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 gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US 
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 Microscopy for gilled fungi  2
#15780737 - 02/08/12 06:24 PM (3 months, 18 days ago) |
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Notes on microscopy and how to get started. Scope!
Identification microscopy for fungi (specifically gilled mushrooms)
Image courtesy http://etc.usf.edu/clipart (Florida Center for Instructional Technology)
A compound microscope will provide you with more specific information to find out what species of mushroom you've collected. You can also share this information with others using sites like Mushroom Observer.
Among the things you can do with a microscope: View, measure, and describe the presence of spores, basidia, cheilocystidia, and pleurocystidia.
A microscope might look like a challenge. It's not. With the right introduction it can very swiftly be understood. The most important thing you need to know if you don't understand a microscope: It simply magnifies what you're looking at.
Below: There are two areas on your microscope that you should first understand:
(1) The "eyepiece" (which usually is a 10x eyepiece or a 20x eyepiece) - (x means magnified that many times. For instance, 20x means 20 times your plain sight).
(2) The four objectives - Most compound microscopes come with four objectives for additional magnifications (a 4x, 10x, 40x, 100x). So if you have the 10x eyepiece in WITH the 4x objective selected that would be 40x total magnification.
Courtesy biology.unm.edu
The objectives are connected to a rotating "nosepiece."
The first thing you need is a microscope that serves its purpose. It should have a 100x oil immersion objective. A friend of mine who's very well versed in microscopy recommends staying at an overall magnification of 1000x, and has said 2000x is not a realistic magnification - 1000x is about the limit for light microscopy, higher numbers will give a larger image but at no increase in resolution. Even 1000x is a stretch unless you are using true Kohler illumination, which few scopes these days have. 2000x is empty magnification, but everything is twice as big so your measurements will be twice as accurate. Most of us on this site have a "(biological) compound microscope." Three of the expensive manufacturers/sellers are Zeiss, Nikon, and Olympus. Three of the less expensive manufacturers/sellers are Amscope.com, Microscopenet.com, and Celestron.com. (Note: Try to get a scope with "PLAN" objectives which provide a wider-focused picture).
Below: The larger "course focus" knob will make significant adjustments to focus (the smaller "fine focus" is used to make final adjustments )
Below: The not-so-bad but slightly lacking 15x Celestron digital imager (which can be used in substitute of the 10x eyepiece).
It is far superior to models like it that are 4-8 times the price as of January, 2012.
Other items you'll want: Get microscope glass slides, slide cover slips, and immersion oil. If you want to store your slides get a "Microscope Slide Box." If you buy immersion oil you will also want some type of microscope objective cloth to remove the oil from the lense. The little pieces of paper that they put between microscope slides works well for this as an alternative, or clean cotton. A trusted mycologist who posts here forgets to clean his oil objective 99% of the time and he mentioned "its fine."
You want to be able to not only see spores, basidia, and cystidia - you'll want to have the ability to measure them. There are two ways to do this - 1. With a camera/digital imager and a pixel measuring program for Mac or Pc (or) 2. A 10mm-ruler linear crosshair reticle inside of a 10x eyepiece.
An imager/camera will allow you to take pics from your desktop and post them. There are other PC/Mac digital imagers/cameras that allow you the freedom to scope straight from your screen without having to look down the microscope. If you don't want to bother with looking down a scope and choose instead to use a digital camera or imager for your computer, you don't have to bother with the reticle (unless you want it as a second method to measure with).
Below: An image of a stage micrometer with its case. We'll get into this further down.
How to calibrate your microscope (ie measure) if you're using a digital imager
Image Below: A digital imager was inserted in place of an eyepiece, then a stage micrometer was placed on the stage. After starting with the 4x objective and gaining focus we moved to the 10x then the 40x and finally the 100x oil objective. An image was taken using PhotoBooth. The image was then opened followed by the app Pixelstick to measure from the middle of one division line to the middle of the next division line.
This measurement above tells me (on my microscope with my 100x objective and my 15x digital imager) that every 159 pixels on my iMac = 10 microns. (A more important note: I then divide 159 pixels by 10 microns for 15.9 pixels=1 micron)
Also remember that all settings need to be consistent from the time you performed calibration (for example the objective, the camera/imager, and the computer screen). Make sure your desktop/screen resolution is also the same as it was for calibration. Also view the pictures in the same picture viewing software as was used and view the photo in the same setting.
The four basic things you need to find with a microscope are: A. Spores (size, shape, wall thickness, color, ornamentation), B. Basidium (which is what produces spores) C. Cheilocystidia and D. Pleurocystidia. Depending on the genus also the caulocystidia, pileocystidia, chrysocystidia, circumcystidia, and gloeocystidia.
Recognizing Common Microscopic Structures
Clamp connections
Spores (Magnified at 900x with a 60x plan objective. Spores viewed within a rehydrated gill using koh).
Basidiospores (Spores still attached to a basidium)
Basidium (pl., basidia)
Basidioles (These look like basidia but lack sterigmata and spores)
Cheilocystidia including forked cheilocystidia
Pleurocystidia
How to view and measure spores
Measure with the 100x oil objective. Take a spore print on a slide, then add a cover slip without any liquid and view it as is. Alternatively, you can add either distilled water or soap water before setting the cover slip on.
Image below: A light spore print taken directly on a glass slide. Taking a spore print in this instance took only 90 minutes roughly. It can be very light as long as there are plenty of spores to view and measure.
Below: A good cluster of spores viewed from the same spore print above.
Below: A couple of spores (on a slide with a drop of soap water and a cover slip) - one of which is measured in length at 199.65 pixels (in PhotoBooth while using my 15x digital imager and the 100x oil objective). I divide 199.65 by 15.9 (the number we got a few paragraphs up) and we now know that this particular spore is 12.6 microns in length. NOTE: Measure the longest length of the spore and the widest width for your numbers and be consistent with this.
Measuring without a digital imager using a reticle to measure spores through a 10x eyepiece:
In order to actually use the reticle, it has to be installed into your 10x eyepiece and calibrated, which is EASY. Calibration is done once a year - its not done every time a measurement is needed. In order to calibrate it you need a stage micrometer. If you buy a typical stage micrometer it will have 10 microns per division. Determine how many reticle divisions = how many stage divisions. Do the math and get it into microns so you know how many microns are in each reticle division. Using my scope with my 100x oil immersion objective and my 10x eyepiece, 1 reticle division = 1 micron exactly.
Explained Again: Calibrating your microscope using a 10mm-ruler crosshair reticle and a stage micrometer
Install your reticle into your 10x eyepiece (unless of course you bought an eyepiece with one built-in).
Place your stage micrometer slide on the stage.
Start with the 4x objective and get focus of where you're at then gradually move up to the oil objective (yes, add oil just before switching to the 100x).
