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An Aloha Medicinals Scholarship month Log


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#41 Seeker2be

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Posted 24 November 2014 - 06:55 AM

Day 14

 

Today was to start doing another inoculation. Unstacking bags, putting them through the window to the inoculation room, inoculating, and then watch the sealing of the bags, the marking of the bags for species, date and lot, the shaking of the bags to distribute the spawn and the restacking of the bags on the cart (250 bags) but on the way to Aloha something occurred to me. Thursday is the day the biochemist spends to do culture sorting, transfers, media mixing and clean room work. While my roommates went to the inoculation room I sought out the biochemist grad student ,a perky, patient, and happy person and asked her if I could help her in anyway. She said “Do you want to pour some agar in petri and centrifuge tubes, make some transfers, do some labeling and assist me?” Graduating from a glove box with poor self taught technique (with a lot of help from mycotopians) I jumped at the chance to learn what I really came here for!!!!  In five hours I poured 40 centrifuge tubes (swirling often the large beakers to keep the agar dissolved and uniform) with precise amounts of agar, made sure the caps were relatively tight and put them along with various agar recipes in the autoclave.  I did about 30 tube to tube transfers using Hailey’s two tube technique.  All surfaces and items to be used in the flow hood were sprayed down with alcohol.  Multiple parafilm strips were made by unrolling about 2 ½ feet and nicking partially opposite sides of the parafilm ever 2 ½ or so inches (after cutting up the center and overlapping them to cut the nicks). The nicks allowed the paper and parafilm to be separated.

 Under the flowhood using sterile technique (mask, gloves, hood, and suit as well as shoe covers) the master culture tube was cleansed with an alcohol soaked paper towel, the parafilm removed from the cap and held between my right thumb and index finger deep into the flow hood maintain my elbows on the edge with the tube mouth opening (covered top slightly facing the hepa filter) next the agar and stick receiving centrifuge tube was placed at a 45 degree angle to the other tube between the index and middle fingers .    The scalpel was placed in the bacticenerator to heat to red and the handle was sprayed with alcohol while in the bacticenerator (sometimes flames ejected which were normal).  I am left handed but held both tubes in my right hand.  The right hand removed the scalpel from the bacticenerator and let it cool horizontally (increased surface area) for 15 seconds.  The cap was removed from the receiving centrifuge tube using my left curled 5 th digit to unscrew it. Moisture was dumped onto a paper towel (decreases contamination) The scalpel was inserted into the agar to provide further cooling (sometimes it hissed).  The cap on the receiving tube was replaced with the left curled 5th digit.  The cap on the master tube held above was removed.  Using the scalpel a straight line was cut in the culture to the side of the slanted agar medium.  This was divided into two parts horizontally and one of the pieces was scooped or skewered with the tip of the scalpel, transported out of the culture tube, cap replaced then the cap of the receiving tube removed with my left curled fifth finger.  The culture piece was inserted slowly and placed face up on the agar.  The scalpel was removed slowly so as not to touch the sides or cause air turbulence.  The cap was replaced and still holding the master between the thumb and index finger, the centrifuge tube was place in a test tube rack.  Two transfers were made before re-sterilizing the scalpel and spraying down myself and the surfaces with IPA.  5 transfers were made from one master.  Keeping the master and tubes paired in the test tube rack.  Each was closed with parafilm then the genus-species , strain,  P value, QC# (to note source of contamination if it occurred later),the date and agar medium type of the tube was noted .

I was elated reaching the center of the journey that I had so wanted to achieve : improving my clean room techniques , making and pouring agar techniques and finally spawn making which I had learned in one of the previous days of inoculation.

The lab director then discussed with us the macro and micro features of identifying wild mushrooms.

 

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A handout from the lab director:

 

“When you identify wild mushrooms….

Make sure to use several identification guides just because some might have incomplete descriptions of some important characteristics used in the identification process.  Also make sure that these guides have pictures of the mushroom species discussed. Another important aspect is the description itself which could be in depth describing both macro-morphological and microscopic features.

Use guides that describe fruit body macro-morphology for easy to identify mushrooms. Use complete guides [with both macro and micro-morphology description] for species that seem to be hard to identify.

