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InvisibleRigVedaLXVII.Soma
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Microscopy Supplies Suggestions?
    #15667142 - 01/14/12 09:21 PM (12 years, 2 months ago)

Hello everyone,

I was wondering if anyone could possibly recomend any supplies I should get for microscopy purposes. Like stains, for example, mounting agents, color removers, whatever you feel that everyone with a compound microscope should have in relation to the study of fungi.
I mainly ask, because I definitely don't think I can get my hands on any Melzers reagent right now.

Also don't feel restricted to the study of fungi, I also have a strong passion for byrology, phycology, bacteriology, and I know this is considered bacteriology, but the study of Cyanobacteria as well.

Another main thing I am curious about, how can I go about viewing mycelium or something similar I may have growing on a culture medium from a petri dish? There is a series of pictures of Cyanobacteria weaving a mat on agar, through a microscope at 400x. One of the photos is in my sig all the way to the right. It's caption actually said it was from a petri dish, laid on the stage of the microscope without a cover slip. So that's one simple method, but I don't believe that would work for Mycellium on agar.. I also have read a journal article about using a piece of clear tape, laying it down on top of the mycellium in the dish, then laying that on the microscope slide, using a mounting agent (cant remember which, but it actually melted away the stickyness to the back of the tape) then a coverslip put over it.

So any suggestions? It would really help since I am about to buy some materials tonight.

Thanks!


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

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OfflineToxicManM
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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma] * 1
    #15675629 - 01/16/12 09:11 PM (12 years, 2 months ago)

A good substitute for Meltzer's is Tincture of Iodine. You can get it in the first aid supplies area in most grocery or drug stores. The difference between Meltzer's and Iodine is that Meltzer's has Chloral Hydrate added to it. Chloral Hydrate is the original date rape drug, and it's federally controlled.

The stain I use second most is Lactophenol Cotton Blue. You'll have to check around chemical supply places to see if any of them will sell it to you. It's widely used for medical mycology, so it's produced quite a bit.

Potassium Hydroxide (KOH) is widely available for soap making. A chemical or science supply place is a good source.

Another possible good source for stains and such can be the chemistry department of a nearby college or university. You may have to take a chemistry or microbiology class or two to convince them that you're not going to do something stupid with the stuff. If you can convince them that you're serious, they can be a good source for things like Meltzer's.


The best reference that's easily available for stains and such is How to Identify Mushrooms to Genus III: Microscopic Features by David Largent, David Johnson, and Roy Watling (ISBN 0-916-422-09-7). It'll also tell you what you should be looking for under the  microscope.


When trying to view something like mycelium on top of agar, the big trick is getting good illumination. The standard microscope setup is with the light shining from below through the specimen. To get good images for this type of setup, get a strong light and use it to illuminate the specimen as closely to above as you can get. You'll have to shine it quite a bit from the side, but that helps show the 3D form by throwing hills and depressions into high contrast.

Looking at the photo you mention of the cyanobacteria, you can see that the light is to the upper left of the specimen.


--------------------
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OfflineAlan RockefellerM
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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15676752 - 01/17/12 05:10 AM (12 years, 2 months ago)

Get cotton blue, congo red, and a reticle to measure sizes.

You can use iodine or Lugol's instead of melzers.  It might be a little different but its 98% the same as melzers.  The chloral hydrate is a clearing agent.  You can use other clearing agents or leave it out, perhaps with slightly different effects.  Peter Werner makes some fake (legal) melzers reagent with a different clearing agent, lactophenol.

Mount in 5% KOH or water with a small amount of dish soap.


You can put a petri dish under the scope at 100x without removing the cover.  For 200x, remove the cover and focus close with bright light coming from the side.

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OfflineLanLord
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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15677146 - 01/17/12 08:44 AM (12 years, 2 months ago)

posting so I can refer back.
excellent information here.


--------------------
Good judgment comes from experience, and a lotta that comes from bad judgment.

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Alan Rockefeller]
    #15677617 - 01/17/12 11:24 AM (12 years, 2 months ago)

Awesome Thank you so much, ToxicMan and Alan Rockefeller.

Both extremely informative posts. Thanks for the help!


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

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Invisibleriverdweller
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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15677651 - 01/17/12 11:31 AM (12 years, 2 months ago)

Indeed, now I also know how to get around the reagent issue.  Thanks for posting your questions.


--------------------
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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Alan Rockefeller]
    #15677797 - 01/17/12 12:18 PM (12 years, 2 months ago)

Also, any suggestions on how to correctly prepare KOH from dry flake crystaline? There is an unlimited supply on ebay of it, but it all mostly consists of Dry flakes, not in solution. I was wondering if you mix it with water? or Alcohol? and what is the Ratio? I don't have a great enough knowledge on chemisty to understand a reaction that may occur, if mixed with the wrong element... Probably all pretty basic knowledge, but I am having trouble finding any information in regards to this situation, considering most information available is directed towards it's use in soap making, as ToxicMan stated.

