Chemical Tests and Stains For Fungi

Transcription

1 Chemical Tests and Stains For Fungi By John Plischke III - Fungi01@aol.com Macro Tests Micro Tests

2 Note Taking Note taking is not requited except for this download link which is a copy of this program 20Tests.pdf It will be available here for the next 2 weeks only

3 Material data safety sheets They are in a folder called Reagent Kits e_sunday/reagent_kits.zip Make certain to print the ones that you have and keep them with your chemicals!!!

4 Additional Material data safety sheets Ammonia %20ammonia%20solutions%2010%20percent.pdf Sodium dodecyl sulfate A/USA_USA_ pdf Methylene blue ne_blue_msds_eu.pdf

5 Ordering Stains, Reagents and Chemicals -USA 1. In the United States -Most chemical companies will not sell to individuals or ship products to home addresses 2. Look on places like Amazon, E-bay and the handful of stores that will ship to individuals. 3. Also make certain that nothing is on a precursor watch list. There are several of them. Some are precursor chemicals to drugs or explosives

6 Ordering Stains, Reagents and Chemicals From Overseas 1. In some other countries they can be easier to get and some can be legally shipped here 2. Make certain none are on a watch list. 3. There have been many cases where people importing glassware for a home chemistry lab from China get raided. The customs form triggers it. Local Police think Meth Lab. The Government is watching you!!!!

7 Ordering Stains, Reagents and Chemicals -Overseas 4. Chinese sellers will often put down on the customs form whatever you ask them so hopefully the package will not get intercepted. They are so nice and agreeable that they will even send the DEA your asking them to relabel you shipment when asked. 5. Occasionally overseas sellers will relabel packages to avoid poison pack fees that can often be in the 40 dollar range. This is done on their own and your package will be lost and not replaced if intercepted. Make certain not to order anything without having the fee paid if this is a must needed item.

8 Ordering Stains, Reagents and Chemicals -UK 1. Chemical sales in the UK are lax. You can even order chloral hydrate (Knock out drops) Don t try having it shipped here The DEA may arrest you 2. UK - easy to get most things 3. Shipping takes about a month sometimes up to 2 months 4. Shipping is high. Often around 12 to 25 dollars or more for a single small item.

9 Ordering Stains, Reagents and Chemicals Turkey, China, Japan 1. Shipping from Turkey and China often cost less than a dollar. Less than me sending the same thing from my house to the neighbors. Expect a 1-2 month wait. Quality of item is sometimes uncertain from China 2. From Japan shipping is expensive, often 15 dollars or so shipping for a small item but they are often high quality items with a 2 month shipping wait.

10 Ordering Stains, Reagents and Chemicals -Overseas At the time the only place I could find Canadian Balsam Really what s going on Looks like I got a tool box. Items were very small I could not read size

11 Premade or mixing your own reagents? There are a number of advantages of purchasing premixed stains and reagents A. Its more convenient. B. Its can be safer by not having heat and other violent reactions occur when chemicals are mixed together Example KOH and water C. There is no need for the extra expense of lab equipment to weigh, mix, and other necessary things D. No complicated formulas to follow

12 Premade or mixing your own? Advantages of premixed stains and reagents E. Often multiple chemical are required for a single formula each often costing 5 or 6 times more than a pre mixed dropper bottle that was ready to use. - Example The List price for Sodium Dodecyl Sulfate 1kg is about $ and it is just a wetting agent for some stains F. No need to store necessary supplies and equipment that is not currently being used

13 Premade or mixing your own? Advantages of mixing your own stains and reagents A. Many chemicals once mixed expire and will not work after a period of time. With having your own bulk supply, if you run out it can be quick and easy to mix some more. (finding and order premixed chemicals can take a while) B. Even though the initial cost is higher because of bulk chemical purchase, the ml price of all that can be made in the long run is cheaper. (like bulk food)

14 Premade or mixing your own? Advantages of mixing your own stains and reagents C. The quality (of premixed) often can not be determined. They can use inferior and impure ingredients. Example would be a stain used rubbing alcohol etc. D. The mixture of premixed ones often can not be determined. It may not be to the right formula that you desire or it could be water based vs alcohol based. Many Asco experts prefer water based formulas for vital (In viro) taxonomy but often they do not keep as long

15 Premade or mixing your own? Advantages of mixing your own stains and reagents E. Some reagents like Melzers are so hard to get its good to keep a stockpile just in case you don t get an opportunity to get more for several years.

16 Safety A Gloves, lab coat and safety glasses, poisonings etc. B Using the proper type bottle 1. Dropper bottles bulbs dissolve 2. Caps for the bottles 3. Types of bottles

17 Safety C chemicals reacting to each other 1. When mixing temperature etc. KOH+Water = lots of heat 2. Which order to add chemicals --Always add your KOH or lye to the water, not the other way around. Adding the water to the KOH can cause a volcano type reaction. This stuff can blind you if it gets into your eyes 3. When applying on a mushrooms cap etc. etc. don t let drops of reactive chemicals touch or run together there may be a dangerous reaction

18 PETE Bottles Polyethylene Terephthalate Products like Ammonia will weaken and make the bottle fail. Not good for a number of chemicals

19 Unlabeled Bottles Don t use them for anything that could cause a problem. I would not depend on them

20 Nalgene Bottles LDPE- SPI resin ID code 4 - these are decent bottles for many chemicals but some will eat through them. A film canister is in the left for scale, do not use it, its not LDPE. I get mine from the REI store search for Nalgene

21 The second one on the left is a nice little dropper bottle for about $2.50 Nalgene Bottles LDPE Low Density Polyethylene. Buy a name brand they are expensive. Read reviews on cheap imports which many have poor reviews for leakage. Read online chemical compatibility charts to see if you chemical is compatible with the type of bottle that you want put in it.

