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Offlineehtdaedlufetarg
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Calibrating Microscope
    #11133509 - 09/26/09 07:32 PM (14 years, 5 months ago)

So i got this microscope a month ago about. Ive been enjoying just looking at different spores but i really need to be able to measure them. I read up from Micheal Kuos article. So my question is 1. My microscope doesnt seem to have any sort of measuring system in the Eyepiece. Do i have to put one in? How?  Its hard to start from next to no Experience and go straight from a article. I would really appreciate some advice from anyone Experienced in mushroom Microscopy. im lost.


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Invisiblejohnnyblaze2316

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Re: Calibrating Microscope [Re: ehtdaedlufetarg]
    #11133522 - 09/26/09 07:33 PM (14 years, 5 months ago)

i believe what you will be needing is called a reticle

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InvisibleBobzimmer
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Re: Calibrating Microscope [Re: ehtdaedlufetarg]
    #11133546 - 09/26/09 07:37 PM (14 years, 5 months ago)

You need a stage micrometer that shows a "grid" when you look through the eyepiece. You have to calibrate that.


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Mr. Mushrooms said:
I will confess something that should be quite obvious, CC.  I love mushrooms, i.e. fungi.  I really do.  I am talking about a strong feeling, i.e. emotion, for them.  I think they are beautiful.  I even dream of them.

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Re: Calibrating Microscope [Re: ehtdaedlufetarg]
    #11133648 - 09/26/09 07:51 PM (14 years, 5 months ago)


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OfflineAlan RockefellerM
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Re: Calibrating Microscope [Re: ehtdaedlufetarg]
    #11133694 - 09/26/09 07:58 PM (14 years, 5 months ago)

You need a reticle, which is a little piece of plastic that goes into the eyepiece, so you have a grid when you look through it.

Most people buy a 10x eyepiece with a reticle installed for about $35, but you can buy just the little plastic bit, maybe, but I don't know where.

I like the grid style better than the crosshairs style, its much easier to measure the spores that way.

Always measure spores at the highest magnification you can.

I usually keep the reticle in all the time but that gives some people headaches if their eyes can't ignore it.

To calibrate the reticle you need a stage micrometer, which is a special microscope slide with fine divisions.  They cost between $30 and $100 on ebay.  You only need it once.  I got a nice one for $30 on ebay that was supposed to cost $400, it is made from mirrored glass and has tiny holes.  Anyone who wants to borrow it should PM me.

The divisions on the reticle are usually 1 or 10 micrometers, but using the stage micrometer I found that my divisions are actually 8.7 microns, which is a significant difference and a real good thing to know when calculating spore size.  Cactu's reticle is actually 9.4 microns per each 10 measured.

If you are using a microscope to look at mushrooms, you need "How to Identify Mushrooms to Genus part III: Microscopic Features".  If you have a microscope but don't have a copy, PM me and I will fix that problem for you.

Measuring spores by hand introduces a lot of error.  Its better to use the Mr. Mushrooms method, where you hold a camera to the microscope reticle to photograph the spores and grid, then use a photo editor like The Gimp or Photoshop to count the number of pixels wide and long each spore is.  Then measure the grid, calculate the number of pixels per micrometer, and use math to turn the number of pixels into micrometers.  This eliminates lots of sources of error and gives you measurements that are exact to the nearest ten nanometers, more or less.  Probably less but that doesn't really matter, its cool to be able to say "this spore is 9.67 microns long".

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InvisibleWorld Spirit
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Re: Calibrating Microscope [Re: falcon]
    #11133860 - 09/26/09 08:27 PM (14 years, 5 months ago)

This was helpful to me as well. What measurement type of stage micrometer should we get? My maximum view is 500x.

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InvisibleWorld Spirit
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Re: Calibrating Microscope [Re: World Spirit]
    #11133862 - 09/26/09 08:28 PM (14 years, 5 months ago)

Woops should've refreshed the page first
Thanks AR

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InvisibleMr. Mushrooms
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Re: Calibrating Microscope [Re: Alan Rockefeller]
    #11134487 - 09/26/09 10:25 PM (14 years, 5 months ago)

Quote:

Alan Rockefeller said:

Measuring spores by hand introduces a lot of error.  Its better to use the Mr. Mushrooms method, where you hold a camera to the microscope reticle to photograph the spores and grid, then use a photo editor like The Gimp or Photoshop to count the number of pixels wide and long each spore is.  Then measure the grid, calculate the number of pixels per micrometer, and use math to turn the number of pixels into micrometers.  This eliminates lots of sources of error and gives you measurements that are exact to the nearest ten nanometers, more or less.  Probably less but that doesn't really matter, its cool to be able to say "this spore is 9.67 microns long".