Thanks again folks. Good luck on your microscopy everyone!



And I found this for anyone interested in Bacterial Microscopy.
Quote:


Stains for Bacteriology
Albert stains - reagents used to demonstrate volutin granules and cytoplasm in diphtheria organisms.
Albert Stain # 1

    Mix 1ml of glacial acetic acid into 100ml of distilled water
    Mix 2ml of 95% ethanol into the solution from step 1
    Dissolve 0.15g of toluidine blue in the solution from step 2
    Dissolve 0.2g malachite green in the solution from step 3

Albert Stain # 2

    Dissolve 2g of iodine in 50ml of distilled water
    Add remaining 250ml of water to the solution from step 1
    Disslove 3g of KI in the solution from step 2

Albert stains are commonly used to demonstrate volutin granules and cytoplasm in diphtheria organisms.


Field's Stain - a rapid stain used commonly on thin films for malarial parasites.
Field's Stain is a rapid stain used commonly on thin films for malarial parasites.

Field's Solution # 1

    Dissolve 1.6 g of methylene blue in 1 liter of distilled water
    Dissolve 2.6g of Na2HPO4 (anhydrous) to the solution from step 1
    Dissolve 1g of Azure 1 in the solution from step 2
    Dissolve 2.6 g of KH2PO4 in the solution from step 3
    Place on mild heat with stirring or shaking for 45 minutes to 1 hour
    Let stand at room temperature for 24 hours
    Filter

Field's Solution # 2

    Dissolve 2g of Eosin Y in 1 liter of distilled water
    Dissolve 2.6g of Na2HPO4 in the solution from step 1
    Dissolve 2.6g of KH2PO4 in the solution from step 2
    Filter


Flagella Stain - allows for the direct observation of flagella
Flagella Stain is a stain used commonly to allow for the direct observation of flagella.

    Dissolve 20g of tannic acid in 30ml of 60% ethanol
    Dissolve 36g of aluminum chloride (hydrated) to the solution from step 1
    Dissolve 20g of ZnCl2 in the solution from step 2
    Dissolve 3g of rosaniline HCl in the solution from step 3
    q.s. to 70ml with 60% ethanol



Gentian Violet Stain Recipe - Reagent used in the gram-stain procedure.
Gentian Violet is one of the stains used in the gram stain procedure. Gram-positive bacteria take up and retain gentian violet stain.

    Dissolve 1g gentian violet in 20ml of 95% ethanol
    Add 60ml 5% formalin to the solution from step 1 and mix completely
    Filter solution to remove any precipitate


Retrieved from http://www.thelabrat.com/protocols/bacculture.shtml





--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

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Edited by RigVedaLXVII.Soma (01/17/12 12:43 PM)

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Invisibleriverdweller
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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15677865 - 01/17/12 12:36 PM (12 years, 2 months ago)

i zeroed my container on the scale, measured 20 parts water to 1 part flakes.


--------------------
I'm still here  :wink:

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: riverdweller]
    #15677912 - 01/17/12 12:45 PM (12 years, 2 months ago)

Awesome, Thank you riverdweller!


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

MyPostImage Gallery  Trade List
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:peace: Solanaceae, Amanitaceae :peace:

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OfflineAlan RockefellerM
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Re: Microscopy Supplies Suggestions? [Re: riverdweller]
    #15678110 - 01/17/12 01:29 PM (12 years, 2 months ago)

Quote:

riverdweller said:
i zeroed my container on the scale, measured 20 parts water to 1 part flakes.





That is what I do too.  When I have a scale.  In Mexico I eyeball it and it works fine.  The concentration doesn't matter much, some mycologists recommend 10% to make sure the reactions are maximized, others recommend 3% for safety reasons.  I tend to make it a bit on the strong side because I strongly disapprove of safety.


I carry KOH everywhere when I am mushroom hunting, you never know when you find a mushroom that needs it.  In Mexico I had a bottle of pretty strong KOH, unknown concentration but probably around 8%.  The bottle unscrewed in my pocket and spilled all over my crotch.  I didn't notice until several hours later when I needed to use it on a bolete and there was barely any left.  No negative effects or skin irritation was observed, but my hand felt soapy whenever I got anything out of my pocket for the next couple days.

KOH is extremely dangerous in pure form, see


I sometimes add a few drops to my beer, which amuses other mycologists greatly but doesn't change the flavor.  It is immediately neutralized by the carbonic acid into potassium carbonate.  They sell very dilute KOH solutions in mexican markets, they use it in cooking for softening frijoles and maize.

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Alan Rockefeller]
    #15678144 - 01/17/12 01:38 PM (12 years, 2 months ago)

Haha nice, Fight Club. Great movie, Throwing that one in right now.
Nearly as basic as sodium hydroxide, huh? Okay, I will definitely be careful with the pure form. Thanks again Alan, your information never ceases to amaze.

