TESTING SOLUTIONS PREPARATION:

Color development reagent

This mixture is the actual solution which when in the presence 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 weighing out 20 gr of the ferric chloride into 100 mil of distilled or Deionized(DI) water in the 250 mil graduated cylinder. Wear gloves. Ferric chloride can be corrosive. Make sure it all goes into solution. If any particles remain insoluble, filter the solu- tion. Store the finished solution in a 125 mil bottle and label. Rinse out the graduated cylinder.
3. Using the 1 ml pipette and the pipette-aid(never suck up corrosive or toxic chemicals in a pipette!) draw up 0.3 mil of the stock ferric chloride solution (#2,above). Add this to the 250 mil beaker with the DMAB already in it.
4. Add 35 mil of distilled or Deionized water to the 250 mil beaker and stir the water to dissolve the DMAB as much as possible.
5. Add 65 mil of Sulfuric Acid from the graduated cylinder to the beaker with the DMAB. Be careful with sulfuric acid. Wear gloves, an apron and preferably a face shield. When adding the acid, set the beaker on the table. Do not hold the beaker. An exothermic reaction will take place when you add the acid to the water present in the beaker and it will be too hot to hold. Gently stir the mixture. The heat of the acid-water mixture will drive the remaining DMAB into solution.
6. Let the mixture cool down and pour this stock solution into a 125 mil bottle and label it. Do not put hot liquids into the bottles; they may break.

Store the DMAB solution in a dark, cool location, preferably in the freezer section of your refrigerator. If you cannot store it somewhere cold, the solution will gradually darken and thus be unusable for colorimetry. I have successfully stored my stock solution in the freezer for over a year and it has not dark- ened noticeably. At room temperature you will probably need to discard the solution after a few months.


Acetic Acid Extraction Solution

This stock solution is used to extract the mushroom of its active tryptamines. Then a sample of this extract is tested using the above DMAB color development solution.

1. Measure out 50 mil of glacial acetic acid in the 250 mil graduated cylinder. Fill the cylinder to the 250 mil mark with DI or distilled water and pour it into the 1000 mil bottle. Rinse the cylinder with 3 X 250 mil amounts of DI water into to the 1000 mil bottle to make a total of 1000 mil stock solution of 5% Acetic Acid.
2. Distilled vinegar (i.e. "white vinegar") can be substituted for 5% acetic acid. Distilled vinegar is approximately 5% acetic acid.

THE para-DIMETHYLBENZALDEHYDE COLORIMETRIC TEST

See figure 6.

1. Set up the table or bench on which you will do your test with all the equipment in the above illustration spread out. Turn on the colorimeter or spectrophotometer to begin warm- ing it up and adjust or set the proper wavelength(570nm or close to this). Most have light sources which need to be on for 20 to 30 minutes to stabilize the spectral output. The older models also have slow-to-warm-up electronics.
2. Turn on the heating plate and begin to heat the water filled quart pan. Do not let the water boil. The boiling action will tip over the test tubes with the samples spilling out into the boiling water.
3. If you have not done so, use a coffee mill to grind up the dried mushrooms which you wish to test. Weigh out 0.5 grams of the ground mushroom. It's best when using a scale to use a piece of paper on which you weigh the mushroom. Find out how much this paper weighs before adding any mushroom powder then add this weight to the required half gram sample. This total weight in grams is what you should set the scale for if you have a triple beam-type scale. With the weighed mushroom 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 preheated near-boiling water. Note the time or start a timer. Heat this suspension for 30 minutes. This time is not critical. If you should heat it for longer than thirty minutes it will not make much difference. Too long will oxidize the psilocin in the solution and cause a false low reading of the sample. Also, the acetic acid solution does evaporate.
   
   Every ten minutes or so stir the suspension with the stirring rod. The mushroom powder tends to float on the acetic acid solution which cannot optimally extract the mushroom powder when it is not suspended. Also, the top of the powder can be exposed to air and will oxidize to a dark blue. Again, this can lead to a false low reading.
   
   The immersion of the glacial acetic acid solution in near boiling water with the mushroom powder during the extraction phase of the test is important to deactivate any enzymes which can cause bluing, and thus tryptamine loss, in the mushrooms.
   
   Without heat and with time the mushroom extract solution will slowly turn blue-green until it becomes dark blue after several days.
   
   As noted in the above section on theory, use a thermometer to make sure that the extraction solution reaches 70 degrees Centigrade for at least ten minutes. This temperature converts all the psilocybin to psilocin which makes for more pure test color.
   
   
5. While the mushroom powder is extracting, set up the next step: the filtering and sample development. For each sample, use one small filtering funnel. In the funnel put a polyester ball. Using the wash bottle, wet the cosmetic ball then press any excess water out of the cosmetic ball with your finger. Discard this water if it went into the collection test tube.
6. After 30 minutes or so, remove the test tube holder from the water bath. Turn off the heat plate(or stove). Just before the initial filtering, stir the suspension one more time. Pour the test tube contents over the top of the polyester cosmetic ball in the small filtering funnel which drains into a large(20X150mm) test tube on which you marked the 20 mil level. Do not worry if you cannot get every last drop of the suspension out of the test tube.
   
