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An Aloha Medicinals Scholarship month Log


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#1 Seeker2be

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Posted 18 November 2014 - 01:15 PM

Aloha Medicinals
Day 1
Today we started by putting on scrubs and helped load 250 lbs milo grain, 750 cc of oyster shell dust and 25 gallons of water in each of 10 large stainless steel containers. After that we talk mushrooms with the director of mycology lab, then on to a 2 hour session with a 25 years teacher with a lot of enthusiasm who taught us about designing simple fruiting rooms and what the basic necessities were to maintain moisture, temperature and CO2 levels below 1000 for fruiting as well as about insulation and design. We had some hands on with low tek techniques. We made 3 trash barrels of 6 lbs each of straw hay (for low tek inoculation) one treated with one cup hand soap, one with 2 cups of bleach and one with 750 cc of lime. We submerged the hay to tomorrow’s inoculation of oyster mushrooms. I’ve read about this and have done some of this including cold formation (not attempted here) but again here it is hands on and the little nuances in 3 D really hit home the concepts. We had a 3 hour lecture and interchange regarding Sterilization and Clean room techniques with specific details on avoiding contamination in commercial product. The instructor the plant manager ,a stickler for sterility discussed the definition of a clean room, the meaning of sterile, the 7 different ways we can unknowing introduce contamination, the details of a HEPA filters (also trouble shooting them) and their importance, how air quality is measured with a Lazer particle counter and petri dish samples of the air, how much contamination is allowable, what involves work space attention (avoiding clutter, foot decontamination (90% of contamination), air filtration functioning, personal hygiene, hand washing clean clothes, minimum movement, dedicated equipment, nothing in the hood, and proper cleaning of the tools and work area every 10 min. We discussed the 7 sources for contamination ( Air, tools, inoculums, personnel, media or substrate, surface and mobile contamination units-MCUs) I had read and tried to practice take notes of all this at home but it is different again to see it and have excellence required. They did a hand washing test with with black light sensitive material that mimicked the adherence of bacteria. This to test our hand washing techniques. Before and after finger prints on agar were done for culture.
We discussed the proper cleaning techniques for clean rooms including wall and ceiling and wall cleaning in one direction with an autoclavable “swiftee” ($250 dollars each).
We are basically employees in training and must do all the chores and techniques of the other employees. No special priviledges will be afforded us and we will assist in all activities.
We ended the day discussing the effects of various medicinal mushrooms and blends.
We have a written test required for clean room certification before we can enter the clean rooms.


Sterilization and Clean Room Techniques and Concepts
Aloha medicinal Inc. runs a number of clean rooms and it is important that anyone working in these cleanroom areas understand the concepts of sterility and the procedures to be followed.
  • What is a cleanroom?
An air-controlled room.A cleanroom is a space in which the air is specially filtered with HEPA filters and the personnel are specially suited to ensure that sterility is maintained and no contamination is present which could affect the products.In all cleanrooms special techniques and cleaning procedures are used to ensure sterility at all times.This allows us to perform sterile operations and to process our finished products in a sterile manner.
  • What is the meaning of Sterile?
We are surrounded at all times with microorganism in the air,water and on Every surface. These organisms are bacteria,virus,molds,yeast, spores and microscopic insects.Normally,this is not a problem. But in the case of Aloha Medicinals we do things and perform jobs that require us to maintain a minimal number of these foreign organisms in our processes and products.An example is when we inoculate mushroom cultures into grain bags, the presence of even one single foreign organism will create problems for us.We are also required by law to have a minimum of foreign organisms in our finished products
Sterile means that all the microorganisms present are killed or removed.

Once something has been sterilized, it cannot get any more sterile.It can only pick up organisms as it is being processed further.This is why it is so crucial for us to maintain cleanrooms, so that the number of organism that get back into the product once it has been sterilized remains at a minimum
  • What are the Different Ways we can unknowingly introduce contaminants into our products?
1. Air
2.Tools
3.Inoculum (packaging for finished products)
4.Personnel
5.Media or Substrate
6. Surfaces
7. Mobile Contamination Units (MCU’s) such as mites (in the personal cultivators space cats, dogs, and insects)
  • What is a HEPA filter and why is a HEPA air filtration important?
HEP A is a special kind of air filter. HEPA stand for High Efficiency Particulate Airfilter. This is a especially made that air passes through, which removes all microorganisms and dust particles down to the size of .3 microns (about 1/3 of a micron).The aire around us is filled with particles that are too small to see. These are dust particles, smoke, spores, bacteria, and other microscopic particles both living and dead.About ½of all the particles in the air around us are alive.If these living things were to land in our sterilized products, that would begin to grow there and then we would have a contaminated product.

