Microscope Slide Preparation is an essential and thought-out methodology that ensures you observe through your microscope at its full potential – read our guide to master how to properly mount, stain and prepare a microscope slide!
But first – what are microscope slides?
What are Microscope Slides?
A Microscope Slide is a flat, thin piece of glass with a thickness of around 1 mm. This makes them great for mounting specimens, ideally sliced thin, for observation at high magnification under a biological or compound microscope.
In addition, Microscopic Slides make storing, preserving and transporting specimens incredibly easy, and are an essential accessory for users of compound microscopes.
Types of Microscope Slides
There are two main types of microscope slides – Flat Slides and Concave (Well/Depression) Slides.
Well (Concave) Slides have an indent to hold liquids. This means that a Cover Slip, which is a smaller section of glass normally used with Flat Slides, isn’t needed when using Concave Slides.
But there are also many more specialised microscope slides. For example, a Graticule Slide is often used to estimate the size of a specimen.
Now that you know more about microscope slides, you must be wondering – what is Microscope Slide Preparation and why do we need to prepare Microscope Slides?
Microscope Slide Preparation – Guide
Microscope Slide Preparation is an essential and thought-out methodology that allows your samples to appear as clearly as possible under your microscope.
The easiest way to start with microscopy is to purchase pre-prepared slides, which normally come in a kit of 50 or 100 slides. However, many scientists and enthusiasts outgrow these kits and soon begin to wonder what else they can see with their microscopes.
If you are one of those scientists or enthusiasts, below I have listed step-by-step instructions for you to follow when preparing your own slides at home.
Required Materials for Microscope Slide Preparation
Step 1
The first step to preparing your microscope slide is to gather all required instruments. You will need the following (Neuhaus et al, 2017):
- Flat Slides or Concave Slides
- Personal Protective Equipment (PPE) such as safety glasses and gloves
- Cover Slips (Optional)
- Distilled Water
- Sterile Forceps
- Sterile Pipette/Dropper
- Inoculation/Smear Loop
You will also need the following if you intend to perform a Slide Staining:
- Stain Solution
- Staining Rack
- Blotting Paper
Microscope Slide Mount
Step 2: Microscope Slide Preparation Guide
Now that you’ve gathered your materials, you can move on to mounting the slide.
It’s important to note that there are a few different types of slide mounts.
Dry Mount
The first of these is a Dry Mount, which is the simplest way to mount a slide. The simplicity of this method makes it an efficient choice for the examination of dry samples such as hair or feathers, dry sections of plants or samples of pollen. (Miami University, n.d.)
However, there is one notable disadvantage to using a dry mount. Because specimens are not suspended in a mounting medium, they result in lower image quality, stemming from a significant difference in refractive index between the specimen and the air that surrounds it.
But this does not detract from the fact that dry mounts are incredibly useful for simple, quick observations and observations where the mounting medium would not be suitable for that specimen.
To Prepare a Dry Mount:
- Place the specimen directly onto a clean and dry slide
- Optional: Place cover slip over the specimen to cover its entirety
A Cover Slip is useful to not only hold the specimen in place, but to also prevent the specimen from coming into contact with your microscope’s objective lens.
Wet Mount (or Temporary Mount)
The second type of slide mount is called a Wet Mount. These are useful for the observation of motile specimens capable of motion, but can be used for a number of observations on samples such as water-bound organisms or bodily fluids like blood. (Centers for Disease Control and Prevention [CDC], n.d. -a)
They are also known as a Temporary Mount as the liquid normally evaporates (Mountain Empire Community College, n.d.).
A Wet Mount is also relatively straightforward. To Prepare a Wet Mount:
- Use your sterile pipette to place a liquid medium (such as water) onto the slide
- Submerge your specimen in the fluid
- If using a cover slip, rest the edge of the coverslip against the glass slide at a 45-degree angle before slowly lowering it over your specimen. (Carlson et al, n.d.)
Figure 2: Lowering a Cover Slip onto a Glass Slide (Danielle 8.1, n.d.)
Using this method to place a cover slip over a specimen will decrease the number of air bubbles between the cover slip and the slide.
But with a Concave Slide, you won’t need to use a Cover Slip as they are capable of holding larger liquid samples.
Smear Mount
A Smear Mount is often used to examine samples of blood.
To Prepare a Smear Mount (CDC, n.d. -b):
- Prepare a small sample of your specimen.
- Transfer the sample onto a slide.
- From a broth culture, use a sterile pipette to transfer 1-2 drops onto your slide. Spread the drop into an even smear spread thinly across the length of your slide.
- From a solid media culture, use a sterile pipette to transfer a drop of sterile water or saline solution to the middle of a slide. Using a sterile inoculation loop, isolate a small sample of the culture and mix evenly into the drop of sterile water or saline solution.
- Spread the drop into an even smear spread thinly across the length of your slide.
