Wet mounts are made by placing a drop of water on a microscope slide and covering it with a cover slip. This can be a tricky process.
The size of the drop (too much and the water jiggles with everything in it), grit in the sample, keeping water from climbing atop the slip and centering the cover slip can add up to frustration when it's time for observation under the microscope. It takes practice to prepare the perfect wet mount slide.
1" x 3" slides can be used twice by placing a sample drop and cover slip to each side of the center, for different sample viewings.
Try not to touch the surface of the slide or cover slip - hold them by the edges
Using fresh clean slides and cover slips is recommended. They cost very little and it avoids the time it takes cleaning them. Cleaning slides and cover slips for reuse leaves too many artifacts on all of the surfaces.
Dispose of old slides and cover slips in an appropriate small sealed container or wrap and tape them in paper to discard.
Recommended Supplies
Fairly decent microscope (Click here for basic recommendations)
Glass microscope slides - 1" x 3" (76.2 x 25.4 mm), 1.0-1.2 mm thick
Glass cover slips - #1 thickness, 22mm x 22mm, 0.13-0.17 mm thick
Small dish to hold a sample from the culture
Eyedropper
Vacuum sucking pen with broad suction header
Long 10mm plastic pipettes
Paper towels
Collecting a Sample from the Culture.
Use a long 10ml plastic pipette to collect a 3-4ml sample from the culture. Gently squirt the sample into a small dish and let it settle. It is ideal to use a curved-bottom watch glass for this so the debris settles in the center. Wipe the pipette clean with a paper towel and dedicate it to that culture only.
Using an eyedropper, collect some water and debris from the bottom of the small sample dish. Hold the eyedropper vertically, with the tip pointing down, above the small dish until the larger debris settles at the tip. Gently squeeze out the larger or heavier clumps of debris.
Move the eyedropper over the slide and squeeze out a small drop or two of the remaining sample.
Video Objective Focus
Keeping focus on moving microscopic organisms is sometimes difficult due to the limited depth-of-field when using higher power magnification as well as keeping up with their swimming speed. Wet mount slides contain a depth of water that is deeper then the organism allowing it to swim closer and farther away from the objective. Lower magnification gives a greater depth of field at the expense of some detail.
Size of the Sample Drop
If the drop is too large, water will climb up over the top of the cover slip or spill out onto the microscope stage when queued.
If the drop is too small, the water will dry up too quickly.
The ideal amount of water will fill the space under the cover slip without floating. No water should be squeezed out along the edges of the cover slip.
A well-made wet mount can be observed for up to an hour depending on the light source beneath. LED lighting is cooler than halogen lighting and the LED bulbs last a long time.
Placing the Cover Slip
Some procedures call for holding the cover slip at a 30 or 40-degree angle, dipping one edge of the slip into the edge of the water drop and letting it fall over on to the sample. This can lead to problems;
If the slip sticks to the thump or forefinger, it might drop unevenly with one corner hanging over the slide. Touching the cover slip to correct the alignment could cause a mess - water can climb up on top of the cover slip or spill out onto the stage.
Alternate method for Placing a Cover Slip
Using a vacuum suction pen to hold the cover slip and suspending it above the drop, without touching the water, at a slight angle allows correct alignment with the slide and an air-bubble free sample. When the suction is released the cover slip falls onto the drop just right. A few tiny bubbles appear occasionally but have no impact on viewing.
Queuing up the slide
Carefully place the slide onto the mechanical stage and move it into position. If you touch the cover slip in any way, water will climb onto the top. If the cover slip is incorrectly placed or making a mess, start over.
Viewing the Sample
Keep in mind that there are four surfaces to a combined slide and cover slip; top and bottom of each. Cover slips are so light that the surface tension of the water keeps them afloat. While focusing on your specimen with the microscope make sure you are focusing on the top of the slide where most of the microscopic life can be found. It takes discipline to keep focused on this plane.
An adjustable X-Y axis mechanical stage is necessary both for scanning slides from side-to-side and chasing protozoans until they slow. Practice to keep from going the wrong way when slight adjustments are made. What you see through the microscope is backward from left to right and top to bottom.
Using the mechanical stage of the microscope, scan from cover slip edge to the opposite edge starting at one corner. Advance the mechanical stage a bit and scan to the opposite edge. Repeat this alternately until you get to the bottom opposite corner.
The combined power of the eyepiece and the objective will determine the viewing field and how fast you complete a sample. 100X or 200x viewing power is recommended for locating protozoa. A higher power might be needed to study them in detail.
Keeping a Wet Mount hydrated
Some wet mount slides are worth keeping hydrated for longer viewing. Two methods may be employed;
1. For short term hydration carefully put a small drop from the same sample you are viewing next to and touching the edge of the cover slip. Capillary action sucks the water under the slide. Try to avoid getting water on top of the cover slip. This can be repeated many times.
2. Build a "wet box" to keep the slide hydrated for days. Pictured below is a plastic lunch box with tight fitting cover and wings that snap into place to ensure a tight seal. Three half-inch PVC pipes are hot glued into place for a stable and raised place to rest the slide. Adding water to just below the height of the pipes and covering the box tightly will keep the slide hydrated. There will be enough oxygen in the box for the protozoa until the next day. When extracting the slide for viewing, the bottom may need to be carefully dried on a lint-free micro fiber cloth to keep the microscope stage dry.
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