0.25 to 0.75 x. The reduction factor is
generally printed on the outside of the tube that connects the
camera to the microscope.
As a general procedure, for any microscope used to take
photomicrographs, one should take a picture of a stage
micrometer with each objective on the scope, and keep these
pictures in a "calibration" file for that camera/microscope
combination. The stage micrometer will be a true "ruler" with
divisions of 0.1 and 0.01 mm, so it is easy to check the true
magnification of prints or slides. If you don't have a stage
micrometer, then use the built in standard: the average diameter
of red blood cells after most processing procedures is
approximately 7 microns. That is not exact, but is a good way to
check that your magnification calculations are in the right
ballpark
Alton D. Floyd, Ph.D.
ImagePath Systems,
(al.floyd[AT]juno.com)
Answer 2.
The best way is to photograph a calibrated slide using the same
objective and other variable things as for the section. Print
the photos at the same enlargement, and measure with a ruler.
If a 100 micrometre distance is 32 mm on the print, the
magnification is 32000/100 = 320.
Calculations based on the optics commonly lead to ridiculous
mistakes. As a rough check, measure something in the photo and
see if it's a sensible size. If there are cell nuclei 50
micrometres across, somebody has made an arithmetic error.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)
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** Can a method be both published and patented?
Questions.
The tyramide amplification system (for showing peroxidase
activity at sites of antibody binding or in situ nucleic
acid hybridization) is sold commercially in patented kits.
The principal reagent (tyramine coupled to biotin or
various fluorescent compounds) can be synthesized in the
laboratory, following quite simple techniques published
in the Journal of Histochemistry and Cytochemistry, and
elsewhere. Is there a risk of being sued by the firm that
sells the kits, for following a published method to make
a reagent in one's own lab?
Answer.
[ There was some rather heated discussion on the HistoNet
listserver in August 1998, involving various individuals
and one of the patent holders. It centered around the
unavailability of individual reagents and a claim that
a company might even sue individuals for daring to
encourage others to carry out the published syntheses. ]
Linda Margraf relayed this to HistoNet. It was from Mark
Bobrow. He is an author of some of the published procedures
and also one of patent holders.
[ Beginning of M. Bobrow's message ]
The patent system goes back over five hundred years when, in
Britain, one could obtain a patent granted by the King. In the
U.S., the first patent commission was headed by George
Washington, who personally signed every patent granted during
his tenure.
A patent is a right granted by the government. Article I,
Section 8 of the United States Constitution states, "The
Congress shall have the power to promote the progress of science
and useful arts, by securing for limited times to authors and
inventors the exclusive right to their respective writings and
discoveries."
It is often misunderstood that the purpose of the patent system
is, as stated in the Constitution, *to promote the progress of
science and useful arts.* The concept is that by disclosing (and
not keeping a secret) an invention, technological innovation
will continue. In the process of obtaining a patent, the
inventor must disclose the invention and the best mode of
practising it (in other words, they can't hold anything back, or
the patent will not be valid).
In return for disclosing the invention, the government grants
the patent holder the right to exclude others from making,
using, or selling the invention. Currently, these rights extend
for 20 years from the filing date. After the term expires,
everyone is free to make, use or sell the product or method
which was disclosed in the patent
The right to exclude others from practising the invention
applies to everyone, including academic investigators. In terms
of being able to use what is in the published literature, U.S.
patents are published after they issue; in Europe the
applications are published 18 months after filing. So, even
though patented products and methods are in the published
literature, using them without proper authorization from the
patent holder is not legal.
There have been some questions as to the extent of coverage of
the tyramide amplification patents. In the spirit of
simplification, four basic concepts are claimed. They are the
enzyme substrates (e.g., tyramides), the product of the
enzyme-substrate reaction, the method of catalyzed reporter
deposition (e.g., detecting an analyte with a reporter enzyme
using the deposition of a reporter), and assays using the method
of catalyzed reporter deposition. If you wish, you may look it
up yourselves. One of the patents is U.S. Patent 5,731,158,
Catalyzed Reporter Deposition. As an added note, the readers
should be reminded that patents are written in a style that is a
hybrid of law and science (perhaps a suspension is more
descriptive).
Patent information is available on the internet. Here is a list
of some sites:
http://www.uspto.gov/ ; This is the US Patent Office site. You
can search for patents here, and get some information about
patents in general. Later this year, or early next year, the
full text and images of patents will be available.
http://patent.womplex.ibm.com/searchhelp.html ; This is an IBM
site where one can search for patents and view the entire
document (it tends to be slow though).
http://www-sul.stanford.edu/depts/swain/patent/patgeninf.html
General patent information.
[ End of reported communication from M. Bobrow ]
Back to Table of Contents
** Books and articles about artifacts in histology
Question.
Can you recommend any books or articles that illustrate and
explain artifacts encountered in sections stained for light
microscopy?
Answers.