If you're using a 10x WF eyepiece with a 10mm-rule crosshair reticle along with your 100x objective (for a total magnification of 1000x), you will probably see that ten divisions on the reticle is equal to one division on the stage micrometer. If each division on the stage micrometer is 0.01mm (or 10 microns) we now know that each reticle division equals one micron.
Some microscopes may vary so make sure this is what you have.
Also, if you notice the stage micrometer lines are thicker than the reticle lines, put the reticle lines on the center of the stage micrometer lines.
Note: All of your measurements whenever possible should be done with the 100x oil objective.
You can put away your stage micrometer now. Calibration is performed when either new equipment is added or to recheck your initial calibration - usually once per year.
Below: A 10mm ruler crosshair reticle (which can be installed inside a 10x eyepiece).
Alternatively, you can buy a 10x eyepiece with a built-in reticle.
Measuring Spores
Measure the length and width of spores that are laying flat - not at an angle. Measure from the tallest (or longest/widest) parts and make sure you're in perfect focus so you can accurately see the edges.
You'll see people measure spores and write for example, "Spores measure 9 - 12 x 5 - 8 µm."
One µm is the same thing as one µ or one micron, or one micrometre/micrometer.
On a typical Stage Micrometer 1 Division = 0.01mm (0.01mm = 10 microns)
You can't universally breakdown how many pixels are in a micron because it varies from screen to screen, but you can determine that measurment for your screen using Pixelstick which will allow for 360 degree angle measurements.
Officially you need to measure 30 spores and drop the 2 highest and lowest measurements. You can measure the third dimension (thickness) by looking for spores that are standing on end on the stipe or pileipellis. If you aren't doing an official description you can of course measure as many as you wish but 10 would be normal. If you don't want to make a print you can get some mature spores from the top of the stem or the top of the cap. Spores on the gills aren't always mature (so they're sometimes smaller than those from a spore print) so your measurements might be inaccurate. When in doubt, go with a spore print.
Tip: With your measurements, remember to tell others how you prepared your slides for viewing (ie Viewed at 1000x. Spores taken from a spore print. Soap water added, etc). If you can't get a spore print out of your mushroom, you may still be able to see spores by looking first on the pileipellis and stipe since those are guaranteed to be mature. If you still can't find spores this way make a gill cross section, press on the cover slip with a pencil eraser, gently crushing and stretching out the gill. Under the microscope, search through all of the tissues for spores--but remember that you may be viewing immature spores if you find them (and note this in your measurements). If you can't find spores with this method, odds are high that your mushroom is simply immature, and has not yet developed spores.
Above: Immersion oil
How to View and Measure Basidia (Not to be confused with Basidioles)
Basidia are structures which produce spores. Each basidium has sterigmata - little, pointy structures which hold spores. It may be difficult to measure basidia because the base is dug into the the gill and isn't well revealed. Measure the best you can from the base to the top of the basidia. Do another measurement from the base to the tip of the tallest sterigma. List both measurements. Note that many basidia will not have spores on their sterigmata because the spores have ejected, which is fine. But don't measure basidioles (which look like basidia but don't have sterigma or spores attached). Measure with the 100x oil objective.
Below: A basidia with four sterigmata
How to view and measure cheilocystidia
There are two methods worth knowing for viewing cheilocystidia.
Below: Two slides, the higher one is a "preparate cut-out" and the lower is a "gill cross section"
Method 1: Preparate Cut-Out Removing a whole, single gill from a cap then taking a specific area out of it and placing it on a microscope slide (images follow)
Below: Remove a single gill using a razor blade then cut out a section in the center as seen below. This section will have the cap on top, pleurocystidia on the side "false edges," and cheilocystidia on the bottom "true edge."
Note: It may be difficult to see the base of the cheilocystidia. Do the best you can and scope around for the best looking, most clear ones. Measure from the base to the tip for the length (looking for the longest measurement in each individual cheilocystidium, then measure the widest width). If visibility is a slight issue you may approximate (but tell others that this had to be done in your notes).
Below: A few cheilocystidia along the true gill edge
Method 2: Creating a gill cross section to view cheilocystidia
- Using a freshly harvested mushroom (or recently harvested mushroom), remove the stem from the cap, then place the cap gills-down.
- Make a a section and discard it. This will allow you access to a perfectly straight area with closer access to the gills.
- Make your actual gill cross section - a very, very thin (almost impossibly thin) section
- Using your razor again, remove any parts that are too thick. Ideally, you like it to be a good-sized section in terms of length but if you're new to this or you're working with particularly small or fragile mushrooms, aim for a section with around five gills in it.
- Place the section in the center of your microscope slide and either examine it without a cover slip and without stains/liquids (using the 4x, 10x, and 40x objectives). You can also use a "dry mount" by only adding a cover slip and no liquids - using a freshly harvested mushroom. Lastly, you can mount the specimen in any number of liquids including 5% Koh/95% Water, Soap Water, Water, Congo Red, Lugol's Solution, etc - followed by a cover slip.
Below: A field image of Stropharia ambigua showing the gills closeup
Below: A double edged razor, seen below, can also be broken in half and used as two razors (carefully). This is the type of razor than many mycologists use for gill cross sections. Each blade is good for a handful of incisions before dulling.
It may be difficult to see the bottom, base area of many cheilocystidia which could inhibit perfect measurements. Do your best and know that others are doing their best with measurements. Measure with the 100x oil objective.
Tip: There are usually multiple shapes of cheilocystidia for a species. It's best to photograph/illustrate the most representative types instead of just one cheilocystidia.
How to view and measure pleurocystidia
Below: A gill cross section with arrows showing where to look for different cystidia types
There are technically two types of locations for pleurocystidia:
1. Pleurocystida located within the lamellar trama
2. "Pleurocystidia near the true gill edge" (near the cheilocystidia)
Don't worry about pleurocystidia near the gill edge for now - some mycologist will still call it cheilocystidia because of its close location to the gill edge cheilocystidia area. You can use either the method pictured in this document (ie a preparate cut-out or a gill cross section), but I'd advise you to embrace both methods. Make sure the pleurocystidia, if visible, is a good distance from the cheilocystidia.
As is the case with basidia and cheilocystidia, it may be difficult to see the base of many pleurocystidia. Do your best with measurements. Remember to tell others how you prepared your slide(s). Some mushrooms do not have pleurocystidia, and in some instances the pleurocystidia may simply not be viewable. If this occurs the best thing to do is simply inform others "no pleurocystidia observed in this collection."
Tip: There are usually multiple shapes of pleurocystidia for a species. It's best to photograph/illustrate the most representative types instead of just one pleurocystidia.
Below: It's important to remember where the "true gill edge" is located so you can distinguish between cheilocystidia and pleurocystidia (pleurocystidia is located along the "false gill edges").
Extra Tip: Anyone examining anything under a microscope should remember that the application of a cover slip (with or without utilizing a crush mount) can alter the appearance of what is being viewed. I strongly recommend using a 60x PLAN objective with slides that are simply prepared with fresh material and no liquids - no cover clip. Then do measurements with the 100x oil objective.
