Mushroom spore research is essential to unraveling more of their biological attributes and, hence, providing more valuable insights to researchers. Keep reading to understand how magic mushroom spores can be viewed with a microscope.
The discovery of the microscope was a very important breakthrough in science since it enabled scientists to have a clear view of so many organisms or objects that were not visible to the naked eye. Take magic mushroom spores, for example. With the study of magic mushrooms now in its renaissance, thanks to their numerous benefits, there is a corresponding need to conduct meticulous microscopic research on them.
Microscopes for Viewing Magic Mushroom Spores
There are several types of microphones out there that can be used to observe magic mushroom spores. If you are at an introductory level of mushroom spore research, you may want to start with either a compound or a stereo microscope (both are optical or light microscopes). Though the former is generally preferred for observing mushroom spores, combining the unique features of both can be more intellectually beneficial. However, the focus here will be on the more popularly used compound microscope.
A compound microscope is an absolute necessity for any serious mycologist. It offers both high magnification and, more importantly, a high resolution that ensures greater visibility of the object under observation. Compound microscopes are designed for transparent subjects mounted on a microscope slide. However, with a little ingenuity, a researcher can use their top lighting to scrutinize or photograph opaque objects.
Most modern compound microscopes are usually binoculars (i.e., have two eyepieces), meaning that the same image is sent to each eye and appears strictly two-dimensional. There are also trinocular microscopes that allow the image to be viewed by a camera and two human eyes. Unlike a monocular microscope, a binocular model offers more comfort for those working for long periods.
What Other Items are Needed?
Like many other pieces of scientific equipment, microscopes have a number of accessories. So if you want an optimal view of magic mushroom spores, you’ll need things like slides, lens paper, coverslips, immersion oil, and extra bulbs. Very sharp razor blades and some of the chemicals mentioned below will also be necessary.
The kinds of slides and coverslips to use will depend on your experience and preferences. For instance, glass coverslips are great but they are pricier and can break easily. Plastic, disposable coverslips are more affordable but do not offer as good a view as glass. The lens paper is for cleaning your oil immersion lens after every use, while the razor is for spore prints (more on that later).
Below are some of the more commonly used chemicals, stains, and reagents in mycological microscopy:
Chemicals and Reagents
- Distilled water (H2O)
- Ammonia (NH3)
- Ferrous sulfate (FeSO4)
- Phloxine (C20H2Br4Cl4Na2O5)
- Potassium hydroxide (KOH)
- Melzer’s reagent
Though it is possible to mount mycological specimens with water, it is often difficult for mycologists to see specimen features more clearly without the use of stains and/or reagents. Unfortunately, it is not very easy to come by some of these chemicals (especially Melzer’s reagent), even for mycologists.
Stains
- Congo red (C32H22N6Na2O6S2)
- Lactophenol cotton blue
- Safranin (C20H19ClN4)
Key Technical Features to Consider in a Compound Microscope
For the amateur mycologist, a compound microscope can be a complex piece of equipment to work with despite its portable size. Here are some important features to consider if you want to view magic mushroom spores with a compound microscope.
Objective Lens
Objectives have been described as the most important imaging component of an optical microscope and are a major influence on the cost of microscopes. Objectives are not only the source of primary image formation but also play a central role in the determination of the quality of images a microscope can produce. Additionally, the magnification of a particular specimen and the resolution under which fine specimen detail is observed also heavily depend on a microscope’s objectives.
For mycology, you’ll mostly use the 40x objective. The 100x oil-immersion objective is the priciest and generates an image that is 2.5x larger than that of the 40x objective, as well as a bit more resolution. This extra magnification makes it much easier to view and measure magic mushroom spores and other specimens through an eyepiece micrometer.
Water-immersion objectives designed to use water in place of immersion oil are also available (though they are not as optically good as oil immersion). Some manufacturers have a 60x dry objective, (sometimes called a high dry), which will often serve as an alternative for the 100x objective. With a combination of an eyepiece camera and a suitable software program (usually provided when you purchase the camera), you can work effectively without much need for the 100x objective.
The camera generates images that are large enough on screen while the software measures mushroom spores precisely using the 40x or 60x objectives. However, if you’re dealing with small ascomycetes or other specimens where spore ornamentation is essential (e.g., Lactarius, Scutellinia, and Russula), then a 100x oil immersion objective is an absolute necessity.