Our HEPA filters clean the air down to the size of .3 micros.How big is that ?One micron is one millionth of a meter or 1,000,000 meters.Another way to look at this is there are 25,500 microns in an inch.A piece of paper is more than 100 micros thick.As you can see, a micron is very, very tiny.A typical bacterium is 5-20 microns in size.A spore from mold or mushrooms or other airborne life forms are also around 5-20 microns in size.Even the particles in cigarette smoke are larger than .3 microns and are too large to pass through a HEPA filter. So by passing the air through such a small , it effectively removes all the particles and microorganism such as bacteria and spores from the air. Essentially everything is removed from the air, leaving just pure air containing no living spores or bacteria that could contaminate our products.This clean filtered air is crucial to the sterility of the cleanroom.
  • How is air quality measure?
We typically measure air quality by two methods:The first is to use a Laser Particle Counter with is done at least once a week in all the cleanrooms at Aloha Medicinals.This Laser Particle Counter is a machine that draws in air and runs it past a special laser beam counter device that can accurately count all the particles of different sizes and tells us how many particles are in each cubic foot of air.It is common to see around 1,000,000 particles per cubic foot in normal outside air.If 50% of these particles are living, that means that there are 500,000 living organisms in each cubic foot or air.Our inoculation room alone contains about 1344 cubic feet of air, or 1,244,000,000 ( ONE BILLION THREE HUNDRED AND FOURTY FOUR MILLION)particles if the air were unfiltered, or if one of the HEPA filters were faulty.Assuming that 50% of the particles are living, this means there would be 672,000,000 (SIX HUNDRED SEVENTY TWO MILLION) organism that could and would get into ur bas as we tried to inoculate them , if we did not use HEPA filter the air going into the room.
The second method for determine air quality is to expose sterile Petri dishes to the ari to see what grows in them.
  • What is contamination in regards to our product?
There are a number of definitions to the word Contamination.As far as our products go through , the term means the introduction of foreign material into the finished product.This contamination can be in the form of microorganisms, or it can be in the form of chemical compounds like alcohol, lead, cleaners ,etc.For the purpose of cleanroom techniques , we are primarily concerned with microorganisms as contaminants.
  • How much Contamination is Allowable?
It would be nice to say no contamination is ever allowable but sometimes this is an impossible task.So to say how much IS allowable is a touch question to answer because it depends on what stage of the process the product is is in. The most critical phase is the cooling and inoculation of the raw material.AT that stage, NO contaminates are allowable, because any organism introduced into the sterilized substrate will find perfect growth condition and will grow through the substrate.In the cooling room and the inoculation room there should be zero organisms present, or as close to zero as we can achieve. Once the bags and bottles have grown through, there is less danger from contamination, but it still must be held to a minimum.
  • How is contamination measured?
Contamination is measured in two main ways: The first is when our growing material becomes contaminated with another organism, it is usually detected visually or by smell.IT IS CRITICAL that we catch this type of contamination and stop it before the material is dried, because it is often impossible to to detect after it has been dried.Anyone who is working in the drying process is expected to pay careful attention to the material taken out of the bags and SMELL EACH BAG CAREFULLY.If anything seems out of place, YOU MUST put the product aside and ask a supervisor if it is OK to dry.

At the DRYING STAGE, there are two ways to detect contamination- SIGHT and SMELL.

