mycological chemistry and visionary experiences related to psilocy-

bin mushroom use. In PM & E vol. 1 we were presented with opti-

mum harvesting/storage techniques. A study of the bluing reaction

with ways to inhibit its onset was presented. In PM & E vol. 2 the

relationships between mushroom pretreatment agents and various

forms of dehydration were presented, with emphasis on optimum

psychoactivity. In PM & E vol. 4 instructions are given for con-

structing a vacuum dehydration system. HPTLC (high performance

thin layer chromatography) comparisons were noted upon samples.

We continue this series with an overview of psilocybin potency test-

ing - both in the laboratory and through implicit meditation and phys-

iological/psychological observation. PM & E is exceptionally proud

to bring this installment of The Mushroom Entheogen to our readers.

So why do you need to test your psychedelic mushrooms for

their potency?

There are two good reasons: either to see the affect of some

experimental procedure on the final concentration of the active

tryptamines(i.e. psilocybin and psilocin) in the mushrooms

(which is pretty much what these articles are about) or more

important, to know the subjective intensity of the dosage you

may plan to take. You may remember the anecdote from the

previous article about my friend who mistakenly took too

much mushroom powder. He came very close to needing

some medical help, because he thought he was losing his mind.

Neither of us had any idea that we had made a measurement

error with our dose of mushrooms and he had taken twice the

amount of what would have been a large dose. I ended with

about half of a large dose. I was fine but he panicked and the

knowledge that I had taken what I thought was the same dose

made it even worse, because as far as I was concerned he was

being totally irrational.

This extreme example of overdose is more likely to be the

rarity. What is more common and frustrating is "under" dosing.

If you are like me, a mushroom trip is a special event for which

I need to plan the time. With family and job responsibilities I

can no longer take a day off on the weekend anytime I feel like

it. Too many times I have planned for a day of tripping only

to end up with a mild buzz and a loaded feeling, not that

altered state of awareness and consciousness which is charac-

teristic of the full mushroom trip. I needed a mushroom testing

procedure. Knowing what the active tryptamine concentration

is before taking the mushrooms can prevent the possible prob-

lem of over or under dosing.

One aim of my research, besides reducing the toxicity of the

mushrooms, is to maximize the psilocybin content of the culti-

vated mushrooms and to stabilize the quantity biosynthesized

from flush to flush of a particular strain of P. cubensis by con-

trolling environmental and nutritional factors. In my own

research I found that as I experimentally changed these

growth-affecting factors, my particular strain's concentration,

as measured by the test procedure described at the end of this

article, increased by a factor of four or five.

In their research, Bigwood and Beung echo this same varia-

tion in the concentration of psilocybin in the controlled cultiva-

tion of P. cubensis. But because of their large variation in

what they felt was a rigidly controlled growth environment, I

am inclined to conclude that they were not controlling all the

possible factors which control the growth and biosynthesis of

psilocybin. They found that in their own cultivation, concen-

trations varied by a factor of four and, even worse, specimens

from other sources varied as much as ten fold.(4)

An upcoming article, using the results of the mushroom

sample testing will show how, by careful control of the mush-

room nutritional and environmental growth factors, one can

minimize this large flush-to-flush tryptamine(the major molec-

ular grouping in psilocybin/psilocin and other related com-

pounds) concentration variation.

Because of even less environmental and nutritional control,

this sample-to-sample variation is further exacerbated if you

collect samples from the wild. Besides strain differences(i.e.

genetic differences), microenvironmental and growth substrate

nutritional differences contribute to large variations between

specimens, even collected close together. Christiansen, et al

found from their studies of the psilocybin concentration of

many different samples of P. semilanceata in Norway, that the

content varied by a factor slightly greater than ten.(7)

If ten-fold variations exist between mushrooms of the same

species, imagine the potential for variation between different

psychedelic genera. Mushrooms which contain the hallucino-

genic tryptamines include the genera Concybe, Panaeolus,

Psilocybe, and Stropharia. (12) If you are collecting any of

these varieties for psychedelic purposes, you may wish to con-

side a test of their relative strength before taking them. If you

plan to take the mushrooms fresh, then with a little experience

with one of the field tests described later you will be able to

estimate their relative concentration. You can tell not only

from the final intensity of color of the reaction but also from

the speed with which the sample develops a color.

A final point on the need for a test: if you happen to be

someone who buys psychedelic mushrooms, you may want to

know just what you are getting for your money. Ideally, if it

were legal to sell, a mushroom dealer should be aware of the

relative strength of his different batches of mushrooms and

should sell the dried mushrooms not by weight but by what is

necessary for a moderate-dose trip.

Subjective and objective testing.

"Okay," you say, "So maybe it would be helpful to be able

to test the mushrooms I buy or grow, but I am not a chemist

and I want something simple.

There are two basic types of testing: subjective and objective.

Subjective testing of mushrooms is descriptive. It is easy and

cheap and requires only attention to one's own mind, but it

does take time.

Objective testing, on the other hand, is quantitative. It is sim-

pie, usually quick, repeatable, but can in some procedures,

require complex and expensive equipment. Although I have

identified two forms of testing, we need both to know the psy-

chedelic effectiveness of an unknown batch of mushrooms or

to communicate what our batch of mushrooms will be like to

someone else.

The problem with subjective testing is standardizing the

method. Because of the vagaries of the mind, one needs to

control the set and setting under which one performs his sub-

jective testing. By controlling these two factors, although very

difficult at that, one can establish a common reaction(e.g.

degree of energy, quantity of hallucinations, their colors and

shapes, the ease of feeling at-one with external objects or con-

cepts, etc.) to a standardized dose. This reaction can be the

gauge which one uses to compare all other subjective tests.

This subjective response to a standardized dose will help one

to know how much to take later.

The problem with objective testing is that no matter what

value of concentration(usually expressed in mg of psilocybin/

gr. of mushroom) one finally arrives at from his test, it does not

tell him what the subjective experience will be like. To know

what the subjective experience is, one still needs to take the

psychedelic. By doing this a few times for various concentra-

tion levels, one can extrapolate a subjective intensity value

from an unfamiliar objective test value, thus dispensing with

the need for subjective testing.

The advantage of having an objective test concentration

value is that it can communicate what the personal experience

will be like to anyone who has taken the time to compare sev-

eral batches of mushrooms subjectively after finding an objec-

tive test concentration value for each batch. By comparing

their experiences with a few corresponding numerical values,

one can infer from a new objective test value the intensity of

the personal experience when taking an unknown batch of

mushrooms.

The subjective effects may vary considerably from one indi-

vidual to another but it is the intensity and duration which will

change linearly with the increase in the objective test value.

The subjective tests need to be done only a few times (and

recorded for future comparisons) when comparing different

concentration levels of mushroom strengths, after which you

can rely solely on the objective test for evaluating new samples

of mushrooms. Whereas, if one opts to use the subjective test

only, then one will need to test each batch by actually taking a

small, standardized dose. Then from this tedious evaluation

one can determine the amount needed for whatever level of

tripping one wishes to reach later.

Besides the problem of taking a sample dose for each and

every batch of mushrooms, the subjective test has another dif-

ficulty when used alone. Its results can be difficult to cornmu-

nicate to someone else because the phenomenal experience

may vary radically from one individual to another. For

instance, someone may describe a reaction to a five gram

mushroom trip as giving him a feeling of strong sexual energy

with a keen awareness of his physical self.

Whereas, you may take the very same mushroom powder

and even expect and perhaps look forward to a similar reac-

tion, only to experience a very introspective trip in which the

last thing on your mind is sex. The objective test value allows

a means of communicating the intensity of the mushroom trip

without describing the experience it evoked.

By itself subjective testing has its problems, but when sup-

plemented with objective, quantitative testing, it can become a

predictive tool for us to use. And also, by itself, objective test-

ing conveys no real meaning to us about the subjective nature

of the trip. For the reason that the objective test tells us no per-

sonally valid information, one can conclude that the foundation

for any quantitative test is our own subjective testing.

I have used the following procedures and guidelines in my

own subjective testing. Use your own guidelines, but the pri-

mary rule is to watch the effects of the psychedelic on your

body and mind. It helps to know your mind well before evalu-

ating the changes caused by a psychedelic. The best way to

understand your own mind is to regularly practice some form

of mental meditation techniques in which the emphasis is on

alert consciousness in an ever-increasingly calm mind.

The Subjective Test.

1. One can argue about the effects of set and setting on the

psychedelic experience, but no matter the outcome of the argu-

ment one generally does have more physical effects, greater

duration and depth of the trip with ever-increasing doses of

psychedelics. Observe these differences in the trip's length and

depth for different amounts of mushroom, preferably from the

same mixture of mushroom powder (all properly stored so that

the interval between tests does not degrade the quality of the

mushroom).

2. Observe your mind with little or no other sensory inputs

during the trip. The best way to do this is to close your eyes or

be in complete darkness, plug your ears or be in an absolutely

quiet environment and lie or sit completely still. After sitting

or lying like this for a few minutes, notice the intensity of the

colors in the mind's eye or projected in the dark. Observe the

sharpness of the edges and forms. Is the nature of the forms

benign or malevolent? Do you experience dreamy hallucina-

tions or patterns? Is your mind clear or dreamy or sleepy?

