Choosing a Microscope for your Yeast Lab

Over the past decade I have seen (and been a part of) the “scientification” of craft and home brewing. Gone are the days of “pitch and pray”, with many brewers – both commercial and at home – taking a highly scientific approach. During this time the humble microscope has gone from a brewers toy to an important tool in the toolbox used by many brewers to manage their yeast. However, the microscope is a complex tool that is built in many configurations, which for the uninitiated can be overwhelming and difficult to understand. And unfortuantly, these details are critical as they determine what the microscope can and cannot do. Choose correctly, and you will have a tool for life. Choose incorrectly and you end up with an expensive paper-weight.

The goal of this post is to do a deep dive into microscopes, with the goal of teaching brewers everything they need to know to select the right microscope for their needs. This is going to be a very long and detailed post, so you may want to grab a brew before diving in. This post is a more detailed and in-depth exploration of microscopy than my previous post and video on choosing a microscope (embedded below).

The Big (Microscope) Picture

The first step in choosing a microscope is determining which capacities you actually need. Microscopes come with a bewildering array of options, so it is important up-front to define what you need. This makes searching for a microscope much easier, and can help to contain costs by ensuring you are not paying for unneeded features – or buying a scope which doesn’t meet your needs.

When it comes to defining features for a brewery, there are generally three things that microscopes get used for – performing yeast counts, using dyes or other methods to check yeast viability/vitality, and relying on cellular morphology to identify yeast, other brewing organisms, and contaminants. Those three purposes are listed in order of required capabilities, with yeast counting requiring a minimalist microscope, while morphology requires a more advanced setup.

In general:

• Yeast counting: All you need for this is a microscope with a basic transmitted light source and enough magnification to resolve individual yeast cells. Almost any microscope with 100x to 200x magnification (more on how to determine this, below) and a light source will suffice.
• Viability/vitality testing: Viability and vitality testing require a bit more than simple yeast counting. These methods use dyes which selectively stain cells based on whether they are dead (viability testing) or based on how metabolically active the cells are (vitality testing). A slightly better microscope is required for this than for yeast counting. Higher magnification makes this testing easier (400x to 600x), and you need a better light source and optics which will reliably and with minimal artifacts, recapitulate the colour of the cells.
• Cellular morphology: Identifying cellular morphology and detecting bacterial contaminants requires an even more advanced microscope. High magnification is required (minimum 600x, ideally 1,000x) alongside quality optical components which make the best use of this high magnification. A tuneable light source (capable of Kohler illumination) is a must. Additional features such as a mechanical stage and phase contrast optics can also be valuable for this use.
• Fluorescence: A cellular morphology microscope should be sufficient for 99.9% of brewers and breweries. However, if running a yeast lab or doing highly experimental work, you may need to move into fluorescence microscopy. This opens up a world of new options, especially for the experimentalist. However, the costs are prohibitive, with even a basic florescence microscope costing several thousand dollars – with some costing over a million! That said, there is a dedicated group of scientists trying to make DIY variants, which can be added onto conventional microscopes, for a more reasonable amount. Fluorescent microscopy is beyond the purview of this post, so I will not be discussing it further.

Lastly, don’t forget to think about the future. If you currently brew exclusively clean beers, but are thinking of making the move into wild or mixed-fermentation beers, it may be worthwhile spending the extra money now for a more capable microscope than purchasing a second microscope in the future.

Microscope Morphology:

Before we can discuss how to select your desired features, we first need to understand the construction and parts of a microscope. To the right is a diagram of a basic microscope, showing the key parts. I’m going to go through these in the order that light passes through the microscope, which is essentially in reverse order:

Light source (F): The formation of an image on your microscope starts with the light source. In the diagram, this is the mirror ‘F’, which is used to reflect the light of an external lamp (flashlight, or even the sun) through the sample (‘C’). While there is nothing wrong with a mirror, most modern microscopes have replaced this with a built-in bulb.

Condenser (D): Between the light source and the sample, many microscopes have a condenser. In its simplest form, this is a series of lenses which allow you to ensure the light is evenly projected onto your sample. But some condensers are more complex, and allow you to control the light in ways which can enhance contrast in the image (e.g. phase contrast or dark field illumination).