The second way contamination is measure is by analytical lab methods. This is the Stand Plate Count (SPC) test, usually called just PLATE COUNT.In this test, a one gram sample of material is put on a Petri dish and grown for 24 hrs.. The number of organisms that grow is then counted.That number is described as the number of Colony Forming Units per gram, written cfu/g. This method is used to evaluation the completeness of our sterilization process.A SPC number of <100 cfu/gram is considered Sterile.Each customer and each country will specify how many cfu/g is allowable, but in any case, a plate count of no more than 10,000 cfu/g is ever allowable. Even if we get 5000 cfu/g it indicateswe have a real problem in our process.
Assume that when we sterilize our dried material we get a plate count of <100 cfu/g then everything we do to that material from then on can only INCREASE the plate count number.There is no way the number can go down, but will always icreas as we handle it. That is why it is so important that we use our very best cleanroom techniques when we work with dry powder in the canning and packaging stage.
  • What are the factors involved in keeping the contamination down?
  • Clean and Sterile suiting including gloves, face masks and foot covers.
  • De-contaminate your feet before entering the cleanroom area. 90% of contaminates come from the floor.
  • Be CERTAIN the air system is working properly.
  • Personal hygiene. Hand Washing, Clean clothes, etc.
  • Minimum movement- and keep your hands above your waist.
  • Attention to work space.
  • Dedicated equipment. Do not take any cleanroom equipment out of the cleanroom. Do not take non-cleanroom equipment into the clean room.
  • What does attention to work space involve?
  • Nothing in the room or work area other than the day’s work EVER
  • Nothing in the hood except your hands and what you are working on. The hoods are not storages place.
  • Proper cleaning of the room before the work starts and throughout the day.
  • Not leaving the work area and re-entering without full re-suiting and de-contamination.
  • Proper cleaning and handling of all implements such as pens, scissors,tape,etc.
  • Be ABSOLUTELY CERTAIN YOU USE THE FOOT WASH TRAYS. No exceptions to this rule.
  • Do not leave any work uncovered, ever. If you do not have your hands in the container it should be covered. This applies to all materials and supplies used in the clean rooms. It does not matter whether it is cans or bottles or powder or cotton or caps, or anything else. Keep them covered.
  • What are the proper cleaning techniques for cleanrooms?
Proper cleaning consists of removing everything that is not essential to the operation of the clean room at that time. This means removing anything that is not being used that work day. Or at least things that are to be used within the week. It is not OK to store things in the cleanroom just because they might be used later.
After the non-essential are removed, the room needs to be 100% cleaned with the designated cleaning sterilants. The particular cleaner will be specified by the supervisor in charge of that area. By 100% cleaned, that means floor, walls, ceiling and all surfaces such as countertops, canning machine, on top of the hoods, everywhere. If you cannot eat off the surface it is not clean enough.
It is extremely important that different sterilants be cycled through on each subsequent cleaning. The sterilants Aloha Medicinals typically uses are Bleach, Nolvasan, Iodine,Quaternary Ammonia, and Pine Sol. These MUST be measured and mixed according to instructions and not just dumped into the water bucket for cleaning. By properly mixing and cycling different sterilants, the maximum cleanliness is maintained.
Mops need to be clean and of the proper type. The mops need to be rinsed thoroughly every few minutes and not just wiped around spreading the dirt from here to there. The idea is to remove everything that is not a part of the room and not jot just to make the surface look wet. Only the broom, dust pans, mops, and mop buckets assign to that particular cleanroom are to be used. DO NOT tak cleaning supplies from one room to another. If some supplies are missing form your work are, INFORM you superviso so he or she can take care of the issue.
Alcohol an /or Nolvasan solution should be available in spray bottles at all times and all surfaces regularly wiped or sprayed with these sterilants every 10-15 minutes. This should be done more often when working under the hoods. If you are out of spray bottle sterilants stops and contact your supervisor to get you some more. This is one of the things you should check when your first set up for the job to ensure you not run out of sterilants or other supplies during the working day.
  • Check lists
A check list of supplies is to be posted at each cleanroom entrance. Before starting a job review the checklist and make sure you have everything you need.

Edited by coorsmikey, 26 May 2016 - 08:16 PM.
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#2 Needles

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Posted 18 November 2014 - 03:51 PM

What an awesome opportunity you have there seeker2be, hands on at a commercial level. What strains are you working with? Enjoy....
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#3 Seeker2be

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Posted 18 November 2014 - 08:44 PM

The strains at aloha were varied but the commercial production was of cortryceps Chinesis and militarias grown as mycelia for supplements.  We , Three, did some primitive teks as noted and grew pleurotus salmoneo, pleurotus citriniopiliatus, pleurotus colombinus, and hypzizygus ulnaris elm. By mid month we had daily sautéed eggs and mushrooms and sautéed mushrooms daily in the apartment harvested from our effortsl


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#4 Seeker2be

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Posted 18 November 2014 - 08:59 PM

Day2

Today we got up at 4:30 am (I got up at 3:30  to study for the state test that would allow me to work in the clean or sterile rooms at Aloha.

It was dark when we arrived at grain prep area with the 60 foot by 8 feet autoclave which cooked grain we had loaded and prepped the day before.   It was now time to load the 2,500 lbs into 11lb portions in special gusseted bags with tyvek vents for the mycelium to breath and for air exchange in the autoclave for sterilization.  We arrived to a bright dawn coming from the East and blaring Mexican rancho music so loud no one could here you talk.  We filled, folded and stacked the bags in metal crates ready to reenter the autoclave at the end of the day.  Four hours of vigorous exercise without a break and this will be repeated many times in the coming weeks.  We were glad to contribute, the 3 of us who received a scholarship. After that workout and great experience at hands on we went to take our test and then to meet with our young mentor.  We had prepared three low tek straw projects that had been soaking all night  (not requiring pressure cooking or high energy consumption).  One pile was straw soaked in hand soap, another bleach, and the third lime (CaOH) all to chemically kill off completing bacteria.  We made plastic bags of straw, tamped them and compressed from each of the three processes using pink mushroom spawn and elm oyster spawn.  We then mixed preportionaly alder sawdust with bran and oyster shell (to provide nutrients and Caclum respectively).  These were mixed in an old bathtub fixed to a rolling metal guernay then transferred to gusetted bags  ready for autoclaving before adding spawn.  We made 30 bags of those.  That took four hours .  We next went to see the  head of the cleanroom laboratory.  He educated us in the proper way of sterile gowning and gave us a tour of the clean lab. We were exhausted from the day and heat >100.  We had little breakfast, no breaks, and no lunch and that added to our weariness .  We were greatful for the knowledge and work .  We look forward to the next 28 days.IMG_2393.JPG IMG_2423.JPG IMG_2426.JPG IMG_2382.JPG IMG_2386.JPG IMG_2387.JPG IMG_2395.JPG IMG_2412.JPG IMG_2415.JPG IMG_2437.JPG