3. Observe the nature of your perceptions in an eyes-open

mode in a well-lighted environment. Notice the rippling effect

around objects. Do you have colored patterns or hallucinations

projected on the environment. This is sure indicator that you

have taken a strong dose.

4. Listen to sounds or music and feel their effect on your

emotions and observe how they change the hallucinations or

patterns.

5. Observe the quality of your consciousness. Are you

cloudy, sleepy, moody, willful, clear, alert? Note all physical

side effects, such as nausea, headaches, muscle aches, stomach

cramping, aching joints and other uncomfortable symptoms.

Many aches and pains can be a result of psychosomatic mani-

festations during a psychedelic trip. But many times impuri-

ties in the mushrooms can initiate the side effects, too. All the

above are indications of the quality of the trip and conse-

quently the mushroom quality. Generally, as I perfected my

growing and storage techniques, the above physical symptoms

diminished.

6. Watch how you respond physically. How is your coordi-

nation, such as your ability to walk and talk?

7. I have noticed that left brain functions--those usually

associated with concrete, analytical thought processes--become

harder to perform with increasing doses of psychedelics. Can

you perform simple math tasks? Do you have trouble express-

ing ideas in speech? Can you give directions to a familiar

location to someone?

8. How fast does the trip come on? How long is the "rush?"

How long before you start to come down from the psychedelic

portion of the trip? I find that the drug effects last no longer

than six to eight hours, but as I increase the dosage or strength

of the mushrooms, the trip comes on faster, the rush lasts

somewhat longer and the psychedelic portion gradually

increases from as little as thirty minutes to as much as five or

six hours. By reassessing your trip as it progresses using

some of the evaluation criteria as described in the above

points, you can observe the course of the trip accurately and

predict its length and intensity.

9. When establishing a subjective baseline for your trips, it

is important to standardize the set and setting of your trip so

that you minimize the results of such variables on the trip. Try

to take it in the same type of environment, preferably at the

same time of day. Do not take an evaluative trip if you are in a

negative mood. My friend recommends jogging, or other aero-

bic exercises, to help elevate and stabilize one's moods.

It is best to take the mushrooms on an empty stomach during

an evaluation because different foods will affect the nature of

the trip. Also, having food in the stomach will slow the

absorption of the psilocybin and give the impression that you

had less. If you are correlating objective test concentration

information with subjective intensities, make sure you always

use the same amount of mushroom. I found that one gram was

enough to test. It usually was not enough to put me on a full-

blown trip, but was plenty to observe all the psychedelic mani-

festations, particularly if I closed my eyes and plugged my ears

and practiced meditation techniques which I know.

Having evaluated your mushroom samples subjectively, you

are well on your way to being able to plan for an entheogenic,

or ecstatic trip because you know how much to take for the

sought-after experience. The emphasis of these articles is pur-

posely limited to the use of the mushrooms for the more intro-

verted and spiritually expanding psychedelic experience. True,

many of the preparation, growing techniques and even some of

the suggestions for directing the energy of the experience(a

later article) can apply to trips which focus on interpersonal

relationships or even for those who just want to have fun for an

afternoon, but you will not need to work as hard for these non-

entheogenic experiences. My background with psychedelics,

and primarily with the "magic mushrooms," has shown me that

the highest quality mushroom experience and states of con-

sciousness come with effort and planning between trips and a

tremendous burst of yearning during the actual trip.

Apparently a change in consciousness takes effort and time.

The more intense the concentration of effort and desire for

such a change, the faster is the change in consciousness.

The next section may be superfluous to your needs if you

are not drawn to such objective evaluation of your mushrooms.

But even if you do not elect to do any chemical testing of your

mushrooms the discussion might help you to understand the

meaning of the values which I will describe in upcoming arti-

cles on environmental and nutritional influences on the growth

and psychedelic tryptamine production in P. cubensis. So I

encourage you to read at least the general theory and interpre-

tation of the test's results. The actual test procedure is at the

end of the article for those who wish to do their own chemical

evaluations.

OBJECTIVE TESTING.

General theory and various detection and measuring

schemes.

Objective test results can give a numerical value which tells

how much, and for some tests, what is in a mushroom. One

can find a relative concentration value or an absolute concen-

tration value. If one has access to the pure psychedelic trypta-

mines then one can derive the absolute concentration by

measuring an accurately weighed amount of the pure sample

and then comparing that test result with the value obtained for

the unknown sample. If one does not have a pure sample it

does not matter because the numerical test results will indicate

that one sample has more of the measured tryptamine than

another and to what degree it has a greater concentration.

These numerical or objective test results greatly help commu-

nicate the relative strength of different batches of mushrooms

to any one else who may have a different subjective interpreta-

tion than yourself.

Each individual chemical behaves differently from all other

chemicals because of its unique structure. Based on this

uniqueness of each chemical, objective tests are possible. One

type of objective testing relies on the unique absorption of spe-

cific wavelengths of light absorbed by each unique chemical.

In other words, each chemical has a different color, although

the "color" is usually not in the visible spectrum. Another

aspect of each chemical which is often used in designing a test

procedure is that it will interact with or react with other chemi-

cals completely differently.

See Figure I.

Tests can be developed which are also based on the similar-

ity of various compounds with the understanding that a related

portion of the molecule will react similarly. For instance, there

are several active molecular components in the entheogenic

mushrooms but the most important component includes the

general family of molecules, called tryptamines. All these tryp-

tamines have in common the indole ring in their molecular

makeup. Tests have been developed which show whether this

indole ring (as part of the larger tryptamine molecule) is

present in a solution and to what degree.

The several different types of tests which are available to the

scientist include spectrophotometry, colorimetry and chroma-

tography . Spectrophotometry measures the degree with which

the molecule under investigation absorbs light at specific wave

lengths. But because most organic molecules absorb best in

the infrared or ultraviolet spectrum and these instruments are

expensive for the average hobbyist, I have not pursued these

techniques.

Chromatography is a technique which separates mixtures of

compounds by passing the mixture while in solution through a

specially prepared media(the "sorbent") of highly refined sand,

called, "silica gel." Silica gel is the most common sorbent

used, but other less common sorbents can be used. The mix-

ture of various molecules interact differently with the mole-

cules on the surface of the silica gel as they pass and thus slow

their movement to a greater or lesser degree.

After migrating through the silica gel for a distance, the mix-

ture of compounds segregate into separate bands of pure com-

pounds. Chromatography generally falls in two modes,

depending on the apparatus used with the silica gel to separate

the sample--thin layer chromatography(TLC) and column

chromatography of which high pressure liquid chromatogra-

phy(HPLC) is another technique subgroup commonly used in

the lab.

As the name indicates, in TLC a thin layer of silica gel is

applied to a glass or plastic plate then the sample is streaked

or spotted near the bottom of the plate. The plate is then put

into a glass tank in which a small amount of a particular sol-

vent mixture, determined by experimentation, has been poured.

The silica gel, being porous, allows capillary action to draw up

the solvent which pulls the sample, too. As the sample moves

each pure chemical in the mixture moves at a different rate

thus causing a separation into bands of the pure component

molecules of the sample mixture. Different techniques are

available to make each pure chemical band visible.

See Figure 2.

For a given sorbent and solvent a particular compound(e.g.

the psychedelic tryptamines) will always move up the TLC

plate the same relative distance when compared to the distance

that the solvent was allowed to creep up the plate. For exam-

pie on one occasion a researcher let the solvent develop on the

TLC plate for one hour and the solvent moved up the plate 10

cm. After using either a chemical dye to detect the chromate-

graphed spots or a UV lamp, he found that the spot he was

most interested in moved 5 cm up the plate or half way

between the starting point and the solvent front. On another

occasion he only let the plate in for 45 minutes and the solvent

moved only 8 cm. Because he knows the compound has an

Rf(an abbreviation referring to relative migration distance up a

TLC plate of a pure compound) of 0.5, then under identical

conditions for the same chemical, the spot of interest will

move half way up the plate or 4 cm. And so it does in practice.

The experimental literature will usually have these relative

distance values for most compounds, which are always less

than 1.0 (An Rf of 1.0 would indicate that the compound

moved with the solvent all the way up the plate; therefore its

relative distance when compared to the distance that the sol-

vent moved is 1.0.) After a TLC separation it is easy to see if a

particular compound is present by looking for a band which

occurs at the correct distance up the plate. The intensity of the

color of the band will indicate the concentration of the com-

pound, or one can actually scrape the band off the plate and

measure the nearly pure compound by some of the other tech-

niques available.

HPLC(or High Pressure Liquid Chromatography) is a form

of column chromatography. The sample is applied at the top

of high pressure capable column with sorbent in it, then the

solvent is pumped through the column at high pressures (usu-

ally 1000 psi or greater). At the other end of the column a

spectrophotometer monitors the solvent for an absorbance

which indicates an organic compound is coming off the col-

umn. The experimenter can view the output from the spectre-

photometer via a graph output or a video screen.