Stage (E): Your sample needs to rest on something, in order to position it correctly between the light source/condenser and the objective lens. The stage serves this purpose. Stages can be as simple as a flat piece of plastic or metal with a couple of clips to hold your slide in place (a stationary stage). But many stages have gearing systems that allow you to accurately move your sample by hand (mechanical stage), or even with a computer (motorized stage).

Sample (C): A microscope is worthless if you don’t have something to look at. The thing you are looking at (usually yeast, if you’re a brewer) is the sample, and it is most often placed on a glass slide for viewing on the microscope.

Objective Lens (B): This is the lens which images the sample, and it is the most critical component of your microscope. So critical that I’m dedicating an entire sub-section to it (below). The objective lens resolves your sample, performs most of the magnification, and determines the limits to the imaging capabilities of your microscope. In most microscopes, multiple objective lenses are attached to a nose piece, which allows you to change magnification by rotating the desired lens into position, all without loosing your focus.

Ocular (A): The ocular (or eye piece) is the final stage in forming an image through your microscope. In simple terms, the focus of the objective lens (e.g. where the magnified image it creates is in-focus) is inside of the microscope. The ocular is focused on the image created by the object lens, further magnifying the image and projecting it into your eye.

That’s it – the major components of a microscope. In simple terms, light from a light source is projected via the condenser onto your sample. The objective lens gathers and magnifies this light, and the resulting image is then magnified a second time by the ocular. You view the sample through the ocular, thus seeing a greatly magnified image of the objects in your sample.

The Simple Choices

The first choices you have to make are the easy ones – microscope format, and how many eyepieces you need.

Format: Microscopes come in two primary formats – upright and inverted. An upright microscope, which is what is shown in the picture above, has the lenses positioned above the microscope stage, and the light source/condenser is below. An inverted microscope has the lenses below the stage and the light source/condenser is positioned above the stage. Factors such as magnification and resolution are not affected by the format of the microscope, but the format can make some kinds of imaging easier. Generally speaking, an upright microscope is what you will want for brewery use. That said, if all you can find is an inverted microscope, it will do fine (in fact, that’s what I have), but some samples may be harder to image than with an upright microscope.

Number of oculars: The second simple choice you have to make is the number of oculars (eye pieces) your microscope has. There are three options – monocular (single ocular), binocular (two oculars) or trinocular (two oculars plus a dedicated camera port). It’s hard to justify a trinocular scope in the brewery – these are targeted more at researchers who need to frequently record samples. Most people find binocular microscopes more comfortable to use, especially if using them over a long period of time. But for most brewers needs, a monocular microscope is perfectly fine. My suggestion is to get a binocular microscope if you can afford it, but conversely, the binocular feature should be the first one you give up should you want to spend a fixed budget on another feature.

Binocular/trinocular microscopes will often have a diopter on one eyepiece. This allows you to adjust the focus of one eye separately from the other – a key feature for people who normally wear glasses and have one eye whose prescription is different than their other eye. To use the diopter, focus on the sample using the eye piece without a diopter, then adjust the diopter such that both eyes are in focus. You should check this before each use, as a mis-set doipter is a recipe for a bad headache.

Optics

More than any other factor, the quality of a microscopes optical components will determine the price of the microscope. A microscope is an optical instrument, and as such, its cost and capabilities are determined by its optical components. Deciding what optics you need is the most important part of choosing a microscope. In this section I’m going to cover the basic principals of microscope optics, and outline what brewers should be looking for. When thinking of optics, microscopists generally concern themselves with three things – numerical aperture (NA), magnification and optical aberration.

Magnification: Magnification is the more obvious aspect of a microscope – it is how much larger an object will appear through the ocular (or camera) than it is in real life. For example, at 1,000x magnification your average yeast cell (which is ~4 microns) will appear to be ~4 mm in size. Magnification is determined by the product (multiplication) of the magnification of the objective lens and the ocular lens. For example, a microscope with a 20x objective lens and 10x ocular would have: 10x X 20x = 200x total magnification. There is a trade-off with magnification, and that is image brightness. All other factors kept constant, the brightness of an image drops exponentially – e.g. an image captured at 200x will be one-fourth as bright as an image captured at 100x; a 400x image will be one-eighth as bright as at 100x, etc.