Edited by Seeker2be, 18 November 2014 - 09:00 PM.

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#5 wharfrat

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Posted 18 November 2014 - 09:02 PM

that's some excellent information seek, what a great class to be taking. keep sharing the knowledge.  :biggrin: 



#6 Seeker2be

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Posted 18 November 2014 - 09:14 PM

Sharing the knowledge is what this site is about and I love all of you for sharing and critiquing when one of us posts something erroneous  Maybe the program at Aloha will be terminated and I wanted others to know what it was about


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#7 Seeker2be

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Posted 19 November 2014 - 07:18 AM

Day3

The day started out with the  chief of the lab. He discussed with us some generalities.  The mother culture is dead if the offspring are not growing.  Different strains or same strains grown on different medium can have different morphologies. There can be two different morphologies in the same culture . Morphology can change with time.

Norospora, a contaminate can form droplets first light then darker. Pencillium green blue and trichoderms, green.  Form of molds don’t matter but color does.

Dr. Holiday discussed medicinal products : Fruit bodes low in bioactive compounds.  Biomass products have high amount of undigested substrate. Total mycelia product are extracellular components only. Goal at Aloha is to produce Full spectrum product. They let mycelium grow until it digests 98% of substrate and just start to pin in the bag.  He showed us independent chromatography analysis of his cortryceps product vs. competitors .  Chinese extracts (of cortryceps) and full spectrum products had the highest active chemicals. He talked about the PF tek being better on ryeflower or sorgum powder.

BE ,Biological efficiency was discussed based on initial weight of dry substrate and yield of fresh mushrooms. Yield should be 100% or more.  Nothing less is acceptable.  
A trick at Aloha is to place chips of mycelium (agar) (when growing out bottles) against the glass to visualize growth then  selecting out the best bottles and doing g2g or g2b(bottle) with those more vigorous colonizers.  MushroomerClub. Com was mentioned as a basic resource.

 

 

The chief of the lab had a discussion regarding laboratory supplies:

Slants: Tube types, advantages and disadvantages

Instruments used to cut mycelium for transfer under a flowhood vary because of the thickness of the mycelium.  Needle, inoculating loop for the softer and perhaps scalpel or dental instrument if some species form crusts.  He preferred a flat top scoop for transfers.  If screw top tubes are used mycelium grow may decrease if screw tops are on too tight decreasing gas exchange.  Screw tops are one block to contamination and adding parafilm (sprayed with IPA) in the glove box or flowhood is the second line of defense against contaminates.  Plastic tubes cloud up with recurrent sterilization.  Colonies are more visible with larger tubes.  Graduation marks and baked in label are advantages


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#8 TurkeyRanch

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Posted 19 November 2014 - 11:42 AM

Thanks and keep it coming! I hope that they keep the scholarship program open, it's a great opportunity for learning. Sounds amazing and totally fun. Thanks for sharing your experience with us.
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#9 bugs

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Posted 19 November 2014 - 07:39 PM

What an opportunity! That's great, working directly with some real pros.

 

 I've always wondered how they grow their cordyceps. Mental picture is acres of dead caterpillars  :tongue: Do I remember seeing on their website (haven't looked at it for a couple years) that they just use the mycelium, or do they actually fruit them?



#10 Seeker2be

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Posted 19 November 2014 - 08:26 PM

They use just the mycelium after most of the substrate is consumed.  Said to be less than 3% of the product (the substrate)



#11 Seeker2be

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Posted 19 November 2014 - 08:35 PM

Day 4

Today was a short day due to a tour from Germany of 30 people.  We inoculated the bags in the clean room , they were sealed and using sterile technique with complete tyvek gowning we shook the bags (approximately 100) to distribute the spawn in the grain.  .  The bags were marked with the days  run as posted on the wall 9/8/14 PO (pleurotis osteatus), PS (pleurotis salmoneo) strain.

