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
So why do you need to test your psychedelic mushrooms for
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
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
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
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
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
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
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
General theory and various detection and measuring
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
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
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-
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
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
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
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
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
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.
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).
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
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 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
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
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
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
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
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-
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
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
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-
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.
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
Quantifying the contribution of non-psilocybin/psilocin fac-
tors which react to DMAB.
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
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
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-
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.)
Balance capable of weighing at least one gram and
accurate to 0.1 gr*
Electric Coffee grinder
25 grams p(para)-Dimethylamino benzaldehyde
1 pt concentrated . Sulfuric Acid
1 pt Glacial Acetic Acid
125 gr Ferric Chloride
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
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-
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
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
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.
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.
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
4) Continue with another set of a few cuvettes and keep
selecting the least variable cuvette until you have six to ten
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
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.
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
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
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%
2. Distilled vinegar (i.e. "white vinegar") can be substituted
for 5% acetic acid. Distilled vinegar is approximately 5% ace-
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
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
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
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
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
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
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.
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."
1. Agurell, S., Blomkvist,S. and Catalfomo, P..
"Biosynthesis of Psilocybin in Submerged Culture of
Psilocybin cubensis: Part I. Incorporation of labeled trypto-
phan and tryptamine," Acta Pharm. Suecica, Vol. 3 (1966), 37-
2. Agurell, S., Lars, J. and and Nilsson, C.. "Biosynthesis of
Psilocybin: Part II. Incorporation of Labelled Tryptamine
Derivatives, Acta Chemica Scandinavica, Vol. 22, No. 4
3. Beung, M.W. and Bigwood, J.. "Quantitative Analysis of
Psilocybin and Psilocin in P. Baeocystis by HPLC and by
Thin-Layer Chromatography, Journal of Chromatography,
Vol. 207 (1981), 379-385.
4. Bigwood, Jeremy and Beug, Michael W., and others, edi-
tors. "Variation of Psilocybin and Psilocin Levels with
Repeated Flushes(Harvests) of Mature Sporocarps of Psilocybe
Cubensis (Earle) Singer," Journal of Ethnopharmacology, Vol.
5 (1982), 287-291.
5. Casale, John F.. "An Aqueous-Organic Extraction Method
for the Isolation and Identification of Psilocin from
Hallucinogenic Mushrooms," Journal of Forensic Sciences,
Vol. 30, No. 1(Jan., 1985), 247-250.
6. Catalfomo, P. and Tyler, V. E., Jr.. "The Production of
Psilocybin in Submerged Culture by Psilocybe cubensis,
Lloydia, Vol. 27, No.l (March 1964), 53-63.
7. Christiansen, A.L. and Rasmussen, K.E., and others. "The
Content of Psilocybin in Norvegian Psilocybe semilanceata,"
Planta Medica: Journal of Medicinal Plant Research, Vol. 42
8. Haard, Richard and Karen. Poisonous & Hallucinogenic
Mushrooms, 2nd Edition. Cloudburst Press, 1977.
9. Leung, A. Y. and Smith, A. H., and others. "Production of
Psilocybin in Psilocybe Baeocystis Saprophytic Culture,
Journal of Pharmaceutical Sciences, Vol. 54, No. 11
(November 1965), 1576-1579.
10. Leung, A.Y. and Paul, A. G.. "Baeocystin and
Norbaeocystin: New Analogs of Psilocybin from Psilocybe
baeocystis," Journal of Pharmaceutical Sciences, Vol. 57, No.
10 (October 1968), 1667-1671.
11. Leung, A.Y. and Paul, A.G.. "The Relationship of Carbon
and Nitrogen Nutrition of Psilocybe baecocystis to the
Production of Psilocybin and its Analogs," Lloydia, Vol. 32,
No. 1(March, 1969), 66-71.
12. Lewis, Waiter H.. Medical Botany: Plants affecting Man's
Health. Chapter 18, Hallucinogens. Wiley-Intersci-ence, John
Wiley & Sons, 1977.
13. Norland, Richard. What's in a Mushroom. Pear Tree
14. Repke, David B. and Leslie, Dale Thomas, and others.
"Baeocystin in Psilocybe, Conocybe and Panaeolus," Lloydia,
Vol. 40, No.6 (Nov-Dec 1977), 566-578.
15. Sarvicki,editor. Photometric Organic Analysis, Vol 31.
16. Snell & Snell,editor. CoIorzetric Methods of Analysis, 3rd
Edition, Volume IV. Van Nostrand, 1954.
17. Stafford, Peter. Psychedelics Encyclopedia. From Chapter
4, "Mushrooms". Berkeley, California: And/Or Press,
18. Weeks, Amold R. and Singer, Rolf, and others. "A New
Psilocybian Species of Coplandia," Journal of Natural
Products, Vol. 42, No. 5 (1979), 469-474.
19. Windholz, Martha, editor. The Merck Index. Ninth Edition,
Entries #7711 and #7712. Rahway,N.J.: Merck & Co., Inc.,
.... To be continued.