Common Magnifications Used In Microbiology:

• 100x – necessary for setting the initial focus, but otherwise not often used. Yeast cells, but not bacteria, are visible at this magnification.
• 200x – a sharp-eyed user can do cell counts at 200x, but most will find a higher magnification more comfortable. Yeast cells, but not bacteria, are visible at this magnification.
• 400x – good for cell counts, and passable for viability/vitality stains. A sharp eyed user may be able to see larger bacteria (e.g. lactobacilli) at this magnification.
• 600x or 630x – probably too high a magnification for cell counts, but this is ideal for viability and vitality stains. Good for yeast morphology, but not for bacterial morphology. Basic morphological features of larger bacteria may be visible; small bacteria will be visible only as small spheroids.
• 1,000x – Can be used for viability and vitality stains, although the smaller amount of light collected by high magnification lenses can make some dyes hard to make out. 1,000x is required for morphological analysis of bacteria and some yeasts.

Most lab-grade microbiology microscopes have a 10x ocular and combine that with a range of objective lenses to give a magnification range of 100x to 1,000x. Cheaper consumer/pro-sumer microscopes often include a 10x and 20x (or 25x) ocular. They will also often advertise the 20x as a feature, and claim up to 2,000x magnification, but this is often more of a marketing gimmick and a way around poor quality optics (or to provide fewer objective lenses), than a useful feature (more on this below). The presence of a 20x ocular is not an automatic sign of a poor quality microscope, but you should be aware that its use will be limited.

Numerical Aperture: Far more important than magnification is the numerical aperture (NA) of the microscope. Magnification determines how much larger an object will appear, but NA determines whether you’ll see the object at all. At 1,000x magnification, a yeast cell viewed through a high NA lens will have all sorts of details visible – yeast buds, cellular structures, and so forth. The same cell viewed at the same magnification but with a low NA lens may appear as nothing more than a vaguely yeast-shaped blob.

In simple terms, NA determines the resolution of the lens – e.g. how small of an object you can see with the lens. As a rule, the total magnification of a microscope (e.g. ocular X objective) should not be more than 500 x the NA. So a 1.25 NA lens is optimal if used at or below 625x (1.25 X 500) magnification. Magnifying past this range will not make smaller features visible. Magnification with insufficient NA is called empty magnification, and while not ideal, empty magnification is not completely useless. For example, 1.25/100X objectives are often combined with a 10x ocular for a total 1,000x magnification. While above the ideal 625x magnification, the “extra” magnification can make it easier to see the smaller features resolved by the lens…even though even smaller features will remain unresolved.

NA also determines the light gathering capability of a lens. As NA goes up, so does the amount of light the lens will collect. Most higher magnification lenses will have a higher NA than lower-mag lenses. This helps counter some of the loss of image brightness that otherwise comes with increasing magnification. With higher NA also comes higher contrast, making it easier to make out objects in your samples.

I think you can now appreciate why the 20x ocular is of little use. The common 1.25NA/100x objective lens found on many consumer microscopes already exceeds by 60% the maximum recommended magnification when paired with a 10x ocular. A 20x will create further empty magnification and drop the brightness by 75%, creating a blurry and dim image if paired with the 1.25NA/100x lens. As such, 20x oculars should be limited to use with lower magnification lenses.

Aberration: The final lens characteristic you need to consider is aberration (or, more accurately, how the lens is corrected to limit aberration). Really cheap lenses may distort the image due to being malformed – you can usually tell a manufacturer is making these kinds of lenses if they don’t print the NA on the lens. You’ll want to avoid these, as they make imaging difficult.

But even properly made lenses have aberration. This comes from a few issues – the most common is chromatic aberration, which is caused by different colour of light having slightly different focal planes. In practice, lenses uncorrected for chromatic aberration create images where objects have blurred, rainbow-coloured edges. Field curvature is the second common form of aberration seen in microscope lenses. This makes the sample appear as though it is being viewed through a bubble (expanded in the middle, compressed at the edges). Field curvature can also appear as variations in the brightness of the image, where the middle of the image is brighter than the edges. Field curvature is a result of how light bends through different parts of the lens.