Hey, that was supposed to be our secret.  :lol:  Seriously though, thanks for the credit.  I've used both methods but prefer mine for accuracy.

Also, after calibrating my way I merely hold a ruler up to the monitor and measure the spores, doing the math.  I can measure 30 spores in less than 5 minutes.  The standard way takes far longer.


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OfflineTwiztidsage
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Re: Calibrating Microscope [Re: Mr. Mushrooms]
    #11134576 - 09/26/09 10:50 PM (14 years, 5 months ago)

Is Ebay a good place to get a microscope? I am guessing no. If not, where?

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InvisibleMr. Mushrooms
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Re: Calibrating Microscope [Re: Twiztidsage]
    #11135419 - 09/27/09 02:59 AM (14 years, 5 months ago)

I think Alan has a link to a decent microscope offered on eBay.


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Offlineehtdaedlufetarg
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Re: Calibrating Microscope [Re: Mr. Mushrooms]
    #11137147 - 09/27/09 12:47 PM (14 years, 5 months ago)

Wow. Soo helpful. Thanks guys.


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InvisibleWorld Spirit
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Re: Calibrating Microscope [Re: ehtdaedlufetarg]
    #11137583 - 09/27/09 02:25 PM (14 years, 5 months ago)

I want to add to this thread for future search inquiries a note or two on the following unrelated material:

(Not listed at www.dictionary.com is the word pleurocystidia)

pleurocystidia
Plural form of pleurocystidium.

Pleurocystidium -
(mycology) A relatively large cell found on the gill face of a basidiomycete


Jump in and add a significant piece of basic knowledge to help upcoming microscopin' fungofamiliacs

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OfflineAlan RockefellerM
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Re: Calibrating Microscope [Re: Twiztidsage]
    #11137762 - 09/27/09 03:06 PM (14 years, 5 months ago)

Quote:

Is Ebay a good place to get a microscope? I am guessing no. If not, where?




Yes.  There are many suitable microscopes on ebay for good prices.  There are also lots of crappy microscopes and also really nice microscopes.  Sometimes for low prices.

This one costs $170 and has achromatic objectives:

http://cgi.ebay.com/BINOCULAR-BIOLOGICAL-MICROSCOPE-w-MECHANICAL-STAGE_W0QQitemZ350179455000QQcmdZViewItemQQptZLH_DefaultDomain_0?hash=item5188527018&_trksid=p3286.c0.m14

There are also many decent microscopes on amscope.com.

Quote:

(mycology) A relatively large cell found on the gill face of a basidiomycete




I wrote that. 

Its now the second google hit when you search for pleurocystidia.  I am so excited.  Its going to make me famous for sure.

Everyone that wants pleurocystidia will have to come to me to get them.  When putting a name on a species its really important to have them yet they can be really hard to find. 

Its easy enough to find them on fresh material - you cut the cap in an extremely thin slice perpendicular to the gills so you have a slice that looks like a very thin saw blade.  The pleurocystidia is on the sides of the saw teeth and the cheilocystidia on the tips. 

But when its dried its a lot harder to find the cells on the gill face.  You can't cut the cap like that, it will just break. Cheilocystidia is no problem.  For the pleurocystidia I guess you can rehydrade and fold the gill over.  But that only gives a narrow section of the gill face and usually there are no pleurocystidia there.  Also the gill usually breaks when you try to fold it over which pretty much sucks.

Maybe the key is to make lots of really thin slices of a bit of gill, and hope that some pleurocystidia shows up exactly where you cut it so you can get a good picture.  Good luck with that.

I guess workman knows how to find pleurocystidia in a dried sample and maybe he has some insight into the problem.

Maybe the sample could be rehydrated and then cut in a similar way.  That would turn the sample into a blob of goo but maybe it would work.

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OfflineTwiztidsage
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Re: Calibrating Microscope [Re: Alan Rockefeller]
    #11137842 - 09/27/09 03:22 PM (14 years, 5 months ago)

Thanks for the info Mr. Rockefeller!

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InvisibleWorld Spirit
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Re: Calibrating Microscope [Re: Twiztidsage]
    #11137916 - 09/27/09 03:39 PM (14 years, 5 months ago)

Amen. Thanks.

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InvisibleMr. Mushrooms
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Re: Calibrating Microscope [Re: Alan Rockefeller]
    #11146507 - 09/28/09 08:50 PM (14 years, 5 months ago)

Quote:

Alan Rockefeller said:
Quote:

Is Ebay a good place to get a microscope? I am guessing no. If not, where?