Don’t rely on pictures too much:  Especially fleshy mushrooms found in different developmental stages show different colors or characteristics.  Colors in fleshy mushrooms might change according to environment conditions such as humidity or temperature.  Therefore this is a less reliable feature.

Pictures sent via internet won’t give you accurate identification results unless the mushroom to identify is and “easy one” for an expert.  In some other cases one picture of a mushroom sent via internet with a request for identification is non-sense since there are so many features that overlap in which case not even experts will be able to tell you what it is.  Besides, one picture is just “one side of the moon” in most cases it doesn’t show all the characteristics that you need in order to identify that mushroom.

For in depth analyses you’ll need a microscope.  In this case spore shape, size, ornamentation or color is import to analyze along with presence of clamps or septa on the hyphae.

 

 

What to look for when you want to identify mushrooms?

 

Habitat: It is quite important since some mushroom species are more likely to appear in some types of forest than in others.  Some species prefer conifers while some others prefer hardwoods or both.  Some other mushrooms prefer grasslands. Some species are commonly found in all types of habitats both in boreal and subtropical or tropical climate types-these are particularly well adapted fungi species to various substrates or climate conditions.

Frequency: If the mushroom is commonly found or not.  Some mushrooms are rather rare but when you’ll find them they will be all over while some others are common but can be hardly found one next to each other.

Season:  This is important because this way you can exclude some species that are considered poisonous, “look alikes” that may pop up in a different seasons.

General aspect: single fruit bodies/clusters/mushroom fruit body outline

Cap surface: color/scale presence/shape/margin

Under-cap: gills/pores/teeth/or combinations. Color?

Stem-shape/color/ring presence or absence/stem base color/rhizomorphs

Flesh-consistency or oxidizing reaction in presence of air are just two of the important characters that one needs to look for.

Spore print-in some species has an important role in the mushroom ID especially in those that present poisonous look- a-likes that present a different spore print color.

Odor: Is an important character because in some species of fungi  they have specific odors that tells us if that are poisonous or not

Possible changes: Refers to the ontogenetic development of a mushroom fruit body or environmental changes that affect cap color or size.

All these features are differently evaluation by various authors-that is why you need to use several guides when trying to get a mushroomed.  Some authors simply might overlook some important features. Reading several descriptions will get you a general description that includes lots of important characteristics.

Other important things to consider..

An expert advice always matters

Inform yourself and study the poisonous mushrooms from you area or areas that you would collect from. Check for mycological societies.  They have checklists comprising the mushrooms species found in the collection area.

Don’t pick up mushrooms that ‘you believe’ that you know them. Being 90% sure of the ID of that mushroom is not what you want especially if you are going to eat them.

Don’t pick up edible mushrooms when you found them near poisonous ones.

If you focus on 5 edible mushrooms that you know very well that narrows the risk of poisoning.

If your 5-7 species* are hard to confuse with poisonous mushrooms that’s ever better.  Make sure that none of them belong to the amanita family.

Several opinions on a mushroom ID matters

Always heat  treat your mushroom crop before eating them.

*1.Fistulina helpatium (no poisonous look alikes) beef steak  fungus, polypore grows on oak or beech.  Lives on stumps. Has the consistency of flesh and has red colored droplets when cut. Temperate climates like Washington.

2. Chanterelle: Summer under conifers and hardwood.  Prefers moist areas like moss

3. Chanterelles tuleforece- corocopileatis (black trumpet)

4. Auricluaria Auricula

5. Honey mushrooms Amilleria Malea under conifers in the hills or mountains.  Boil before eating

6. Marasamus spring to fall in grass and forests (Has some poisonous look alikes )

7 .Macrolepiote procera small scales Rachodes has bigger scales. Stem not edible has bottom bulb but not a vulva.

 

 

 

DNA identifications

We cannot rely only on DNA analyses in order to identify fungi and this is because of several reasons:

Lack of sequences available in data bases like Genbank for some fungi species (especially rare fungi)

Poor sequence availability for some other fungi.

Single publications listing several sequences for the same species.  Usually for a more accurate identification you need to consider and compare sequences resulting from multiple authors.

Sequences resulting from basidiocarps collected in different geographical regions of the worlds might show a bit of genetic shift.

We should take into consideration DNA sequences resulting from the analyses of several gens and not just one.