The area under the peaks which represent each different

molecule are proportional to the concentration. With samples

of the pure tryptamines, one can calculate the absolute concen-

tration of the compounds investigated. An HPLC solvent is

pumped through the column until most of the sample has been

washed off. Instead of distance traveled through the silica gel

as in TLC, the time it took to wash the compound off the col-

umn before it was detected by the spectrophotometer is used to

determine what the molecule is.

If you read the technical literature you will see HPLC men-

tioned often. Its advantage is far greater sensitivity and ability

to resolve many more compounds which may be present in an

unknown sample. Its disadvantage is cost. The average HPLC

set up may cost $10,000. Whereas one can buy pretty much all

he needs to perform TLC for about $100 to $200.

Which brings us to the last general detection technique and

the one of choice for most of my research--colorimetry.

Colorimetry is similar to spectrophotometry in that a solution

of the sample absorbs specific wavelengths of light and the

wavelength and the degree of absorbance can tell much about

the sample. But in colorimetry the light absorbed and the con-

sequent color of the solution measured falls within the visible

spectrum. Also, because the absorption peaks cover a much

broader range of wavelengths than in the ultraviolet (UV) or

infrared (IR) regions, the spectrophotometer used can be much

less sensitive and can use coarser methods of breaking up the

light spectrum to irradiate the sample. The instrument used for

colorimetry is called not a "spectrophotometer," but a "color-

imeter" and can use filters rather than the much more expen-

sive diffraction grating monochrometer used in

spectrophotometers.

The trick with colorimetry is making the sample molecule

which normally does not absorb in the visible spectrum(i.e.

400 to 700 nanometers, which is the wavelength of light from

the deepest reds to the faintest violets) visibly colored.

Chemists have found that there are molecules which by them-

selves are uncolored but when combined with certain other

molecules will form a color. These chemicals are called "chro-

mophores" and the presence of this color indicates that the

molecule under test exists in the solution and the intensity of

the color tells one how much of the compound is in the

solution.

The problem with colorimetry is that most chromophores do

not combine specifically with a unique molecule but with a

portion of the molecule under test. For example, in the test

which I used in my research, the chromophore reacts with the

indole ring of the psychedelic tryptamines to form a blue or

purplish color. Indole rings are not specific to psychedelic

tryptamines. There are many molecules other than the psyche-

delic tryptamines which have indole rings.

And to further complicate things, the chromophore will react

with other nitrogen containing centers, although without the

usual blue or violet color. The net result can be a hodgepodge

of color which overlaps to a greater or lesser degree with the

specific wavelength, or color, which the colorimeter is view-

ing. The colorimeter is dumb; it does not know the difference

between an absorbance at 570 nanometers which is caused by

urea or psilocybin or a little of both. Generally, the non-active

compounds have absorbances far enough away in the light

spectrum so that they do not interfere with the psychedelic

tryptamine readings, but this is not always the case. I will dis-

cuss this interference and how it relates to the interpretation of

the test's results in a later section.

Spectrophotometric theory tells us that the measured absor-

bance of a compound is directly related to its concentration in

solution. In other words as absorbance increases so does the

concentration. If we test one mushroom for active tryptamines

and find that it has an absorbance of 0.600 and then test

another and find that it has an absorbance of 0.900, we can say

that the latter one has a greater concentration of tryptamines

than the first. Sure, we do not know the absolute concentration

in mg/gram of the psilocybin/psilocin, but who cares? We can

as easily relate to 0.600 A as we could relate to 2 mg/gram in

our own subjective experience.

What is in a mushroom? What are we measuring?

Because of the potential for ambiguity in the test results

through the chromophore's multiple color reactions, it may be

appropriate to review the literature to see what constituents of

P. cubensis others have found in their research. Some of these

other natural compounds will react with the test and add to the

test value even though they are not active tryptamines. And

others are active tryptamines which because of their somewhat

different psycho-physiological activity can modify one's trip

significantly for better or worse. We need to know what these

are, too.

As far as active tryptamines in the mushroom, the two with

the greatest concentration in P. cubensis are, of course, psiloc-

ybin and psilocin. (13 p.109) The "tryptamine derivatives" are

called such because of their similarity to serotonin. This class

has an INDOLE group and a DIMETHYLAMINE group. The

tryptamine derivatives include the brain transmitter substance,

serotonin, the essential amino acid, tryptophan, the fast acting

but short lasting psychedelic, DMT, and of course psilocin and

psilocybin. "Active tryptamines" refers to the various psyche-

delic tryptamines, including psilocybin, psilocin and all their

analogs. See Figure 1 for examples of the various tryptamines.

Repke points out that any psilocin detected in mushroom

samples may in fact be an artifact caused by hydrolytic cleav-

age of the phosphate group off the psilocybin molecule in the

handling and sample preparation.(l4) In fact, other analogs

can be easily formed by the various enzyme systems and the

presence of oxygen. I tried to make this point in the first article

when I emphasized the importance of low temperature, vac-

uum drying when preparing the mushrooms for storage or the

care needed in preparing the mushrooms for ingestion, for it is

these analogs and breakdown products which are most likely

the cause of the headaches, mental cloudiness and achiness

which are not normal side effects of synthetic psilocybin.

Most literature references which I read noted that little psilo-

cin was present in mushrooms. They may not have been able

to find any psilocin with the psilocybin, because it is an artifact

or perhaps because of the ease with which psilocin is oxidized.

In the work of Bigwood and Beug, however, they found that

after the second flush the psilocin levels are significantly high.

In fact, they range from about ten to thirty percent of the total

active tryptamine concentration(concentration of psilocybin

and psilocin in this paper).(4)

Apparently, baeocystin, which is another psychedelic analog

to psilocybin and psilocin, plays a major role in the natural bio-

synthesis of the psilocin and psilocybin in the mushroom and it

is present in small but significant levels in P. cubensis. Based

on the various samples tested in the cited literature, the range

extends from 0.001% to 0.02% baeocystin of the mushroom's

dry weight. Repke and the others found that baeocystin was

never found in mushrooms which did not already have psilocy-

bin present also. To put this in perspective, psilocybin usually

makes up about one percent of the dry mushroom weight.

(Baeocystin forms a pink to purple to blue color reaction in the

presence of Ehrlich's reagent, which is similar to the test rea-

gent which I describe at the end of this article.) (14)

The researchers, Beung and Bigwood found through their

TLC work that they could isolate 12 distinct spots(i.e. different

compounds) on the silica gel plate. Besides psilocybin, psilocin

and baeocystin, their tentative conclusion is that the other spots

represent N-methyl and tryptamine analogs of psilocin and

psilocybin.(3)

Another tryptamine found in mushrooms is tryptophan.(9)

This will react with the test reagents with a similar color as

psilocin and will consequently add a small amount of absor-

bance to any test result, giving a slightly false high reading.

The test which I used to quantify the amount of psilocybin

and psilocin in the P. cubensis mushrooms reacts with other

nitrogen containing compounds, although it is most sensitive to

indole containing compounds when read at the prescribed

wavelength.(l0) The common amino acid, glycine, is one such

nitrogen containing compound found in abundance in the

mushroom.(l8) Another nitrogen based compound which has

been found in the mushroom is urea. The yellow color change

of the test indicates the presence of one of these ubiquitous

compounds. Luckily, yellow adds only a small amount of

absorbance to the value of the active tryptamines when read at

570 nm, the test wavelength.(3)

Casale, in reviewing the literature, mentions that besides the

compounds already mentioned, ergosterol, ergosteral peroxide

and a,a-trehalose have also been found in the methanol extracts

of the Psilocybe mushrooms.(5)

I could find no other mention in the literature about other

tryptamine analogs, toxins, enzymes or hormones which may

be present in P. cubensis and thus affect one's subjective expe-

rience. Agurell, Blomkvist and Catalfomo (1) identified a

lengthy list of possible tryptophan metabolites which might

show up in the psilocybe mushrooms: 6hydroxytryptophan;

kynurenine; tryptophan; kynurenic acid; xanthurenic acid; psil-

ocin; tryptamine; methyltryptamine; dimethyltryptamine; 3-

hydroxyanthranilic acid; anthranilic acid; N-acetyltryptophan;

and indoleacetic acid. Agurell and Nilsson (2) demonstrated in

their paper a tentative biosynthetic route for psilocybin for

which any of the intermediates could exist in the mushroom,

too. The synthetic pathway proceeds from tryptophan to tryp-

tamine to N-methyltryptamine to N,N-dimethyltryptamine to

psilocin to psilocybin.

Any and probably all precursors to psilocybin can be found

at one time or another in P. cubensis. Some may be bound to

proteins or enzymes which may tie them up for any chemical

reaction or assimilation in the body. The consequence of the

presence of all these other non-active tryptamine or nitrogen

containing compounds which react with the test reagent is to

artificially raise the absorbance or apparent concentration. In

testing, the change in the absorbance does not necessarily

mean a change in the active tryptamine (i.e. psilocybin/

psilocin) concentration at all.

Some quick and easy tests for field or the non-technically

inclined.