Lower magnification objectives are comparatively less pricey. It’s good to have a low magnification objective, such as 10x, for searching the slide and setting up the illumination. Though the 4x is also fairly standard and much less costly, it is not essential. Objectives often come as a set, but you may want to check out all the magnifications and types available before settling for a set. There are three main types of objectives: achromats, fluorite achromats, and apochromats.
Oil Immersion Objectives can only be used by adding a drop of microscope immersion oil between the objective and the coverslip. The oil increases the numerical aperture (NA) and hence the resolution. The numerical aperture of the objective is a key parameter for the optical image and determines the resolving power of the objective and the brightness of the image. The larger the numerical aperture, the narrower the focal spot, and hence the higher the resolving power.
Oil immersion is usually meant for the 100x objective. One drop of oil (or two) is placed on top of the coverslip, and the 100x objective lens is brought into position so that it touches the oil and creates a ‘bridge’ of oil between the slide and the objective lens. It has a refractive index very close to that of glass. This allows very little refraction of the light rays as they go through the slide, specimen, coverslip, oil, and the glass objective lens of your microscope.
The physical properties of the medium through which light rays travel determine the extent to which the light will be refracted. The refractive index (n) is a numerical value (without units) that expresses the degree to which light will refract when passing through a medium. Air has a refractive index of 1.0, while glass, which many microscope slides and coverslips are made of, has a refractive index of 1.5.
Taking this difference into account, the purpose of the immersion liquid in optical microscopy is to match (as closely as possible) the refractive index of the glass between which the specimen is mounted and, therefore, increase the number of light rays that will form the final image. Immersion oils are very suitable for this purpose because most of them have a refractive index of 1.51.
In other words, there will be a reduction in resolution if the mountant has a low refractive index, e.g., temporary mounts in water, Melzer’s, or lactic acid. Theoretically, a resolution reduction will also occur if you do not apply oil between the condenser top lens and the underside of the slide (to achieve the intended numerical aperture and resolution). Water immersion objectives are similar to oil immersion objectives, although the aperture and resolution gains are not as significant (water has a lower refractive index of 1.3). In theory, water immersion objectives are more suitable if the subject is also mounted in water, as is often the case with mycological preparations. In practice, many people routinely use the 100x oil immersion objective to observe and measure spores or examine their surface ornamentation.
Eyepieces
Older eyepieces often vary from 5x to 20x, while more modern models are usually 10x or 15x. The total magnification is derived by multiplying the eyepiece magnification by that of the objective. The higher the magnification, the more the field of view will be restricted. An exception to this is wide field eyepieces which, as their name suggests, provide a much wider view of the object and are therefore really worth their higher cost. The eyepiece is also known as the ocular lens
Graticules
An eyepiece graticule (also known as an eyepiece micrometer) is a vital part of the measurement of mushroom spores and other objects (though it has no units of its own). It is a small glass disc designed with a numbered scale that fits inside the eyepiece in a way that ensures it is in focus. The scale appears superimposed over the image like a ruler, allowing you to measure objects beneath.
To measure length and width, rotate the graticule by rotating the whole eyepiece. This rotation shouldn’t lose the eyepiece focus, but if the eyepiece focus rotates more easily than the entire eyepiece, then secure the focus with sticky tape. Alternatively, you can measure by photography, in which case you may forgo the eyepiece graticule.
Whether graticule or photography, you’ll need a calibration slide (or stage micrometer) to calibrate the measurements. Calibration is usually done once, when you first set up your microscope. Why calibrate? Calibrating your microscope is simply the process of comparing the units on your microscope with the measuring yardstick of a stage micrometer. Though the graticule in your microscope’s eyepiece is evenly divided into what seems like units, those units do not correspond to any particular measurement. Therefore, you’ll need to compare and covert the measurement on your microscope graticule to the unit of a stage micrometer, which is formally recognized.
Most microscopic mushroom spore measurements are expressed in micrometers (or microns). The symbol for a micrometer is µm, and one micrometer is equal to 0.001 millimeters. Once you have a conversion basis, you may need to do a little math every time you measure something. For example, if each unit on your eyepiece graticule equals 1.3 µm on a stage micrometer when you’re on the highest magnification, an object that measures 9 units long on the graticule will actually be 11.7 µm long (1.3 x 9). Sometimes, you may be lucky to find that your highest magnification conversion is 1:1, in which case, no math conversion will be needed since both units are exactly equal (though it will still be necessary to calibrate your other lenses).