DRIED MATERIAL versus FRESH FUNGI (rehydration and examination techniques)
It is an appropriate question to ask, "Are microscopic observations identical in rehydrated material and freshly harvested material?" My answer is, no.
There are at least two popular liquids which will allow you to rehydrate dried mushrooms. The first is to use 70% isopropanol and the second is using 95% water with 5% koh. Here's a step-by-step procedure:
1. Let's start with a whole, dried mushroom and remove a whole, single gill with a new razor
2. Place the gill on a microscope slide and place enough drops to provide a submersion using either the isopropanol or 95/5 water/koh mixture
3. After a short time the entire gill should be rehydrated - some will take longer than others
4. Place a napkin corner (or paper towel corner) at the edge of the excess liquid and it will soak into the paper
Other liquids used for rehydration that are recommended by mycologists: Ethanol (70%-95%)
Rehydrating material after its fully dried
Break off the stem then break the cap in half so you have full access to one nice looking gill.
Using a razor blade, slowly press in between two gills and break off a piece (actually a whole gill) of your favorite fungi. 2-3 drops of koh are added (95% water and 5% koh approximately)
After two minutes or more the koh should rehydrate the specimen
Any excess koh is soaked into the edge of a napkin or paper towel
A drop or two of congo red is placed directly on the specimen
After approximately 3-4 minutes the material has absorbed the congo red sufficiently to view cystidia. A cover slip is gently applied...
Soap water in a dropper is provided near the edge of the left area of the cover slip while being exhumed underneath the cover slip
A napkin edge is used on the opposite side to slowly absorb the exiting liquid
Below: The slide is now ready for observation. The gill has been stained, the excess stain has been removed, and water is seen throughout the entire cover slip area
True gill edge at distance
Cheilocystidia at 900x
Rehydrating a gill, making a preparate cut-out and adding a cover slip illustrations...
A slightly dehydrated gill is placed on a slide and a drop or two of koh is added
Within a couple of minutes the gill is rehdrated.
The excess koh - at least a lot of it - is soaked up using the edging of a napkin.
The same cardboard that originally encased a razor blade is used to hold the gill in place as a preparate cut-out is made
The cutout will eventually reveal the cheilocystidia on the true gill edge, while the pleurocystidia (if present) will show itself on either of the left or right false edges
Either before or after the cover slip is used, you can then add about four drops of soap water or congo red as seen below. If you add the soap water or congo red after the slip is ontop just add the drops along the perimeter of the slip and the liquid will get sucked underneath.
Multiple methods for preparing slides
Above: Three highly effective liquids to have next to your microscope. The congo red can be used exactly as it is. The koh is prepared with approximately 95% distilled water and 5% koh. The soap water is about 99.5% distilled water and 0.5% liquid dish soap.
Fresh mount/dry mount method
Using the term "dry mount" is basically a way to describe putting a gill cross section (or any other section) of the mushroom directly on a microscope slide and viewing it without anything else - and that means not even a drop of water and no cover slip - as long as the mushroom itself is freshly harvested and naturally hydrated. You will not be able to use oil immersion with this method and therefore you will almost definitely not be able to accurately measure cystidia (if you try to make absolutely certain that you have an immaculately thin section sitting perfectly flat on the slide and insure you can see the entire outline of the cystidia). In some mushrooms, while utilizing a dry mount, you will see an entirely different world of cystidia than you will in any type of wet mount I've seen practiced as of early 2012.
Water or Soap Water
Simply place your gill cross section on the slide and using a dropper, add a few drops of either distilled water or 99.5% distilled water with 0.5% dish soap (approximately). Add a cover slip so its sitting very flat. There will be excess liquid still on the slide. Take a napkin and clean up any excess liquid.
Stains (5% koh/95% water, Congo Red, Cotton Blue, Lugol's Solution, Melzer's Agent, etc)
The reason you'd use a stain instead of water (or soap water) is to improve your sight of the image, or in the case of KOH you can also use it to rehydrate a dried sample. Different species and different microscopes will have their own needs, so its up to you what route to go. You can add these stains the same way as with soap water.
Below: A gill cross section is placed on a slide and gets a drop or two of congo red stain
(which will help you see several features of the section) - allow the stain to settle in for several minutes
A cover slip is placed on top
A soap water dropper is used from one side while the a napkin is used on the opposing side, exhuming the extra congo red
The slide is ready (the section is stained and water is underneath the entire cover slip
Getting air bubbles out of your microscope slides
Koh is good for dried material but its better to use soap water for fresh material. The soap in the water destroys the surface tension and makes it 100 times easier to get rid of the air bubbles. You can usually hold the slide vertically and the air bubbles will float up away from your sample. Put a little liquid dish soap in your dropper bottle of water when filling it - a few drops.
When using fresh or dried material there are a couple of recommended liquids to have ready: Phloxine (sometimes added to ethanol when working with dried material), and Teepol for fresh material. As noted earlier, if you are making a simple wet mount slide you may be able to use 99% water with 1% dish liquid soap.
Using your slide w/cover slip and the 100x oil immersion lense
The first time you do it don't make it complicated. Just prepare your slide, make sure the cover slip is on as flat as you can possibly get it. Then get focus with the 4x and move up to the 10x, then the 40x. Once you have focus with the 40x, rotate the objective nosepiece a few degrees so that the slide is more or less in between the 40x and 100x objectives. Add one drop of immersion oil ontop of the cover slip where the objective will go and then rotate into the 100x objective.
How to view and measure caulocystidia
Caulocystidia can be found by making a thin cut to the stem. Some mushrooms will not reveal caulocystidia. Scope the edging of the material. It is ideal to do this using freshly harvested, fully hydrated fungi.
How to view and measure pileocystidia
Pileocystidia can be viewed in the same section that is made from a gill cross section. Look along the margin of the pileipellis for clamp connections extending outwards.
Question: I've heard the term "crush mount." What is it and why use it?
A crush mount sounds a bit violent. Its not. Here's how to do it:
Put the best material available on a slide with a drop of KOH solution (or a liquid of your choosing) then add a cover slip. Press it down with a pencil eraser to gently flatten the material and vanish 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 several attempts.
Crush mounts are tricky and require the exact right amount of pressure. Too little and the cells are not freed, too much and the cystidia are crushed. It may allow you to see basidia and cystidia more properly and therefore make you more confident in the measurements you take.
Photographing your microscope's images - without a digital imager
You may be able to get better pictures by holding a small digital camera up to the eyepiece instead of using a microscope digital imager (unless you spend a lot of cash). One trusted mycologist recommends a Canon SD1300 or similar. It is good to use a camera that has manual focus.
When making micrographs the focus on the camera doesn't matter much, its the fine focus on the microscope that matters. However if you have a reticle, the focus on the camera matters or else the scale will be blurry. The scale will come into focus if you set the manual focus to one meter.