A simple test described in High Times to determine whether

one has inadvertently purchased LSD laced mushrooms is to

mash the mushroom in some methanol and let it sit overnight.

Decant the methanol the next day and hold the extract up to a

black light. If the liquid glows blue then you have LSD con-

taining mushrooms, which, as far as I know, do not exist.(l7 p.

252)

Norland describes a few colorimetric tests which can be

used to identify mushrooms which contain tryptamine deriva-

tives.(l3 p.116) You may find them more useful than the

longer and more complicated test procedure at the end of this

article. You do not require a colorimeter for test results and if

you can live with an eyeball color comparison and your mem-

ory, you can at least estimate the concentration differences

between mushroom flushes.

1) A simple test for indole-containing compounds and tryp-

tamines is to crush a small piece of mushroom into 1/2 ounce

of vodka or ethyl alcohol("denatured alcohol" or the hardware

store "shellac thinner" is fine) and mix. Add 3-4 drops of

hydrochloric acid(or the hardware store variety called, "muri-

atic acid") then drop a pine tree shaving into the solution which

will turn "cheny red" in the presence of indoles.

2) Another test for indoles uses a small crushed piece of

mushroom in 1/2 oz. of either methanol or ethanol(or Vodka)

If you are interested in testing for psilocybin use methanol; if

psilocin use ethanol or vodka. The difference in solubility

between the two active tryptamines account for the difference

in the solvents used. Mix well then filter. Let evaporate over-

night or use a steam bath or a hair dryer to dry. Spot the resi-

due on filter paper and let dry. Spray or drop on the following

developer. In order for the test to work effectively the devel-

oper must be made fresh. To make the developer, add one

drop of 37% formaldehyde to 15 drops of concentrated sulfuric

acid. Psilocin should turn green to black where as psilocybin

should turn yellow to green-yellow; green is normal. Orange-

brown indicates amphetamines or LSD.

3) Ehrlich's reagent is a name of a mixture which is used to

detect indole compounds which have been separated on a TLC

plate. After spraying the test solution on the plate, a colored

spot will form where such an indole-like compound lies. The

reagent is made from p-dimethylaminobenzaIdehyde(5%)

(abbreviated DMAB) in concentrated hydrochloric acid (HC1).

(9)

Another variation of the Ehrlich's reagent is 2% DMAB in

HC1-ethanol (1:1). This reagent gave the following color

changes: psilocybin turned reddish-purple then faded to violet

whereas psilocin yielded a strong blue color which faded to

violet.(l8)

4) If you are interested in pursuing a TLC testing procedure,

see Leung, Smith and Paul for the various solvent systems

which can be used to separate out the constituents of the mush-

room. Also, you will be able to use the information about the

expected relative distances (Rf values) which psilocin and psi-

locybin will travel up the plate for each solvent system. This

information plus a test reagent such as the Ehrlich's will help to

establish if psilocybin or psilocin or both are present in the

mushrooms you test.(9)

For my own TLC use, I found that the solvent system which

Beung and Bigwood used in their research worked best. They

found that they could obtain the greatest resolution of the most

spots by using a solvent mixture of n-butanol-acetic acid-water

(12:3:5) with silica gelplates.(3) In my use of this solvent sys-

tem, T found that the solvent ratios mix well. Some of the

other literature suggests solvent mixtures which are not home-

geneous after shaking, but instead quickly separate out into

two layers, making these other solvent mixtures difficult to use

in TLC.

In my own separations, I used a UV light and the fluorescent

version of the TLC plates for detection. I did not have access

to a fine mist sprayer which is required if using the Ehrlich's

reagent. I could only distinguish six spots in contrast to the

twelve which Beung and Bigwood found when they used both

detection schemes(i.e. Ehrlich's reagent and UV lamp).

In another test procedure I made a test paper which can

detect the presence of indole compounds by using p-DMAB

(para-dimethoxybenzaIdehyde) as the detection reagent or

chromophore. Although the paper is not sensitive to low levels

of indoles, I found it useful for quick checks of mushroom

extracts for the presence of psilocybin/psilocin .

p-DMAB Test Paper

1. Add 1.0 gram of p-DMAB to 28 mil of ethanol(dena-

tured). Stir until dissolved.

2. Add water to the above mix until 60 mil total volume

achieved.

3. Soak some filter paper in the solution and let dry com-

pletely. One can use a hair dryer to speed the drying but do

not use too hot of an air flow. The heat will destroy the p-

DMAB and consequently the test paper's usefulness. Store the

paper in a tight jar in the refrigerator.

4. To use the paper add a drop of the extracted sample to

the paper and let dry. Hold the test paper over the mouth of a

bottle of concentrated hydrochloric acid. The fumes will

develop the purplish/blue color quickly. If the fumes do not

develop the color try adding a drop of hydrochloric acid on

the paper next to the sample spot. As the hydrochloric acid

diffuses into the filter-test paper and comes close to the sample

spot, the color, purple or blue will form if the sample is posi-

tive for indole compounds.

The theory behind the reference colorimetric test.

In the preceding sections I have outlined the general parame-

ters of colorimetric testing and in the last section listed the pro-

cedures for several test which are easy to set up and read. The

test which I have selected for measuring the active tryptamine

content in the various experimental samples from the last four

years, is based on a color reaction with para-

dimethylaminobenzaldehyde (DMAB). DMAB is the common

reagent ingredient in several other tests mentioned above

including the Ehrlich's test.

To review the general colorimetric test again, the indole part

of the tryptamines reacts with DMAB in a solution conducive

to driving the reaction to completion thus forming a colored

complex which can be visualized or read with a colorimeter or

spectrophotometer. The intensity of the color(i.e. absorbance)

is proportional to the concentration of the tryptamine content

of the solution.

The use of this test is common in the literature in slightly

different formats. The test which I developed for measuring

the indole-like compounds, psilocybin and psilocin, was origi-

nally used in a slightly modified form to measure ergot alka-

loids.(16) Lysergic acid and ergotamine tartrate are ergot

derivatives--both precursors to LSD and other pharmaceuticals

and can be tested using DMAB.

Besides the more sensitive but more complex HPLC (High

Pressure Liquid Chromatography) testing procedures, various

researchers have used other means of quantifying psilocybin

and psilocin. Leung and Paul used a quantitative TLC(Thin

Layer Chromatography) method in which the least amount of

chemically pure psilocybin to cause a reaction with Ehrlich's

reagent (a 5% solution of p-DMAB in hydrochloric acid ) was

compared to the least amount of a test sample from extracted

mushroom tissue needed to induce a color change. One

assumes that the psilocybin concentration is equal in both

cases and then computes the percent concentration of the psi-

locybin in the mushroom by knowing the amount of mushroom

which was extracted.(11)

OTHER POTENTIAL CHROMOPHORES

The test does not have to be restricted to DMAB as a chro-

mophore. Although in the following testing procedure and in

all the technical literature, para- dimethylaminobenzaldehyde

has been used as a color developing agent, another chemical,

para-dimethylaminocinnamaldehyde may be used. This partic-

ular developing agent will yield a much more intense color

than the DMAB and will consequently be more sensitive.

This additional sensitivity may be necessary if you are using

a more crude colorimeter with a broad bandpass filter(i.e. more

than 30 NM). Instead of the accepted reading wavelength of

570 nm used for the DMAB test, you should use 625 nm for

para-dimethylaminocinnamaldehyde .(15) But in most applica-

tions this increased sensitivity will cause too dark a reaction

color to develop and thus be hard to read on the colorimeter

scale.

Still another reagent used similarly to the DMAB reagent

(Erlich's reagent) is the Pauley reagent. This reagent uses dia-

zotised sulphanilic acid. It is more specific than DMAB in that

it does not react with psilocybin or urea, but only with psilocin,

giving a deep red-orange color.(l8) (I do not have details on

which wavelength to measure this color reaction.)

THE CORRECT WAVELENGTH TO MEASURE THE

DMAB CHROMOPHORE.

A few researchers used DMAB in their colorimetric test and

generally the wavelength they used to read the indole contain-

ing compounds has been 570 nm. The spectrophotometric

peak of both psilocybin and psilocin after reacting with DMAB

is sufficiently broad so that one can use another wavelength

close to 570 nm without affecting the sensitivity of the test.

This could be important if you use a filter colorimeter and the

available filters do not include the specific wavelength of 570

nm.

In figure 3 I have constructed a spectral absorbance graph

for a positive DMAB test for some mushroom powder. I took

transmittance readings every 10nm from 400 nm to 610 nm

and then converted the transmittance values to absorbance,

which I later plotted.

The best wavelength to read a colorimetric test is usually at

one of the absorbance peaks. As you can see, the test could be

read at more than one wavelength based on this criteria.

Another possible absorbance maxima besides the approximate

580 nm peak is on the 550 nm peak. It is probable that these

two peaks represent psilocin and psilocybin. The large peak

around 410 nm may be a secondary peak for psilocybin, which

is usually seen as purple--a combination of red and blue.

When analyzing the graph remember that this represents spec-

tral absorbance not transmittance. Therefore for the color blue

one would look for an absorbance peak in the red area of the

spectrum(i.e. near 600 nm).