Yes.  There are many suitable microscopes on ebay for good prices.  There are also lots of crappy microscopes and also really nice microscopes.  Sometimes for low prices.

This one costs $170 and has achromatic objectives:

http://cgi.ebay.com/BINOCULAR-BIOLOGICAL-MICROSCOPE-w-MECHANICAL-STAGE_W0QQitemZ350179455000QQcmdZViewItemQQptZLH_DefaultDomain_0?hash=item5188527018&_trksid=p3286.c0.m14

There are also many decent microscopes on amscope.com.

Quote:

(mycology) A relatively large cell found on the gill face of a basidiomycete




I wrote that. 

Its now the second google hit when you search for pleurocystidia.  I am so excited.  Its going to make me famous for sure.

Everyone that wants pleurocystidia will have to come to me to get them.  When putting a name on a species its really important to have them yet they can be really hard to find. 

Its easy enough to find them on fresh material - you cut the cap in an extremely thin slice perpendicular to the gills so you have a slice that looks like a very thin saw blade.  The pleurocystidia is on the sides of the saw teeth and the cheilocystidia on the tips. 

But when its dried its a lot harder to find the cells on the gill face.  You can't cut the cap like that, it will just break. Cheilocystidia is no problem.  For the pleurocystidia I guess you can rehydrade and fold the gill over.  But that only gives a narrow section of the gill face and usually there are no pleurocystidia there.  Also the gill usually breaks when you try to fold it over which pretty much sucks.

Maybe the key is to make lots of really thin slices of a bit of gill, and hope that some pleurocystidia shows up exactly where you cut it so you can get a good picture.  Good luck with that.

I guess workman knows how to find pleurocystidia in a dried sample and maybe he has some insight into the problem.

Maybe the sample could be rehydrated and then cut in a similar way.  That would turn the sample into a blob of goo but maybe it would work.




Yeah, that's the link I was talking about.

And yeah, thanks for posting all of that.  I was just thinking we should share a few more tips on microscopy here for those interested in it.

When I am looking for pleurocystidia from a dried specimen I rehydrate a gill with 3% KOH solution and lay it flat on a slide.  Then I use a razor blade and slice off the thinnest slices I can.  When I have a few of them I put a cover glass over the mess and start looking around.  Most of the time I can find them. Photographing them is my problem.  I use a camera with a smaller lens than the ocular like you suggested.  Still I have problems focusing, even more problems holding the camera steady.  I think I might have a holder special made for it.

Maybe Workman will share his method with us like you said.


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OfflineWorkmanV
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Re: Calibrating Microscope [Re: Mr. Mushrooms]
    #11146709 - 09/28/09 09:14 PM (14 years, 5 months ago)

I pretty much do the same thing as Mr. Mushrooms on the slicing, but I only put the best slice on the slide with a drop of KOH solution.  I then press it down with a pencil eraser to flatten the gill slice and expell any air bubbles.  You have to mash it enough to get good visibility but not so much that you destroy the sample.  It often takes a few trys.

I've found that the less material on the slide, the sharper the image.  There is something about extra material on the slide, even if its not directly under the objective, that muddies the image.


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InvisibleMr. Mushrooms
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Re: Calibrating Microscope [Re: Workman]
    #11146741 - 09/28/09 09:17 PM (14 years, 5 months ago)

Sweet.  I'll have to try that.  I was afraid of getting the section on its side so I used a few of them.  If you mash it like you do that should solve that problem.


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OfflineWorkmanV
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Re: Calibrating Microscope [Re: Mr. Mushrooms]
    #11146878 - 09/28/09 09:32 PM (14 years, 5 months ago)

If your slice is thinner than the gill is wide it naturally floats to the right orientation (in theory) :wink:


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InvisibleinskiM
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Re: Calibrating Microscope [Re: Mr. Mushrooms]
    #11146888 - 09/28/09 09:33 PM (14 years, 5 months ago)

I think the problem with too much material on the slide is that it is too wide creating a greater depth of focus, hence the importance of creating as thin a section as possible, it takes practice.
A good way of telling if your sections are thin enough is if your slide cover sits flat against your slide when applied!
A problem with the crush mount technique is the distortion of the cells which in turn gives you inaccurate measurements.
I have worried about the same thing you describe Mr. Mushrooms where the section tips on it's side, this is another reason for the importance of as thin a section as possible!

Workman, do you have any microscopy images of the basidia of penis envy, I would be very interested in seeing some!
inski..


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Edited by inski (09/28/09 09:35 PM)

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