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.
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.