See Figure 3.

LIGHT SPEEDS THE TEST REACTION

It was from the paper of Agurell, Blomkvist and Catalfomo

which I discovered the principle outline of the test which T

present here.(l) In that paper they suggested the use of a UV

light which will speed the reaction with the DMAB chrome-

phore. I have substituted that step by letting the reaction

develop under fluorescent lights for a longer period of time.

Even at that, the reaction continues to develop for 24 hours

after initiation. The researchers mentioned also produced a

calibration curve which could prove useful. But these

researchers not only extracted the psilocybin but also purified

it to some extent before testing their samples. You may be

able to get a "ball park" absolute concentration in mg/gram of

mushroom by extrapolating from this curve and by purifying

your mushroom extract. Consult the reference for more details

on purification if you are interested. (A future article will out-

line a purification procedure.)

The DMAB reaction requires at least thirty minutes to reach

a plateau of color development under fluorescent light. Note

figure 4 which shows this. Because the reaction actually con-

tinues for up to 24 hours, you will need to accurately time the

development period and then standardize this time for all tests.

I use 30 minutes because the greatest color changes have

occurred by this time and I prefer not waiting too long for the

results. Another good reason for using a shorter development

time period is that psilocin and other related tryptamines which

are present do gradually degrade, thus altering the value of

concentration for the active tryptamines if allowed to sit in

solution while the test is developing.

See Figure 4.

DEFINING THE EXTRACTION SOLVENT

The researchers, Agurell, et al., tested the extracted and

somewhat purified psilocybin. They did not test for psilocin.

To avoid a tedious and lengthy sample preparation, I wanted

to extract, then read the mushroom sample without purifica-

tion. Ideally, I wanted to use an aqueous solution to avoid

organic solvents and to test for both psilocybin and psilocin

during the same test. But psilocin has difficulty dissolving in

water, and psilocybin is easily dissolved in water. Since psilo-

cin is especially unstable in alkaline solution, I felt that an

acidified aqueous solution would be the best to use as a sol-

vent.(l9) Many TLC tests confirmed that the acetic acid-water

solution which I finally decided on did, in fact, extract both the

psilocybin and psilocin.

I use an acetic acid-water extraction solution to help extract

the psilocin and psilocybin more completely and also, to lower

the pH so that the active tryptamines will be more stable.

Without the acetic acid the solution will quickly react with

atmospheric oxygen in the presence of endogenous enzymes to

form a strong blue product and in the process destroy some of

the psilocybin/psilocin. Also, the color blue itself will inter-

fere with the test results, since the reaction yields a blue or pur-

ple color for tryptamines. Specifically, psilocin yields a

brown-deep blue and psilocybin a yellow-green and purple

color. In contrast, LSD will react with DMAB to form a blue-

purple color.(l7)

Apparently, others have also found that a dilute acetic acid

solution is an excellent solvent for both psilocin and psilocy-

bin. Not only does the solution completely extract both trypta-

mines but the solution extracts other interfering substances to

a lesser degree. Casale also notes that if one heats the extrac-

tion solution of dilute acetic acid to 70 degrees centigrade for

ten minutes, then the psilocybin is completely converted by

dephosphorylation to psilocin.(5)

I have found on my own that heating the acetic acid solution

eliminated whatever bluing reaction was occurring in the

enzyme denaturing environment of the low pH extraction solu-

tion. That psilocybin is converted to psilocin is a plus, too. It

means that the color reaction will form a more pure color and

is therefore easier to interpret the test results.

Besides measuring the color of the developed reagent when

it has stabilized somewhat, it is also important to measure a

sample of the mushroom extraction as soon as possible. The

dilute acetic acid slows the degradation of the psilocin-like

tryptamines but does not totally inhibit this degradation. The

longer you wait to perform the test on your sample, the lower

the value will be. I found the reduction to be approximately

10% after 20 hours. Interestingly, the greater the concentration

of active tryptamines as measured on a fresh sample, the

greater the effect of time in reducing the apparent

concentration.

SAMPLE WEIGHT

I arrived at the sample weight of 0.5 gram powdered mush-

room by running a test on four sample masses: 0.2 grams, 0.5

grams, 1.0 gram and 1.5 gram. For the extraction volume of

20 milliliters, the 0.5 gram sample works best. The larger mush-

room samples tend to float on top of the extraction solution in

the large test tube and have to be constantly stirred so that the

powder remains in the extraction solution. The smaller

amounts of mushroom powder become increasingly harder to

weigh accurately and precisely. Also, the smaller samples

have less of a color in the developed reaction making it harder

to read the spectrophotometer.

OTHER INFLUENCES ON THE TEST

An interesting but unexplained influence on the color test

came from a slight, but apparently significant, change in my

standard procedure. If for some reason I used solvents in the

preparation of the mushroom material and did not extract the

mushrooms, but then totally evaporated the solvent leaving the

original mushroom powder ostensibly unchanged, the test

color shifted to a more pink color and consequently changed

the absorbance from 570 nm. I noted this color shift primarily

when I used methanol. Perhaps methanol reacts with some-

thing in the mushroom and this product in turn reacts with the

DMAB. The point is that the test results cannot be compared

to other test results for which you have modified the test proce-

dures. Common sense may tell you that a particular modifica-

tion may not matter, but in fact, the modification may change

the results dramatically

A more dramatic example of trying to compare apples and

oranges as far as the colorimetric test for psilocybin/psilocin

happens when I have tried to pre-purify a mushroom powder

sample by extraction. My extractions were attempts to clean

up the mushroom powder of non-active tryptamines. The col-

orimetric test becomes a more pure color but because other

chemical entities have been removed which also react with

DMAB, the overall absorbance drops, giving one the immedi-

ate impression that not as much psilocybin/psilocin exist in the

mushroom powder sample when, in fact, just as much psilocy-

bin/psilocin is present.

INTERPRETATION OF THE DMAB TEST.

The DMAB test reacts with other than psilocin/psilocybin.

The DMAB colorimetric test is not a perfect test. The

numerical results can be somewhat misleading when used to

indicate the concentration of psilocybin/psilocin, the two most

common psilocybian analogs in P. cubensis. In the above sec-

tion on "what is in the mushroom and what are we measur-

ing?", I made the point that the test is not specific to just these

two tryptamines. The test reacts with nitrogen-containing

indoles, of which the tryptamines are a larger molecule which

incorporates the indole group. The test is most sensitive to

these indole-containing compounds but still can react with

other indoles besides tryptamines.

DMAB reacts to form different colors with other indole con-

taining compounds or other reactive nitrogen-containing mole-

cules. For instance, psilocin typically is blue and psilocybin is

purple or purple-green. And tryptophan which is chemically

similar to both develops a deep blue color which is different

enough from both to make it difficult to use as a standard as I

had hoped.

The evidence one can obtain from the different color reac-

tions for different indole compounds can help to evaluate the

test results. Note the color mixture after the test has devel-

oped. Record the various colors. How pure a color are they?

The more pure the color the greater the purity of the trypta-

mine present in the mushroom.

In my own TLC work I have consistently seen four separate

bands which react with DMAB:

Zone / Rf / Color with DMAB

1 / 0.137 / Dark Violet

2 / 0.275 / Pure Violet

3 / 0.550 / Grey Blue

4 / 0.965 / Pink Orange

Each of the above "zones" represent a different chemical

compound in the mushrooms which I tested.

By knowing that the DMAB test reacts with other indole

compounds besides psilocin/psilocybin, one can conclude that

the numerical results of the test represent the summed absor-

bances(i.e. concentrations) of the various tryptamines or other

reactive compounds present in the mushroom, not just the

absorbance of psilocybin or psilocin.

One needs to keep in mind that the DMAB test can lead to

ambiguous results when trying to make conclusions about

environmental or nutritional influences on the biosynthesis of

psilocybin/psilocin. What may be occurring is a change in the

mixture of the various tryptamines in the mushroom along with

an increasing or decreasing or static test result. The ultimate

confirmation would be the subjective test, because a change in

the tryptamine concentration will change the nature of the psy-

chedelic trip.

One can mistakenly interpret the test results in other ways.

In addition to the extra absorbance because DMAB reacts with

other compounds in the mushroom extraction besides the ones

we are most interested, the general enzyme class of phenol oxi-

dases which are widely distributed in different species of

mushrooms can use DMAB as a substrate and can thus reduce

the absorbance of the test artificially. By reducing the concen-

tration of DMAB, these enzymes effect a falsely low value for

the tryptamines. This is another reason I use a low pH extrac-

tion solution(i.e. acetic acid in water) and heat the extraction

mixture. Both of these procedural details should reduce the

likelihood of losing DMAB through enzymatic activity.(8,p.

108>

As noted in the article on harvesting and storage, some ions

will interfere with the color development of the DMAB. In

particular, my experience shows that the bisulfate ion inhibits

the reaction. Sodium bisulfite can be an alternative to vitamin

C as an antioxidant and enzyme inhibitor for mushroom

storage.

