Miscellaneous Stuff

** Disposal of used diaminobenzidine (DAB) solutions


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


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


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


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.


Battle Creek, MI 49015


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


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


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** Dilution of concentrated acids: formula etc.


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%



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


The formula for making one litre of a particular normality, N,


100 X M X N

V = ————


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


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


John A. Kiernan,

Department of Anatomy & Cell Biology,

The University of Western Ontario,

LONDON, Canada N6A 5C1


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** Disposal of waste from “special stains.”


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


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


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


Battle Creek, MI 49015


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


Tim Morken,

Centers for Disease Control

Atlanta, GA 30333, USA


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** Magnification of a photomicrograph


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


Alton D. Floyd, Ph.D.

ImagePath Systems,


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


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** Can a method be both published and patented?


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?


[ 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


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


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).


General patent information.

[ End of reported communication from M. Bobrow ]

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** Books and articles about artifacts in histology


Can you recommend any books or articles that illustrate and

explain artifacts encountered in sections stained for light



“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


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


The web site of Roy Ellis has many informative images of

artifacts, with quizzes and explanations. Highly recommended!


John Kiernan, London, Canada


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** How dangerous is picric acid?


Older colleagues tell of picric acid exploding with great

violence, but always in other labs. Is there really a risk

of explosion?


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


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** Which color print film for photomicrography?


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.


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


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