What happens to cheilocystidia and pleurocystidia on a freshly harvested mushroom as it begins to dry?
Most gilled mushrooms are roughly 90% water when they're freshly harvested and in prime condition. That's why they get so small after they fully dry. The same size reduction process that occurs to the overall mushroom definitely impacts the size (and visibility) of both cheilocystidia and pleurocystidia. During the drying process, some cystidia will literally fall over (initially) like a cut down tree (only in a more elastic way). Others will swiftly begin reducing in size. Overall physical characteristics will also undergo transformation. Make a note when you show your images to others - whether the specimens were freshly picked, almost dry, or rehydrated.
What if I view what I believe is pleurocystidia but other mycologists have viewed the same species and concluded it is not present?
Remember there are two basic methods in this document for viewing pleurocystidia - a gill cross section and a preparate cut-out from one individual gill. Go with both methods and use multiple gills from multiple mushrooms. Check using fresh fungi, freshly harvested. Compare to dried/rehydrated material a few days later. Anomalies are possible. Multiple collections from different locations will help. If you view fresh mushrooms under the scope (as opposed to rehydrated, dry samples) you may see more pleurocystidia. Regardless, observations should be noted along with micrographs for comparison (ie microscopy performed on freshly harvested mushrooms).
Going deeper: What's going on inside each individual spore?
Text. Text.
A very brief word about DNA sequencing
DNA sequencing is the current method of ultimate identification of mushrooms - not microscopy. For those of us who don't have the equipment needed to do DNA sequencing, microscopy is still very helpful and its a peaceful activity. If you have any interest in dna sequencing, reference the PDFs of Dr. Borovicka. DNA sequencing is used after (with) macroscopic and microscopic identification - it does not replace microscopy.
Herbarium Notes: Sending an Undescribed Species to a Local Herbarium
The 1958 Mycologia Issue (Volume 50 Page 442) shows the old school instructions for gilled mushrooms. The method at that time was to place each individual, fully dried mushroom into a fitting envelope and glue the envelope(s) to 11x17 herbarium mounting paper, and label them. The other method mentioned in this same article shows that most herbariums were taking deposits that were first fully dried and then placed into labeled boxes.
Another method given to me by a local university (in 2011) is to fully dry the mushrooms and then place them into ziplock bags. The bags are then placed into a fitting box for extra protection. The box is labelled appropriately and each bag gets a label inserted into it as well. The ziplocs keep out air and pathogens.
A good collection for one herbarium will be 15 specimens in ideal condition. They should represent as much diversity of the species, including very young mushrooms, middle aged mushrooms, and fully adult mushrooms - but avoid decaying ones if possible. Include the entire mushroom including the base of it (you can pull very gently and wiggle it free from the ground to do this).
Please photograph your collection as thoroughly as possible and post them on a site of your choosing. Include the link for the herbarium. Lastly, make any helpful notes and include them in the online post or in a letter with the collection (ie habitat, what they were growing in, location, date, Google Earth altitude, and any thing you think is interesting).
Notes regarding the criteria for describing a species
The organization that heads the official international taxonomic descriptions for fungi is called the International Code of Botanical Nomenclature (for algae, fungi, and plants).
I emailed one of the three top contacts at the ICBN regarding how a species is described according to international standards. I received this response:
Whereas there are rules governing the requirements for valid publication of a legitimate name of a new species of fungus, and amongst these is the requirement to provide a “description or diagnosis” in English or Latin, there are no rules, and hence no definitive list, of what constitutes a description for the purposes of the Code of Nomenclature for algae, fungi, and plants.
There is a definition of a diagnosis (“A diagnosis of a taxon is a statement of that which in the opinion of its author distinguishes the taxon from other taxa.”) but that is not much in the way of detailed content.
What is appropriate in describing a new taxon, or indeed in any description of a taxon, is a matter of good taxonomic practice and for that you should turn to text-books of systematic mycology, of which you will be more familiar than I am, as I work with vascular plants.
So far as the requirements, to which I referred above, for valid publication of a legitimate name, these are for the most part to be found in the Vienna Code (McNeill & al. in Regnum Veg. 146. 2006 and online at http://ibot.sav.sk/icbn/main.htm). ; However, from 1 January 2013, there will be significant additional requirements for names of fungi, that will be set out in the Melbourne Code, currently in preparation. A summary of these along with other changes appears in McNeill, J, & Turland, N.J. (2011) Major changes to the Code of Nomenclature – Melbourne, July 2011. Taxon 60: 1495–1497. ( http://www.ingentaconnect.com/content/iapt/tax/2011/00000060/00000005/art00030 ) and those dealing particularly with fungi in Mycotaxon 116: 481–490. 2011.
I hope this answers your enquiry at least in part.
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Cleaning your microscope objectives and eyepieces
How to Clean 4x, 10x, and 40x Microscope Objectives
You can use the the tissues that separate microscope slides and a very tiny amount of an approved objective cleaning liquid. I've personally used Kimtech Kimwipes with a touch of lens cleaner without any issues - but others discourage using kimwipes. It is also possible to use a tiny amount of 70% isopropanol and cotton or kimwipes. Keep in mind that your objectives are expensive and sensitive. Another possibile methods to get dust off the optics is with an air blower like the PISEN Camera Lens Air Dust Blower.
How to Clean the Oil Objective
Tip: Try to clean your oil objective after each session, but don't worry if you forget. Use lens paper or clean cotton to remove the oil (try and mop it up rather than spread it out). Don't over-do it though as rubbing the objective when it is completely dry might scratch it (even if you are using lens paper). Nothing further is required.
How to Clean the Microscope Eyepiece(s)
Blow or use an air blower to remove dust before wiping lens
Clean the eyepieces with a cotton swab moistened with 70% ethanol or an approved optical cleaning material
Clean in a circular motion inside out
Wipe the eyepieces dry with lens paper
Celestron Digital Imager Cleaning Technique
Clean the outside surfaces with slide paper and optic lens cleaner.
Blow off dust with a camel’s hair brush or air blower off the optical surfaces.
When cleaning do not rub in circles on this particular imager.
Mini-Glossary
Apical germ pore = Germ Pore : "Apical germ pore" is a term applied to mushroom spores which have a pore (hole) at one end. See image below.
Basidiole (pl. basidioles): Basidioles are still not definitively understood and are therefore worth studying casually. They are believed to be immature basidia by some. It seems worthwhile to consider that some basidioles are immature basidium while others are providing a different function.
Basidium (pl., basidia): is a microscopic, spore-producing structure found on the hymenophore of fruiting bodies of basidiomycete fungi. The presence of basidia is one of the main characteristic features of the Basidiomycota. A basidium usually bears four sexual spores called basidiospores; occasionally the number may be two or even eight. In a typical basidium, each basidiospore is borne at the tip of a narrow prong or horn called a sterigma (pl. sterigmata), and is forcibly discharged upon maturity.