22 Nalgene Bottles Compatibility Chart Caution use other charts for different brands of bottles Retrieved from CP Lab Safety m/chemicalcompatibility-bottlescontainers/

23 TricorBraun Boston Round LDPE dropper Colors and Bottle reviews The 15mm black ones are 37 cents each from United States Plastics Corp

24 Which is the best squeezable Bottle It depends on which chemical some chemicals can only be used in a certain type of material but Teflon bottles FEP seen like they have a wide range of resistance to many but not all items. Plastic type bottles like this often cost dollars each!!!! That why they are not often used. PFA is also good.

25 Glass Dropper Bottle Sizes 30 and 15ml Glass tip on left and plastic which is made from?

26 CAUTION KOH can Etch Glass- Borosilicate glass by Pyrex may be more resistant than the cheaper glass bottles Glass Dropper Bottles Rubber top dropper bottles caps are not made for long term storage!!! They are only for short term use Many chemicals will eat away the rubber or plastic squeeze top

27 Black Polypropylene Ribbed Cap Chemical Resistant Phenolic Cap with PolyCone Liner Made from LDPE plastic

28 Qorpak Green Melamine Thermoset Plastic Caps with PTFE Teflon Lined Cap Much more resistant but does not work with everything

29 Misc. Storage items

30 Cap and Bottle Measurer They will mail you this for free or you can download and print a copy from HowtoSelecttheRightSizeCapfory ourbottle Often needed to match lid sizes to bottle threads etc.

31 How to make the Solution A balance or a scale will be needed to weigh any solids A graduated cylinder/volumetric flask to measure the water or liquid Temperature resistant glassware A glass / Magnetic stirrer

32 Time It should take about an hour to measure and combine 2 to 4 different solutions

33 Some Mushroom Clubs Make Scientific Kits The WPMC

34 Some Mushroom Clubs Make Scientific Kits The WPMC does these kits at cost

35 3 Groups A Macro-chemical tests B Stains etc. for under the scope C oil etc.

36 Macro-chemical Tests For Field Testing A drop of the chemical is often applied to the mushroom to produce a color change Many mushrooms have been chemically tested but many have not. For Boletes there is a Bolete Filter That is starting to collect a more extensive list of chemical tests and uses online choice key to help identify Boletes

37 Ammonium Hydroxide NH 4 OH and Household Ammonia NH 3 MIXING solutions of 2%, 3%, 10% and 20% are commonly used but lesser concentrated ones more common for scope work

38 Ammonium hydroxide This Xanthoconium turns olive with a drop of Ammonia. Ammonia produces a number of different color reactions on different bolete species and other fungi On Leccinum 10%

39 Ammonium hydroxide and Ammonia CAUTION: Do not mix it with iodine can cause explosions 10% Selected Macro-testing examples : Omphalotus can turn greenish when ammonia dropped onto the cap cuticule Also used on Russula, Lactarius, Cortinarius and others Always use the clear Ammonia not the yellow lemon scent

40 Hypocrea pallida Red with Ammonia

41 A 3% solution is often used Crystals are used to make a few scope solutions Carbolic acid Crystals (=Phenol crystals) C 6 H 5 OH Used on Russula and Cortinarius Russula reactions are often shades of purple, violet, shades of browns or chocolate or current colored. Used on A few Amanita, Amanitaceae, Leccinum, Lactarius, Tricholomataceae. If the result does not show in 20 minutes its a negative reaction

42 A 3% solution Reactions are often shades of purple or browns or current colored here on Lactarius, Carbolic acid Crystals (=Phenol crystals) C 6 H 5 OH

43 Carbolic acid Crystals How to Mix: Keep away from light To make a 3% colorless solution, put 3g of the white colored crystals in a Volumetric flask or graduated cylinder and fill it with double distilled water until it reaches 100ml. Then stir and bottle. We need to convert this to a smaller quantity which is not easy. Some people use a 2 percent solution Shelf Life: about 2 years but use reactions to verify - Very Dangerous

44 Ehrlich s Reagent Made by mixing p dimethylaminobenzaldehyde, ethanol and Hydrochloric acid. There are several formulas others may use phosphoric acid instead of Hydrochloric acid Used to test for LSD, Psilocybin etc Lasts a few months and best stored in a freezer

45 Ehrlich Reagent Fails just about all of the time, I was very disappointed but my picking friends were very pleased

46 Ehrlich Reagent

47 Iron(III) chloride Ferric Chloride FeCl 3 Used on Boletes - the book that has a lot of results from it is California Mushrooms - A Field Guide to the Boletes by Harry Thiers available online at b.com/boletes/index.

48 Iron(III) chloride Ferric Chloride FeCl 3 Pistillarin test A piece of the fungi (Ramaria) etc is macerated and mixed with a Ferric sulfate solution. Pistillarin can cause GI upsets like with Ramaria poisonings. If pistillarin is present in a Ramaria a bluish green color will be produced. This test also can be used on Lyophyllum connatum. Connatin and other chemicals that the fungi contains react with the ferric Chloride to produce a violet color reaction.

49 Iron(III) chloride Ferric Chloride FeCl 3 It can be used to test for Orellanine in Poisonous Cortinarius species, a grayish blue color suggests a positive result -See Schumacher, T. and Hoiland, K. Mushroom Poisoning Caused by Species of the Genus Cortinarius. Orellanine test In the North East C. armillatus has tested positive see Toxicon feb 2016 Arch. Toxicol. 53: enus_cortinarius_fries

50 Iron(III) chloride Ferric Chloride FeCl 3 Chanterelle Vs Jack-O-Lantern

51 Ferrous sulfate Used on A.Boletes, FeSO 4 *7H2O Iron (II) sulfate heptahydrate B.Russula gray, pinkish becoming green, blue green, salmon, green, brownish and no reactions C.Coral fungi D.Cortinarius E. Lactarius Ramaria 10% solution F. Tricholoma. G.Lyophyllum, etc

52 Ferrous sulfate Most people use a 10 percent solution but a few use a 20 or 30 percent solution by weight which gives faster and brighter results - Others use large cm sized crystals and rub them on the fungi- they have a longer shelf life Chemistry Set Refill Shelf Life: sol. about a few weeks to several months if non acidic. It oxidizes over time and becomes orange colored. Soon reactions will not work or cant be see under the orange colored rust. Replace it frequently.