Quantifying the contribution of non-psilocybin/psilocin fac-

tors which react to DMAB.

When I began subjectively testing the effect of nutritional

factors on the growth of my variety of P. cubensis, I noticed

that the increase in absorbance of the DMAB test, which

relates to an increase in concentration, did not seem to be pro-

portional to the large increase in the subjective effects of the

mushroom powder. A small apparent increase in the concen-

tration(absorbance value) of the DMAB test doubled the sub-

jective effects of the mushroom. My earliest concentration

values for the mushrooms tested were 0.6 A(absorbance units).

When I subjectively tested mushrooms which had increased to

0.8 A, I was surprised to have a trip which seemed twice as

strong.

After careful thought, I concluded that the initial concentra-

tion value was composed of two or more colored constituents

which added to the total value and that the active tryptamines

which made the trip possible were a comparatively small per-

centage of this total color intensity.

To obtain evidence to confirm or deny this hypothesis, I

used the process of paper chromatography to separate out three

different mushroom powder samples measuring approximately

0.60 A, 0.80 A and 0.85 A by the DMAB test. After separat-

ing the mushroom extracts, I cut out the zone which corre-

sponded to psilocybin, using its known Rf value for paper

chromatography and a 2% solution of DMAB in ethanol and

hydrochloric acid(1:1) to conclusively identify the width of the

zone by its color reaction. After cutting out the psilocybin

zone, I extracted it in 5% glacial acetic acid at room tempera-

ture overnight. I retested this extraction and compared the

results with the extraction and test of the remainder of the cut

up paper chromatogram which held the other factors in the

mushroom extraction.

After correcting for variations in the sample volumes

streaked on the paper and in spite of the poor separation

achieved on the paper as compared to silica gel TLC, the

results clearly showed that the other unknown but DMAB

reacting factors in the mushroom increased their concentration

with the increasing absorbance at a slower rate than psilocybin.

In other words, the concentration of psilocybin increased

with the increase in absorbance, which follows colorimetric

theory, but as the absorbance increased, the psilocybin concen-

tration contributed a greater percentage to the total color inten-

sity of the various constituents which react with DMAB. This

is in direct conflict with colorimetric theory which states that

the concentration of a solution with an absorbance of 1.0 is

twice that of a solution with an absorbance of 0.5. But colori-

metric theory is for a solution of a single absorbing molecule

and as stated previously, there could be as many as twelve dif-

ferent DMAB reactive compounds in the mushroom, each

increasing its concentration at a different rate as stimulated by

nutritional or environmental changes.

So at this point one can only say that as the absorbance of

the test increases, the concentration of the active tryptamines

increases--but we still do not know by how much. To try and

answer this, I put together a graph at the end of my four years

of research based on my subjective experiences of the strength

of a trip as compared to the absorbance of the DMAB test. My

log shows that I had at least several trips at several different

concentration levels: at 0.5 to 0.6 absorbance; at 0.7 to 0.9

absorbance; at 0.9 to 1.1 absorbance and at greater than 1.1

absorbance. By using the intensity, duration and phenomenal

experiences of the 0.5 absorbance experience as my reference,

I compared each plateau with the previous one and then con-

verted the increase into units of the base reference experience.

For example, I know from written records of my experiences

that the second plateau(0.'ir A) feels twice as strong as the first

(0.5 A) and the third level is 1.5 times as strong as the second,

and so on. The following graph (Figure 5) shows these results.

See figure 5.

There are several points that we can glean from the graph:

1) The increase in the active tryptamines is in fact linear for

this range and this variety of P. cubensis. Apparently for every

increase in the mushroom test value of 0.2 to 0.3 A the experi-

ence intensity increases by the equivalent of the 0.5 A mush-

room experience.

2) The paper chromatography extraction experiment dis-

cussed earlier obtained a value of the other-than-psilocybin

factors' absorbance as 0.35 A for the 0.6 A mushroom powder.

The test was inconclusive on this matter because paper chrom-

atography is such a rough separation procedure, but it suggests

that the value for the absorbance for the other-than-psilocybin

factors increases only slightly as the absorbance increases.

Interestingly, if one extrapolates the subjective experience

graph to the "0" experience point, the absorbance obtained cor-

responds to the point after which one will begin to experience

the psilocybin. This extrapolated absorbance approximates 0.3

A which supports the paper chromatography results that

showed that the first 0.3 A of a mushroom test value is color

from non-active tryptamines and other DMAB reactive com-

pounds. The value also means that all the samples had at least

0.3 A of non-active, but DMAB reactive, junk in the mush-

room. Of course some of the active tryptamines, which can

intensify the trip, may not necessarily be a pleasant addition to

the trip either.

3) Although my work on increasing the concentration of

active tryptamines through environmental and nutritional

manipulation was successful, my secondary goal of reducing

the concentration of the junk in the mushroom was not particu-

larly successful Apparently, the level of non-active, but

DMAB reactive substances, stayed about the same. It is

impossible for me to know without more extensive testing

using HPLC whether as the concentration increased, the mix-

ture and relative concentration of the various active trypta-

mines, such as psilocybin, psilocin, baeocystin and their

analogs, changed in the mushroom powder.

In conclusion, then, when reading the upcoming article(s) on

growth factors for q. cubensis, keep in mind that as the test

values go up the subjective experience increases in intensity,

too. But, an increase in the objective test value cannot be used

to predict an equivalent percentage increase in the subjective

experience. In fact my experience has shown that for a modest

increase in the test absorbance, I increased the subjective

amount of active tryptamines two or even three times.

THE COLORIMETRIC TEST

EQUIPMENT AND SUPPLIES

(Items marked with an "*" have expanded notes and

explanations following the equipment and supplies listing.)

EQUIPMENT

Colorimeter/Spectrophotometer*

Hot Plate

Quart Pan

Balance capable of weighing at least one gram and

accurate to 0.1 gr*

Electric Coffee grinder

CHEMICALS*

25 grams p(para)-Dimethylamino benzaldehyde

1 pt concentrated . Sulfuric Acid

1 pt Glacial Acetic Acid

125 gr Ferric Chloride

Deionized water

SUPPLIES

1 ea. 250 mil plastic graduated cylinder

1 ea. borosilicate 250 mil beaker

2 ea. 125 mil amber narrow mouthed bottle with caps

1 ea. 1000 mil bottle with cap

12ea 20 X 150mm borosilicate glass culture tubes*

6ea matched cuvettes for the colorimeter*

Polyester cosmetic balls (available at most supermarkets)

6 ea. Small(60mm) plastic funnels

1 ea. Multiple funnel rack

1 Box Filter paper: 9 cm Coarse(like Whatman 4) and 9 cm

Medium( 1 or 2)

1 ea. Plastic test tube rack (to hold about 10 of the 20 mm

tubes)

1 ea. Plastic test tube rack (Nalge 5900-0007)$

1 ea. Test tube cleaning brush

1 ea. Volumetric pipette 20 mil

1 ea. Pipette 10 mil in 1/10 mil

1 ea. Pipette 1 mil in 1/100 mil

1 ea. Pipetting Aid (either Bel-Art F-37898 or Nalge 3780-

0100>

1 ea. glass stirring rod

1 ea. stainless spatula, double blade-rounded and squared

1 ea. 500 mil plastic wash bottle

1 ea. bright color nail polish or airplane glue

1 ea. small polypropylene plastic sheet about 4" square*

1 ea. thermometer, 0 to 110 degrees Centigrade

*NOTES:

Colorimeters

I acquired a used Bausch & Lomb Spectronic 20. I under-

stand that not everyone is in the position to so easily buy such

equipment. Try the yellow pages for used laboratory equip

ment dealers. If not specifically listed, try laboratory suppli-

ers. Usually, they have a service department and may have

used spectrophotometers or colorimeters. Some brand names

to look for are older models of the Bausch & Lomb Spectronic

20, the Coleman Jr. and the Turner. These companies sold

many of these lower cost spectrophotometers for many years

and you might be able to buy one comparatively inexpensively,

say 100 to 200 dollars.

Another option is to buy a new colorimeter but without all

the expensive features that the above units have. For the appli-

cation of reading at a fixed wavelength and a broad color peak,

such as in this test, a low cost colorimeter is all that is neces-

sary. All it needs is the proper filter (i.e. as close to 570 nm as

possible), a cuvette or test tube holder and a scale in per cent

transmittance,

There are three companies that I know of which can offer a

low cost colorimeter:

1) Chemtrix(P.O. box 1359, Hillsboro OR 97123; 800-821-

1358) has a colorimeter (20A) for $269;

2) Hach(P.O.Box 389 Loveland, Colorado 80539; 800-525-

5940) has a single parameter DR100 colorimeter for about

$200(contact Hach about supplying a transmittance scale and

the proper filter)

3) Hellige(877 Stewart Ave., Garden City, N.Y. 11530; 516-

222-0302) has a meter readout photometer for about $125.