Some DNA sequences resulted from some genes are more reliable then from others (compare D2 to ITS4)

In some cases you cannot rely on DNA sequences just because they can be easily modified and adapted according to the desire of the person working with them.

Sometimes researches cannot get a complete sequence of a gene.

 

According to my opinion we should always try to identify fungi according to classical mycology methods [Macro and  Micro morphological aspects]- this works pretty much for most fungi (It is also less expensive than DNA isolation)  However, some fungi species require additional analyses such as morphological aspects of colonies in pure culture (e.g. Phellinus baumii and Ph. Linteus- in which you wont see difference in fruitbody morphology but they are different when grown on solid media )  For some rather hard to identify fungi I would recommend data collection from classical morphological analyses and DNA.  “

 

We are reaching the end of our month at Aloha and our fridge is full of oyster mushrooms (citrinopileatus, salmoneo, and blue oyster), ear mushrooms, and elm mushrooms

I have made a determined leap over the last month to understand the secrets of the magic trick that so many people won’t tell you about.

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Edited by Seeker2be, 24 November 2014 - 12:11 PM.

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#42 Seeker2be

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Posted 24 November 2014 - 11:45 AM

Day 15

 

Autoclaves and Pressure cookers

 

Dr. Holiday discussed with us for 4 hours the Autoclave, pressure cookers, the piping and will discuss the boiler with us during the next session.

Some concepts: Steam is a dry gas

Heat has two properties Quantity (BTUs) and Intensity (Temperature)

(Like a piston example )Same quantity different intensity based upon compression

If pressure goes up then temperature goes up

15 psi , 1 kg/cm or 1 ATM

At 15 psi =121 c or 252 F=pure steam.  Steam transports heat

Two problems of autoclave (Does not refer to pressure cookers except to attention to detail-that is venting air for 10 min until steam forms)

95% of the problem of sterilization is insufficient air venting with autoclaves and pressure cookers.

Goal: Introduce steam at top not bottom of autoclave and do it in a way that prevents turbulence (mixing air and condensation with steam) by using a distribution pipe and trough.  Vent from bottom the air and condensation.  Goal 100% steam

Have a sheet metal tent over jars to prevent condensation on filters (which would aid contamination) In the case of a pressure cooker and jars: aluminum foil

Steam traps always at bottom of autoclaves  keeps steam in and water and air out.  Use redundant check valves.  Thermostatic disk check valves and F and T check valves (float and thermostatic disk check valves and inverted bucket type valves.  They Close when steam is introduced and open for cooling condensation.

2nd problem of autoclaves and pressure cookers : 4%  Vacuum breaking (applies to PC also)  As heat is loss with cooling , decreased BTU’s  zero PSI creates a vacuum that unaddressed could be a source of inoculated contamination especially if steam trap vented to sewer better to be vented to clean air room with hepa filter and condensation vented back to boiler.  A bleach filled rag over the steam valve or opening the PC in front of a flow hood is the way to avoid this source of contamination.

Dr. Holiday reinterated the way to measure failure or contamination is by the per cent of biological efficiency you have.  Again biological efficiency is dry weight of substrate vs weight of mushroom fruits produced.  Should be at least 100%

Reasons for contamination

1.Air

2. Tools, equipment

3. Personal hygiene and improper hand washing and inadequate suiting up, failure to use alcohol frequently, etc.

4. media or substate

5. Surface area

6.Mobile contamination units.

The unicorn bags most used with the single or double patch have a 10% level of contamination.  The mycobags (referenced earlier and described on the Aloha medicinal cultivation equipment section of the web site) that Aloha uses and sells have filters 500 times more precisely and have a zero contamination .  Again reference that information in previous post and links to Aloha Medicinals cultivation equipment site.

Back to Vacuum breaking problem on pressure cookers: 3 ways to prevent contamination on cooling:

1. When gauge gets near zero place cloth with bleach solution on top of valve port.

 

 2.Put in glove box to cool with sprayed bleach. 3. Put in fron of flow hood to cool.

To sterilize tools : place in heat sealed bag with 2 or 3 drops of water for steam sterilization. The heat converts the water to steam for sterilization.  Or wrap instruments in newspaper with cover of aluminum foil to sterilize in PC.