53 Ferrous sulfate Used How to Mix: crystalline form -10% solution A. put 100ml of double distilled water in larger Volumetric flask. B*. C. Then mortar 10g iron sulfate and add it to the water. Stir and bottle. An hour in a magnetic stirrer can be helpful. B*= some people add 20 drops of 50% sulfuric acid to the mix. This will make the shelf life about 1 year since it will oxidize slower. In this form it is called a 10 percent acidic sol. Also a alternative formula in the same ratio substitutes the white colored Anhydrous ferrous sulfate for the green above 1g of crystals in 10ml distilled water Solution Ph 2-3?

54 Ferrous sulfate Exixor an iron supplement from the drug store - It will not work and should not be used I used United Research Laboratories brand. Its inactive ingredients are citric acid, FD&C yellow # 6, Flavor, propylene glycol, Saccharin sodium, sodium benzoate, Sorbitol solution, sucrose, and water. The label says that each 5ml contains: ferrous sulfate 220mg (44 mg of elemental iron) alcohol 5% by volume. It is used as an iron supplement and can be purchased at a drug store over the counter. It costs about $5.00 for a 1-pint bottle. I discontinued testing it because it can stain differently than the 10% ferrous sulfate by having a yellowish, orangish or other color of stain which can be the same or different. It can also fail to stain all or any part or parts green on some mushrooms unlike the ferrous sulfate or it can stain them much lighter in color or have the same color. Its results are not consistent with the 10% ferrous sulfate and it should not be substituted for it.

55 Formaldehyde - Formalin A drop is applied to the fungi approximate 37% solution is used Used on Boletes the flesh of Tylopilus felleus turns pinkish, also used on Cortinarius, Lactarius, Russula, Tricholoma, Gomphidius and Leccinum Example of a reaction would be reddish as with L. scabrum, some reactions take 5-20 minutes

56 Formaldehyde It is toxic and corrosive Keeps several years, and sometimes older solutions can be brought back to life by using a special procedure Leccinellum crocipodium test

57 Guaiac Has been used macroscopically by placing a drop of it on Russula and Cortinarius. Example a drop on Russula can have a bluish green to grayish reaction or even purplish On gills and stalk base

58 Guaiac Clitopilus prunulus flush is not supposed to turn blue while the toxic C. dealbata and a few others have flesh that turn blue

59 Guaiac Some Tricholoma have a reaction The orange to blue reaction took a few minutes

60 Guaiac How to Mix: put 10g Guaiac resin in a Volumetric flask and bring to 100ml with 75 percent Everclear or ethanol. (If the Everclear is 95 percent first dilute it with double distilled water) Put on magnetic stirrer for a few hours then filter and bottle. Some say it should be changed every year and others say they have no problem after several years of using it. Use the reaction. Caution the solution is flammable

61 Metol A chemical typically used in Photography Used on Cortinarius. A drop is placed on the mushroom. Reaction colors can range from none to violet-purple, red becoming purple, and yellow.

62 Metol How to Mix: Formula A: Put 5g of powdered Metol in a flask and add 50ml of almost 195 degree f hot double distilled water so it dissolves. Shake and put on the magnetic stirrer for 5 minutes. Then bottle once it cools. Keeps about 1 year. Formula B: put 50ml formaldehyde, then 1g powdered Metol, then 1g carbolic acid crystals, and put on the magnetic stirrer for 30 minutes, then bottle. Keeps about 5 years. Must be put in Amber bottles - light sensitive

63 Metol A good example of a negative reaction on Cortinarius evernius Not all reactions are positive

64 Muriatic acid -bad lid Hydrochloric acid is what is used for testing it is 37percent Muriatic is usually less pure with more contaminants. Both have the same Formula HCl. Muriatic may be 31.5 but varies greatly Acid Magic brand supposedly a lot safer but maybe about 20% CAUTION: Nasty stuff Severe Burns and Even the fumes can get you

65 Muriatic acid -hydrochloric acid Used for the Meixner test for amatoxins The test involves squeezing juice from the mushroom onto newspaper that has a high lignin content. Drawling a circle around the spot and letting the juice dry. Apply 2 drops of hydrochloric acid in room less than 80 degrees and out of the sun- allow up to 20 minutes for low amounts of amatoxins Typically false positives can be present

66 Muriatic acid -hydrochloric acid Used for the Meixner test for amatoxins Only 1 in 7 of the sample tests produced a positive blue spot and the Galerina marginata were from different logs at different locations A Blue Green reaction indicates Amatoxins Left By Christine

67 Muriatic acid -hydrochloric acid CAUTION Very dangerous if mixed with Ammonia or sulfuric acid and others. If its even put close to Ammonia a poisonous gas can be formed The middle spot is it on the cap of Xanthoconium purpureum

68 Para-cresol Used for macro testing on Amanitia Used in an aqueous solution Positive results are orange to red in color. It is test for tyrosinase See Mycologia Marr et. Al. 78(2) 1986 pages Available for free at

69 Potassium Hydroxide KOH It is used for both macrochemical and macroscopic reactions but in different percentages For Macro-chemical tests a 10 percent solution is common

70 Potassium Hydroxide KOH EXAMPLES It can make the upper flesh tissue of Fomes fomentarius turn deep blood red Hapalopilus croceus Reddish Purple with KOH Hapalopilus nidulans - Violet

71 Potassium Hydroxide KOH KOH is used to test many different types of mushrooms. I probably use it most for Bolete testing. Others include Cortinarius deep brown or green or red or yellow, Lactarius, Russula, Amanita section Phalloideae etc. I order several pounds at a time and repack it into smaller LDPE or HDPE SPI resin code 2- containers because it attracts moisture and the flakes lock up and stick together easily when water is absorbed. KOH Slowly Eats glass

72 Potassium Hydroxide KOH MIXING 10 Percent solution A 10% aqueous solution of KOH in water is made by taking 10 grams of KOH and dissolving it in water up to 100 ml according to a mass vs. volume formula (m/v)%. But this would be a incorrect formula, because there are other things to consider But its not that simple this reaction also creates heat so temperature resistant glass must be used and add the chemicals in the proper order

73 Potassium Hydroxide KOH MIXING 10 Percent solution But KOH is hygroscopic and absorbs water from the air - for the most accurate formula. A 10% aqueous solution of KOH in water is made by taking 11.7 grams of KOH and dissolving it in distilled water up to 100 ml. That would figure out using 85.4 percent purity KOH. It is common for tech grade KOH to be 85 percent pure, the rest is water etc.