Balance

The Ohaus triple-beam balance is a recognizable standard

school lab scale. Presently these cost about $80 to $90 from

almost any lab dealer or even hobby shops or some specialty

hardware stores. But for this test and most work with mush-

rooms, you will not need the capacity, or ability to weigh large

as well as small masses, offered by this triple-beam. I do not

have company names but I have seen in High Times advertise-

ments for small, Inexpensive scales which can weigh accu-

rately up to 30 grams.

Large borosilicate test tubes.

Of the twelve test tubes set aside six. These will be used

"as-is" with out matchin8. The other six need to be volumetri-

cally marked for the standard extraction volume of 20 milliliters.

To do this, simply fill up the 20 mil volumetric pipette with

water then add it to the test tube to be marked while standing

in the test tube holder. Then mark the bottom of the meniscus

with an indelible marking felt pen. Do the same with the other

five test tubes.

Matched cuvettes.

Cuvettes should be one centimeter ID, but because of varia-

tions in the extrusion Process for the tubes the ID differs

slightly. This irregularity in the internal diameter and the occa-

sional streaks or variations in the thickness in the glass walls of

the tubes will result in cuvette-to-cuvette differences in the

amount of light from the colorimeter filter passing through the

cuvette and solution. This variation can be as much as five per-

cent transmittance. To increase test-to-test precision, some of

the manufacturers of the colorimeters mentioned above offer

matched cuvettes as an option. Matched cuvettes have been

selected so that the amount of light which is absorbed by the

walls of the cuvette is essentially the same from cuvette to

cuvette. For the price it is usually easier to buy them. If you

have to prepare matched cuvettes follow this procedure:

1) 13 mm OD culture tubes will have a nominal ID of 10

mm. Buy at least a couple of dozen.

2) Chose several and fill them with water. Insert them into

your colorimeter and set the wavelength to 570 nm or as near

as your filter will allow.

3) Set the meter to an arbitrary mid-meter position. Then

slowly rotate the cuvette. Notice the variation as you rotate the

cuvette the full 360 degrees. Choose the culture tube with the

least variation.

4) Continue with another set of a few cuvettes and keep

selecting the least variable cuvette until you have six to ten

cuvettes.

5) Now match the cuvettes by making an arbitrary mark

with nail polish or airplane enamel on the open lip of a cuvette

such that this mark can be used to align the cuvette in its

holder. Note the meter readout.

6) Take another pre-selected cuvette and rotate it until it

matches the transmittance of the first marked cuvette. Mark it

with nail polish or paint, too.

7) Continue until all the cuvettes are matched and marked.

When using the cuvettes to measure the transmittance of the

DMAB test reaction, be sure to always align the mark with the

pre-determined alignment position in the cuvette holder.

Usually the holder itself has a mark or ridge to reproduce the

correct cuvette position.

8) It is possible that the initial cuvette which you arbitrarily

marked may not match any or only a few other cuvettes. Just

start over but use another cuvette as your initial reference

cuvette.

Test Tube Holder.

The particular holder I use is an open style which is one tube

deep and seven across. I use this holder for heating the extrac-

tion solution in a one quart pan. To make it fit in the pan, I cut

the holder in two sections of three test tube capacity each.

When using this holder with the test tubes make sure you bal-

ance the weight of the tubes in the three slots. The holder

tends to float. For instance if you put a single test tube in the

holder's end, the holder will float up dumping the tube into the

heated water and thus losing your mushroom sample. To

avoid losing your sample, when extracting a single sample put

the tube in the center of the holder, or if using two test tubes,

put them on either end of the holder.

Chemicals.

I have used technical grade chemicals throughout all my

testing. Technical grade is somewhat less pure than reagent

grade and usually less expensive. The sulfuric acid could be

reagent grade which would be somewhat clearer than the

slightly yellow-brown technicalgrade. Sulfuric acid is used in

the color development solution and is read on the colorimeter,

so clarity and lack of particles floating around would help

readability and reproducability. I have had no problems with

the less expensive technical grade by always using a sample

blank to compare all mushroom test samples against it. A sam-

ple blank is used in colorimetry to set the "zero" point. It is

usually the test reagent with a distilled water sample rather

than the material or extraction to be tested. If the test reagent

is colored in the spectral region of the test before any reaction,

then by using a sample blank one can offset the colorimeter by

this degree of color so that the test result is not artificially high.

Plastic sheet.

All that is necessary is a piece of plastic to cover your thumb

so that when you shake the culture tube with the test reaction

mixture the sulfuric acid will not burn your skin. A piece a

couple inches square should suffice. Most plastics are unaf-

fected by sulfuric acid.

TESTING SOLUTIONS PREPARATION:

Color development reagent.

This mixture is the actual solution which when in the pres-

ence of an indole or indole-like compound will develop a

color from a near colorless solution.

1.Weigh out 0.2 gr of p-dimethylaminobenzaIdehyde

(DMAB) and put it into the 250 mil beaker.

2. Make a 20% aqueous ferric chloride solution by weigh-

ing out 20 gr of the ferric chloride into 100 mil of distilled or

Deionized(DI) water in the 250 mil graduated cylinder. Wear

gloves. Ferric chloride can be corrosive. Make sure it all goes

into solution. If any particles remain insoluble, filter the solu-

tion. Store the finished solution in a 125 mil bottle and label.

Rinse out the graduated cylinder.

3. Using the 1 ml pipette and the pipette-aid(never suck up

corrosive or toxic chemicals in a pipette!) draw up 0.3 mil of

the stock ferric chloride solution (#2,above). Add this to the

250 mil beaker with the DMAB already in it.

4. Add 35 mil of distilled or Deionized water to the 250 mil

beaker and stir the water to dissolve the DMAB as much as

possible.

5. Add 65 mil of Sulfuric Acid from the graduated cylinder

to the beaker with the DMAB. Be careful with sulfuric acid.

Wear gloves, an apron and preferably a face shield. When

adding the acid, set the beaker on the table. Do not hold the

beaker. An exothermic reaction will take place when you add

the acid to the water present in the beaker and it will be too hot

to hold. Gently stir the mixture. The heat of the acid-water

mixture will drive the remaining DMAB into solution.

6. Let the mixture cool down and pour this stock solution

into a 125 mil bottle and label it. Do not put hot liquids into

the bottles; they may break.

Store the DMAB solution in a dark, cool location, preferably

in the freezer section of your refrigerator. If you cannot store

it somewhere cold, the solution will gradually darken and thus

be unusable for colorimetry. I have successfully stored my

stock solution in the freezer for over a year and it has not dark-

ened noticeably. At room temperature you will probably need

to discard the solution after a few months.

Acetic Acid Extraction Solution.

This stock solution is used to extract the mushroom of its

active tryptamines. Then a sample of this extract is tested using

the above DMAB color development solution.

1. Measure out 50 mil of glacial acetic acid in the 250 mil

graduated cylinder. Fill the cylinder to the 250 mil mark with

DI or distilled water and pour it into the 1000 mil bottle. Rinse

the cylinder with 3 X 250 mil amounts of DI water into to the

1000 mil bottle to make a total of 1000 mil stock solution of 5%

Acetic Acid.

2. Distilled vinegar (i.e. "white vinegar") can be substituted

for 5% acetic acid. Distilled vinegar is approximately 5% ace-

tic acid.

THE para-DIMETHYLBENZALDEHYDE

COLORIMETRIC TEST

See figure 6.

1. Set up the table or bench on which you will do your test

with all the equipment in the above illustration spread out.

Turn on the colorimeter or spectrophotometer to begin warm-

ing it up and adjust or set the proper wavelength(570nm or

close to this). Most have light sources which need to be on for

20 to 30 minutes to stabilize the spectral output. The older

models also have slow-to-warm-up electronics.

2. Turn on the heating plate and begin to heat the water-

filled quart pan. Do not let the water boil. The boiling action

will tip over the test tubes with the samples spilling out into the

boiling water.

3. If you have not done so, use a coffee mill to grind up the

dried mushrooms which you wish to test. Weigh out 0.5 grams

of the ground mushroom. It's best when using a scale to use a

piece of paper on which you weigh the mushroom. Find out

how much this paper weighs before adding any mushroom

powder then add this weight to the required half gram sample.

This total weight in grams is what you should set the scale for

if you have a triple beam-type scale. With the weighed mush-

room powder on the weighing paper, cup the paper to form a

trough and pour the sample into an empty, large

(20mmX150mm) unmarked test tube.

4. Add 20 mil of 5% Glacial Acetic Acid to the sample. Stir

the sample with a glass stir rod making sure all the mushroom

powder is wetted with the acetic acid solution. Put the test

tube in the plastic holder and immerse the holder in the pre-

heated near-boiling water. Note the time or start a timer. Heat

this suspension for 30 minutes. This time is not critical. If you

should heat it for longer than thirty minutes it will not make

much difference. Too long will oxidize the psilocin in the

solution and cause a false low reading of the sample. Also, the

acetic acid solution does evaporate.

Every ten minutes or so stir the suspension with the stirring

rod. The mushroom powder tends to float on the acetic acid

solution which cannot optimally extract the mushroom powder

when it is not suspended. Also, the top of the powder can be

exposed to air and will oxidize to a dark blue. Again, this can

lead to a false low reading.