Caulocystidium (pl., caulocystidia): Cystidia located on the stipe.
Cell (See Fungal Cell Below):
Cell Wall:
Cheilocystidium (pl., cheilocystidia): A cystidium (plural cystidia) is a relatively large cell found on the hymenium of a basidiomycete (for example, on the surface of a mushroom gill), often between clusters of basidia. Since cystidia have highly varied and distinct shapes that are often unique to a particular species or genus, they are a useful in the identification of basidiomycetes. In general, the adaptive significance of cystidia is not well understood. Cheilocystidia, as noted well above, are located on the true gill edges.
Chitin (pronounced ky-tin): The main component of the cell walls of fungi. The structure of chitin was solved by Albert Hofmann in 1930.
Chrysocystidium (pl., chrysocystidia): Chrysocystidia are cystidia whose contents contain a distinct refractive yellow or golden body, that becomes more deeply yellow when exposed to koh, ammonia or other alkaline compounds. Chrysocystidia are characteristic of many (though not all) members of the agaric family Strophariaceae.
Below: Two different types of chrysocystidium viewed in koh
Clamp Connections: A clamp connection is a structure formed by growing hyphal cells of certain fungi. It is created to ensure each septum, or segment of hypha separated by crossed walls, receives a set of differing nuclei, which are obtained through mating of hyphae of differing sexual types. It is used to create genetic variation within the hypha much like the mechanisms found in crozier during sexual reproduction.
Cystidia: A cystidium is a big, funny-looking end cell that sticks out of a gill surface but doesn't look like a basidium. Since cystidia have so many shapes, and these shapes hold (fairly) constant for a given species, they are useful in identifying mushrooms. What they are and what they do is still being investigated within each species.
Fungal Cell: http://en.wikipedia.org/wiki/Eukaryote#Fungal_cell
Germination: Germination refers to the emergence of cells from resting spores and the growth of sporeling hyphae or thalli from spores in fungi. With many mushroom species, germination begins in the dimpled depression on the spore. The process initially looks like a seed sprouting.
Germ pore (in reference to a spore): A germ pore is a small hole (or pore) in the outer wall of a fungal spore through which the germ tube or hypha exits - upon germination. It can also be referred to as "apical germ pore," which means the identical thing in mycology.
Germ slit (in reference to a spore): If the cell wall of an individual spore is divided from one end to the other, this is called a germ slit (meaning there's a line travelling from one pore end to another pore end). Alternatively a slit does not have to reach the full length of the spore.
Germ tube: When a spore begins to create hypha it is sometimes initially referred to as a germ tube.
Gill Trama: The inside tissue of a gill.
Hymenium: A specialized layer of cells from which arise basidia and their spores, as well as cystidia and "other cells." Alternatively put, hymenium is the spore-bearing surface of a mushroom . The gills constitute the hymenium of gilled mushrooms.
Hymenophoral Trama: Another synonym for lamellar trama (ie gill trama)
Hypha (plural hyphae): A long, branching filamentous structure. In most fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium.
KOH: Potassium hydroxide. It has multiple uses for mycologists including rehydration of dried material, a mounting medium for microscope slides, and to observe color changes in either a microscope preparation or in the field on a fresh specimen.
Lamella (plural lamellae): A euphonic synonym for "gill."
Lamellulae: The short gills originating from the outer peripheral edge of the cap but fully extending to the stem.
Meiosis: The process of reduction division by which a single cell with a diploid nucleus subdivides into four cells with one haploid nucleus each.
Mitosis: The nonsexual process of nuclear division in a cell by which the chromosomes of one nucleus are replicated and divided equally into two daughter nuclei.
Mycelium (pl., mycelia): A branching network of hyphae. This is oftentimes a white, strand-like material which produces the mushroom body.
Nucleus (pl., nuclei): Concentrated mass of differentiated protoplasm in cells containing chromosomes and playing an integral role in the reproduction and continuation of genetic material.
Pileocystidium (pl., pileocystidia): Cystidia located on the pileipellis.
Pileus: A euphonic synonym for "cap."
Pileus Trama: This area is located between the pileipellis and the subhymenium (ie above the gills and above the stipe trama). Hyphae of the pileus trama branch downward to form the hymenophoral trama.
Plasma Membrane: The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell from outside forces. It consists of the lipid bilayer with embedded proteins. Cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signaling and serve as the attachment surface for several extracellular structures, including the cell wall, glycocalyx, and intracellular cytoskeleton.
Pleurocystidium (pl., pleurocystidia): See definition above. Pleurocystidia can closely resemble the appearance of a particular mushroom's cheilocystidia. Pleurocytidia occur within the gill - not along the true gill edge. By cutting into the gill pleurocystidia can been seen on the "false gill edges." They are usually far less numbered and more spaced out than cheilocystidia.
Protoplasm: From Wikipedia - Protoplasm is the living contents of a cell that is surrounded by a plasma membrane. Protoplasm is composed of a mixture of small molecules such as ions, amino acids, monosaccharides and water, and macromolecules such as nucleic acids, proteins, lipids and polysaccharides.
Resting spore: A resting spore is a spore created by fungi which is thickly encysted (has a thick cell wall) in order to survive through stressful times, such as drought. It protects the spore from biotic (microbial, fungal viral), as well as abiotic (wind, heat, xeric conditions) factors.
Septum: A partition dividing filamentous hyphae into discrete cells in fungi.
Spore quotient (q): Calculate the spore quotient dividing the spore length by the spore width.
Spores (from gilled mushrooms): Spores are the reproductive structure of gilled mushrooms - like seeds from a plant in many ways. Meiosis is used in order to develop. Spores are specialised cells of the fungus that can function as resting or dispersal propagules. Each spore has the potential to generate another individual of the species.
Sterigma (pl., sterigmata): The extension-tips located at the tops of basidia which hold spores until they are ejected.
Sterigmal appendage: A nipple-like protuberance on the spore where it was connected to a sterigma. With many species, the opposing end of the spore is dimpled with a germ pore.
Stipe: A euphonic synonym for "stem."
Stipe Trama: Although the stipe trama is continuous with the pileus trama, an imaginary line conveniently separates the stipe from the pileus. Below this line the hyphae of the stipe trama run more or less longitudinally yet they frequently intertwine. Many mushrooms have stipe strama composed of typical thread-like hyphae interlaced with laticefers, oleiferous hyphae, or sphaerocysts. The hyphae of the pileus and stipe trama can vary in the following ways: the thickness of the wall, the type of branching, the presence or absence of clamp connections and their presumed functions. They can also possess a variety of pigments, and can react with different chemical reagents.
Sulphidium (pl., sulphidia): Pleurocystidium that have a yellowish coloration (similar to that of of a chrysocystidium). So far this term has been used extremely infrequently and is not so much as listed in a single online dictionary (May 2, 2012).
Trama (as defined in Mycelium Running): The internal layers of cells between the gills of mushrooms.