   Let the filtrate pass through the small filter completely by gravity. Sometimes pressing the cosmetic ball will succeed in driving more water extract off the polyester fibers. This step is important because if you don't get all of the original, undiluted extract in the final test sample, your concentration value will be falsely low. I have occasional-ly forgotten to do this squeezing step and have had readings which were off by 25%.
   
   Although you could get more consistent results by just using filter paper instead of the cosmetic ball, by filtering the thinly mucilaginous mushroom suspension with the cosmetic ball you can save literally hours of waiting for all the suspension to get through the paper filter.
   
   
7. Now use the wash bottle filled with distilled (or deionized) 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 20 ml mark you are forced to discard the water which may contain some of your sample. The results may then be artificially low. Solution: add smaller amounts of rinse water with the squeegee bottle, so that you will not leave any of the liquid mushroom extraction on the filter that will then not be part of the test.
   
   If in the process of rinsing the sample on the cosmetic ball and adjusting the 20 mil volume, you should over-fill the test tube beyond the 20 mil mark, just correct the final absorbance by multiplying the absorbance by a fraction consisting of the volume which was filtered (over 20 milliliters) as the numerator and 20 as the denominator.
   
   
8. This rough filtered extract is your test sample but needs to be filtered again so that it is clear and will not add absorbance to the colorimeter reading because of its cloudiness. Pour this extract through another small plastic funnel with a folded piece of filter paper in it. Fold the filter paper on itself three times (i.e. into eighths).
   
   Most extracts will have some colloidal suspension in them, making them look slightly cloudy. Even filtering these with a fine filter will not remove all the turbidity. For most filtering you will need to remove only coarse particles and the Whatman #4 filter paper will suffice. For heavier suspensions use the finer, but slower, Whatman #2 filter paper. The idea here is to obtain a reasonably clear sample which has the least amount of turbidity. Turbidity, if excessive, will give false high colorimetric readings.
   
   For the receiving container for this filtrate, use another large test tube as the receiver. After a few milliliters of fluid has passed through, you can stop the process if you wish. I usually like at least enough prepared sample for a couple of tests(each tests requires 0.5 or 1.0 ml of sample), just in case I wish to repeat a reading.
   
   
9. The preceding two-step filtering process should take 10 to 15 minutes. While the filtering is proceeding, using the pipette-aid and the 10 mil pipette, draw up 2 mil of the DMAB reagent and dispense it into a matched cuvettes/small test tube. Continue dispensing the DMAB reagent into a matched cuvette for each sample or test you may wish to run. Now you are ready to develop the color and read the final absorbance. If you store the DMAB test solution in the freezer, allow it to return to room temperature before using it in a test.
10. Using the pipette-aid and the 1 mil graduated pipette, draw up a 1 ml sample of the mushroom extract to be tested. If you anticipate an absorbance reading above 0.700 A or so, then draw up only 1/2 mil of sample, but before doing so add a 1/2 mil of Deionized or distilled water to the small reaction test tube/matched cuvette.
    
    The reason for this dilution is that for colorimeters with a transmittance scale, the most readable portion of the scale lies above 20% transmittance. Lower transmittance than this corre- sponds to higher absorbance and greater possibility of reading errors and poor precision. I checked to make sure that both ways of testing equaled the same value after making the necessary 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 sample will become turbid. If so, dilute with 3ml of DI water, read and divide the absorbance result by two. This dilution will usually water down the turbidity enough to be able to read the test.
    
    It is important to test the mushroom extraction solution soon, otherwise the bluing and other oxidation reactions will breakdown the active tryptamines and cause you to have a false low value for the test's absorbance. By testing the mush- room extract solution at regular intervals, I found that no sig- nificant reduction of the active tryptamine concentration occurred until after six to eight hours.
    
    I have always conducted this test under two 40 watt fluorescent 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 incandescent lights or sunlight coming through a glass window. The absence of such lighting in your test area may slow the reaction 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 reading 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 transmittance 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 absorbance. The formula is: Absorbance Log (1/.01 X Transmittance in %).
    
    The easiest way to calculate this is to buy an inexpensive calculator with a "log" and "l/x" or reciprocal functions. Then calculate by entering the transmittance as a number between one and one hundred, divide by 100, push the "1/X" key then push the "Log" key. The result will be the absorbance which is proportional to concentration.
    
    
    

CONCLUSION

It is my hope that the above helps to explain the test values which will be used in the upcoming articles. The next article on the environmental and nutritional influences on the growth and biosynthesis of psilocybin in P. cubensis will make use of this test extensively. The last section of this article, of course, is for those people inclined to do their own testing. I encourage anyone with any amount of chemistry background to try this test. The test values will help immensely in planning your own entheogenic experiences with the "magic mushroom."


BIBLIOGRAPHY

1. Agurell, S., Blomkvist,S. and Catalfomo, P.. "Biosynthesis of Psilocybin in Submerged Culture of Psilocybin cubensis: Part I. Incorporation of labeled tryptophan and tryptamine," Acta Pharm. Suecica, Vol. 3 (1966), 37-44.

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 (1968), 1210-1218.

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, editors. "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 (1981), 229-235.

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 Publications, 1976.

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. Wiley-Interscience, 1970.

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, 1977.

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., 1976.

.... To be continued.