The Measure of the Mushroom
C.B. Gold
Taken from PM&E Volume Five
(OCR'd by GluckSpilz
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 absorbances(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 psychedelic trip.
One can mistakenly interpret the test results in other ways. In addition to the extra absorbance because DMAB reacts with other compounds in the mushroom extraction besides the ones we are most interested, the general enzyme class of phenol oxidases 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 concentration of DMAB, these enzymes effect a falsely low value for the tryptamines. This is another reason I use a low pH extraction solution(i.e. acetic acid in water) and heat the extraction mixture. Both of these procedural details should reduce the likelihood of losing DMAB through enzymatic activity.(8,p. 108>
As noted in the article on harvesting and storage, some ions will interfere with the color development of the DMAB. In particular, my experience shows that the bisulfate ion inhibits the reaction. Sodium bisulfite can be an alternative to vitamin C as an antioxidant and enzyme inhibitor for mushroom storage.
Quantifying the contribution of non-psilocybin/psilocin factors 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 concentration(absorbance value) of the DMAB test doubled the subjective effects of the mushroom. My earliest concentration values for the mushrooms tested were 0.6 A(absorbance units). When I subjectively tested mushrooms which had increased to 0.8 A, I was surprised to have a trip which seemed twice as strong.
After careful thought, I concluded that the initial concentration 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 percentage 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 separating the mushroom extracts, I cut out the zone which corresponded to psilocybin, using its known Rf value for paper chromatography and a 2% solution of DMAB in ethanol and hydrochloric acid(1:1) to conclusively identify the width of the zone by its color reaction. After cutting out the psilocybin zone, I extracted it in 5% glacial acetic acid at room tempera- ture overnight. I retested this extraction and compared the results with the extraction and test of the remainder of the cut up paper chromatogram which held the other factors in the mushroom extraction.
After correcting for variations in the sample volumes streaked on the paper and in spite of the poor separation achieved on the paper as compared to silica gel TLC, the results clearly showed that the other unknown but DMAB reacting factors in the mushroom increased their concentration with the increasing absorbance at a slower rate than psilocybin. In other words, the concentration of psilocybin increased with the increase in absorbance, which follows colorimetric theory, but as the absorbance increased, the psilocybin concentration contributed a greater percentage to the total color intensity 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 colorimetric theory is for a solution of a single absorbing molecule and as stated previously, there could be as many as twelve different 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:
THE COLORIMETRIC TEST
EQUIPMENT AND SUPPLIES
(Items marked with an "*" have expanded notes and explanations following the equipment and supplies listing.)
EQUIPMENT
25 grams p(para)-Dimethylamino benzaldehyde
1 pt concentrated . Sulfuric Acid
1 pt Glacial Acetic Acid
125 gr Ferric Chloride
Deionized water
SUPPLIES
Colorimeters
I acquired a used Bausch & Lomb Spectronic 20. I understand 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 suppliers. 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 application of reading at a fixed wavelength and a broad color peak, such as in this test, a low cost colorimeter is all that is necessary. All it needs is the proper filter (i.e. as close to 570 nm as possible), a cuvette or test tube holder and a scale in per cent transmittance,
There are three companies that I know of which can offer a low cost colorimeter:
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 mushrooms, 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 advertisements for small, Inexpensive scales which can weigh accurately 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 volumetrically marked for the standard extraction volume of 20 milliliters. To do this, simply fill up the 20 mil volumetric pipette with water then add it to the test tube to be marked while standing in the test tube holder. Then mark the bottom of the meniscus with an indelible marking felt pen. Do the same with the other five test tubes.
Matched cuvettes.
Cuvettes should be one centimeter ID, but because of variations in the extrusion Process for the tubes the ID differs slightly. This irregularity in the internal diameter and the occasional 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 percent 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:
The particular holder I use is an open style which is one tube deep and seven across. I use this holder for heating the extraction 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 balance the weight of the tubes in the three slots. The holder tends to float. For instance if you put a single test tube in the holder's end, the holder will float up dumping the tube into the heated water and thus losing your mushroom sample. To avoid losing your sample, when extracting a single sample put the tube in the center of the holder, or if using two test tubes, put them on either end of the holder.
Chemicals.