Pressure measuring is meaningless .  The temperature achieved  is what matters.  Temp is 121c

On endospores: They are produced by bacteria esp baccilus cereus to survive though dry or drought conditions and have no water inside so cannot be sterilized.  Dr. Holiday did a demonstration of broom corn in the microwave.  The kernals popped due to the water inside just like bacteria but not the endospores present on the grain.  One cannot avoid heat resistant organisms.

To avoid this ( Aloha’s Grain prep method )  Either have clean grain or wash grain (does not lose much nutrients) which reduces microorganism load.

He drew a graph with Temperature/pressure on the vertical line and time on the horizontal. Effective sterilization period is the area under the graph.  The lower the pressure used the more time to sterilize and visa versa.  The enemy is carmalization which reduces the sugars available to the mycelia .  The lighter the malt used in agar the less carmelization.  He demonstrated carmelization with a blow torch and sugar on a metal plate.

 

Time is everything .  The longer it takes for colonization the more time other contaminating organism have to grow.  So minimize carmelization vs. optimal sterilization

Sugar in the substrate is no problem for oysters but it is for Shitake and agaricus so avoid carmelization.  Goal shortest time colonization possible to get best BE

Pre cooking grain forces hydration of the endospores and germination.  Soaking grain overnight increases biological load. Cooking grain and draining it wastes valuable nutrients. He did not agree with Roger Rabbits method of boiling and draining the water.

Dr. Holiday’s Aloha process based on BE (Biological efficiency). Most Grain has 10-12% water so adjust added water to total of 50%.  Rinse if necessary in cold water.

Best method: put a pot of grain with the water desired in the PC elevate it so it is off the bottom with rocks or some other objects.  Add lime (Ca carbonate) or oyster shell dust. Put lid on pot of Pc and cook for 30 min at 5 psi.  The water should incorporate into the grain, if wet too much h2o added and if not chewy too little water or not cooked long enough. Experiment!.  This process hydrates the endospores, let cool 12 hours add gypsum to keep grains from sticking together, load into bottles and pc at 15 psi for 1 ½ hours.

Cellulose filters for bottles are better than Teflon because Teflon pores sizes increases with sterilization and can cause contamination problems.

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Edited by Seeker2be, 24 November 2014 - 12:04 PM.

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#43 kcmoxtractor

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Posted 24 November 2014 - 10:50 PM

i think it is kind of funny that there is a coconut agar, then list coconut as

a no-no because it is anti-fungal. google scholar lists coconut as a beneficial

additive ingredient and it is part of the super cake recipe.


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#44 Seeker2be

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Posted 25 November 2014 - 01:43 PM

Well one of the things that occur with mycelia is some dont grow on some media and some grow on other media so the variability exists.  I believe for the strains Aloha Medicinals grow the coconut didnt work for them.  I am reminded of a topic that Dr. Holiday discussed in telluride when B. thuragenesis was injected into coconuts and then they were thrown into swamps.  The b. thuragenesis would invade the mosquito and kill the malaria organism. 
So some things grow and some things dont.



#45 Seeker2be

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Posted 27 November 2014 - 10:10 AM

Day 16

Today we went out with one of the lab instructors, a researcher, and mycoartist to an area around Trout River near lake Tahoe, Nevada.  We hiked through an area of some what appeared to be lightening fire struck trees and trees felled by woodsman. There was lots of of substrate for the next mushroom season .  It had rained for 2 days .  We found soggy Bolete edulis (boletes),Amanitas, Macrolepiota sp (Shaggy parasol), Ganoderma sp. (Reishi), calvatia sculpta (Sierra Puffball,) and lentiporus sulphorosa (chicken of the woods)  The researcher described on the ride his efforts to make paper mache art and hats inoculated with mycelia.


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#46 Seeker2be

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Posted 27 November 2014 - 10:11 AM

Day 17  Our last day. 

Dr. Holiday discussed piping from boiler to autoclave .  Goal is 100% steam and zero water.  He said always trace a pipe with your finger and note check valves, steam traps, manual valves, and pressure release valves.  Top should be steam and bottom water.

He talked about boiler types: The low pressure boilers  15 psi and the Power Boilers >15psi.

Low pressure types don’t require a permit and minimal regulatory requirements. These are the type for in -home heating, apartments and restaurants.

