JOURNAL OF FORENSIC SCIENCES
American Academy of Forensic Sciences
(1948)
Volume 45 - Number 3 - May 2000 - JFSCAS 45 (3)513-754 (2000)
Susan T Gross, B.A.
Forensic Scientist, Minnesota Forensic Science
Laboratory, 1246 University Ave. St. Paul, Minnesota.
This project was
supported by the Federal Bureau of Investigation (FBI). Minneapolis Field
Office.
Received 26 Feb. 1999: and in revised form 12 May 1999: accepted 23
Aug. 1999.
REFERENCE: Gross ST. Detecting psychoactive drugs in the developmental stages of mushrooms. J Forensic Sci 2000;45(3):527-537.
ABSTRACT: The following questions regarding the detection of psychoactive drugs in mushrooms are addressed: At what stage of the mushroom development can the psychoactive drugs psilocyn and psilocybin be identified, and what effect does light have on the growth of these mushrooms. To answer these questions. Psilocybe cyanescens Wakefield mushrooms were grown from their spores in a controlled setting. At various times of their development, samples were taken and analyzed for psilocyn and psilocybin. Knowing what stage of development the psychoactive drugs can be identified may be useful to law enforcement personnel and forensic chemists. Methanolic extracts of various samples were analyzed by TLC and by GC/MS. It was determined that the mycelium knot stage of the mushroom was the earliest stage at which the psychoactive drugs could be detected. It was observed that light affected the time of development and the appearance of these mushrooms.
KEYWORDS: forensic science. psilocyn. psilocybin. psychotropic mushrooms
Law enforcement agencies in Minnesota are beginning to see an increased number of mushroom growing operations. Knowing what stage of development the psychoactive drugs can be identitied may be useful to law enforcement personnel and forensic chemists. This information is important because in the state of Minnesota it is illegal to possess any material, compound, mixture or preparation which contain any quantity of psilocyn and/or psilocybin (I).
The word mushroom is a general term used to describe the relatively large and fleshy fruiting bodies of fungi, particularly all gill fungi. They are fungi that differ from plants in that these lack roots,stems, leaves, flowers, seeds and chlorophyll. Since mushrooms lack chlorophyll, they depend upon their surrounding medium for their nutrients. The vegetative portion of the fungus accumulates a reserve of food from the immediate surroundings in order to develop fruiting bodies (2-3).
Fungi are categorized as follows: kingdom, phylum, class, order, family, genus, and species. Mushrooms containing psychotropic drugs are classified in the kingdom Mycota, the phylum Basidiomycota, the class Hymenomycetes, and the order Agaricales. There are four families of mushrooms, Strophariaceae, Bolbitiaceae, Coprinaceae, and Cortinariaceae, that contain psilocybin, psilocyn, or related alkalojds with an indolic nucleus. The genus and species of Psilocybe mushrooms that were grown were identified as Psilocybe cyanescens. The pleurocystidia sizes noted in the keys describing the Psilocybe cyanescens mushroom varied slightly from the mushrooms grown. This may indicate a variant of this species (communication with Dr. David McLaughlin, Plant Biology Department, University of Minnesota) (2,4,5-8).
The four stages making up the life cycle of a mushroom are the spores, the mycelium, the primordia, and the mature fruit. The spores are the reproductive cells or "seeds" of the fungi (Fig. I - photo of spores under magnification). Germination of the spores takes place when a suitable substrate and correct environmental conditions are present. These spores grow outward seeking nutrients and branch out forming a complex "cob-like" system. This "cob-Iike" system is the vegetative portion of the fungus which is called the mycelium (Fig. 2 - photo of mycelium spreading in PF jar). The mycelium absorbs water and nutrients from the substrate which is used in the production of the fruiting bodies. The ability of a fungus to begin fruiting is affected by genetic competence and various environmental factors including moisture, temperature, light, and aeration. The formation and growth of the fruiting bodies is known as primordia and has been referred to as "mycelium knots" and "pinheads." The "mycelium knot" is referring to the initial fruiting body that is formed when the mycelium clumps together and seems to form a "knot" (Fig. 3 - photo of birthed PF cake and pins). This knot eventually grows into the 'pinhead,' a plump growth, yellow in color and with a brown tip (Fig. 4 - photo of invitro primordia in PF jar). The fruit is considered mature when it is able to disperse spores and begin this life cycle over again (Figs. 5-6 - photos of mature PF race shrooms on cakes) (2,9).
The spores used in this experiment were obtained legally through an advertisement in High Times Magazine from Psylocybe Fanaticus (PFTek Seattle, WA). The spores were received in 10 mL syringes in an aqueous solution. The spore solutions were each viewed using a 1250X-magnification microscope.