 

Day 5 Started at 6:30 am rotating through stations of adding spawn to the bags we had made last week 2 runs of  Pink oyster and 2 runs of lenticula edodes about 1000 lbs including special orders.  We took turns pouring the spawn with the proper bag opening technique (a flip of the index finger under the double folded flap so as not to touch the top edge of the bag and then alternate finger technique to open the semi-fused bags)  We spent a lot of time cleaning off the aluminum tables with rotating types of sterile disinfectants (Isopropal alcohl towel wipes then spraying down with volsana between groupings of bags.  The bags to be sent over to the clean room for packing had to be arranged folded flap up and then quickly passed through a plastic flap (like in the cold section of a market hanging divided vertically). Another station was to shake the bags after the genus and special name , date and run serial number was noted.

In the Afternoon the director of the culture lab discussed techniques of taking a sample from petri dishes and transferring to another using the central or mid portion of the growing mycelia.  He showed us how to open the petri dishes in the palm of your hand like a clam shell facing the flow hood, the size of the pieces to be selected and how to scope them into the agar mycyelium side up.  We talked about the best tool for making transfers, a spatula not a scalpel or a inoculation ring and why it was best.  He discussed where to place the transferred piece in the Petri  dishes (center) and the tubes (midway) to prevent contamination.  His preparation for the flow hood placing everything you would need (dishes, test tubes, test tube rack, parafilm (cut and measured in the flow hood prior to tissue transfer, pens , insider and outside the flow hood designated alcohol spray bottles.  The need to spray down the all the implements including the parafilm in easy preparation before the transfers.  He reminded us to loosen the caps of receiving containers and to keep them slightly lose for air exchange before the parafilm was placed so as not to kill the growth.  We did a mock transfer outside the lab and then went into the lab to do transfers of L. edodes from tube to tube and from petri to petri critiquing our learned technique.  He spent 2 hours with us critiquing and improving our techniques.


Edited by Seeker2be, 19 November 2014 - 08:36 PM.

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#12 wharfrat

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Posted 19 November 2014 - 08:56 PM

awesome



#13 August West

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Posted 19 November 2014 - 11:24 PM

Great share, man. This will be a useful post. Kudos.

#14 Seeker2be

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Posted 20 November 2014 - 05:54 AM

Day 6:  Today we got up at 5:15 Am to continue the process we started 1 week ago but with different bags of colonized cortryceps mycelia dated 7/3 (the day they were inoculated G2G).  As noted the completed product was < 3% grain unlike other products from other companies.  This would be a blend of multiple different cortryceps species.  Full spectrum product equal to or exceeding Tibetan cortyrceps.  Before cleaning the pans  (About 250) to dry the mycelium, which would later be milled, we spoke with one of our mentors about the vexing problem of evolving a fruiting chamber gaining success before investing big capital in any mushroom growing operation.  He, himself, had gone from a fruiting box to a stand of shelves in his apartment to designing a building to manipulate temperature, humidity and co2 exchange .  We talked about the colocybe indica mushroom that would grow on straw with a casing layer (from India)

In the processing are we dunked all the pans in bleach water and dried them off placing a paper mat on each.  The grain was brought to a semi clean area and in full sterility tyvek suits we poured the grain onto the 3 ½ foot by 2 foot pans (or so).  The pans were next loaded into the dryer for drying at 100 degrees.  There were 15 doors with 10 racks holding 2 trays each.  When finished the equivalent of an 11 lb bag would be 2lb and would then go to the grinder.  It was hot exhausting work for 4 hours in 100 degree temps and everyone was sweating profusely.  Tomorrow we will be working with a biochemist and mixing up agar.



#15 esculenta

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Posted 20 November 2014 - 08:39 AM

congrats on your internship.  sounds like they are working you to death, but that's par for the course if you're going to start a mushroom farm!  welcome to the grustle!  (grind+hustle)


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#16 Seeker2be

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Posted 20 November 2014 - 07:55 PM

Day 7 We spent the morning with a young biochemist, who talked to us about media preparation.  It was a hands on day making (roughly )120 tubes of media-slants  We made up various formulas that Aloha has been expanding on.  Anything from ground up crickets, ground up shrimp shells and earthworms.  It seems that adding activated charcoal to agar mixes may speed up colonization and may limit contams. We measured the components on a scale and mixed them and loaded the tubes.  We made malt yeast peptone agar with charcoal and 7 other mixtures. 20 petri dishes can be made out of 500cc although we were making slants for our transfers.  If agar contains yeast then one must autoclave for 45 min to assure yeast is killed.  When using test tubes with screw tops prior to autoclave make tops tight but not so tight you can’t loosen them. They add stir sticks to all their slants for added nutrition to the mycelia. The biochemist will supply us with an updated list Aloha has for agar recipes. In a large flask the agar is swirled and microwaved for 1 min intervals and swirled until just about boiling , a total of about 3 min. This prevented undisolved agar from remaining and uneven mixtures from producing non gelling agar in some tubes or plates thus guaranting uniformity.