74 Sodium Hydroxide LYE MIXING A 10 Percent solution for macro tests. Many sources say it can be substituted for KOH for both macro and Micro testing. When it is follow the mixing instructions listed for Potassium Hydroxide It is corrosive. Keep away from the eyes & skin

75 Sodium Hydroxide LYE - NaOH On Amanita bisporigera It also should be stored in tightly sealed bottles just like KOH because it absorbs water from the air. It also reacts with it which causes the formula to change

76 Syringaldazine Mixed in an ethanol solution 0.1g of syringaldazine is added to 100ml of 95%ethanol Used to test Amanitas. The drop is left on it for 20 minutes and the color reactions recorded. Positive test results are pinkish, or lavender or purplish in color. There are 106 test results for it listed on the Amanita study site ringaldazine Its reaction occurs because of the laccase in the fungi

77 Sulfo-Vanillin Sulphovanillin Sulfovanillin Used both Macro and Microscopically Used on several mushrooms macroscopically Russulas can have purplish to carmine (pinkish red) colored reactions It has been used on Psathyrella and Panaeolus It is reported to turn the gills of Amanita phalloides / Amanita virosa purplish. Amanita bisporigera

78 Sulfo-Vanillin Sulphovanillin Sulfovanillin Amanita bisporigera gills have a purplish reaction

79 Sulfo-Vanillin -Sulphovanillin Smells like Vanillia Formula = 1 cc distilled water, 2cc sulfuric acid (some russula formulas use 1cc), and 2.5 mg vanillin. OR less precise one forceps pinch of vanillin to a drop of 50 percent sulfuric acid mix with a tiny glass rod It should be made in small quantities each day before use, because it does not keep once mixed. Sometimes it will last a couple of days

80 Sulfuric acid MIXING A 50% or concentrated solution is used to test boletes and other fungi. Amanita phalloides gills can turn purplish or vinaceous but also has reactions with others

81 Sulfuric acid Stropharia Chemical test Yellow with Sulfuric acid Auto Store and Hardware store but do not get colored or impure A 50% VERY DANGEROUS it will burn the hell out of you!

82 Macrochemicals Where and costs 1. Ammonia Hydroxide Ace hardware about $3 for quart 2. Carbolic acid Crystals-Phenol Ebay about $17 for 100g 3. Ehrlich Reagent Ebay about $11.00 for 3ml 4. Ferric Chloride Radioshack liquid Ebay about $11 for 4oz 5. Ferrous sulfate Ebay about $9 for 4oz 6. Formaldehyde Ebay about $7 for 1oz 7. Guaiac 8. Metol Ebay about $12 for 80g 9. Hydrochloric acid ebay $8 for 1oz as Muriatic acid Hardware store $5 10. Potassium Hydroxide KOH Ebay $8 for 8oz 11. Sodium Hydroxide LYE 12. Sulfuric acid Hardware store as Lye ebay $10 for 16oz 13. Vanillin Ebay $5 for 25g

83 Stains and Micro-chemical Testing and other Microscope items STAINS - a drop of the reagent is often applied to the slide to highlight a feature or make one stand out or produce a color change Reagents Also used under the scope often to help show a feature

84 Aceto Carmine See Swiss book V3 It is used to stain the basidia contents, which turn violet black to dark purple black. Used on Asterophora, Calocybe and Lyophyllum in that manor. Useful to view Siderophilious basidia in Tephrocybe and basidia contents in Asterophora

85 Aceto Carmine There are several different formulas but a more commonly used mycological one from (based on Henderson, Orton, and Watling, 1969) is: Boil 45 ml of 45% acetic acid in 55 ml of distilled water with 1 to 4g or carmine for 30 minutes then filter. In a different container mix 45ml of 45% ethanol with 55 ml of distilled water. Then add the 2 solutions together. A drop of Ferric hydroxide per 30ml of solution can be added to intensify the color reaction. This will keep several years in a sealed dark container. I also tape

86 Aceto-Orcein The natural form of this stain is made from 2 lichens. It can be used to stain Furia ithacensis spores. It will stain the chromatin that is in the spores multiple nuclei a reddish color

87 Aceto-Orcein To make it fresh for each use (reportedly has better contrast) 45ml of boiling acetic acid is poured over 1 gram of the powdered orcein. Then once cool 45 ml or double distilled water is added then everything is filtered. There are several formulas sucrose and gelatin for a longer shelf life It is best to buy this premixed because mixing it is to dangerous unless in a lab setting under a flow hood with proper safety knowledge

88 Ammonia /Ammonium hydroxide Ixocutis - The outer surface of the fungi, 2-3% Useful for brownish spores Photo Renée Lebeuf

89 Ammonia /Ammonium hydroxide When used on some Conocybe such as C. tenera, long needle looking shapes are formed when looking at the gills under the scope. They are not seen without ammonia 2-3% but can measure um in length. (it takes about 15 minutes for the reaction to occur) Useful to see chrysocystidium of Hypholoma, Pholiota and Stropharia and Marginal cystidia on Hypholoma and Stropharia