Directions for preparing the growing media were received with the spores. Supplies used for the growing media were half-pint wide-mouth jars (KerrGroup, Inc. Jackson, TN), horticultural vermiculite (Schultz, St. Louis MO), brown rice powder and distilled water. The canning lids were prepared before the mixture was added to the jars. The rubber sealing edge of the canning lids were turned upwards and four holes were punched symmetrically around the outer edge. A mixture of one fourth cup brown rice powder, one half cup vermiculite and one fourth cup distilled water was prepared for each half pint jar. This mixture was placed into the jars and covered with dry vermiculite. The lids were screwed on tightly and aluminum foil was used to cover the lid to prevent additional water from entering the jars during sterilization.
Since growing media is susceptible to contamination, the top layer of dry vermiculite was used to keep airborne contaminants from the wet substrate and absorb and regulate moisture transpiration and condensation (10). The jars with the growing media mixture were sterilized at 120¡C for 20 minutes. The jars were cooled before inoculation. Contamination was detectable through various colors from pastels to black. The growing media that became contaminated was observed but was not analyzed.
Eight jars per week were inoculated with I0 mL of spore solution. This was done for 9 weeks for a total of 72 inoculations. In addition to the eight jars inoculated per week, one jar per week was not inoculated and was used as a control blank. During the first four weeks, all samples were allowed to grow under indirect light. The last five weeks, half of the samples were allowed to grow under indirect light while the other half were kept in the dark. The jars kept in the dark were exposed to light only when samples were taken. Each jar was covered with parafilm after inoculation to keep airborne contaminates from the substrate.
The samples were transferred to terrariums after the pinheads became came too large for the Jars they were growing in. Two different terrariums were used for this experiment. The first one consisted of a styrofoam cooler with a piece of plexiglas inside of it. The second terrarium consisted of a 2-1iter pop bottle with the middle portion cut out. To maintain a high level of humidity, both terrariums were sprayed with distilled water two to four times a day. Fanning the chamber with the lids two to four times a day also kept the terrariums well ventilated.
The growth and colonization was monitored for the samples grown under indirect light. The mycelium was sampled 13 days after inoculation. Samples were also taken from each jar at various stages of growth of the mycelium, primordia and the mature fruit. The growth of the mycelium, primordia and the mature fruit was monitored and compared for the samples grown under indirect light and in the dark simultaneously.
Samples were allowed to soak in methanol overnight. The methanol was decanted
into a shell vial which was then condensed to near dryness (
TLC was carried out on 5 X 10 cm silica gel plates (Analtech Newar, DE).
Psilocyn (Alltech State College, PA) and psilocybin (Alltech State College, PA)
standards were spotted on each plate along with the sample extracts. The plates
were developed to 6 cm at room temperature in a covered development tank with a
9: I chloroform/methanol solution. A beaker containing 3 mL of ammonium
hydroxide was placed in the tank to assist in development. The plate was dried
with low heat and visualized with a paradimethylarninobenzaldehyde (p-DMAB)
spray reagent. (The p- DMAB reagent consisted of 2 9 of p-DMAB in 50 mL of
ethanol and 50 mL of hydrochloric acid.) The relative Rr value of psilocybin is
0.00 and the relative Rr value of psilocyn is 0.85.
The lower limit of detection was determined by serial dilutions of the
psilocyn standard and spotting/developing it until the spot associated with the
standard was not seen. The lower limit of detection for the TLC method was
determined to be approximately 0.03 mg/mL.
The Hewlett Packard gas chromatograph 5890 Series II interfaced with the
Hewlett Packard 5970 series mass selective detector (MSD) and the Hewlett
Packard GI800A gas chromatograph detector system (GCD) were used for the
detection of the analytes. These two instruments are equivalent and samples were
run on specific instruments depending upon their availability. An HP-1 12 m
column (film thickness 0.33 um, column id 0.2 mm) was used for the gas
chromatography (GC). The parameters for the GCD were as follows: injection port
250¡C and detector temperature 280¡C. Method SCAN70-Low mass 35, high mass 425,
initial temperature 70¡C, ramp rate 25¡C/min and final temperature 300¡C hold
for 3.0 minutes. The parameters for the MSD were as follows: injection port
265¡C and detector temperature 280¡C. Method SCN90-Low mass 35, high mass 400,
initial temperature 90¡C, ramp rate 25¡C/min, and final temperature 300¡C hold
for 4.0 minutes. Sample volume was approximately 3 uL with the split ratio of
30:1.