I greatly appreciate the input! :smile:


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

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:peace: Solanaceae, Amanitaceae :peace:

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: ToxicMan]
    #15679361 - 01/17/12 05:28 PM (12 years, 2 months ago)

Would a 0.1% Aqueous solution of Congo Red be adequate for this purpose? Seems I am having trouble finding good enough quantities of this, and Lactophenol Cotton Blue.

Kind of hard to find these things other than from Sigma aldrich, who I highly doubt will sell to me..


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

MyPostImage Gallery  Trade List
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:peace: Solanaceae, Amanitaceae :peace:

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InvisibleByrain


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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15679393 - 01/17/12 05:36 PM (12 years, 2 months ago)

Quote:

RigVedaLXVII.Soma said:
Would a 0.1% Aqueous solution of Congo Red be adequate for this purpose? Seems I am having trouble finding good enough quantities of this, and Lactophenol Cotton Blue.

Kind of hard to find these things other than from Sigma aldrich, who I highly doubt will sell to me..




http://www.amazon.com/Congo-Red-Stain-500-mL/dp/B0017UAJHQ/ref=sr_1_1?ie=UTF8&qid=1326847021&sr=8-1

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Byrain]
    #15679408 - 01/17/12 05:40 PM (12 years, 2 months ago)

Yeah, the only one I found is on Amazon. From the same company as in the link you just provided. There are multiple sizes. But they all say 0.1% solution. and this one in your link, doesn't state the percentage, but trying to look close to the small picture, it looks like maybe .4-.5% solution? but would these percentages be adequate though?


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

MyPostImage Gallery  Trade List
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:peace: Solanaceae, Amanitaceae :peace:

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InvisibleByrain


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Re: Microscopy Supplies Suggestions? [Re: RigVedaLXVII.Soma]
    #15679413 - 01/17/12 05:42 PM (12 years, 2 months ago)

I still haven't gotten any stains, but that is what I have seen being recommended for mycology.

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Byrain]
    #15679448 - 01/17/12 05:48 PM (12 years, 2 months ago)

Okay cool, I will give it a try.
I appreciate the answer buddy.


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

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:peace: Solanaceae, Amanitaceae :peace:

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OfflineTerry M
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Re: Microscopy Supplies Suggestions? [Re: Alan Rockefeller]
    #15679469 - 01/17/12 05:52 PM (12 years, 2 months ago)

Quote:

Alan Rockefeller said:
You can use iodine or Lugol's instead of melzers.  It might be a little different but its 98% the same as melzers.  The chloral hydrate is a clearing agent.  You can use other clearing agents or leave it out, perhaps with slightly different effects. 




But Alan, what about this work by Lawrence Leonard:
http://www.fungimag.com/archives/Melzer__Lugol.pdf

For mushroom identification, substitutions for Melzer's appear to be a significant problem. A good percentage of the species Leonard tested did not respond the same between Melzer's, Logol's, and tincture of iodine. In these cases, identification would have been wrong.

Table 2 shows reactions for 35 samples. Of these, 20 of 35 (57%) reacted differently with Lugol's compared to Melzer's. And for the straight Iodine, this figure was 15 of 35 (43%).

I would LOVE to find a reliable substitute for Melzer's, which I don't have.


--------------------
Liberté, égalité, humidité.

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OfflineLuckOfTheFryish
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Re: Microscopy Supplies Suggestions? [Re: Terry M]
    #15679536 - 01/17/12 06:10 PM (12 years, 2 months ago)

well hello Terry! whats a classy dame like you doing in a place like this? :lol:


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OfflineTerry M
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Re: Microscopy Supplies Suggestions? [Re: LuckOfTheFryish]
    #15679555 - 01/17/12 06:14 PM (12 years, 2 months ago)

Quote:

LuckOfTheFryish said:
well hello Terry! whats a classy dame like you doing in a place like this? :lol:



:lol:


--------------------
Liberté, égalité, humidité.

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InvisibleRigVedaLXVII.Soma
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Re: Microscopy Supplies Suggestions? [Re: Terry M]
    #15679600 - 01/17/12 06:21 PM (12 years, 2 months ago)

Yeah, but we aren't necessarily talking about staining for field identification, as much as we are referring to staining for microscopic observation.


--------------------
"While clinging to the moronic belief that they constitute a "counterculture," they share our society's overriding urge for expediency. They make no attempt to learn about the organisms they eat and it always struck me as ironic that people with such a low level of consciousness should be seeking "higher consciousness." -David Arora

"Man is placed in the middle between two infinities - the infinitely great and the infinitely little - both of which are equally incomprehensible to him." -Pascal

MyPostImage Gallery  Trade List
Spores I Am Looking For  Seeds I am Looking For
:peace: Solanaceae, Amanitaceae :peace:

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