Mounting medium is usually very easily available commercially, but can a mounting medium be prepared at the lab? Are there different types of mounting mediums?
In this article I will discuss all these questions and describe how you can make a mounting medium yourself.
In general terms, microscope mounting medium can be made in one’s lab using glycerol as the base and a saline-based buffered solution like PBS which maintains the pH of the medium. Depending on planned use, additional ingredients may include antifade agents, adhesives, and anti quench agents. Let’s discuss in detail.
Let’s very briefly go over what a mounting medium is.
Mounting medium is a water-soluble or organic-solvent-based chemical that is used while preparing histological slides. Mounting is one of the last steps in the protocol of preparing a slide that is intended to be studied under the microscope. A mounting medium is used in this step over a sample already placed on the slide, and then a coverslip is put on top of it to make everything set in one place.
You can prepare your mounting mediums yourself using various ingredients. In fact, it is also believed that preparing the medium yourself sometimes gives better results than the commercially prepared ones. Most labs have their own recipe which they prepare using the ingredients of their choice and the purpose they have in mind— for example, whether they want to fix a slide temporarily or permanently, and the type of protein/tissue being examined. Therefore, the recipe usually varies from lab to lab.
As i stated earlier, the most commonly used mounting medium is composed of glycerol as the base and a saline-based buffered solution like PBS (phosphate-buffered saline) that maintains the pH of the medium. Other ingredients may include things like anti-fade agents (like 97% thiodiethanol or commercially prepared anti-fade kits), adhesives (epoxy resins, crystal bond thermoplastic resin, etc), anti-quench agents (free-radical scavengers; p-phenylenediamine, propyl gallate, etc), fluorophores (prevents photobleaching), and agents for ringing the coverslip like, glycerine (liquid), and vaseline or simple nail polish (solid).
How To Prepare A Mounting Medium?
Mounting medium is prepared using glycerol and PBS in the ratio of 9:1. Northern Arizona University says various studies have proved that the optimum pH of the medium should be maintained at 8.5 to 9.0, as at this pH the sample on the slide can be preserved for a longer duration of time.
A wide range of ingredients can be used to prepare a mounting medium, but the simplest recipe includes 3 ingredients: glycerol, an anti-fade agent, and a buffer. Here’s a recipe recommended by Northern Arizona University and the Harvard Medical School:
- Glycerol: 90 ml in 50-90% concentration. Glycerol raises the refractive index (RI) of the sample and helps in the formation of a brighter and higher resolution image. It is important to match the refractive index (RI) of the mounting medium to the RI of the sample. When the light passes from one medium to another, it bends (refraction). So, if the RI of both the mounting medium and the specimen is the same (or almost similar) the scattering of light would be less. Glycerol is used in the concentration of about 50-90% depending on which type of imaging is being done. Harvard Medical School suggests 90% glycerol should be used for fluorescence images, while 50% glycerol should be used for DIC images. This will give you the best image in the respective setting.
- Buffer: 10 ml 0.1M phosphate buffer, or 10 ml 0.1 M TRIS buffer.
- Anti-fade agent: p-phenylenediamine hydrochloride- 100 mg. This is considered to be the best anti-fade agent. But it is known to react with blue dyes and destroy it slowly, and so, over time the image may weaken. Therefore, this anti-fade agent is not recommended if you are planning to store your slide for a longer period. Also, it is toxic in nature. Wear all protective gear and avoid inhaling it. Its toxicity can cause serious organ damage and can even be fatal. Alternatively, you can use 0.5% n-propyl gallate– 500 mg. This agent can be used with living cells as well, and you should be able to store your sample for a longer time with this agent when compared to p-phenylenediamine hydrochloride. However, it interferes with the apoptosis of the cells, and therefore it may not be the most suitable anti-fade agent if you are preparing the medium to study biological processes. Other agents that can be used are ascorbic acid and 1,4 diazabicyclo (2,2,2) octane (DABCO).
- Mix these ingredients and shake well. You may need to warm it up to 37 degrees Celsius, especially if you are using n-propyl gallate as the anti-fade agent since it is not readily soluble. Heating it up will help with that. Alternatively, you can also leave it to dissolve slowly overnight.
- The mounting medium should then be stored at 4 degrees celsius and can be used for 1-2 weeks.
- An ideal mounting medium is a transparent one. So, if you notice a color change, discard it. Also, if you see some discoloration while preparing the medium, discard it, as that would mean some contamination has happened.
This recipe will help you form a temporary slide. Glycerol helps preserve the slide for 3 months at least. You can store it for a longer period if you store it at -20 degrees Celsius in the dark to prevent any kind of photobleaching.