Both chromatic and field aberration can be corrected, but doing so is expensive. In my lab we have a single 100x lens that is worth $40,000 – yes, $40k, as in the price of a nice car. The microscope next to it has a 100x lens worth a measly $500. The difference between the two is simply the degree of aberration correction applied to each lens. For the home or commercial brewery I’d suggest looking for achromatic lenses. These are corrected for chromatic aberration at two prime visual wavelengths, and flat-fielded in one. For standard microbiological use – including your home or craft-scale yeast lab, this is more than sufficient. There are increasingly corrected lenses, which add corrections for chromatic and flat-field aberrations at additional wavelengths, but every correction essentially doubles the price of the lens.

Objective Lenses

The previous section went into lenses and their features in some depth, but it is worth spending a bit more time discussing objective lenses. Hopefully I’ve imparted on you the importance of numerical aperture, but there are a few other features to consider:

Parfocality: Most microscopes are configured so that you can set the focus using your lowest magnification lens, and then turn the nose piece (the part which holds your objective lenses) to swing a higher magnification lens into place without loosing focus. This is important as it allows you to “zoom” in without loosing focus – a key feature as focusing high magnification lenses is difficult, and it is easy to smash a high magnification lens into your sample when seeking the focal plane. All but the cheapest of microscopes should be parfocal. This can be more of an issue if you’re adding additional lenses to a microscope, or if you are building one from parts scavenged from second-hand units.

Magnification Range: The magnification range of your microscope is determined by the possible combinations of ocular and objective lenses. For microbiological uses, a range of 100x to 1,000x is ideal, with the highest magnification lens ideally being a 100x lens with an NA of at least 1.20. If you are only doing cell counts of viability staining you can get away with a lower maximum magnification. My preferred setup is a 4-lens system: 10x, 40x, 60x and 100x objective lenses, all paired with a 10x ocular. This allows you to focus the system easily (100x mag) while having ideal lenses for cell counts (400x), viability staining (600x) and morphological work (1,000x). Magnifications below 100x are not useful for microbiology work (but are great for looking at bugs, leaves, feathers and the like), while magnification above 1000 will likely be empty magnification and result in very faint, low-contrast images.

Oil and Water Immersion Lenses: The final feature to consider when selecting objective lenses is whether the lens is a dry lens (sometimes called an air lens) versus an immersion lens. Dry lenses view the sample through the air. the other options are water or oil lenses, in which the lens is immersed into a droplet of water or oil placed on the sample. The water or oil acts as an optical “bridge” between the lens and the sample. The advantage to this is two-fold – it allows for a higher NA lens than does a dry lens (which maxes out at an NA of 1.0, versus 1.33 for water and 1.51 for oil), and there tends to be less aberration with an immersion lens. As a rule you want your lower magnification lenses to be dry lenses, but your 100x objective must be an oil lens – water or dry lenses are useless at this magnification for microbiological uses. Since you cannot use a water and oil on the same sample, you shouldn’t get a water lens for a microscope with an oil lens.

A few notes on oil lenses:

1. You must use microscope-grade immersion oil. Vegetable, mineral or other oils will not work and may damage your lens.
2. You must be very careful to not get oil on your air or water lenses, and if you do, immediately stop work and clean the lens with rubbing alcohol or an appropriate lens cleaner. Oil will very quickly, and irreversibly, damage air and water lenses.
3. Oil should be removed after you are done working with the oil lens. Oil will oxidize over time, creating a layer of partially polymerized oil on your lens – this ruins the lens. Excess oil should be dabbed (not wiped) off with lens paper, and then isopropyl alcohol (rubbing alcohol) or an appropriate lens cleaning solution used to clean the lens.
4. Only a tiny droplet of oil should be used. Too much oil is wasteful, will make it hard to move your sample, and can damage your lens. Even worse, it can drip off of the lens and onto your condenser, permanently damaging your microscope.
5. To use your oil lens, focus on your sample using your lower magnification lens, then switch to the next higher magnification and adjust the focus. Repeat this until you are at the highest magnification dry lens. At this point, rotate your lenses so that the oil lens is almost in-place, then put a teardrop-sized drop of oil on the sample. Rotate the oil lens into place, adjust your focus, and then start your work. Once a sample has been oiled you should never go back to a dry lens as – at best – the oil will distort the image. At worst, you may get oil on your dry lens, permanently ruining the lens.