90 Ammonia /Ammonium hydroxide It is also used on dehydrated fungi. First the piece is wetted in ethanol then they are put into ammonia hydroxide to make them swell to close to their original size 2-3% Caution Do not mix with iodine -

91 Brilliant Cresyl Blue Aqueous solution The BMS formula is.5 to 1g in 99.5 to 99ml of distilled water, stir and wait 10 minutes then filter for a aqueous solution that will have a shelf life of a few weeks. An alternate French formula also uses 0.5g of Sodium dodecyl sulfate as a wetting agent There is also an Alcoholic Formula

92 Brilliant Cresyl Blue Alcholic solution Formula is 2g of Cresyl Blue, 98ml ethyl alcohol and 0.5g sodium dodecyl sulphate, and 100ml of distilled water. Stir in magnetic stirrer for a couple of hours then filter if needed. This formula keeps several years. There is also a Clemencon formula

93 Brilliant Cresyl Blue Geoglossum glutinosum - Hyphes It is Useful to view species with Gelatinous hyphae Photo Renée Lebeuf

94 Brilliant Cresyl Blue A metachromatic reaction can take place that can cause spore walls and some hyphae to turn violet to reddish or reddish violet in color. Positive reactions that have the color reaction can happen for example in Macrolepiota, Leucoagaricus, Leucocoprinus spores, and to the hyphae of only specific species of Mycena. Agrocybe, Boletus, and Resupinatus gill trama can have the reaction and so can the basidia of tricholoma A Orthochromic reaction can also happen it is dark blue colored Photo Renée Lebeuf

95 Carbol Fuchsin It is used to stain acido-resistant structures parts. An example would be with Russula sp. It would be stained for about 5-10 minutes then washed with weak (10 percent or less) Hydrochloric acid, then washed in water. Then the hyphae or macrocystidia/ would be examined under the scope.

96 Carbol Fuchsin There are at least a half dozen recipes An example: for mixing it. Basic Fuchsin 0.5g, 90 percent Alcohol 5ml, distilled water 50cc, carbolic acid crystals 2.5g. Typically once stained a drop of 10 percent hydrochloric acid is added then distilled water It is much easier and safer to get it premixed. Don t mix it if you don t know the dangers. Also the cost of the ingredients to mix this one is on the expensive side. Very Toxic and can cause severe burns

97 Congo Red It is one of the most commonly used mycological dyes. It is an excellent stain for staining cell walls of fungi. (Hyphae walls and Spore Walls)

98 Congo Red Cheilocystidias - Lepiota Photo Renée Lebeuf

99 Stir and bottle - It is reported that SDS Congo red should not be used with KOH but Congo red can 1% Congo Red Aqueous and SDS solution Put the following into a volumetric flask A. 1/2g of Congo red powder B. 0.25cc clear dishwashing soap This is an optional ingredient (a wetting agent) or 0.25cc or g (SDS) Sodium Dodecyl Sulfate or Kodak photographic wetting (Photoflo) agent can used instead of the dish soap C. Then add distilled water -bring it up to 50ml

100 1% Congo Red Ammonia Put the following into a volumetric flask A. 1/2g of Congo red powder B. Then add full strength Ammonia bringing the volume up to 50ml Stir, filter then bottle lasts about a year Danger Do Not mix solutions with Ammonia with iodine - explosive

101 1% Congo Red Ammonia ften the stain once applied needs ashed to get nicer contrast. First make our slide and add the piece of fungi, ongo red stain and add the cover slip. If etter contrast is needed once you bserve you specimen just add a drop of mmonia hydroxide to one side of the over slip, on slide across from it use a mall piece of absorbent paper towel to raw the ammonia under the cover slip nd remove the excess liquid.

102 Crystal Violet It works well under the scope with some Inocybe Mycologist use it like Gentain Violet For one formula see A519&lpg=PA519&dq=Crystal+Violet+mycology&source=bl&o ts=midntiblpt&sig=c1oqmc8- qkmt1pejrp7qgtak9h4&hl=en&sa=x&ved=0ahukewjaqlcuk PzMAhWHGj4KHZn4DRYQ6AEIKDAB#v=onepage&q=Crystal% 20Violet%20mycology&f=false

103 Eosin Y Stain Similar to Phloxine stain How to Mix: To make a 0.5 aqueous solution -0.5g Eosin Y and distilled water to 100ml. Some use a few drops of chloroform to preserve it microscope. com/access ories/slidesstains/blank - slides/oman o-eosin-y- stain-1-15ml.html#d escription_t ab

104 Erythrosin B Stain Used to help see the spores and make them stand out. Useful for some Epispores. Useful for Hyphal protoplast.

105 Erythrosin B Stain A.5 percent solution is made in 10 percent Ammonia hydroxide. Other Formulas are 1% with 0.1g or it in 100ml of distilled water and a couple of drops of chloroform is sometimes used to preserve it. Lasts a few years once mixed.

106 Formalin Nigrosin Solution Nigrosin is a difficult formula to prepare and is used occasionally in mycology. When formalin is added to the solution it keeps for many years There are several formulas both water (aqueous) and alcohol (alcoholic) based, both use 5g of it with 100cc of liquid

107 Hydrochloric acid - Muratic Used several ways 1% and 5% percent are used under the scope. 5% is used on russula. Used on Cortinarius cells and hyphae- a positive reaction is red or green. Coprinus also has different staining reactions on some parts DANGER: Mixing creates heat, poisonous gas. It can ruin your lungs, eat your skin and permentaly blind you. It must be done outdoors or under a flow hood with proper knowledge. Uses full strength 37% as a macro-chemical test see other section

108 Hydrochloric acid How to Mix 5%: 100ml double distilled water and 5ml of 37% Hydrochloric acid. But add in the proper order and only a little at a time because of heat and other reactions. Stir and bottle. 10 percent solutions are also used Uses full strength 37% as a macrochemical test see other section