Focus Controls
The adjustment knobs serve as the operating mechanism for refining the image resolution. The location of the adjustment knobs may differ according to the type of microscope you are using. In some microscopes, the coarse focus knob sits more centrally on the arm of the microscope, with the fine focus knob positioned below it. In others, both knobs are located on the same axle (and are termed coaxial microscopes), with the fine focus knob on the outer side while the coarse focus knob is closer to the body of the scope. Whatever the design, the fine focus knob is smaller in diameter than the coarse focus knob.
The coarse focus knob is used to quickly bring the specimen into approximate or near focus. Use the fine focus knob to sharpen the focus quality of the image (including under progressively higher magnifications) after it has been brought into focus with the coarse focus knob.
Some coarse and fine adjustment knobs are designed with a numeric dial that provides the researcher with precise adjustment settings. Such dials are typically found on more advanced compound microscopes.
Stage
Look out for a mechanical stage since it ensures that a slide can be moved accurately in either direction by turning thumbwheels. Make sure it runs smoothly and is neither too slack nor too stiff, especially if you’re buying a fairly used product. Verify that no dead space appears when you change direction and ensure that the stage holds the slide securely; any sticky motion of the mechanical stage while measuring mushroom spores under the 100x objective can be annoying.
Light Source
The majority of modern microscopes are designed with built-in illumination (a light source built into the microscope’s base). LED is usually the most popularly used bulb, although 100w halogen is also used in several high-end microscopes to support differential interference contrast (DIC) and dark field illumination, which demand brighter illumination than currently offered by LED technology.
Substage Condenser and Köhler Illumination
Köhler illumination is one feature lacking in most entry-level or basic microscopes. Consider spending a little more to obtain it since it ensures the best illumination while also improving the microscope’s resolution. Köhler illumination is all about providing a controllable cone of light to make the best use of the objective optics, ensuring even illumination for a good image. It uses an adjustable field iris diaphragm immediately above the spot where the light comes up out of the base.
There’s also a substage condenser that can be moved up and down to focus the field iris in the subject plane, as well as a condenser iris that controls the cone of light coming into the objective. The condenser helps to supply bright light to the field of view, filling the small area needed by the objective.
Correctly setting up Köhler illumination will ensure that both the field iris and the subject stay in focus, with the field iris exerting control over the diameter of the illuminated spot on the slide. In other words, the field iris controls the size of the illuminated spot – from relatively large for the 10x or 20x objective to a tiny spot suitable for 100x. However, it is not possible to make the spot of light large enough to fill the field of view of very low magnification objectives, such as 4x. So when such an objective is being used, there is usually a flip-out top lens on the condenser that can be moved out of the light path in order to create a wider beam of light.
Some condensers are factory-set to align perfectly, while others come with a pair of centering screws that work against a third fixed pin to ensure movement in both dimensions. Be cautious when adjusting these since there may also be a central locking screw that completely releases the condenser when unscrewed. If it is a condenser that supports phase contrast illumination, there may also be a second pair of screws to center the phase rings. Be sure to use appropriate condensers for advanced imaging techniques such as phase contrast, dark field, or DIC.
Filter Tray
At the bottom of the condenser is a swing-out ring known as the filter tray. The tray enables the researcher to experiment with various types of lighting through the addition of filters. It is not a feature of all compound microscopes. If your microscope lacks a filter tray, you can equally lay filters over the light port where the light comes out of the base.
Orientation
Earlier microscope models had mirrors and an external light source. The stand was always towards the viewer with the front of the instrument on the side, away from the user. This arrangement made the microscope a bit inconvenient to use since you always had to peer around it to insert a slide or determine the objective position.
Things have since changed with modern models that have a built-in light source that allows the microscope to be turned around so that the front is towards the researcher, making the instrument much easier to use (though a nosepiece full of objectives can still pose some vision challenges, for example, when adding immersion oil or focusing). A notable advancement in microscope development is the turning of the revolving nosepiece such that the unused objectives stay within the microscope’s body, enabling the user to have a clear view of the current objective and the slide.