Vesicles: In fungi, a bubble-like sac produced by zoosporangium, and from where zoospores mature and are eventually released.
Zoospores: A zoospore is a motile asexual spore that uses a flagellum for locomotion. Also called a swarm spore, these spores are created by some algae, bacteria and fungi to propagate themselves.
Full Circle with a Mushroom's Life Cycle
There are exceptions to the life cycle, so this example can be considered for "many" gilled mushrooms. Starting with an adult mushroom body, the gills have basidia with their spores. These spores are ejected, sometimes reaching fertile ground. If successful in the life cycle, a spore can "asexually" produce through its hole in its cell wall (the germ pore) - which releases initially a single "thread" of hypha which becomes hyphae and thus mycelium. This can occur with two or more spores separately and their hyphae can enjoin, forming one mycelium network. The mycelium is capable of then producing new bodies of mushrooms.
Also see the following link for a very brief overview of how a spore germinates: http://www.fungionline.org.uk/2spores/3germination.html
Other sites & references:
How to Identify Mushroom to Genus III: Microscopic Features
Mycelium Running: How Mushrooms Can Help Save the World
www.mushroomobserver.org
www.mushroomexpert.com
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
Edited by gsharpnolack (05/11/12 01:33 AM)
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 Fungi01
John Plischke
 
Registered: 06/29/08
Posts: 826
Loc: Western Pennsylvania
Last seen: 1 day, 13 hours
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really nice work.
John
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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Re: update to microscopy notes [Re: Fungi01]
#15781933 - 02/08/12 10:56 PM (3 months, 18 days ago) |
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only 30 views and 1 post? I hate to see such informative posts get lost...
Unfortunate..
Really great work. Looking good man.
You are a good user. You contribute some good threads. Definately keeping track of your posts. Good Job.
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     "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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bloodworm
cube con·nois·seur
Registered: 05/22/10
Posts: 4,073
Loc: 352
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Quote:
RigVedaLXVII.Soma said:
only 30 views and 1 post? I hate to see such informative posts get lost...
Unfortunate..
Really great work. Looking good man.
You are a good user. You contribute some good threads. Definately keeping track of your posts. Good Job.
true.
thanks for sharing man. 
will definitely help me out... 
thanks again.
peace and love
bloodworm
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knarkkorven
Entheoholic
Registered: 06/22/05
Posts: 394
Loc: Sweden 
Last seen: 1 day, 7 hours
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Re: update to microscopy notes [Re: bloodworm]
#15782174 - 02/09/12 12:51 AM (3 months, 18 days ago) |
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Very good guide!
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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Re: update to microscopy notes [Re: knarkkorven]
#15782311 - 02/09/12 02:32 AM (3 months, 18 days ago) |
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By the way, anyone have any tips or suggestions on cross sectioning or microscopy methods needed for observing polyporaceae? Especially specimen with maze-like pores? such as Daedalea species?
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"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 ListSpores I Am Looking For Seeds I am Looking For Solanaceae, Amanitaceae
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 suchen
Mushluminary
Registered: 06/28/11
Posts: 2,904
Loc: Shangri-la
Last seen: 3 minutes, 15 seconds
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Stiiicky! Stiiiicky! 
Fantastic post, man.
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Bobzimmer said: "I'm just a guy with a hard-on for fungi photos."
koraks said:
"Nice chanterelle. Nice girlfriend too  "
maynardjameskeenan said:
"I wish when I was growing up someone would have educated me about the respect that you need to give to something that can be so deadly and so delicious."
amilibertine said:
"Best go find whoever is out there in the wild with a purple sharpie marker coloring mushrooms. I'll bet he has what you're looking for.  "
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Re: update to microscopy notes [Re: Fungi01]
#15786548 - 02/09/12 11:26 PM (3 months, 17 days ago) |
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Thank you all for the positivity - I hope it benefits our members
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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hungrygoldfish
Registered: 10/23/10
Posts: 641
Loc: UK 
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I bought some shop mushrooms to practice on  Am still new to using a microscope so I'm going to have quite a few questions but this thread has already answered quite a few!
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fux234
Mycophil xD
Registered: 09/22/10
Posts: 992
Loc: Westwood
Last seen: 16 hours, 35 minutes
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another nice work gsharp 
thanks for sharing this very good descriprion!
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"Carpe diem!"
P.Semilanceata 2011- Germany
   
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riverdweller
Misanthropic Voyeur
Registered: 08/19/09
Posts: 1,585
Loc: Oregon, USA
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Quote:
hungrygoldfish said:
I bought some shop mushrooms to practice on Am still new to using a microscope so I'm going to have quite a few questions but this thread has already answered quite a few!
Man, what a great idea! Nice to see you doing so well w/ your scope!
Gsharp, thanks for the time you spend sharing your thoughts and processes on microscopy. Creative and giving, good traits!
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I'm still here 
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hungrygoldfish
Registered: 10/23/10
Posts: 641
Loc: UK
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I just tried to find the Pleurocystidia using a gill from a shop bought mushroom- did I find it right?:
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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That may be a basidium.
Keep looking along the false edges and remember not all species have pleurocystidia.
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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The Thinker
Registered: 09/01/10
Posts: 2,609
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Quote:
gsharpnolack said:
Then make an incision as seen above. The cheilocystidia will be on the bottom-view edge. Pleurocystidia will be on the left and right "false" edges. Make sure the pleurocystidia, if visible, is a good distance from the cheilocystidia.
wouldn't this show you mostly cut lamellar hyphae? you may be able to see pleurocystidia that fold over the side of the "false" edges but if I didn't see any I wouldnt conclude the species didnt have pleurocystidia.. to check decisively for pleurocystidia I think you'd need to make (at least)1 good gill cross section. you can also crush mount and look into the gill face to find pleurocystidia(not so decisive but you can get some good pictures this way)
Quote:
There are technically two types of pleurocystidia, and depending on your organization's standards, you should be aware of the two types.
1. Pleurocystida located within the lamellar trama
2. "Pleurocystidia near the true gill edge" (near the cheilocystidia)
Don't worry about pleurocystidia near the gill edge for now - some mycologist will still call it cheilocystidia because of its close location to the gill edge cheilocystidia area. You can use either the method pictured above for finding cheilocystidia or you can do a gill cross section, but I'd advise you that the first method is more trustworthy.
here is a graphic that may show how cheilocystidia can be found slightly into the gill face- or maybe some pleurocystidia are pigmented(couldnt tell, roughly same shape pleuro and cheilo for this species)..
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The Thinker
Registered: 09/01/10
Posts: 2,609
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Re: update to microscopy notes [Re: The Thinker]
#15788547 - 02/10/12 01:13 PM (3 months, 17 days ago) |
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nice guide by the way
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Re: update to microscopy notes [Re: The Thinker]
#15788855 - 02/10/12 02:26 PM (3 months, 16 days ago) |
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Quote:
wouldn't this show you mostly cut lamellar hyphae?