109 Lacto-Fuchsin add photos See - Carmichael, J.W. (1955) Lacto-Fuchsin a New Medium for Mounting Fungi. Mycologia, 47, page 611 read for free at Cyberliber How to Mix: 0.1 g of Acid Fuchsin to 100 ml of percent Lactic acid. Great for seeing cell walls and better than cotton blue for that because the refractive index is more different than the cell wall - lasts about a year and stains the cells red

110 Lactophenol Cotton Blue Cotton blue is also called Methyl blue but not Methylene blue It is one of the most popular and useful mycological stains I was told that when the club purchased it was so expensive. This is all I got. An unused almost empty bottle

111 Lactophenol Cotton Blue Used on Ascos essential for Scutellinia and others because it helps visualize spore ornamentation. Also useful with Ramaria and others. Often Useful to see Hyphae contents

112 Lactophenol Cotton Blue Cyanophiles spores Lyophyllum connatum Lepiota can have Cyanophiles spores see the dark colored spore walls Photo Renée Lebeuf

113 Lactophenol Cotton Blue How to Mix: Formula mix in order Methyl Blue = Cotton Blue A. Carbolic Acid/ Phenol Crystals 20g/or 20 ml of crystals that have been melted B. Lactic acid full strength - 20g C. Glycerin 40g/ml D. Distilled Water - 20g/ml E. Methyl Blue / Cotton Blue 0.5g Mix with a glass stir rod solution to thick to filter may need to stir several times over a few hours

114 Lactic acid Cotton Blue Formula mix in order Safer because the phenol has been removed There are a few other formulas, tell about mixing but less acidic and may not keep as well A. Distilled Water - 30g B. Lactic acid full strength - 20g C. Glycerin 50g D. Methyl Blue / Cotton Blue 0.5g Mix with a glass stir rod solution to thick to filter may need to stir several times over a few hours or days

115 Cotton Blue Lactic acid Cyanophilous Reaction This reaction happens when the normally hyaline colored spore walls get stained blue from the stain. Basically the spores are put onto a slide in the stain and brought to a quick couple second boil without drying them out. Then the cover slip and another drop are reagent is added and the spores are examined.

116 Lactic Acid Although commonly used in different stain mixes it can also be used on its own as under the coverslip. A drop or so of percent lactic acid helps see dark colored spores and other structures such as dark colored cell walls.

117 Lugols (IKI) It does not work for dextrinoid reactions Hemiamyloid reactions can be seen with this that Melzer s will not show. Unlike Melzer s this is a vital stain where spores are living and not killed this is added to water where it should first be observed

118 Lugols (IKI) Moser s -Formula 150ml distilled water 2g potassium iodide 1g iodine Mix let dissolve for 10 minutes and mix with a glass rod again then bottle. Caution Do not mix with metal

119 Melzer s One of the most widely used and desired mycological reagents - It is very difficult to obtain because chloral hydrate is a controlled substance. This is Myco Gold. Formula 100ml distilled water 20ml 2.5g iodine 0.5g 7.5g Potassium iodide 1.5g 100g Chloral Hydrate 20g

120 Melzer s Dextrinoid (reddish brown to brownish reaction) spores Melzer Lepiota Spores with amyloid (blue/black reaction) ornementation Melzer Lactarius argillaceifolius Spore Photos Renée Lebeuf No to a yellowish color = no reaction = inamyloid

121 First Water Melzer s amyloid Peziza varia The reaction typically takes a few seconds

122 Melzer s amyloid Besides telling if spores are amyloid or dextrinoid Other parts may react. Some Boletes have amyloid septa, Some Mycena dextrinoid and Chroogomphus react both ways also have hyphae that react It lasts at least a few years, some people have reported it keeping over 12 years

123 Melzer s Reagent Some people try to get it with a prescription. The Chloral Hydrate typically comes in 100g bottles and will need to be mixed at a local compounding pharmacy. 2/3 of them seem to decline to mix it. Hopefully you have a club member(s) that can help

124 Melzer s Substitutions #1 Visikol - A Proprietary formula. See instructions and ordering info on the companies web site

125 Melzer s Substitutions #2 IKI in Lactophenol -Based on A formula from A. Phenol 8g B. Lactic acid 8ml C. Glycerine 16ml D. Distilled Water 8ml E. Potassium Iodide (KI) 1.5g F. Iodine (I) 0.5g It is said that the staining reaction between this and Meltzer s are almost indistinguishable

126 Methyl Violet =Gentian Violet It can be made into water or alcohol based stains By mixing with aniline or phenol the stain will allow the staining process to work better and keep much longer

127 Methyl Violet =Gentian Violet Formula A From the French site Gentain Violet 2.5g 95% Ethanol 5cc Aniline 1cc Distilled water 44cc Stir and Filter

128 Methylene Blue Use like Brilliant Cresyl Blue Aqueous Formula: Distilled Water 99ml Methylene Blue 1g Optional formula In addition add (as a wetting agent) Sodium dodecyl sulfate 1g Stir a couple of hours on the magnetic stirrer and Filter

129 Methylene Blue Some steps

130 Methylene Blue Some steps Safety First Don t do this

131 Patent Blue Useful to see chrysocystidium of Hypholoma, Pholiota and Stropharia useful on some Ascos See - A Simple Technique for Staining Chrysocystidia with Patent Blue V in Mycologia Volume 76, No pages Can stain deutoroplasm of chrysocystidia

132 Phloxine B An excellent mycological stain. It can be used to view cytoplasm, cystidia and other features such as hyaline structures. Used to stain the cell contents not the cell walls. Can be used to help see spore ornamentation. Useful with some Tricholoma & Laccaria spores. Helps to see what s inside the Hyphae. Once applied often a drop of Ammonia hydroxide is added and any excess is removed with a paper towel.