The lower limit of detection for both instruments was determined by serial
dilutions of the psilocyn standard and analyzing it until a peak at the correct
retention time containing the prominent ions 44, 58, 77, 159, and 204 was not
detected. The lower limit of detection was determined to be approximately 0.1
mg/mL for both instruments.
Identifcation of the mushrooms grown in this project was made by examination
of the spores, fruiting bodies and the mature mushroom. Spores were examined for
their color, shape, and size. The spores were purple to brown in color and
elliptical to oblong elliptical in shape. They ranged in size from 6.7-8.2 um by
12.6-15.0 um. The fruiting bodies were examined mainly for color. The mature
mushroom was examined for shape, size, color, texture, gill characteristics, and
general appearance.
The original spore solutions were analyzed by TLC and by GC/MS. No psilocyn
or psilocybin were detected in any of the spore solutions.
Mycelium growth was observed from 4 to 6 days. Fruiting bodies were observed
from 24 to 48 days. The average amount of time for the primordia to appear was
32 days. Samples of mycelium were taken after 13 days of growth, 20 days of
growth, and at various other days of growth. A total of 29 samples of the white
mycelium growth were analyzed. No psilocyn or psilocybin was detected in any of
these 29 samples. Nine of the 29 samples were confirmed by GCIMS, and again no
psilocyn was detected.
Samples were analyzed after the first sign of growth of mycelium knots. A
total of 22 mycelium knot samples were analyzed by TLC. Samples were considered
to be consistent with a standard if their relative Rr value and their color
matched the standard also spotted on the plate. Samples were considered to
indicate a standard if their relative Rr value matched the standard but the
color was not as dark as the standard spotted. Of the 22 mycelium knot samples,
17 were consistent with psilocyn. Of these 17 samples, 8 were also consistent
with psilocybin and I indicated there was psilocybin in the sample. Four samples
were consistent with the psilocybin standard spotted on the TLC plate, and one
of these samples also indicated there was psilocyn in the sample. There was no
psychoactive drugs detected in one of the samples.
Samples were analyzed after the first pinheads of the fruiting bodies were
observed. A total of 25 samples of the pinheads were analyzed by TLC. All 25
samples were with the psilocyn standard spotted on the TLC plate. Of these 25
samples, 3 were also consistent with the psilocybin standard spotted and 3
indicated there was psilocybin in the sample (Table I).
The 22 mycelium knot samples were also analyzed by GC/MS. In the inlet system
of the gas chromatograph, thermal dephosphorylation of psilocybin occurs. As a
result of this degredation of psilocybin to psilocyn, one is unable to
differentiate the two by GC/MS. With this inability to differentiate psilocyn
and psilocybin, it is unknown if the starting material contains psilocyn,
psilocybin, or a mixture of both drugs. For this project, only a psilocyn
standard was analyzed by GC/MS (Figs. 7-9). Samples were considered to be
consistent with psilocyn if their retention time and mass spectral fragmentation
pattern matched that of the psilocyn standard. Samples were considered to
indicate psilocyn if their retention time was consistent with the psilocyn
standard and contained the prominent ions, but were lacking ions in the total
fragmentation pattern. Of these 22 mycelium knot samples, 12 were consistent
with the psilocyn standard. Seven samples were found to indicate psilocyn, and
there were three samples where psilocyn was not detected.
The 25 "pinhead" samples were also analyzed by GC/MS. Of these 25 samples, 19
were consistent with the retention time and mass spectral fragmentation pattern
as psilocyn. Three samples were found to indicate psilocyn and psilocyn was not
detected in 3 samples (Table 2).
Samples of the mature mushroom were also analyzed. Eleven samples were
analyzed by TLC and by GC/MS. All eleven samples were consistent with the
psilocyn standard spotted on the TLC plate. Of these 11 samples, 2 also
indicated psilocybin in the sample. All 11 samples analyzed on the GC/MS were
consistent with the psilocyn standard (Figs. 10-12).
There were some noticeable differences in the samples grown under indirect
light versus the samples grown in the dark. All samples started to show mycelium
growth at 4 days. The first signs of fruiting bodies were observed to be from 19
to 25 days in the samples that were grown under indirect light with the average
being 21 days. The first signs of fruiting bodies were observed from 23 to 45
days for the samples that were grown in the dark, with the average being 26
days. The samples that were grown under indirect light had primordia which grew
faster and larger. They were plump, yellow in color with brown tips. The samples
that were grown in the dark had small white primordia that were skinny and long.