An interesting aside on Day 1 we planted our finger prints on a petri dishes before and after hand washing. A solution of black light imaged liquid was used before and after handwashing. We were deficient.  Looking at the cultures today showed us we need to address this personal hygiene issue more seriously than we thought.  Good hand washing needs to be vigorous, repeated and using plenty of soap with scrubbing sponges and nail cleaning despite gloves. A no brainer for sure but taken for granted by many of us.

 

A Handout:

 

Instructions for Media Preparation

 

Pour 20 ml of media in each 50 ml tube

Pour 7 ml of media in each 15 ml tube

Agar should face the sticker on the tube

 

Wooden stick (coffee stir sticks) (aids growth)

 

Has to be 4" long for both 50 and 15 ml tubes

Note: torn stick are not acceptable!

 

Tissue transfer

 

Culture Check

 

Before doing transfers check tubes for contamination:

1. Broken seal

2. Molds under tube cap

3.Molds growing inside the tube

4. Bacteria

5. check tubes for viability

 

Note: If you notice any suspicious tubes let the QC know about them

 

Clean Culture

 

Before taking tube into the lab please wipe them with sterilizers (alcohol,etc.) then place them in zip lock bag

 

Before taking tubes under the flowhood for transfers please take tubes out of the zip lock bag and wipe them again with sterilizers.

 

Technique

 

When cooling your scalpel.....

 

In the tube medium-simply cool your scalpel in the tube medium that your going to do the transfer in.

 

In the plate- make sure that you have a freshly done medium in the plate.  And make sure your transferring plate is free of contaminants.  Use a plate for a maximum of 10 transfers.

 

Never  ever cool scalpel in alcohol!! Only in the fresh plate!!

 

 

Rotate sterilizers when disinfecting objects or surfaces

 

 Parafilm

 

When sealing tubes, please cut the parafilm sealing tape under the hood and spray with alcohol before beginning any transfers.

 

A minimum of 2 wraps of sealing tabpe should be enough tin order to seal one tube. Make sure screw top tube are loosened slightly for gas exchange prior to placing parafilm.  Clean with alcohol in place in zip lock bag any work you complete.

 

Hand writing on 50 ml tubes:

 

Species name- the complete latin name, readable and without any shortcuts.  Has to be written on the side facing the agar medium.

 

Strain ID- uppercase, readable and the same as on the mother culture

 

Date: Has to be readable.  Make sure that you know the correct date when inoculating tubes.  Has to be written in the first line under the latin name.

 

P-value- Has to be the correct P-value #.  Single numerical value only!{ NOT P33-116!}  Even if the mother cultures is written in a range value

 

Media type-Check the bag that the tube was stored in- on it you will see the media type.  Write that down on the tube under the date.

 

Hand writing on the 15 ml tubes:

 

Species name

Strain Id

Date

P value

Media type. 

 

Note: Double check the label afer it has been written

 

Colony growth

 

Transferred tubes should stay in the labe 4-5 days until colonies grow.  They are then checked by the QC

 

Tube Taping

 

Please make sure that tape covers the whole hand written area on the 50 ml tubes

 

Box Filling

 

Next the tubes will be wiped with sterilizer and inserted into zip lock bags in the culture bank.


Edited by Seeker2be, 20 November 2014 - 08:02 PM.

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#17 CatsAndBats

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Posted 20 November 2014 - 08:19 PM

Seeker, I really appreciate your very detailed posts/threads. Keep up the great work buddy. Thanks for the heads up on radical mycology too.

-catattack



#18 Seeker2be

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Posted 20 November 2014 - 08:39 PM

Another Tip:  Tradd Cotter's book Organic mushroom growing and remediation will be the new bible: Simpler that Stamets' to read and very practical


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#19 Seeker2be

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Posted 20 November 2014 - 08:55 PM

Media recipe book part 1  The purpose of these varied recipes is to challenge your mycelia with different food and prevent or delay senescense or aging of said strain. 