I have used technical grade chemicals throughout all my testing. Technical grade is somewhat less pure than reagent grade and usually less expensive. The sulfuric acid could be reagent grade which would be somewhat clearer than the slightly yellow-brown technicalgrade. Sulfuric acid is used in the color development solution and is read on the colorimeter, so clarity and lack of particles floating around would help readability and reproducability. I have had no problems with the less expensive technical grade by always using a sample blank to compare all mushroom test samples against it. A sample blank is used in colorimetry to set the "zero" point. It is usually the test reagent with a distilled water sample rather than the material or extraction to be tested. If the test reagent is colored in the spectral region of the test before any reaction, then by using a sample blank one can offset the colorimeter by this degree of color so that the test result is not artificially high.
Plastic sheet.
All that is necessary is a piece of plastic to cover your thumb so that when you shake the culture tube with the test reaction mixture the sulfuric acid will not burn your skin. A piece a couple inches square should suffice. Most plastics are unaffected by sulfuric acid.
[ NEXT SECTION ]
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 absorbances(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 psychedelic trip.
One can mistakenly interpret the test results in other ways. In addition to the extra absorbance because DMAB reacts with other compounds in the mushroom extraction besides the ones we are most interested, the general enzyme class of phenol oxidases 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 concentration of DMAB, these enzymes effect a falsely low value for the tryptamines. This is another reason I use a low pH extraction solution(i.e. acetic acid in water) and heat the extraction mixture. Both of these procedural details should reduce the likelihood of losing DMAB through enzymatic activity.(8,p. 108>
As noted in the article on harvesting and storage, some ions will interfere with the color development of the DMAB. In particular, my experience shows that the bisulfate ion inhibits the reaction. Sodium bisulfite can be an alternative to vitamin C as an antioxidant and enzyme inhibitor for mushroom storage.
Quantifying the contribution of non-psilocybin/psilocin factors 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 concentration(absorbance value) of the DMAB test doubled the subjective effects of the mushroom. My earliest concentration values for the mushrooms tested were 0.6 A(absorbance units). When I subjectively tested mushrooms which had increased to 0.8 A, I was surprised to have a trip which seemed twice as strong.
After careful thought, I concluded that the initial concentration 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 percentage 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 separating the mushroom extracts, I cut out the zone which corresponded to psilocybin, using its known Rf value for paper chromatography and a 2% solution of DMAB in ethanol and hydrochloric acid(1:1) to conclusively identify the width of the zone by its color reaction. After cutting out the psilocybin zone, I extracted it in 5% glacial acetic acid at room tempera- ture overnight. I retested this extraction and compared the results with the extraction and test of the remainder of the cut up paper chromatogram which held the other factors in the mushroom extraction.
After correcting for variations in the sample volumes streaked on the paper and in spite of the poor separation achieved on the paper as compared to silica gel TLC, the results clearly showed that the other unknown but DMAB reacting factors in the mushroom increased their concentration with the increasing absorbance at a slower rate than psilocybin. In other words, the concentration of psilocybin increased with the increase in absorbance, which follows colorimetric theory, but as the absorbance increased, the psilocybin concentration contributed a greater percentage to the total color intensity 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 colorimetric theory is for a solution of a single absorbing molecule and as stated previously, there could be as many as twelve different 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:
- 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 mushroom experience.
- The paper chromatography extraction experiment discussed 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 chromatography 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 corresponds 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 compounds. The value also means that all the samples had at least 0.3 A of non-active, but DMAB reactive, junk in the mushroom. Of course some of the active tryptamines, which can intensify the trip, may not necessarily be a pleasant addition to the trip either.
- 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 particularly 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 mixture and relative concentration of the various active tryptamines, such as psilocybin, psilocin, baeocystin and their analogs, changed in the mushroom powder.
THE COLORIMETRIC TEST
EQUIPMENT AND SUPPLIES
(Items marked with an "*" have expanded notes and explanations following the equipment and supplies listing.)