"An Atlas of Artifacts Encountered in the Preparation of
Microscopic Tissue Sections" by Samuel Wesley Thompson and
Lee G. Luna. Publisher: Charles C Thomas, Springfield,
Illinois, U.S.A. (1978).
There is also a wonderful section on Artifacts (and photographs)
in "Histopathologic Methods and Color Atlas of Special Stains
and Tissue Artifacts" by Lee G. Luna, 1992, printed by Johnson
Printers, Downers Grove, IL.
Marilyn S. Gamble
(Marilyn.S.Gamble[AT]kp.ORG)
I agree with the value of Lee Luna's book "Histopathological
Methods and Color Atlas of special stains and tissue artifacts,"
especially the value of the colour photomicrographs.
The most comprehensive paper I have seen is: Wallington EA.
"Artifacts in tissue sections" Medical Laboratory Science.
1979;36:1-61 (that's right, sixty one pages!) It is the paper
which won the Memorial Prize of our institute - Institute of
Biomedical Science. In those days, unfortunately, published
photos were in B&W only, but there is plenty of text and
explanation. Eric was a real gent, a master of histological
technique and perhaps the greatest authority on artifacts.
Please don't ask me to send a photocopy!
Russ Allison, Wales
(Allison[AT]cardiff.ac.uk)
The web site of Roy Ellis has many informative images of
artifacts, with quizzes and explanations. Highly recommended!
http://home.primus.com.au/royellis
John Kiernan, London, Canada
(kiernan[AT]uwo.ca)
Back to Table of Contents
** How dangerous is picric acid?
Question.
Older colleagues tell of picric acid exploding with great
violence, but always in other labs. Is there really a risk
of explosion?
Answer.
From the late 19th Century until the First World War, picric
acid was used as a high explosive in military shells. Its
melting point (122C) is quite well separated from its
exploding temperature (above 300C). Picric acid can be
ignited by a nearby spark at temperatures above its
flash point of 150C. More sensitive explosives can be formed
by chemical reaction of picric acid with other substances.
An example is ammonium picrate (which has been used in
histology to fix vital stainings with methylene blue).
In 1915 a French freighter, the Mont Blanc, full of expired
explosives, caught fire in the harbour of Halifax, Nova Scotia.
The largest man-made, non-nuclear explosion followed, and it's
customary to blame it on picric acid, which probably accounted
for much of the cargo.
When you buy a bottle of picric acid for the lab, the yellow
powder is mixed with 10% to 40% of its weight of water (varies
with the supplier), so it is impossible for the temperature to
go above 100C, let alone the 300C required for an explosion.
If a jar of picric acid were to dry out, as a result of neglect,
it's conceivable that a high temperature might develop from
friction when unscrewing a tight bottle cap, but 300C seems
highly improbable. Nevertheless, it's usual to loosen a tight
cap by standing the jar upside down in water for a few minutes
before applying force to it. Percussion can cause a locally
high temperature, so you shouldn't hit dry picric acid with
a hammer. One of its uses is in matches. Stories of picric acid
explosions in labs are like sitings of ghosts: always second-
or third-hand.
Various toxic effects are described, especially skin reactions.
Oral LD50 values range from 60 to 250 mg/kg depending on the
animal. (This puts it in the same league as ferrous sulphate.)
Sources: Various chemistry textbooks; Merck Index; Lange's
Handbook of Chemistry; MSDS sheet.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
(kiernan[AT]uwo.ca)
Back to Table of Contents
** Which color print film for photomicrography?
Question.
What brand of color 35mm film and ASA (film speed) is best
suited for photographing H & E sections? I would like to
produce prints, not projection slides.
Answer.
Fuji or Kodak, use the slowest speed, lowest ASA you can. ASA 25
is good, 100 will produce good results.
If there is much vibration where your camera is, you may need to
go to a faster film to shorten your exposure times.
Use professional film, not consumer. The difference is that pro
film is refrigerated after it's made, so there is no color shift
with aging. Keep used film in your lab refrigerator for this
reason.
You don't have to worry much about daylight vs tungsten film
because you're shooting negatives and not transparencies. If
your photomicroscopy set up controls color temperature, then try
to shoot at 5500K (5500 deg), because color film likes sunlight.
Use neutral density filters to lower light levels if needed.
Also: who's doing your printing? A film lab or someone used to
histo shots? If it's a film lab, then they won't know how to
balance the color of your sections, and you're likely to get
weird results. If your camera back comes off the scope, take the
first one or two shots of a Caucasian person outdoors, sun
behind the camera. The automated developing and printing
machines are set to correctly balance Caucasian skin tones, and
should keep this setting for the rest of the roll. If your
camera cannot come off of the scope, then when you send your
film to be printed, include an image of an H & E section with
correct color balance. This will give the photo lab a reference
to use for balancing the colors of your film when printing.
Middleton, WI 53562
(oshel[AT]terracom.net)
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[ End of FAQ document ]
Return to John Kiernan's home page
Link to the Biological Stain Commission
----------------------------
Last updated: January 2007