Microscope Illumination

The next thing to consider is the illumination system of your microscope. The simplest illumination system is a mirror that reflects light onto your sample. While adequate, this is not likely to be sufficient for high magnification work. It is rare to see this setup today, with most manufacturers including a lamp in their microscopes for more consistent and even illumination. More advanced microscopes will have illumination systems which allow you to equalize brightness across the field, or perform special imaging tricks…all at an added cost, of course.

Lamps: Lamps are generally bulb or LED based. LED’s have a longer life, require less maintenance, and tend to be cheaper. Bulbs tend to produce a softer light that is easier on the eye, but will need more frequent replacement and may not get as bright as an LED (brightness is important for high-magnification imaging). There are high intensity lamps available for professional-grade instruments (halogen and mercury lamps), but I’d recommend avoiding these for home and brewery use due to their cost, complexity, and use of heavy metals.

Condenser: Not all simple microscopes will have a condenser, but chances are high that there will be a condenser on any system with achromatic lenses. Condensers can vary greatly in design, but unless you wish to do specialized forms of imaging (e.g. phase contrast), a standard Abbe condenser is all you need. An Abbe condenser consists of three parts – a focus mechanism, a lens, and a diaphragm. The diaphragm is used to cut-out any unwanted light, the lenses gather and focus the light, and the focus mechanism allows you to move that focused light onto your sample. This ensures even illumination and maximizes contrast. When adjusting your you condenser you are trying to achieve Kohler illumination; the instructions that come with your microscope should provide additional information on how to configure your condenser properly. As a rule you should configure your condenser at the beginning of each imaging session, but once set, it should not require adjustment between samples.

The reason for using a condenser is straight forward – it maximizes the resolution of your microscope, produces a brighter image, and improves contrast. While not required, a condenser is a feature you will want if you plan on doing anything more advanced than cell counting.

Advanced Illumination: While a condenser-less or Abbe-condenser microscope is more than enough for the average yeast wrangler, there are some other interesting options out there. The first of these is phase contrast (see image above). These microscopes tend to be expensive, but offer huge advantages over conventional microscopes when it comes to imaging samples without dyes. Phase contrast is a way of magnifying the contrast in your image, making it much easier to see small objects and cell morphology. Another interesting option is dark field imaging. In this form of imaging, cells appear as bright dots on a black background. This only works at lower magnifications (generally below 200x), but may be a real boon to performing cell counts. There are a range of other imaging methods (DIC, reflection microscopy, polarized light microscopy, and more), but most of these are specialized methods not applicable to your yeast lab.

The Microscope Stage

At this point you’ve picked the major features you need – namely, light source and lenses, but there are a few other factors to consider. One of these is the microscope stage. For the home/craft brewer there are only two stage options worth considering – a fixed/stationary stage, or a mechanical stage.

Fixed Stage: A fixed stage is simply a plastic or metal plate with a hole in it to allow the illumination light to pass through the sample, and sometimes small clips to hold a slide in place. For yeast counting or viability testing these are fine, but it can be hard to move a slide a small distance by hand – something which is useful when working at high magnification.

Mechanical Stage: Mechanical stages use a gearing system to allow you to move the stage in a precise and controlled manner. For most of these systems you turn one knob to move the stage left-to-right, and another to move the stage front-to-back. Some even have double-knobs for each direction, one providing course (e.g. fast) movement, and the other providing finer movement. These stages are a must for high magnification imaging, as they allow you to easily and evenly scan the sample and to centre objects of interest.

Other Options: Scientifically-orientated microscopes have a plethora of stages, most of which are overkill for a yeast lab. These include computer- or joystick-controlled mechanical stages, heated live-cell imaging stages, stages which eliminate any extraneous light, and other stages custom-built for complex microscopy tasks. None of these are needed for your yeast lab, and some may even interfere with some types of microbiological microscopy.

Cameras

For most brewers a camera is an unneeded luxury, but it can be useful if you want to keep a record of your yeasts’ morphology, or the presence of contaminants. Microscopes tend to produce dim images, so quality cameras are required in order to collect even the most basic of images. Some microscopes incorporate a cheap USB camera – I’d suggest avoiding these as their quality tends to be low. Moderate-quality cameras can be purchased for a few hundred dollars that do a good job. But for most users there is a better option – your cell phone. Cell phone cameras have come a long way, and many can produce good-quality images. Focusing a cell phone through an ocular can be difficult, but is possible. If you have access to a 3D printer, you can print a specialized holder that will reliably align your phone with the ocular, for less than a dollar. This model is one I’ve used with great success.