133 Phloxine B Formula A aqueous solution Distilled water 98-99ml Phloxine 1-2g Mix to make a 100ml solution Then stir, filter and bottle Formula B Alcohol solution Same as above but substitute the same ml of Alcohol in place of the distilled water

134 Potassium Hydroxide - KOH It is used for both macro-chemical and macroscopic reactions but in different percentages. Here for scope work we would use it is less concentrated solutions of 3-5% Can be used to rehydrate a tiny piece of a mushroom for microscopic examination CAUTION: Dangerous to eyes and can burn your skin.

135 Potassium Hydroxide - KOH Russula a 3-5 percent solution is used to view them under the scope Selected Examples of positive reactions A. Some species of Cystoderma pileipellis hyphae become reddish brown B. The cystidia contents of Hypholoma turn yellowish Shelf Life: 1 year or sometimes longer but use reactions to verify or replace when cloudy

136 Potassium Hydroxide - KOH Psathyrella bipellis Spores 400X Scuttelinia_Ascii_Paraphyses_1000X Spore Photos by Rocky Houghtby

137 Potassium Hydroxide - KOH A 10% aqueous solution of KOH in water is made by taking 11.7 grams of KOH and dissolving it in water up to 100 ml A 10 percent formula is typically used for macroscopic testing. For microscope use the below formulas 1%1.17g KOH 2% 2.34g 3 percent 3.51g 5 percent 5.85g

138 Potassium Permanganate (KMnO4) Formula Distilled Water Potassium Permanganate 99ml 1g It is reported to color hyaline spores such as Tricholoma spores or amanita spores. I need to mix it stronger and retry

139 Potassium Permanganate Hard to get DEA regulated? Cocaine It has been used as a disinfectant, to treat fish diseases, purify water, CAUTION DANGER Very dangerous if mixed with glycerin can cause fire and melt glass bottles. Danger Sulfuric acid. Also dangerous with Alcohol. With hydrochloric acid, chlorine gas can be produced. When diluted with water the dangers may lessen. It can stain you.

140 Safranin O It can make the nuclei red. Formula. Safranin 0.5g Distilled Water 100ml Store in amber bottles

141 Sudan III A good and often used Mycological stain Only soluble in ethanol or methanol not water Used to stain oil drops inside the spores

142 Sudan III It stains the walls of the fungi cells and it does a good job staining the lipids that are found in some ascomycetes There are multiple formulas some involve Carbolic acid crystals, Lactic acid, Distilled water Sudan III an Glycerin and a heating process others use ethanol and distilled water

143 Sudan IV Used to stain oil drops inside the spores Use like Sudan III and the formula is similar

144 Sulfo-Vanillin Sulphovanillin Sulfovanillin Used both Macro and Microscopically Once you make your slide with it you should not take a break, the acid can destroy the specimen after a little time looks like everything melts away. For Microscopic on Russula the cystidia, dermatocystidia and vascular hyphae are observed. Color changes or lack of a change and the intensity of a change can be important for some groups of russula. An example of the reaction can be a black to dark blue s colored reaction

145 Sulfo-Vanillin Sulphovanillin Sulfovanillin CAUTION Sulfuric acid can burn you and your clothing. It can blind you if it gets into your eyes Panaeolus -Sulphidium by Rocky Houghtby

146 Sulfuric acid It has been used on Psathyrella and Panaeolus to see if the spores fade or not CAUTION It can burn you and your clothing. Dilute not full strength Psathyrella_bipellis_Spores_400X Spore Photo by Rocky Houghtby

147 Toluidine Blue O Formula 1 from How to Identify Mushrooms to genus III Distilled water Toluidine Blue 99ml 1g Formula 2 from other sources Distilled water 100ml Toluidine Blue 1g Glycerin 25g 95% alcohol 50ml

148 Toluidine Blue O An excellent and commonly used mycological stain spore wall layers Good for staining fungi species that have gelatinous hyphae such as Flammulina, Gomphidius, Gymnopilus, Hebeloma, Hygrocybe, Limacella, Psilocybe, Resupinatus, Rhodotus, Stropharia, Tylopilus, etc

149 Trypan Blue An excellent and commonly used mycological stain It can be useful in staining some spores and is good for staining the fungi hyphal wall

150 Trypan Blue Formula 1 Distilled water 50ml Trypan Blue powder 0.1g Stir 30 minutes, filter and bottle Formula 2 More commonly used It is made into a solution with lactic acid

151 Distilled Water Don t use tap water it has to many impurities. Either buy distilled water Water-Laboratory-Reagent- Liters/dp/B015D6NX54, use high quality filtered water from the grocery store - ion exchange membrane technology, or make your own double distilled water using a pot and bowl and ice or stop at a university and see if you can get some Milli-Q water.

152 Distilled Water Used both to mix some stains and for making water mounts.

153 Microchemicals Where and costs 1. Aceto Carmine - in Multi chemical pack Amazon 2. Aceto Orcein Ebay about $13 for 15ml 3. Acid Fuchsin ebay about $56 for 25g 4. Brilliant Cresyl Blue Amazon about $7 for 30ml 5. Carbol Fuchsin Ebay about $22 with 2 others for 30ml 6. Congo Red - Ebay about $15 for 100ml 7. Crystal Violet - Ebay about $7 for 15ml 8. Formalin Nigrosin Ebay about $14 with 2 other stains 30m 9. Hydrochloric acid 1% - Ebay included with above 10. Lactophenol Cotton Blue Amazon about $75 for 50 pack single use - Hardy

154 Microchemicals Where and costs 11. Lactic acid cotton Blue $ 12. Lactic acid less than $10 on ebay and Amazon 13. Lugols Sol. ebay $8 for 1oz 14. Melzer s Solution ebay $ uk and other source 15. Methyl Violet = Gentain Ebay $7 for 30ml 16. Methylene Blue Ebay about 4 dollars for 30ml 17. Phloxine B 18. Potassium Hydroxide premixed Ebay about $20 for 30ml 19. Potassium Permanganate Ebay about $5 for 1oz 20. Safranin O - Ebay about $6 for 15ml Note shipping charges not included in prices

