Materials
Below is a list of items and materials you will need to in order to collect, prepare, and examine your wet mount sample.
Compound Optical microscope
See Microscope Overview for more information on operation of an optical microscope
First off, you will need a decent (doesn't need to be expensive) bright field (there are different kinds, bright field is the classic type that we are all familiar with) optical microscope to view your samples. These can range in price depending on the brand, quality, and available functions. You can find many options from Amazon and other online stores. Another option, if you have the time, is to look around for lab and university auctions or sales as they will often sell there old equipment for cheap.
There exists a special kind of bright field optical microscope called an inverted microscope - these are expensive so I doubt you will be buying one. While these are somewhat superior to the normal microscope for biological imaging it is not needed and overkill for wet mount samples. Don't waste your money on this variant
At a minimum you will need a microscope that meets the following requirements
- Magnification rating of at least 400X. Lactobacilli are 0.5-1.5 μm in width and 1.0-10.0 μm in length and the 400X allows you get up close to clearly see what's going on.
- Course and Fine focus adjustments. It can be challenging at first to get the focus just right and having a fine adjustment makes this much easier.
- Adjustable (focus adjusting) eyepiece lenses. This allows you to adjust the focus on the eyepiece which is important for those who have less than perfect eyesight. If you are looking at binocular microscopes (two eyepieces rather than one) then get one that can adjust the distance between the eyepieces. We all have different spacing between our eyes and it can be a real headache to use a microscope that is either too large or too small for your face.
- A solid construction and stage. Cheaper microscopes will work in a pinch but they can be frustrating to get clear images of your sample as they won't hold your sample firmly and could drift when trying to focus on the sample. Its helpful to use a cellphone to capture images of the samples to share with your doctor, and you may be left disappointed when trying to get a good image on a cheap (build quality here, not price) microscope.
Other things I recommend but are NOT required
- Mechanical sample stage. This allows you to move the sample around while viewing in a controlled manner without using your fingers on the slide.
- Sample stage that grabs the slide by pinching it on the edges. There are types that clamp down on the top of the glass slide and while these work they are not something I personally like using due to their sometimes finnicky nature. Also, if you are being careful with your resources and want to set up multiple samples on the same slide (up to 3) then this reduces the slide real estate available. See Sample Preparation for more information.
- Rotating DIN Objectives. This makes it faster and easier to increase or decrease the magnification.
- LED light source. These are better than incandescent bulbs as they do not output as much heat. The heat from the light source will dry out your sample faster limiting your working and viewing time.
Below is a link to the optical microscope that I use.
OMAX M82E Series LED Binocular Compound Lab Microscope 40X-2000X
Cotton Swabs
Next up are the swabs that you will use to collect your sample.
PLEASE GET SANITARY MEDICAL GRADE COTTON TIP APPLICATORS. I cannot emphasize this enough, you do not want to introduce other harmful biota into your body or contaminate your sample.
Medical applicators are individually wrapped (2 in a package usually) and this keeps them more sanitary then if they are all open to the environment.
You will need ones that are at the very least 6 inches (~15 cm) long in order to get a good sample. See Sample Collection for more information.
Below is a link for some applicators. There are many more options online elsewhere.
Dynarex 6-Inch Sterile Cotton Tipped Applicators
Glass Slides and Covers
This is fairly simple - you just need glass microscope slides and covers. They are usually sold together. There are a bunch of options online but I have put a link below to a set.
Stains
You don't need to stain your sample but you will need a clean solution to apply to your sample. If you don't want to use a stain I recommend you use a sterile saline solution. These can be purchased at local medical supply stores or online.
Despite not needing to stain your sample I do recommend using Methylene Blue stain as this makes viewing the sample much easier. Methylene blue can be found in saline or alcohol solutions. Saline won't kill the bacteria on the sample but I have found it is usually darker than the alcohol solutions so it can be a little bit harder to get a good image of your sample if you are trying to take a photo with your phone. So I usually opt for the alcohol solution base as it allows more light to transfer through the sample. It kills the bacteria in the process but we aren't concerned with that - our goal is to see the quantity and structure of the lactobacilli and epithelial cells. See Sample Preparation for more information. Little word of warning Methylene blue stains everything so be careful where you put it. It will eventually wash off your hands and its not harmful but best to be aware so you don't stain your table or clothes.
One neat thing is you can also look for Candida infections with wet mount microscopy as well using a KOH (potassium hydroxide) solution. I won't cover that in this tutorial for now but if anyone is interested in learning more I can add a section for that, just let me know.
Links for stains online. Again there are many sources for these stains online and locally (In the US) just be sure to get a sterile stain from a reputable source.
Other
If your microscope has a 100X objective lens then you will likely need an immersion oil. Read what your microscope requires but this is typical. The oil is applied to the top of the glass slide cover.
After each time you use the immersion oil be sure to clean the objective lens with a kimwipe or microfiber cloth to ensure the lens doesn't become damage by the oil.