Try it out.
Quote:
you may be able to see pleurocystidia that fold over the side of the "false" edges but if I didn't see any I wouldnt conclude the species didnt have pleurocystidia
I agree - but you are being more particular than the average mycologist.
This goes back to doing both methods: A preparate cut-out first, then for confirmation a gill cross section. If neither show pleurocystidia, get a new gill and repeat the process.
Quote:
you can also crush mount and look into the gill face to find pleurocystidia
This is something I should do this coming week and I will. Let's continue to compare methods over the next few days.
--------------------
One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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hungrygoldfish
Registered: 10/23/10
Posts: 641
Loc: UK
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I was wondering if anyone used a 60X objective and would it be useful to buy one?
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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I use one and love it. Its a 60x plan objective and I can get a good image and use it to avoid oil immersion.
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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Are you guys referring to a Water immersion lens? I know there is a 65x water immersion and a 100x water immersion. They are supposedly quite superior to oil immersion lenses in a resolution sense.
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"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 ListSpores I Am Looking For Seeds I am Looking For Solanaceae, Amanitaceae
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suchen
Mushluminary
Registered: 06/28/11
Posts: 2,904
Loc: Shangri-la
Last seen: 3 minutes, 15 seconds
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For the very thin/flat pieces of tissue used in mycology I don't think water immersion would be beneficial. I believe the usefulness of water immersion objectives is primarily seen in thicker pieces of tissue or tissue suspended in some media.
--------------------
Bobzimmer said: "I'm just a guy with a hard-on for fungi photos."
koraks said:
"Nice chanterelle. Nice girlfriend too "
maynardjameskeenan said:
"I wish when I was growing up someone would have educated me about the respect that you need to give to something that can be so deadly and so delicious."
amilibertine said:
"Best go find whoever is out there in the wild with a purple sharpie marker coloring mushrooms. I'll bet he has what you're looking for. "
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Re: update to microscopy notes [Re: suchen]
#15808552 - 02/14/12 01:57 PM (3 months, 12 days ago) |
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I'm not familiar with water immersion. I use an achromatic plan objective (60x) just like I use my 40x objective.
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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Re: update to microscopy notes [Re: suchen]
#15808616 - 02/14/12 02:10 PM (3 months, 12 days ago) |
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Aha, I see. But Wouldn't it be beneficial to give the section more depth of field, or are you saying it is too overkill for this purpose?
As shown in this image, even though it isnt a gill section, it isnt too much thicker than a gill section. C. in the figure is was captured with a hoffmann phase contrast and water immersion lens.
Either way, I think I could find many uses for it besides strictly fungal microscopy.
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"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 ListSpores I Am Looking For Seeds I am Looking For Solanaceae, Amanitaceae
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Terry M
Stranger in a Strange Land
Registered: 06/18/10
Posts: 800
Loc: Rhode Island
Last seen: 4 hours, 12 minutes
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Re: update to microscopy notes [Re: suchen]
#15808633 - 02/14/12 02:14 PM (3 months, 12 days ago) |
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Great info in general. I like the stain step-by-step, because I'm just a beginning microscopist and haven't seen the mounting and staining process shown in this much detail before. Got my Congo Red on the shelf and ready to go.
I also found it particularly interesting about observing a gill rather than a gill cross section. This looks like a nice quick technique. I still want to practice cutting cross sections, though. I've only cut my thumb once so far!
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Obsessed with edibles all my waking hours.
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hungrygoldfish
Registered: 10/23/10
Posts: 641
Loc: UK
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Re: update to microscopy notes [Re: Terry M]
#15819072 - 02/16/12 01:28 PM (3 months, 10 days ago) |
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I contacted the seller I got my microscope from to buy some other bits for it and about the 60x objective he said:
Quote:
The 60x objective will work on your scope but you need to re-focus as it is not parfocal with your Kyowa objectives. That 60x is a DIN type and is longer than yours.
What does that mean? Is it difficult to do?
Also in terms of measuring spores would it be better to get a Calibration slide or Eyepiece graticule or both?
Also for the oil lens is This the right type of oil?
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Quote:
hungrygoldfish said:
I contacted the seller I got my microscope from to buy some other bits for it and about the 60x objective he said:
Quote:
Quote:
The 60x objective will work on your scope but you need to re-focus as it is not parfocal with your Kyowa objectives. That 60x is a DIN type and is longer than yours.
What does that mean? Is it difficult to do?
He's just saying that after you rotate from a less-powered objective (ie 10x) into the 60x you'll have to use the focus knobs to refocus. Piece of cake 95 percent of the time.
Quote:
Also in terms of measuring spores would it be better to get a Calibration slide or Eyepiece graticule or both?
For measuring spores you can get this: http://store.amscope.com/ep10x23r.html or another similar one
Quote:
Also for the oil lens is This the right type of oil?
This is what I use for immersion oil:
Your stage micrometer will work.
--------------------
One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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yeah man, Idk If I would buy immersion oil from the link you posted. They list it in Pet Health/care section, and have NO description whatsoever.
--------------------
"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 ListSpores I Am Looking For Seeds I am Looking For Solanaceae, Amanitaceae
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Byrain
-
Registered: 01/07/10
Posts: 1,819
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Cargille is great.
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hungrygoldfish
Registered: 10/23/10
Posts: 641
Loc: UK
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Re: update to microscopy notes [Re: Byrain]
#15819710 - 02/16/12 04:11 PM (3 months, 10 days ago) |
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Quote:
gsharpnolack said:
He's just saying that after you rotate from a less-powered objective (ie 10x) into the 60x you'll have to use the focus knobs to refocus. Piece of cake 95 percent of the time.
I have to always refocus anyway!
Quote:
RigVedaLXVII.Soma said:
yeah man, Idk If I would buy immersion oil from the link you posted. They list it in Pet Health/care section, and have NO description whatsoever.
That's exactly why I was asking, I have a £20 amazon voucher to use up so I was hoping that this product would be good 
Quote:
Byrain said:
Cargille is great.
Thanks for the tip!
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TimmiT
Registered: 03/23/10
Posts: 2,431
Loc: Victoria 
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Quote:
What does that mean? Is it difficult to do?
I've never had to add an objective, so I'm not sure what's involved. I imagine that it would be fairly straight forward though. He means what gsharpnolack said... that you will have to refocus when changing objectives. In an ideal world when you change objectives the subject will remain in focus because they're made so that the focal distances match. More often the focus will need fine adjustment. If you used that 60X the focus would not match the other objectives and it would take more than fine adjustment
Quote:
Also in terms of measuring spores would it be better to get a Calibration slide or Eyepiece graticule or both?
You need both. An eyepiece with a reticle (probably easier to buy an eyepiece with the reticle already installed) and a stage micrometer (calibration slide). You will only need the stage micrometer once so try borrowing one if you can. It's purpose is to measure the distance between the graduations of the reticle. Once you know the distances for each objective you don't need the micrometer again.