The coloring was off-white with only a few having dark brown tips. The mushrooms
that were grown under indirect light had thick stipes with yellowish to chestnut
colored caps. The mushrooms that were grown in the dark had lighter stipes that
were much skinnier than the mushrooms grown in the light. The caps of the
mushrooms grown in the dark were also lighter in color than the mushrooms grown
under indirect light. Psilocyn and/or psilocybin was detected in the mycelium
knots, the pinheads and the mature mushrooms of all samples grown either in the
dark or the light.
The psychoactive drugs psilocyn and psilocybin were not detected in the
mycelium, the earliest stage of development of the mushroom. These drugs were
identified in the mycelium knots, the earliest stages of the fruiting body of
the mushroom.
Light affects the growth of the Psilocybe cyanescens mushroom. This affect is
apparent in the time of development and the appearance of the mushroom. Light
affected the color and size of both the fruiting bodies and the mature mushroom.
Light did not affect the presence of psilocyn or psilocybin in the early stages
of the primordia or the mature mushrooms, nor did it affect the ability to
detect these psychotropic drugs. It appears that the Psilocybe cyanescens
mushrooms are not photosynthetic, but are photosensitive.
The author wishes to acknowledge Dr. David McLaughlin, Plant Biology
Department, University of Minnesota for his time and assistance in identifying
the mushroom, and his explanations about the classifIcations of fungi and the
development of the mushroom. This project was supported by the Federal Bureau of
Investigation, Minneapolis Office which generously provided the supplies.
I. Minnesota Statues Chapter 152.02. Schedules of conlrolled substances;
Subdivision 2, Schedule 1. The following ilems are listed in Schedule I:(3) Any
material, compound, mixture or preparation which contains any quantity of the
following hallucinogenic substances, their salts, isomers and salts of isomers,
unless specifically excepted, whenever the existence of such salts, isomers, and
salts of isomers is possible within the specific chemical designation:
3,4-methylenedioxyamphetamine; 4-bromo-2,5-dimethoxyamphetamine;
2.5-dimethoxyamphetamine; 4-methoxyamphetamine; 5-methoxy-3,
4-methylenedioxyamphelamine; Bufotenine; Diethyltryptamine; Dimethyltryptamine:
3,4,5-trimethoxyamphetamine; 4-methyl-2.5-dimethoxyamphetamine; 4 Ibogaine;
Lysergic acid diethylamide; Marijuana; Mescaline; N-ethyl-3-piperidyl benzilate;
N-methyl-3-piperidyl benzilate; Psilocybin; Psilocyn; Tetrahydrocannabinols;
1-(1-(2-thienyl) cyclyohexyl) piperidine; N-ethyl-I-phenyl-cyclohexylamine;
1-(I-phenylcyclohexyl) pyrrolidine.
2. Kaul TN. Introduction to mushroom science. Enfield, New Hampshire: Science
Publishers, Inc., 1997.
3. McKnight KH and McKnight VB. A field guide to mushrooms. Boston: Houghton
Miffiin Company, 1987.
4. Rumack BH and Salzman E. Mushroom poisoning: diagnosis and treatment. West
Palm Beach, Florida: CRC Press, Inc., 1978.
5. Ammirati JF, Traquair JA. and Horgen PA. Poisonous mushrooms of the
Northern United Stales and Canada. Minneapolis: University of Minnesota Press,
1985.
6. Guzman G. The genus Psilocybe. Nova Hedwigia: Beih, 1983;74:1-439.
7. Guzman G. Supplement to the monograph of the genus Psilocybe. In: Petrini
O and Horak E, eds., Taxonomic monographs of Agaricales. Bibliotheca Mycologica
1995;159:91-141.
8. Singer R and Smith AH. Mycological investigations on teonanacatl, the
Mexican hallucinogenic mushroom: Part II. A taxonomic monograph of Psilocybe,
section Caerulescentes. Mycologia 1958;50:262-303.
9. Starnets P and Chilton JS. The mushroom cultivator. Olympia, Washington:
Agarikon Press, 1983.
10. Psylocybe Fanaticus (PFtek), 1996.
FIG. 1 - microscope photo of spores
There are several interesting points In the first article, "Detecting
Psychoactive Drugs in the Developmental Stages of Mushrooms".
1. The article was the result of a full FBI investigation of PF in 1998. What
saved PF was this; "The original spore solutions were analyzed by TLC and by
GC/MS. No psilocyn or psilocybin were detected in any of the spore solutions".
If these drugs would have been detected, PF would certainly be history and the
new spore syringe phenom would be over..
2. The article gave PF credit for the source of the spores, "The spores used
in this experiment were obtained legally through an advertisement in High Times
Magazine from Psylocybe Fanaticus (PFTek Seattle, WA)". This is quite amazing
because the FBI did not have to give any credit for this research, but they did.