The high pressure types require regulations and permits.  These are the ones that cause the flash steam explosions at Chernybol and Fukishima because of a failure of water lever control.

Low pressure boiler (Bailey) when purchased require new blow off valves and have a heat switch or differential switch.

Misconception : Pressure means something.  NO, temperature is what you want to measure and is the effective modality in sterilization.

Pressure cookers need to be vented, for 10-15 minutes, of steam to make sure that most of the air is out otherwise compressed air does not sterilize but steam does. If you don’t vent the will have contamination and not recognize why. This is due to cold spots in the PC if not air vented at the beginning.

Cleveland boilers (cheap) are low pressure can be used to run autoclaves (also called steam ovens) Can handle 6-8 grain bags per chamber and up to 3 stacked chambers.  Gas is the only way to go.  Electric ones are 100x more expensive to run. Dont buy them. Buy gas or oil ones on ebay.  Propane can be switched to natural gas or visa versa with a cheap adapter.  These are what restaurants use.

Companies Cleveland, Crown, Goen.

Steam  kettles can be had cheaply and provide sterilization via a double jacket.

 

 

 

 

Last meeting.

Extraneous questions on growing mushrooms with Dr. Holiday:

Button mushrooms are complicated to grow. Need to make specific compost first takes a lot of time.

Straw substrate does not need to be soaked in water (in a full barrel) which gives the problem of waste products and waste of water instead water and soap, lime, ashes, or bleach can be added slowly and dripped gradually on the substrate saturating it.  Wood ash seems to produce the most oysters as a low tek method.

Coprinus mushrooms can have a shelf life of 10 days if dipped for a few minutes in cold salt water.  This breaks the enzymatic reaction leading to desqualescence.

The Mushroom growers handbooks one and two extensive booklets printed in Korea which can be downloaded from the Aloha Medicinals site free:  These books (out of print) discuss growing oyster mushrooms and shitake mushrooms by low tech methods in 3 rd world countries.

Aloha uses one quart juice jars for sterilization and they stay vacuumed for use  when preparing agar recipes.

About rinsing grain and soaking it for 12-20 hours: Don’t do it because the water soluble nutrients are drained away and the same is true with boiling grains and draining the water.

Goal is to keep the biological load low. Better to put the desired water in with oyster shell dust and cook at 5-10 psi for 1 hour.  This hydrates the grain and the endospores.  Let sit for 8-12 hours, mix with gypsum and load into jars for sterilization.  You can start with 50:50 water:grain mixture. Reduce water or cook time based on your experiments.  Use only husk on only grain

Supplements produces better results but increase contamination risk. 

Buy spawn gradually don’t stock it up.

One half a juice jar of agar makes 20 plates

Don’t allow mycelium to become accustomed to one type of media to prevent senescence and promote vigor.

Maintain cultures in at least 2 places.

Can use mayonnaise jars to grow mushrooms.  Cheap. With holes in lids and filters

On Spore prints.  Plate 10 plates with one spore print and you will get different strains. Select for speed of growth and other characteristics. It is rare that they get infected if done properly.

Mix different strain spore prints on same dish to get new strain development.

Mycelium run away from contaminates but may carry bacteria from plate transfer to plate transfer.

Ways to clean up petri dishes of contamination

  1. Use antibiotics to decrease the bacteria that ride with mycelia.
  2. Filter sterilization with syringe to clean antibiotic of bacteria (See Pall Life Science or Gellman for details)
  3. Grow underneath the agar in sandwich with another plate of agar or use sterilized butter knife to lift agar.  The moment the mycelium pokes through the agar and make another transfer and leave slower bacteria behind (usually one or 2 days)
  4. Glass cylinder method with sawdust and 2 open ends plugged and sterilized.  Inoculate and when mycelium reaches the distant side plate it leaving bacteria behind.
  5. Freezing the culture which may kill bacteria.
  6. Consider pouring hot agar over the growth that is contaminate.  If it doesn’t kill it it will clear it.

On yield:  Mark a graph weight the bags and weigh each flush for biological efficient to see if some different conditions or treatment made a difference in BE.