EQUIPMENT
- Colorimeter/Spectrophotometer*
- Hot Plate
- Quart Pan
- Balance capable of weighing at least one gram and
- accurate to 0.1 gr*
- Electric Coffee grinder
- 1 ea. 250 mil plastic graduated cylinder
- 1 ea. borosilicate 250 mil beaker
- 2 ea. 125 mil amber narrow mouthed bottle with caps
- 1 ea. 1000 mil bottle with cap
- 12ea 20 X 150mm borosilicate glass culture tubes*
- 6ea matched cuvettes for the colorimeter*
- Polyester cosmetic balls (available at most supermarkets)
- 6 ea. Small(60mm) plastic funnels
- 1 ea. Multiple funnel rack
- 1 Box Filter paper: 9 cm Coarse(like Whatman 4) and 9 cm Medium( 1 or 2)
- 1 ea. Plastic test tube rack (to hold about 10 of the 20 mm tubes)
- 1 ea. Plastic test tube rack (Nalge 5900-0007)$
- 1 ea. Test tube cleaning brush
- 1 ea. Volumetric pipette 20 mil
- 1 ea. Pipette 10 mil in 1/10 mil
- 1 ea. Pipette 1 mil in 1/100 mil
- 1 ea. Pipetting Aid (either Bel-Art F-37898 or Nalge 3780- 0100>
- 1 ea. glass stirring rod
- 1 ea. stainless spatula, double blade-rounded and squared
- 1 ea. 500 mil plastic wash bottle
- 1 ea. bright color nail polish or airplane glue
- 1 ea. small polypropylene plastic sheet about 4" square*
- 1 ea. thermometer, 0 to 110 degrees Centigrade
Colorimeters
I acquired a used Bausch & Lomb Spectronic 20. I understand 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 suppliers. 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 application of reading at a fixed wavelength and a broad color peak, such as in this test, a low cost colorimeter is all that is necessary. All it needs is the proper filter (i.e. as close to 570 nm as possible), a cuvette or test tube holder and a scale in per cent transmittance,
There are three companies that I know of which can offer a low cost colorimeter:
- Chemtrix(P.O. box 1359, Hillsboro OR 97123; 800-821-1358) has a colorimeter (20A) for $269;
- 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)
- 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 mushrooms, 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 advertisements for small, Inexpensive scales which can weigh accurately 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 volumetrically marked for the standard extraction volume of 20 milliliters. To do this, simply fill up the 20 mil volumetric pipette with water then add it to the test tube to be marked while standing in the test tube holder. Then mark the bottom of the meniscus with an indelible marking felt pen. Do the same with the other five test tubes.
Matched cuvettes.
Cuvettes should be one centimeter ID, but because of variations in the extrusion Process for the tubes the ID differs slightly. This irregularity in the internal diameter and the occasional 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 percent 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:
- 13 mm OD culture tubes will have a nominal ID of 10 mm. Buy at least a couple of dozen.
- 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.
- Set the meter to an arbitrary mid-meter position. Then slowly rotate the cuvette. Notice the variation as you rotate the cuvette the full 360 degrees. Choose the culture tube with the least variation.
- Continue with another set of a few cuvettes and keep selecting the least variable cuvette until you have six to ten cuvettes.
- 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.
- 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.
- 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.
- It is possible that the initial cuvette which you arbitrarily marked may not match any or only a few other cuvettes. Just start over but use another cuvette as your initial reference cuvette.
The particular holder I use is an open style which is one tube deep and seven across. I use this holder for heating the extraction 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 balance the weight of the tubes in the three slots. The holder tends to float. For instance if you put a single test tube in the holder's end, the holder will float up dumping the tube into the heated water and thus losing your mushroom sample. To avoid losing your sample, when extracting a single sample put the tube in the center of the holder, or if using two test tubes, put them on either end of the holder.
Chemicals.
I have used technical grade chemicals throughout all my testing. Technical grade is somewhat less pure than reagent grade and usually less expensive. The sulfuric acid could be reagent grade which would be somewhat clearer than the slightly yellow-brown technicalgrade. Sulfuric acid is used in the color development solution and is read on the colorimeter, so clarity and lack of particles floating around would help readability and reproducability. I have had no problems with the less expensive technical grade by always using a sample blank to compare all mushroom test samples against it. A sample blank is used in colorimetry to set the "zero" point. It is usually the test reagent with a distilled water sample rather than the material or extraction to be tested. If the test reagent is colored in the spectral region of the test before any reaction, then by using a sample blank one can offset the colorimeter by this degree of color so that the test result is not artificially high.
Plastic sheet.
All that is necessary is a piece of plastic to cover your thumb so that when you shake the culture tube with the test reaction mixture the sulfuric acid will not burn your skin. A piece a couple inches square should suffice. Most plastics are unaffected by sulfuric acid.
[ NEXT SECTION ]