155 Microchemicals Where and costs 21. Sudan III - Ebay about $5 for 15ml 22. Sudan IV Toludine Blue - Ebay about $8 for 15ml 24. Trypan Blue Water distilled Ebay about $35 for 1 gallon 26. Canada balsam as Un-cement from Hometrainingtools.com about 12 dollars for 15ml Note shipping charges not included in prices

156 Rehydrating Dried Fungi Many people use water but once on the slide for example KOH or Ammonia Hydroxide can be added

157 Ammonium hydroxide Useful for brownish spores 2-3% It helps the dried fungi to swell up to close its normal size. The same is true with KOH. In this manor it would be used after ethyl alcohol was used. The ethyl shrinks the features that we look at under the scope. The Ammonia or KOH swells the features back close to the normal size Once the alcohol is applied then drained just add a drop of ammonia hydroxide to one side of the cover slip, on slide across from it and use a small piece of absorbent paper towel to draw the ammonia under the cover slip and remove the excess liquid.

158 Other Microscope items Here such things as Oil etc are grouped

159 Acetone Found in the paint store and in some fingernail polish removers. It can be used to clean smudges and fingerprints from glass microscope slides

160 Canada Balsam Is conifers species Abies balsamea Used to make permanent microscope slides in the past it was used to cement lenses. Formula Pure Canada Balsam Xylene 50ml 25ml

161 Softening Solution - Clemencon s There are several different softening liquids Formula Concentrated Ammonia 20ml Ethanol 96% 80ml Glycerin 1g An entire mushroom may have to soak in the solution for an hour, a small piece could soak as little as 15 minutes. Fragile mushrooms or pieces of them will become soft like wax and easy to section. It can also be used to rehydrate dried fungi. Once Rehydrated 10%ammonia is sometimes used to swell the material.

162 Gum Arabic Media It is made from the sap of Acacia trees. art3c.html Also useful for making Hoyer s Medium but it is difficult to obtain

163 Glycerine Used several ways by itself and also in several slide mounting mixtures such as Hoyer s Medium. Also used in Softening Liquids Used several ways mixed with stains such as some formulas of Brilliant Cresyl Blue, Lactophenol cotton blue, and Toluidine Blue A 90 percent distilled water 10 percent glycerine solution can be used in place of water to help slides from drying out as fast

164 Heet Used several ways Yellow Bottle heet is different than the red one. The yellow is 99% methanol. It has been used but has impurities. Both methanol=methyl Alcohol here and ethanol= Ethyl Alcohol are used for the Alcohol in different stain formulas. In many formulas they can be interchanged. Typically try to use the highest quality one you can find that has the least impurities. Ethyl is typically taxed higher. isopropyl alcohol should not be used

165 Methyl Alcohol =Methanol Like Heet used in several ways. It can be more pure that heat, but the type of Methyl alcohol found in pharmacy's is impure and should not be used same as with stove alcohols. (any blue colored methyl has been stained by methylene blue and is also impure ) Get the pure stuff from a chemical supplier

166 Methyl Alcohol =Methanol It can be found in things like wood stove alcohol or model racing fuel at hobby shops. Lab stuff is extreamly expensive. Amazon has it from Duda Diesel that will work well. It is Poisonous, don t drink it. I prefer Everclear 95 if you can get it.

167 Everclear 95 Everclear is Ethanol= Ethyl Alcohol. It can be found in the liquor store. This is 95 percent but also available as 151 which is 75% Tell about permit Like Heet which is methanol it is used in several ways including as alcohol that is included in many stains

168 Everclear 95 or 75 Ethanol= Ethyl Alcohol. Do not use denatured forms for microscopy they are not pure It can also be used as a wetting agent Used on dried mushrooms To convert 95% to 75 percent take 100 cc of the alcohol and add cc of distilled water For a conversion chart see

169 Isopropyl Alcohol To impure to use for stains but good and cheap less than a dollar - for sterilizing and cleaning microscope slides. 91% works better than 70 percent but both do the job. Some people use it to rehydrate large pieces of dried collections

170 Immersion Oil Used with the microscope 100 oil lens

171 Lens cleaner Used microscope objectives

172 Nail Polish Has been used on its own to seal coverslips onto slides for preservation and later use Also used to seal the edges of coverslips onto slides made with Hoyer s for preservation and later use Not as good as Polyvinyl Alcohol and other slide sealing methods but for a dollar at the dollar store its inexpensive

173 Sodium Dodecyl Sulfate Used as a wetting agent for some stains

174 Toluene Used several ways, can be used like Xylene

175 Xylene Used several ways, mixed with Canada balsam, lens cleaner of last resort. Use Chemical resistant gloves

176 Lab Equipment

177 Scale Used to weigh

178 Magnetic stirrer and Used to mix certain stains

179 Stoppers Used on bottles when mixing stains on the magnetic stirrer

180 Beakers Used to

181 Funnels Great for putting stains into tiny bottles

182 Graduated Cylinder Used to measure

183 Teflon Tape Can be used to seal lids onto bottles for longer storage and to help prevent leaks and evaporation

184 Electrical Tape Can be used to seal lids onto bottles for longer storage and to help prevent leaks and evaporation but not approved. Talk about heat seal on lids

185 P touch Labels Nice for labeling bottles Fisher label

186 My setup works with desktop and laptop Asterophora lycoperdoide s shot with the Leica microscope camera

187 Places that sell Chemicals

188

189 Places that sell Microscope stains and reagents 1. Brunel Microscopes (UK) log/staing_solutions 30mls.html 2. Micro-Science UK 3. Myko-shop DE =%2FShops%2F %2FProducts%2FZChem %2FSubProducts%2Fzchem-0100

190 Places that sell Microscope stains and reagents -US 4. Home Training Tools in the US 5. Electron Microscopy Sciences products/chemicals/acetate.aspx

191 Mycological Places that sell Basic Macro chemicals - Premixed The Hoosier Mushroom Society m/commerce/products / /?ri d= &rt=6 &hsi=1&hc_location=uf i

192