Most likely, it was because a lot of money was invested for the research and
they achieved valid results. They even spelled Psylocybe Fanaticus correctly
incorrect with a Y, and not an I. Also, the FBI performed the PF TEK exactly as
written (credit given in the references section and footnotes) and because they
followed the PF TEK, even they were able to get a first time success, making
their research project and money spent, successful.
3. The paper identified the shroom grown (PF race) as a Psilocybe Cyanescens.
Everyone knows that the PF race is Psilocybe Cubensis and not Psilocybe
Cyanescens. Why then identify it as a wrong specie? The clue is here, "The genus
and species of Psilocybe mushrooms that were grown were identified as Psilocybe
cyanescens. The pleurocystidia sizes noted in the keys describing the Psilocybe
cyanescens mushroom varied slightly from the mushrooms grown. This may indicate
a variant of this species (communication with Dr. David McLaughlin, Plant
Biology Department, University of Minnesota) (2,4,5-8)". For the last few years,
PF has taught the concept of "spore race". What the scientists did was to ignore
what PF said about the identity of the shroom and went to the books and keys to
do an objective ID, and what they obviously saw was that the PF race shroom
looks more like a Psilocybe Cyanescens than a Psilocybe Cubensis. They refer to
it as a "variant of the species". This is more vindication of PF's new concept
of spore race as opposed to the common designation - "strain", which falls short
of describing these various races that do not mate, but stay unique and
separate.
4. They found no drugs in the young mycelium. This is very surpising, because
after ingestion of "tea" made from boiling down mycelium engulfed grain, a
slight "psilocybian buzz" can be felt for a brief time. The answer is that the
lab equipment could not detect an amount of psilocybin that the human psyche
can!
HALLUCINOGENIC MUSHROOMS ON THE GERMAN MARKET - SIMPLE INSTRUCTIONS FOR
EXAMINATION AND IDENTIFICATION
"The cultivation or possession of whole Psilocybe mushrooms and its spores
are restricted by German law since 1998"
Psilocybin and psilocin measurements (%) for 18 specimens of Psilocybe Cubensis,
9 specimens of Ps. Semilanceata, 6 specimens of Paneaolous cyanescens
and 4 specimens of Ps.Tampanensis.
Extreme variations in potency of a given collection of magic shrooms has been
reported ever scince reports have been done about these shrooms. And similarly,
this report also shows the extreme variability of psilocybin content amongst the
dried samples. So if one wants potent and satisfying Cubensis magic shrooms,
they should be grown for potency. And that is done by harvesting them in their
young stage, before sporulation begins. When that is done, even the most
different appearing spore races look about the same. When the caps aren't fully
expanded, all of the races look similar. The visual differences emerge when the
shrooms mature, but then when they mature, they are only good for spore
printing. These are weak in potency and unsatisfying for tripping. So the word
of wisdom is, grow them PF style, harvest them when they are young and cool dry
them with desiccant. When this is done, they are an entheogen of the highest
nature.
THIN-LAYER CHROMATOGRAPHY
GAS CHROMATOGRAPH/MASS SPECTROMETER
LOWER LIMIT OF DETECTION
RESULTS AND DISCUSSION
CONCLUSION
ACKNOWLEDGMENTS
REFERENCES
PHOTO AND CHART DESCRIPTIONS (figures and table-charts)
FIG. 2 - mycelium invitro
FIG. 3 - birthed PF cake with fungal pins
FIG. 4 - PF jar with invitro primordia
FIG. 5 - PF race shrooms on cake
FIG. 6 - PF shrooms on cake
FIG. 7 - 12 - GC/MS readout charts
Table 1 - TLC chart
Table 2 - GC/MS chart
PF comments
Chapter two
Potency comparisons of 4 species of "Dutch over the counter" Magic Mushrooms
excerpt from THE FORENSIC SCIENCE INTERNATIONAL journal. 113 (2000) 389-395
Psilocybe Cubensis Psilocybe Semilanceata
psilocybin psilocin psilocybin psilocin
none .14 .01 .48
none .05 .16 .13
none .10 .25 .08
none .10 .27 .24
none .11 .30 .03
.01 .05 .42 .04
.02 .09 .51 .12
.17 .09 .72 .01
.31 .23 .91 .90
.50 .12
.87 .04
.98 .03
1.07 .01
Panaeolous Cyanescens Psilocybe Tampanensis
Psilocybin Psilocin Psilocybin Psilocin
.02 .56 none .02
.44 .14 .01 .03
.47 .22 .03 .03
.51 .64 .19 .01
.54 .09
1.15 .90
PF comments