Permanent slides are excellent for teaching processes and organism anatomy to students. They take away the worry and effort of preparing the slides repeatedly. However, making permanent slides requires specific techniques and reagents.
This article tells you how to make permanent slides of fungi, insects, or others.
Table of Contents
What are Permanent Slides?
Permanent slides have organisms preserved in a mounting medium to help students study and observe the specimen for longer. They contain the samples without changes in the tissue structure, allowing you to examine the samples in their original form. Most permanent slides have a mounting medium that solidifies to preserve the specimen for up to a century!
How to Make Permanent Slides
The preparation of slides of insects, fungi, animals, or other organisms requires killing them before fixing and hardening them. Next, you must stain and dehydrate the samples, followed by clearing and mounting them.
Here’s the process in detail for easy understanding:
Killing the Specimen
Killing the specimen is the first step in preparing permanent slides. The killing stops the living activity of the organism and any further physiological changes. You can use 0.1% osmic acid or ether to stop the activity without changing the internal or external structure of the tissue. Some people also use alcohol, but it may shrink the tissues.
- Spread a thin film of Mayer’s albumin on the slide by rubbing a drop of the albumin with your index finger.
- Next, place the killed organism onto the slide.
- Put a few drops of alcohol or ether on the slide and let them evaporate. Alternatively, place the slide inverted on the mouth of the bottle containing osmic acid crystals and distilled water.
Fixing and Hardening
Fixing and hardening the slide is critical to organisms that require further treatment after killing the samples. It helps stop changes in tissue form and makes them suitable for staining. You need to use a fixing agent to stop further alterations in the structure.
The most important characteristics of a fixing agent include:
- The fixing agent must preserve the tissue and structure of the specimen without deformation.
- It must kill the specimen quickly (but remember that some agents can be toxic and harmful).
- The agent must penetrate the sample well to ensure that it hardens all parts of the sample.
Fixing agents are categorized into four groups, including alcohol and acetic acid, aldehydes, tanning agents, and oxidation agents. Alcohol and acetic acid are less harmful and suitable for hobbyists. Conversely, formaldehyde is fast but it can be toxic and requires extra care.
Mostly, the process is done on sections of larger animals using fixing agents like formalin, acetic acid, mercuric chloride, Bouin’s fluid, osmic acid, 70% alcohol, and potassium dichromate.
After hardening and fixing the sample, make sure to wash the sample with a reagent to avoid damage to the tissues. Commonly used reagents for specific fixing agents are:
|70% alcohol + iodine
0.12% chloral hydrate
|Osmic acid and K2CrO7
When learning “How to make permanent slides,” staining is critical to know how to differentiate tissue parts. Various acidic, basic, or neutral dyes are used to stain the specimen for permanent slides. The basic dyes stain the nuclear part of the cell, whereas acidic dyes color the cytoplasm.
However, using the single staining technique, you can simultaneously stain the cytoplasm and nucleus with a basic or neutral dye. Also, combining two or three stains helps color different cell parts uniquely.
Single staining typically uses pircrocarmine in water or borax carmine in 70% alcohol.
- Pass the specimen through alcohol gradually increasing the percentage of the staining medium after thorough washing (30% alcohol, then 50% alcohol, then 70% alcohol for three minutes each when using micro-indigo-carmines or borax carmine).
- Next, transfer the material to the stain for three minutes until it obtains the dye (dark green for pircrocarmine and dark red for borax carmine).
- Wash the material with the solvent or medium (70% alcohol in the case of the dyes mentioned above).
- Leave the tissue in acid alcohol for a minute or two to remove excessive stains if needed.
- Repeat the process every thirty seconds and check until the tissue has the desired color.
Double staining usually uses two dyes, usually Eosin in 70% alcohol and Delafield’s Haematoxylin in distilled water.
- Put the sample material in Haematoxylin for 5 minutes after washing; it might take a few more minutes sometimes to obtain a blackish-blue hue.
- After staining, pass the material through the solvent or stain in which you have prepared the second stain (30% ale, 50% ale, and 70% alcohol for Eosin).
- Next, keep the material in the prepared stain for one minute.
- Now, wash it in 90% alcohol.
The next step of “How to make permanent slides for a microscope,” is dehydration, which helps remove excess water from the slides to improve their shelf life. The process replaces the water content with alcohol as the mounting medium solvent and clearing agents are alcohol-soluble.
Dehydration follows the same basic process as staining (except the stain). Pass the tissue through gradually increasing percentages of absolute alcohol (30%, 50%, 70%, 90%, and 100%) to avoid shrinking.
Next, dip the tissue in fresh absolute alcohol for one minute each at least three times. You can replace alcohol with ethylene glycol monoethyl ether as it easily mixes with water, alcohol, and clove oil. Use cavity blocks or staining tubes to prevent the entry of moisture while avoiding the evaporation of alcohol.
Now, it’s time to remove excess chemicals from the sample and give it a clear appearance for easy observation. Making permanent slides substitutes the dehydrating agent by the mounting medium solvent. Typically, xylene and benzene are used to clear the tissues. However, it is better to use clove oil or cedarwood oil as they do not cause shrinkage, unlike xylene.