MALT AGARS

Malt Extract Agar

 

For the premixed malt agar: Use 25 g

ME

H2O

500 mL

Agar

10 g

Malt

10 g

 

Malt-yeast Extract

MYA

H2O

500 mL

Agar

10 mL

Malt

10 mL

Yeast

1 mL

 

Malt Extract with Activated Carbon

MEAC

H2O

500 mL

Agar

10 g

Malt

10 g

Activated Carbon

1 g

 

Malt Extract with Peptone

MEP

H2O

500 mL

Agar

10 g

Malt

10 g

Peptone

1 g

 

Malt Extract with Yeast and Peptone

MYAP

H2O

500 mL

Agar

10 g

Malt

10 g

Yeast

1 g

Peptone

1 g

 

Malt Extract with Yeast, Peptone, and Activated Carbon

MEPYAC

H2O

500 mL

Agar

10 g

Malt

10 g

Peptone

1 g

Yeast

1 g

Activated Carbon

1 g

 

 

 

Wheat Malt-yeast Extract

WMYA

H2O

500 mL

Agar

10 mL

Wheat Malt

10 mL

Yeast

1 mL

 

Wheat Malt Extract with Peptone

WMEP

H2O

500 mL

Agar

10 g

Wheat Malt

10 g

Peptone

1 g

 

Wheat Malt Extract with Yeast and Peptone

WMYAP

H2O

500 mL

Agar

10 g

Wheat Malt

10 g

Yeast

1 g

Peptone

1 g

 

Malt Extract with Milo

MEM

H2O

500 mL

Agar

10 g

Malt

10 g

Ground Milo

10 g

 

Malt Extract with Corn

MEC

H2O

500 mL

Agar

10 g

Malt

10 g

Ground Corn

10 g

 

Malt Extract with Honey

MEH

H2O

500 mL

Agar

10 g

Malt

10 g

Honey

16 g

 

ATTC Medium 597

597

H2O

500 mL

Agar

10 g

Malt

 3.75 g

Yeast

0.3 g

Peptone

0.5 g

 

MYAP with Multi-vitamin

 

Add Multi-vitamin after sterilization.

MYMP

H2O

500 mL

Agar

10 g

Fresh ground crickets

8 g

Multi-Vitamin

0.01 g

Peptone

1 g

Yeast

1 g

Malt

10 g

 

Can use just 1 or 2 elements, so label MYPZ or MYPCZ, etc.

MYAP with elements

MYPMCZ

H2O

500 mL

Agar

10 g

Malt

 10 g

Yeast

1 g

Peptone

1 g

Calcium

0.01 g

Magnesium

0.01 g

Zinc

0.01 g

 

Yeast Malt Extract

YME

H2O

500 mL

Pre-mixed Malt Agar

25 g

Yeast

1 g

 

Peptone Malt Extract

PME

H2O

500 mL

Pre-mixed Malt Agar

25 g

Peptone

1 g

 

MYA with CS4

CS4MY

H2O

500 mL

Agar

10 g

Malt

 3.75 g

Yeast

0.3 g

Ground Cordyceps Sinensis 4

10 g

 

MYA with CSG2

G2MY

H2O

500 mL

Agar

10 g

Malt

 3.75 g

Yeast

0.3 g

Ground Cordyceps Sinensis G2

10 g

 

 

Chitin Method

:ch

Add 0.3 g chitin to any malt agar

 

 

For 24 test tubes (1 rack) use:

 432 mL H2O

8.6 g Agar

8.6 g nutrient

0.86 g yeast/peptone

 

 

 

 

 

 

 


ANIMAL RELATED MEDIA

 

Blood Worm Agar

BW

H2O

500 mL

Agar

10 g

Blood Worms

5 g

 

 

Krill Agar

KA

H2O

500 mL

Agar

10 g

Krill

10 g

 

Fish Flake Dextrose Agar

FDA

H2O

500 mL

Agar

10 g

Fish Flakes

3 g

Dextrose

10 g

 

Meal Worm Agar

MW

H2O

500 mL

Agar

10 g

Meal Worms (ground)

10 g

Dextrose

3 g

Yeast

1 g

 

Fishy Wax Agar

FWA

H2O

500 mL

Agar

10 g

Krill

3.3 g

Tubiflex Worms

3.3 g

Fish Flakes

3.3 g

Bee's Wax

1 g


Edited by Seeker2be, 20 November 2014 - 09:05 PM.

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#20 Seeker2be

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Posted 20 November 2014 - 08:58 PM

The media recipe book Part 2

 

 

 

 

Potato, Ammonium citrate, Thiamin Agar

 

Cordyceps militaris known to fruit from this and brown rice in jars.

PAT

H2O

400 mL

Agar

10 g

Potato Broth

100 mL

KH2PO4

1 g

MgSO4

0.5 g

Peptone

1.5 g

Ammonium Citrate

0.5 g

Dextrose

15 g

Thiamine (B1 vitamin)

0.025 g

 

In the 500 mL of water boil the 150 g of potatoes, and use this broth (not the potatoes). Bring solution back to 500 mL with water then add remaining ingredients. Potato flakes can be used as a substitute (5 g in the 500 mL of water).

Potato Dextrose Agar

PDA

H2O

500 mL

Agar

10 g

Dextrose  or

Honey/Corn syrup

7 g or

10 mL

Diced potatoes

150 g

 

In the 500 mL of water boil the 150 g of potatoes, and use this broth (not the potatoes). Bring solution back to 500 mL with water then add remaining ingredients. Potato flakes can be used as a substitute (5 g in the 500 mL of water).