- Allow the smear to dry completely before undergoing fixation to adhere the smear to the slide via heat fixation or methanol fixation.
- A cover slip is optional.
Now that you understand how to perform a microscope slide mount, you also need to consider whether you need to stain your samples.
Staining a Sample
Step 3: Microscope Slide Preparation Guide
Using a stain helps improve a specimen’s contrast, making features more distinguishable under a microscope. Staining a sample is especially useful for classification as it will highlight a specimen’s shape, as well as distinct features such as a nucleus in eukaryotes.
By staining a sample, you can also identify whether a cell is living or dead or even visualise the metabolic processes of a specimen (Bruckner, n.d.).
There are many different stains that you can use, with each designed for a different purpose (Alturkistani et al, 2016). To demonstrate, listed below are commonly used staining media.
| Iodine | Used on animal and plant specimens to stain carbohydrates. Stains dark-blue when starch is detected. |
| Methylene Blue | Used on animal specimens (such as blood cells) and bacteria. Stains nuclei and other acidic cell parts blue. |
| Crystal Violet | Used to classify bacteria in Gram stains by staining them purple. |
| Eosin Y | Used on plants and animal specimens (such as red blood cells). Stains cell membranes, cytoplasm and extracellular structures either red or pink. |
| Toluidene Blue | Used to demonstrate mitosis in plant cells by staining acidic cell structures (such as nuclei) dark blue. |
The method you would use to stain a sample grossly depends on your specimen and which of its structure(s) you hope to highlight.
Staining Bacteria with Methylene Blue
For example, here is how you would stain bacterial samples with a methylene blue stain.
- Perform a bacterial smear onto a slide.
- Using a sterile pipette, flood the slide with a methylene blue solution. Leave the solution on the slide for approximately 1 to 3 minutes.
- Using distilled water, rinse the slide and drain any excess water.
- Blot the slide between two sheets of blotting paper before allowing it to air dry completely.
Once your bacteria sample has been stained correctly, the stainable structures of each cell should appear blue. This will allow you to visualise the morphology of each cell.
Figure 3: Photomicrograph of a Francisella Tularensis Bacterial Sample Stained with Methylene Blue (Smith, n.d.)
Conclusion: Microscope Slide Preparation Guide
Now that you have read this beginner Guide, you’ll know all about Microscope Slide Preparation. If you’re still new to all this and find it all a little intimidating, that’s totally okay! You might consider starting off with a kit of pre-prepared slides before preparing your own slides.
What are you waiting for? Start preparing your own slides!
REFERENCES
Alturkistani, H. A., Tashkandi, F. M. & Mohammedsaleh, Z. M. (2016). Histological Stains: A Literature Review and Case Study. Global Journal of Health Science, 8(3). 72-79. https://doi.org/10.5539%2Fgjhs.v8n3p72
Bruckner, M. Z. (n.d.). Microscopy. Microbial Life Educational Resources. https://serc.carleton.edu/microbelife/research_methods/microscopy/index.html
Carlson, M., Coombs, B. & Yeo, D. (n.d.). Preparing a Wet Mount – Experimental Skill and Investigation. University of Manitoba. https://www.umanitoba.ca/outreach/crystal/Support%20Files/Experimental%20Skill%20Development/Preparing%20a%20Wet%20Mount%20-%20Experimental%20Skill%20and%20Investigation.doc
Centers for Disease Control and Prevention. (n.d. -a). Preparing a Wet Mount. CDC DIVISION OF LABORATORY SYSTEMS. https://www.cdc.gov/labtraining/docs/job_aids/routine_microscopy_procedures/Preparing-a-Wet-Mount_508.pdf
Centers for Disease Control and Prevention. (n.d. -b). Smear Preparation. CDC DIVISION OF LABORATORY SYSTEMS. https://www.cdc.gov/labtraining/docs/job_aids/routine_microscopy_procedures/Smear-Preparation_508.pdf
Danielle 8.1. (n.d.). How to produce a wet mount [Online image]. https://danielle81.weebly.com/using-a-microscope.html
Miami University. (n.d.). Temporary Dry Mount Slides. College of Arts and Science | Miami University. https://www.cas.miamioh.edu/mbi-ws/microscopes/drymount.html
Mountain Empire Community College. (n.d.). Slide Preparation for Microbial Examination. Water/Wastewater Distance Learning Website. https://water.mecc.edu/courses/Env108/lab1b.html
Neuhaus, B., Schmid, T. & Riedel, J. (2017). Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations. Zootaxa, 4322(1). 1-173. https://doi.org/10.11646/zootaxa.4322.1.1
Smith, Dr. P. B. (n.d.). Tularemia Francisella tularensis [Online image]. US Center for Disease Control and Prevention. https://pixnio.com/science/microscopy-images/tularemia-francisella-tularensis/photomicrograph-of-francisella-tularensis-bacteria-using-a-methylene-blue-stain