Putting it Together

This was likely a little overwhelming, but to simplify things, I’ve outlined some specs for four “levels” of microscopes, and provided links to consumer-grade equivalents over at AmScope. These shouldn’t be considered endorsements, but rather representative examples of what is available at a given price range. Always check a microscopes specs closely before your purchase.

Entry-Level/Yeast Counting: This outlines what the mast basic microscope can be that has a use in the yeast lab. This model is good for counting yeast but not much else. On the other hand, these kinds of microscopes often cost less than $50, making it a budget friendly option.

• Magnification: 100x to 400x, NA is not critical
• Illumination: Lamp or mirror
• Example: AmScope M100C-LED

Yeast Counting and Viability Testing: This is what I would consider the minimal microscope for someone serious about yeast ranching and using microscopy to improve their brewery operations. This model is good for yeast counting, but also has the magnification and optics to allow for viability/vitality staining and basic morphological analysis. Bacteria will be visible on this microscope, but it is unlikely you will be able to do bacterial counts or assess their morphology.

• Magnification: 100x, to 600x or more. All lenses are dry lenses; the highest magnification objective should be an apochromat with a minimum NA of 0.80.
• Illumination: Lamp, with (preferred) or without an Abbe condenser.
  
• Example: AmScope M500

Morphology and Imaging Bacteria: You are going to need a workhorse if you are planning on doing serious work, including characterizing mixed ferments, checking for infection, tracking cell and/or bacterial morphology, plus cell counts and viability testing. An quality Abbe condenser and a mechanical stage are a must.

• Magnification: 100x, to 1,000x, all apochromat lenses. Highest magnification lens should be a 100X oil lens with a minimum NA of 1.20, and ideally 1.25 or 1.30.
• Illumination: Lamp, with a full (rack & pinion + diaphragm) Abbe condenser.
• Mechanical stage
• Dual-stage focus (fine and course focus knobs)
• Example: AmScope B300B

The Cadillac: If you have some extra money burning in your pocket, why not upgrade the morphology microscope to a full-on phase contrast unit? You can still do all the regular imaging that can be performed with the morphology microscope, and have the option of switching to phase at any magnification to bring out those otherwise hidden details. Stats are the same as above, except the objectives and condenser have phase optics incorporated. Example: AmScope B390A-PCS

What About Used?

There are a lot of used microscopes out there. Some are available through companies which refurbish microscope, others through universities or labs getting rid of surplus equipment. If you are careful, you can find a working microscope worth several thousand dollars (new) for a few hundred bucks. Of course, if you can test it before you buy it, that’s always the best option.

If buying used, I’d suggest a few things:

1. Your best option is to buy in-person, so you can test out the microscope before you buy it. Most cities will have a company or two who deal in used scientific equipment. Often, these companies can bring in equipment from other cities if they don’t have what you’re looking for. If you’re lucky you may even find a hobbyist or local company selling their used equipment – check your local virtual marketplace (kijiji, etc).
2. If buying on-line, buy through a company that refurbishes microscopes. While you may luck out and get a working unit off of a random ebay seller, if you get a broken unit it can cost you thousands to repair it – and microscope repairs require tools beyond what most of us have in our workshops. Many refurbishers will offer a warranty on their repairs.
3. Buy a microscope purpose-built for microbiology. These will typically have high NA lenses, mechanical stages, quality illumination, and often include phase contrast and/or darkfield optics.
4. Look for a microscope from one of the “big four” – Zeiss, Leica, Nikon or Olympus. These are very reputable manufactures, their microscopes are well built and stand up to rough use, and they are common enough that parts are usually available. I have a 60 year old Nikon in my research lab – it works as well today as a scope fresh out of its box, and I can still find parts for it when I need them.
5. Don’t settle. Wait to find the right microscope for you.

Coming Up Next?

I’m pretty sure that this is my longest blog post in my history of blogging…so I’m going to cut it off here.

This article is (hopefully) the start of a series on using a microscope in the brewery. Watch my blog for upcoming articles on sample preparation, proper microscope use, and other microscopy topics!