How to View Magic Mushroom Spores With a Compound Microscope
Many mycologists will consider observing and measuring magic mushroom spores as probably among the easiest of all the microscopic procedures in mycology. Easy or not, it is a useful procedure that helps in the identification of certain traits, for instance, whether magic mushroom spores are smooth, “ridged,” “pitted,” “ornamented,” etc., as well as to measure their dimensions.
The most widely adopted microscopy technique for observing magic mushroom spores and other fungi is to start at low magnification and progressively increase to high magnification. Moving from low to high magnification is the only way to first identify the spores and then bring them into greater focus.
Here are the steps to follow:
Move Your Magic Mushroom Spores to the Microscope’s Slide
For spore prints: Use a clean, dry razor blade to lightly scrape on the spore print. If the spore print is on paper, scraping too hard can introduce paper particles into the specimen, which will yield a confusing microscopic observation. You can place a piece of glass underneath the paper before scraping in order to have an even and hard surface to work on. After scraping, gently tap the spore dust off the razor onto a neat slide.
For spore syringes: simply inject spore solution onto a neat slide
In either case, ensure that the spores you use are mature enough (field guides and technical mycological literature prefer mature spores).
Staining
Put a drop of potassium hydroxide or Melzer’s reagent on the spore dust, add a coverslip, and gently tap on it with a small object (e.g., a pencil eraser) to clear any air bubbles
Fungal spores may experience diagnostic changes in color when stained with Melzer’s reagent. The following are the terms associated with stained spores based on color changes:
- Amyloid if the color changes to blue-black
- Dextrinoid if the color changes to reddish-brown
- Inamyloid (or negative) if the color becomes yellowish (or if there is no change in color)
Mount the Slide Onto the Microscope Stage
Position the slide on your microscope’s stage and start viewing with low magnification.
Progress the Magnifications
Because the mushroom spores may still be very tiny, you will have to undertake your magnifications progressively, bringing them into focus as you progress.
Move to the Oil Immersion Lens
Add a drop of immersion oil (very carefully) to the coverslip before moving to the highest magnification (oil immersion lens).
Use the Focus Knobs to Bring the Mushroom Spores Into Full Focus
Gently manipulate the coarse focus knob till the spores are in near focus, then turn the fine focus knob to attain complete visibility. You may want to jot down your observations on paper. You can also capture images of them with a digital camera through the microscope’s eyepiece (though this may need some iterative practice with your camera’s settings).
Measuring Magic Mushroom Spores
To measure the viewed mushroom spores, use the graticules in your eyepiece (converting the values to microns with the method you formulated during calibration). To be certain that your mushroom spores are completely in focus, adjust the fine-focus knob until the dimensions eventually become as small as possible.
If you wish to have an enhanced idea of the size of the spores, then it will be necessary to measure many of them. How many, precisely? Well, the answer tends to differ among mycologists. While a majority insists on between 10 and 20 measurements, others advocate for larger numbers.
Whatever number you decide on, record the various spore sizes and shapes alongside other data you collected about the mushroom(s) of interest. Note the dimensions of the biggest and tiniest spores you found. However, if these large and small spores seem like outliers (e.g., if you find only one small spore amongst hundreds of spores), it may not be necessary to include their dimensions in your record.
For spores with significant, measurable ornamentation (such as spines), it’s best to measure their length or breadth without the ornamentation and then take a separate measurement of the ornamentation (because the spines, for example, may have varying lengths).
The dimensions of the spores can be expressed as two ranges of possibilities: length and width. For instance, 8–11.5 x 3–4.5 µm implies that:
- the thinnest spore you viewed is 3 µm wide,
- the widest is 4.5 µm,
- the shortest is 8 µm, and
- the longest is 11.5 µm.
Mushroom Spores Syringes vs Mushroom Spores Prints
Mushroom spore syringes have an edge since they make microscopic investigation easier. This is because they come already hydrated and suspended in distilled water. Therefore, all that is required from the researcher is to inject the spore solution into the microscope’s slide and add the necessary stains for greater visibility.
A spore print investigation follows a similar process. However, rather than quickly injecting the spores, they are first scraped from the print onto the microscope slides before the stains are added.