The immersion of the glacial acetic acid solution in near

boiling water with the mushroom powder during the extraction

phase of the test is important to deactivate any enzymes which

can cause bluing, and thus tryptamine loss, in the mushrooms.

Without heat and with time the mushroom extract solution will

slowly turn blue-green until it becomes dark blue after several

days.

As noted in the above section on theory, use a thermometer

to make sure that the extraction solution reaches 70 degrees

Centigrade for at least ten minutes. This temperature converts

all the psilocybin to psilocin which makes for more pure test

color.

5. While the mushroom powder is extracting, set up the next

step: the filtering and sample development. For each sample,

use one small filtering funnel. In the funnel put a polyester

ball. Using the wash bottle, wet the cosmetic ball then press

any excess water out of the cosmetic ball with your finger.

Discard this water if it went into the collection test tube.

6. After 30 minutes or so, remove the test tube holder from

the water bath. Turn off the heat plate(or stove). Just before

the initial filtering, stir the suspension one more time. Pour the

test tube contents over the top of the polyester cosmetic ball in

the small filtering funnel which drains into a large(20X150mm)

test tube on which you marked the 20 mil level. Do not worry

if you cannot get every last drop of the suspension out of the

test tube.

Let the filtrate pass through the small filter completely by

gravity. Sometimes pressing the cosmetic ball will succeed in

driving more water extract off the polyester fibers. This step is

important because if you don't get all of the original, undiluted

extract in the final test sample, your concentration value will

be falsely low. I have occasional-ly forgotten to do this

squeezing step and have had readings which were off by 25%.

Although you could get more consistent results by just using

filter paper instead of the cosmetic ball, by filtering the thinly

mucilaginous mushroom suspension with the cosmetic ball

you can save literally hours of waiting for all the suspension to

get through the paper filter.

7. Now use the wash bottle filled with distilled (or deion-

ized) water and rinse the sides of the test tube used to extract

the mushroom sample with a few milliliters of water. Note

that the cosmetic ball filtered extract does not reach the 20 mil

mark on the test tube. This loss of fluid came primarily from

evaporation when you were heating the mushroom suspension.

For reasons of standardization all our test solutions need to

have a total volume of 20 mi. Shake the unmarked test tube

which you just added some rinse water to and pour an esti-

mated amount of the rinse water which will fill up the test tube

to the 20 mil mark over the cosmetic ball filter. This rinse will

remove most of the residual extract left on the polyester ball

and will dilute the initial filtrate up to the 20 mil mark. Watch

the filtering carefully and remove the filter to the sink once the

level has reached the 20 mil mark.

The largest errors can occur in the filtering and dilution

steps. If in the process of washing the filtrate and bringing up

the filtered volume to the 20 milliliter mark, you add too much

water to the top of the funnel filter you will dilute some of the

extracted sample which will not then pass through the filter.

Since you have reached the 20ml mark you are forced to dis-

card the water which may contain some of your sample. The

results may then be artificially low. Solution: add smaller

amounts of rinse water with the squeegee bottle, so that you will

not leave any of the liquid mushroom extraction on the filter

that will then not be part of the test.

If in the process of rinsing the sample on the cosmetic ball

and adjusting the 20 mil volume, you should over-fill the test

tube beyond the 20 mil mark, just correct the final absorbance

by multiplying the absorbance by a fraction consisting of the

volume which was filtered (over 20 milliliters) as the numerator

and 20 as the denominator.

8. This rough filtered extract is your test sample but needs to

be filtered again so that it is clear and will not add absorbance

to the colorimeter reading because of its cloudiness. Pour this

extract through another small plastic funnel with a folded piece

of filter paper in it. Fold the filter paper on itself three times

(i.e. into eighths).

Most extracts will have some colloidal suspension in them,

making them look slightly cloudy. Even filtering these with a

fine filter will not remove all the turbidity. For most filtering

you will need to remove only coarse particles and the

Whatman #4 filter paper will suffice. For heavier suspensions

use the finer, but slower, Whatman #2 filter paper. The idea

here is to obtain a reasonably clear sample which has the least

amount of turbidity. Turbidity, if excessive, will give false

high colorimetric readings.

For the receiving container for this filtrate, use another large

test tube as the receiver. After a few milliliters of fluid has

passed through, you can stop the process if you wish. I usually

like at least enough prepared sample for a couple of tests(each

tests requires 0.5 or 1.0 ml of sample), just in case I wish to

repeat a reading.

9. The preceding two-step filtering process should take 10

to 15 minutes. While the filtering is proceeding, using the

pipette-aid and the 10 mil pipette, draw up 2 mil of the DMAB

reagent and dispense it into a matched cuvettes/small test tube.

Continue dispensing the DMAB reagent into a matched cuvette

for each sample or test you may wish to run. Now you are

ready to develop the color and read the final absorbance.

If you store the DMAB test solution in the freezer, allow it

to return to room temperature before using it in a test.

10. Using the pipette-aid and the 1 mil graduated pipette,

draw up a 1 ml sample of the mushroom extract to be tested. If

you anticipate an absorbance reading above 0.700 A or so, then

draw up only 1/2 mil of sample, but before doing so add a 1/2

mil of Deionized or distilled water to the small reaction test

tube/matched cuvette.

The reason for this dilution is that for colorimeters with a

transmittance scale, the most readable portion of the scale lies

above 20% transmittance. Lower transmittance than this corre-

sponds to higher absorbance and greater possibility of reading

errors and poor precision. I checked to make sure that both

ways of testing equaled the same value after making the neces-

sary multiplication of the diluted sample by two.

The total combined test volume must be 3 mil for both the

sample and the DMAB reagent. The aqueous sample will not

immediately mix with the much denser DMAB reagent but

will rest on top until mixed.

11. Make sure your timer is ready. Hold your thumb over

the reaction tube with the plastic sheet between the thumb and

the test tube and turn the tube upside-down then back again.

Do not shake because the shaking will add bubbles which can

remain on the walls of the test tube and interfere with the col-

orimetric reading later. Either start the timer or note the time

immediately. After exactly 30 minutes, read the transmittance

on the colorimeter(or absorbance if your instrument has a

direct liner conversion to absorbance from transmittance).

Sometimes the reaction mixture in spite of filtering the sam-

ple will become turbid. If so, dilute with 3ml of DI water, read

and divide the absorbance result by two. This dilution will

usually water down the turbidity enough to be able to read the

test.

It is important to test the mushroom extraction solution

soon, otherwise the bluing and other oxidation reactions will

breakdown the active tryptamines and cause you to have a

false low value for the test's absorbance. By testing the mush-

room extract solution at regular intervals, I found that no sig-

nificant reduction of the active tryptamine concentration

occurred until after six to eight hours.

I have always conducted this test under two 40 watt fluores-

cent lamps. In light of the research paper which used UV to

accelerate the DMAB color reaction(6), fluorescent lamps may

have more of their spectral output in the near UV than incan-

descent lights or sunlight coming through a glass window. The

absence of such lighting in your test area may slow the reac-

tion down and thus cause you to have lower readings after the

30 minutes than mine. I have not confirmed this, but be aware

of this possibility.

12. At the end of 30 minutes, read the colorimeter scale and

note the value for later reference. When reading the meter

(unless the meter is digital) look directly down on the needle to

the scale. If you should read the needle at an angle, your read-

ing will not be as accurate, nor reproducible. As the value of

the transmittance becomes smaller, errors in reading make a

greater difference in the final absorbance(or concentration)

value. Some meters have a mirror below the scale. By looking

down on the needle such that you cannot see the needle's

reflection, then you know that you are looking directly down

on the needle and meter. Try to interpolate or "guess-timate"

the value if the needle should fall between two divisions and

show this as part of your recorded value(e.g. 14.6%)

If you have a colorimeter or spectrophotometer with a direct

absorbance output, your test value will be directly proportional

to concentration of the tryptamines present in the mushroom

sample. If, on the other hand, your colorimeter has a transmit-

tance scale(i.e. 0-100%) and only a log absorbance scale(i.e.

the distance between integers gets smaller as the numbers get

larger), you will need to convert your transmittance to absor-

bance. The formula is: Absorbance Log (1/.01 X

Transmittance in %).

The easiest way to calculate this is to buy an inexpensive

calculator with a "log" and "l/x" or reciprocal functions. Then

calculate by entering the transmittance as a number between

one and one hundred, divide by 100, push the "1/X" key then

push the "Log" key. The result will be the absorbance which is

proportional to concentration.

Conclusion.

It is my hope that the above helps to explain the test values

which will be used in the upcoming articles. The next article

on the environmental and nutritional influences on the growth

and biosynthesis of psilocybin in P. cubensis will make use of

this test extensively. The last section of this article, of course,

is for those people inclined to do their own testing. I encour-

age anyone with any amount of chemistry background to try

this test. The test values will help immensely in planning your

own entheogenic experiences with the "magic mushroom."

BIBLIOGRAPHY

1. Agurell, S., Blomkvist,S. and Catalfomo, P..

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2. Agurell, S., Lars, J. and and Nilsson, C.. "Biosynthesis of

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.... To be continued.