Quote:
Also for the oil lens is This the right type of oil?
That oil looks fine.
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"Reality leaves a lot to the imagination" ~ John Lennon
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Terry M
Stranger in a Strange Land
Registered: 06/18/10
Posts: 800
Loc: Rhode Island
Last seen: 4 hours, 12 minutes
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Re: update to microscopy notes [Re: TimmiT]
#15822767 - 02/17/12 09:16 AM (3 months, 10 days ago) |
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There are Type A and Type B of immersion oil commonly used. I asked a pathologist friend what kind he uses. He said a mixture of both types, though he didn't know the proportions used in his hospital because someone in his lab mixes them for him.
Type B is more viscous than Type A. I bought both types, but so far I've just used Type A, which appears to work fine as far as I can tell.
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Obsessed with edibles all my waking hours.
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Re: update to microscopy notes [Re: Terry M]
#15833648 - 02/19/12 05:34 PM (3 months, 7 days ago) |
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Updated above
--------------------
One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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RigVedaLXVII.Soma
Bacteri/Phyc/Bry/Myc/ology
Registered: 11/21/11
Posts: 380
Loc: Michigan
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I was given the permission of my very kind friend gsharpnolack to post my relevent thread, "DIY Microtome; For those thin sections you just cant get yourself" in hopes that some may find it useful in sectioning, and so that the new people who gain interest in microscopy dont lose hope or aspiration just because they cant get thin sections by hand. Now everyone can get almost paper thin sections with ordinary houshold items. No reason that one cant get at least 10 micron thin sections.
Thank you gsharpnolack. I appreciate your kindness.
And dont give up folks! Whether you try my DIY microtome tek, or section by hand, DO NOT GIVE UP! Practice Practice Practice!!
Happy Microscopy!!
Good Luck!
There is a whole other world under that scope!
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"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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Alan Rockefeller
Mycologist
Registered: 03/10/07
Posts: 24,722
Last seen: 4 days, 19 hours
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Re: update to microscopy notes [Re: Terry M]
#15835939 - 02/20/12 12:42 AM (3 months, 7 days ago) |
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Quote:
Terry M said:
There are Type A and Type B of immersion oil commonly used. I asked a pathologist friend what kind he uses. He said a mixture of both types, though he didn't know the proportions used in his hospital because someone in his lab mixes them for him.
Type B is more viscous than Type A. I bought both types, but so far I've just used Type A, which appears to work fine as far as I can tell.
I like type A because when I use type B, it has a tendancy to slide the cover slip over when I move the objective away.
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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I'm trying to find an ideal stain for specifically working with dried/rehydrated gilled mushrooms and found these stains:
http://www.mycology.adelaide.edu.au/Laboratory_Methods/Microscopy_Techniques_and_Stains/
Do any of you have an opportunity to try one of these out and share your results?
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One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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Alan Rockefeller
Mycologist
Registered: 03/10/07
Posts: 24,722
Last seen: 4 days, 19 hours
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My favorite stain is cotton blue. It is extremely blue.
I wonder how well food coloring would work.
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TimmiT
Registered: 03/23/10
Posts: 2,431
Loc: Victoria
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The Adelaide Uni site covers medical mycology, so the techniques and stains are more relevant to that field. You probably won't find them used outside a diagnostics lab.
I have pics of PAS and silver stains (and a few other histo stains) in my gallery if you want to see what they look like (not fungal though).
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"Reality leaves a lot to the imagination" ~ John Lennon
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bloodworm
cube con·nois·seur
Registered: 05/22/10
Posts: 4,073
Loc: 352
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Re: update to microscopy notes [Re: TimmiT]
#15846650 - 02/22/12 08:23 AM (3 months, 5 days ago) |
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i found an excellent stain.
ink stamp refills work awesome...and they are cheap (you can find them at any hobby or office store).
here are some pics where i used ink stamp refills as the stain.
  
Pluteus cervinus cystidia @ 400x in a crush mount
just be careful not to get any of the ink on your lenses.
peace and love
bloodworm
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Alan Rockefeller
Mycologist
Registered: 03/10/07
Posts: 24,722
Last seen: 4 days, 19 hours
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Re: update to microscopy notes [Re: bloodworm]
#15849378 - 02/22/12 06:52 PM (3 months, 4 days ago) |
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Quote:
Pluteus cervinus cystidia @ 400x in a crush mount
Thats not a crush mount. Or if it is, it isn't crushed enough to free the cells.
In a crush mount you should be able to see the whole cell, not just the part that is sticking out of the gill.
The stain looks really good, but I wonder why there is an aura around the cystidia. I have never seen that before.
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falcon
In the green
Registered: 04/01/02
Posts: 5,150
Last seen: 20 hours, 18 minutes
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Thanks!
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gsharpnolack
Registered: 09/05/11
Posts: 657
Loc: Washington State US
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Quote:
In a crush mount you should be able to see the whole cell, not just the part that is sticking out of the gill.
Can you share a few examples?
--------------------
One thing that can improve our planet: Personally creating a small amount of special atmosphere within the home place.
http://en.wikipedia.org/wiki/Four_Noble_Truths
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bloodworm
cube con·nois·seur
Registered: 05/22/10
Posts: 4,073
Loc: 352
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Quote:
Alan Rockefeller said:
Quote:
Pluteus cervinus cystidia @ 400x in a crush mount
Thats not a crush mount. Or if it is, it isn't crushed enough to free the cells.
In a crush mount you should be able to see the whole cell, not just the part that is sticking out of the gill.
The stain looks really good, but I wonder why there is an aura around the cystidia. I have never seen that before.
damnit.
i have to get the pressure down i guess.
u would agree that with some estimation this is enough to measure?
thanks.
peace and love
bloodworm
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bloodworm
cube con·nois·seur
Registered: 05/22/10
Posts: 4,073
Loc: 352
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Re: update to microscopy notes [Re: bloodworm]
#15849516 - 02/22/12 07:24 PM (3 months, 4 days ago) |
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also, i believe the aura is because there is another one directly behind it, just out of focus.
you can see it with the out of focus one in front.
peace and love
bloodworm
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Alan Rockefeller
Mycologist
Registered: 03/10/07
Posts: 24,722
Last seen: 4 days, 19 hours
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Re: update to microscopy notes [Re: bloodworm]
#15852365 - 02/23/12 11:50 AM (3 months, 4 days ago) |
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Quote:
gsharpnolack said:
Quote:
In a crush mount you should be able to see the whole cell, not just the part that is sticking out of the gill.
Can you share a few examples?
Psilocybe cyanescens cheilocystidia
Differential interference contrast microscopy microscopy by Peter Werner
Quote:
bloodworm said:
i have to get the pressure down i guess.
Not down, up.
Quote:
u would agree that with some estimation this is enough to measure?
No, you never know how deeply the cystidia is embedded in the gill.
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