We made transfers of the laetiporus sulphureus , Coniferousus? (Chicken of the woods we collected  Saturday in the Mountains near Lake Tahoe) to petris and checked our cultures for colonization/  


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#47 Seeker2be

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Posted 03 December 2014 - 05:56 PM

This log should be valuable for both newbies and the experienced who want to accelerate their understanding and knowledge of mushroom cultivation. The Insights and tips within the log provided surely will be used in the future whether or not you are into advanced techniques now or not..  I hope you look at this vicariously shared experience. Seeker2be


Edited by Seeker2be, 03 December 2014 - 06:01 PM.

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#48 stoffel

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Posted 08 December 2014 - 08:58 AM

very nice! thanks for sharing this valluable information, and your experience. i enjoyed reading it

 

greets

 

stoffel



#49 Seeker2be

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Posted 08 December 2014 - 08:54 PM

Thanks !!! This LOG contains a lot of knowledge for the beginner and for the advanced. I hope everyone looks at this information.  It dispels misconceptions that I  and probably others have had. The log provides a succinct way to be cleaner in techniques for more success. There is a more extensive agar list than anywhere I have found and it has been tested by people who's livelihood depends on it.


Edited by Seeker2be, 08 December 2014 - 08:57 PM.

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#50 Viridis420

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Posted 08 December 2014 - 10:41 PM

Wow thanks for this gold mine of knowledge Seeker !!..Just stumbled onto this thread tonight. Sounds like an amazing class, Cheers



#51 Seeker2be

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Posted 09 December 2014 - 01:08 PM

If anyone would like a Agar Recipe Handout (the most extensive and complete I have seen and in a better format than able to be posted here) please PM me with an an email address and I will forward it to you.  Very valuable even if you think youll never get into agar....you will. It is the only road available to advanced production and cleaning up your cultures.


Edited by Seeker2be, 09 December 2014 - 01:09 PM.

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#52 wharfrat

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Posted 09 December 2014 - 11:53 PM

awesome thread brother.. wow what a great experience you must have had... very cool!



#53 CatsAndBats

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Posted 10 December 2014 - 08:28 AM

this is archive material! Thank you so much my man!



#54 Seeker2be

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Posted 10 December 2014 - 01:30 PM

I wish you had all had the experience that I had and that we could have brainstormed everyday to catapult ourselves to a whole other level.  I hope everyone gets something from this. Its an honor to share with you all.


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#55 Juthro

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Posted 10 December 2014 - 02:36 PM

Thanks again for sharing your amazing experiences and knowledge learned friend, and I also agree that this is destine for the archives .



#56 Seeker2be

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Posted 13 December 2014 - 01:09 PM

I would hate for this information to get lost to the many people down the road that will need the knowledge


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#57 Heirloom

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Posted 28 December 2014 - 02:43 PM

Seeker2be, I have read what you wrote about aloha medicinals as well as gone to the website.

they have some things like cold pasteurization using either low magnesium hydrated lime or  soap.
more effective & efficient that hot water.

I answered a question about a guy having trouble going from print to print and I suggested using
different substrates to avoid being accustomed to 1 media loseing vigor and leading to senescence.

I understand they offer a training video, thought about buying it .

I think we are still interested in brainstorming. got an idea an will post on it later , based on aloha info.



#58 Seeker2be

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Posted 28 December 2014 - 03:15 PM

Brainstorming is what I am about.  I figured posting all of this would generate some thought and move us all forward. They are in business to produce consistent products though they have experimentation going on all the time.  Thanks Heirloom!!!!



#59 happy4nic8r

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Posted 14 February 2015 - 04:53 PM

Brainstorming is what I am about.  I figured posting all of this would generate some thought and move us all forward. They are in business to produce consistent products though they have experimentation going on all the time.  Thanks Heirloom!!!!

I seriously wish I had seen this when I got started, it would have saved the embarrassment of asking so many stupid questions. This is a gem, and what a ton of info to take in, much less take notes and remember.

 

If I hear you call yourself a "newb" again, I am going to apply for the caveman job in the auto insurance commercials!!



#60 Seeker2be

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Posted 15 February 2015 - 07:13 PM

Thank you.  I want to share this Aloha Medicinal scholarship with as many people as possible to hopefull help themy avoid are the mistakes and frustrations that I have had along this road..   We are all here at topia to share. I am enjoying the people on this site and their ideas and collaboration daily.  I will always be a humble student.  I am happy to learn from all of you






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