(boiled in 500 cc water and use the brothnot the potatoes) bring the solution back to 500cc then add the rest of the ingredients

Potato Dextrose Yeast  Agar

PDYA

 

H2O

500 mL

 

Agar

10 g

 

Dextrose  or

Honey/Corn syrup

7 g or

10 mL

 

Diced potatoes

150 g

 

Yeast

1g

 

Yeast

1 g

       

 

 

 

 

 

Potato Skin Dextrose Agar

PSDA

H2O

500 mL

Agar

10 g

Dried Potato skins

10 g

Dextrose

7 g

 

For potato agars made from real potatoes label

PP so PPPDA or PPPDY for example

For long term storage/ wood decomposers

Add 10 g sawdust and label with :SD

 

Cornmeal Malt Agar

CMMA

H2O

500 mL

Agar

10 g

Cornmeal

10 g

Dextrose

7 g

 

Spore Germination Agar #1

 

10 g cornmeal and 10 g agar can be substituted for the Difco Cornmeal agar.

 

SGA1

H2O

500 mL

Agar

10 g

Difco Cornmeal Agar

8.5 g

Glucose (Dextrose)

1 g

Yeast

0.5 g

 

10 g cornmeal and 10 g agar can be substituted for the Difco Cornmeal agar.

Spore Germination Agar #2

SGA2

H2O

500 mL

Difco Cornmeal Agar

8.5 g

Glucose (Dextrose)

3.5 g

Sucrose

5 g

Yeast

0.5 g

KH2PO4

0.5 g

 

Spore Germination Agar #3

SGA3

H2O

500 mL

Agar

10 g

Cornmeal

10 g

Malt

0.8 g

 

10 g cornmeal and 10 g agar can be substituted for the Difco Cornmeal agar.

 

Spore Germination Agar #4

SGA4

H2O

500 mL

Difco Cornmeal Agar

8.5 g

Glucose

1 g

Sucrose

1.5 g

Yeast

0.5 g

 

 

 

Potato Dextrose Agar

PDA

H2O

500 mL

Agar

10 g

Diced and boiled Potatoes or

Potato starch

150 g or

10 g

Dextrose

7 g

 

Potato Dextrose Agar

PDY

H2O

500 mL

Agar

10 g

Diced and boiled Potatoes or

Potato starch

15 g or

10 g

Dextrose

7 g

 

CHEMICAL MEDIA

 

Malt with Activated Carbon and KOH

R7

H2O

500 mL

Agar

10 g

Epsom Salts

0.25 g

Activated Carbon

1.3 g

Malt

12.5 g

Humus

2.5 g

KOH 1%

20 mL

 

Organic Medium Agar

OMA

H2O

500 mL

Agar

10 g

Glucose

5 g

Peptone

0.5 g

Yeast

0.05 g

KH2PO4

1 g

MgSO4x7H2O or

MgSO4

0.15 g or

0.075 g

 

Czapek's Medium

Czapek's

H2O

500 mL

Agar

10 g

NaNO3

1.5 g

K2HPO4

0.5 g

MgSO4

0.25 g

KCl

0.25 g

FeSO4 or

Fe SO4x7H2O

0.0187 g or

0.0094 g

Dextrose

15 g

 

JUICE/BROTH MEDIA

V8 juice Agar

V8A

H2O

500 mL

Agar

10 g

CaCO3

1.5 g

V8 Juice

100 mL

 

Beef Broth Agar

BBA

H2O

500 mL

Agar

10 g

Beef Broth

20 mL

Malt

5 g

Yeast

1 g

 

Chicken Broth Agar

CBA

H2O

500 mL

Agar

10 g

Chicken Broth

20 mL

Malt

5 g

Yeast

1 g

 

Butternut Squash Soup Agar

BNSA

H2O

500 mL

Agar

10 g

Butternut squash soup

20 mL

Malt

5 g

Yeast

1 g

 

Beef Yeast Agar

BYA

H2O

300 mL

Agar

10 g

Yeast

2 g

Beef Broth

200 mL

 

 

MISCELLANEOUS MEDIA

 

Brine Agar

BA

H2O

500 mL

Agar

10 g

Brine

10 g

 

Sea Water Agar

 

Dissolve beef extract and peptone by heating H2O. Adjust pH to 7.8, and boil for 10 minutes. Readjust pH to 7.3 and add agar.

SWA

H2O

125 mL

Agar

10 g

Sea Water

375 mL

Beef Extract

5 g

Peptone

5 g

To Make Artificial Sea Water

 

NaCl

14.065 g

KCl

0.385 g

CaCl2

0.60 g


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