To prepare a permanent slide, organic solvent-based chemicals containing xylene or toluene are used.
Why Is Mounting Medium Used?
Mounting medium has a few advantages:
- The most important role that any mounting medium plays in the maintenance of an appropriate refractive index (RI) for the formation of a good quality image under the microscope. The recommended RI of the mounting medium is around 1.53. The closer the value of RI of the tissue and the mounting medium to this value, the brighter and clearer the image would be. Glycerol does this job in a mounting medium, with an RI of 1.47.
- Secondly, the mounting medium fixes the specimen in one place and prevents it from drying out.
- It helps preserve the slide for a longer duration of time. A specimen is very finely sliced, and is, therefore, very delicate. By putting a mounting medium on top of it and then covering it with a coverslip protects the structures of the specimen. It also prevents moisture from coming in contact with the specimen which would promote growth of bacteria. Hence, it prevents its degradation by bacteria. Anti-fade agents protect the specimen from damage caused by exposure to light.
Types of Mounting Media
There are two types of mounting media.
- Organic Solvent Based Media: These are composed of natural or synthetic resins dissolved in solvents like toluene, benzene, xylene, etc. They are used when the sample needs to be dehydrated. They are also known as the adhesive medium; meaning it sticks the specimen to the slide and then it hardens which permanently adheres the coverslip on top of the sample. For this reason, permanent slides can be prepared using these. Some of the examples of a solvent-based media are Canada balsam, phenol balsam, Euparal, etc.
- Water-Based Media: These are composed of ingredients that are water-soluble in nature. These are easier to use and are less toxic. But they can’t be used to prepare permanent slides. They can be used to prepare temporary slides that last for a few months. Examples include glycerol-based media, Apathy’s medium, Farrant’s medium, etc.
A slide that is prepared using a liquid medium is known as a wet mount slide. But in some cases, like when spores and pollens are the study subject, no mounting medium is used. A coverslip is directly put over the specimen. In this case, the mounting medium is air, as air surrounds the specimen under the coverslip. This type of slide is known as a dry mount slide. But dry mount usually produces a poor quality image because the RI of air is 1.0, which is pretty far off from 1.53, and this promotes scattering of light to a great deal.
How To Use Mounting Medium?
Once the initial steps of the slide preparations are done— fixing, embedding, sectioning, staining, etc, the mounting medium is put on top of the specimen. Use 6-8 microL of this solution per 18 mm coverslip. Too much of the solution will spill out of the slide.
Glycerol stays in the liquid state, i.e. it never hardens. So, it’s essential that you seal the coverslip (especially if you are planning to keep the slide for a few months) so that the glycerol stays as it is, and there is no entry of moisture or contaminants inside the slide. Sealing the coverslip at its edges to achieve this is known as the ringing of the coverslip. To seal the coverslip a variety of agents can be used. For example, paraffin wax and vaseline— which will form a temporary slide.
A more permanent sealant can be formed using paraffin wax (10 parts) and colophonium resin (40 parts). This is known as the Du Boyer wax-colophonium resin mixture. This is a more permanent sealant. Lastly, clear nail polish can be used as a sealant too. It’s easily available and easy to use. Sealing with nail polish can preserve the slide for a few months. Asphalt-based cement is another option if you are going for a permanent slide.
If you are using a solvent-based medium, you might not need to apply a sealant as it usually contains natural or synthetic resin, which hardens, thereby preserving the specimen within it.
Make sure that the mounting medium you are using is transparent in color, and has a RI of 1.53. If you see any color change— which can range from yellowish, brownish, or black— discard the medium.
Here’s a video that will help you understand the process of using a mounting medium and making permanent slides:
Difference Between A Permanent And A Temporary Slide?
As the name suggests, a permanent slide is a slide that is prepared to be preserved for a very long period— years. Such slides when prepared well can even survive for a century. The permanent slides are prepared using the solvent-based medium, as they contain the resin component which eventually hardens and protects the specimen from damage.
The disadvantage of a permanent slide is that it cannot be used to study a live cell since the specimen is fixed on the slide.
On the other hand, a temporary slide is prepared using a liquid mounting medium like the one mentioned in this article. They can be used to prepare a temporary slide. As mentioned above, to increase its longevity, ringing of the coverslip can be done using a sealant.
The advantage of a temporary slide is that it can be used to study a living cell since the specimen stays in a liquid medium.
The Bottom Line
You can easily prepare a mounting medium using three simple ingredients. The recipe mentioned in this article is water-soluble, and therefore will be useful in preparing a temporary slide. Depending on your goals, you can choose other ingredients. Choosing a solvent-based medium will be ideal if you want to prepare a permanent slide.
Click here to read about the maximum magnification of a confocal microscope.