After dehydration, place the material in the clearing agent until it becomes transparent. You may need to repeat the process twice or thrice if the clearing agent turns white or turbid. Put the material in fresh absolute alcohol for five minutes before dipping it in the clearing agent again.
The last step of “How to make permanent slides” for long-term storage is mounting the material onto a slide. You can use an aqueous or solvent-based mounting media. Glycerine gelatin is a widely used water-based mounting medium, but it is challenging to use due to bubble formation. Alternatively, use a solvent-based medium, but make sure your specimen is free from water.
Commonly used mounting agents include Euparol or Canada balsam in xylene.
- Place a drop of mounting medium (of the same refractive index as the coverslip, i.e., 1.5) in the center of the slide using a glass rod.
- Transfer the transparent material onto the drop of mounting medium on the slide using a brush.
- Cover it with a cover slide slowly, with one end touching the mounting medium and the other side held at an angle using a needle.
- Slowly drop the end of the coverslip to cover the material avoiding air bubbles; make sure the coverslip is in the center of the slide.
- Use a blotting paper to clean excessive mounting medium from the slide.
Lastly, label the slide with the specimen name and your name on the other edge of the slide. Place it under the microscope for observation and learning.
How to Make Permanent Slides: The Easy Way
Preparing permanent slides might seem difficult and complicated, especially for young microscope enthusiasts. If you want to prepare permanent slides easily at home, choose dry specimens or use nail polish to preserve the samples.
Opting for a small dry specimen makes it easy to prepare a permanent slide, just like a wet mount. Choose small insects or parts of insects like wings to make permanent slides without too much effort.
- Add a drop of the mounting medium on the slide.
- Place your dry specimen onto the slide.
- Put another drop of the mounting medium on the sample.
- Slowly place the cover slip on the slide to avoid air bubbles.
- Keep the slide in a dry area to help it dry properly, and store it in a well-ventilated space.
You can also use clear nail polish as a substitute for the Euparal mounting medium. However, the material may shrink when you add clear nail polish.
How to Make Permanent Slides of Plankton?
Preparation of plankton slides comprises fixing, staining, color separation, oil blackeite, dehydration, clearing, and mounting. Making permanent slides of plankton requires considering the following to preserve the liquid-based specimens for long-term preservation:
- Fix the animal plankton using 5% formalin
- Use haematin as a coloring agent to stain the sample
- Apply sour alcohol as the color-splitting agent
- Next, use tap water as an oil blackeite agent
- Use gradient concentration alcohol for dehydration to increase longevity
- Now, clear the specimen using glycerin for the animal plankton material
- Use neutral gum as the mounting agent
This method improves contrast and sharpness, providing a better observation.
How to Choose the Right Mounting Medium?
Now that you know how to make permanent slides of insects and other specimens, choosing the right mounting medium is important. So, which mounting medium is the best for your specimen?
The mounting medium also depends on the medium of the specimen, as specimens in water must be transferred to a water-based mounting medium. At the same time, a solvent-based mounting medium must go into a compatible solution. For example, Euparal and xylene go with alcohol. Otherwise, the resin may get a cloudy appearance.
Non-water-based mounting media often shrink the specimen, so solvent-based mounting media are preferred. It is more common with thick specimens. So, choose mounting media that does not impact the tissue structure.
The refractive index plays a major role in preparing permanent slides to view the specimens clearly. The refractive index of the mounting medium and specimen should be the same for bright-field microscopy. Conversely, a phase contrast microscope requires a difference in the refractive index of the specimen and mounting medium.
Not in all cases, but sometimes, mounting media may degrade the pigments over time. While it is desirable for some samples to be observed better, it may impact the visibility of others; use a mounting medium that does not react with the pigments.
Permanent slides are made to last years and decades. Thus, carelessness in choosing the mounting media can sometimes lead to early deterioration of the samples. The samples may crystalize, the mounting media may run out, or it may react with the pigments. Mounting media like Canada balsam can help improve the longevity of the slides.
Cost is another major factor when looking for the right mounting media for the preparation of permanent slides for insects. You might find an all-rounder mounting media like Canada Balsam, but it is typically expensive. Hobbyists can use clear nail polish and glycerol jelly instead.
Popular Double Staining Methods
Staining is another critical step in preparing permanent slides, and the most commonly used three double staining methods are:
Crystal Violet-Erythrosin Staining
Select the specimen you want to stain and stain it with an aqueous crystal violet stain followed by dehydration in alcohol of a gradually increasing percentage. Then, stain it with erythrosin in alcohol.
Safranin-Fast Green Staining
Stain the specimen with aqueous Safranin for three to four minutes. Now wash it in the water until the water remains colorless. Next, dehydrate and stain it with fast green prepared in 90% alcohol.
This staining method uses Haematoxylin first, followed by Safranin. Stain the sample with Haematoxylin and wash it with ammonia water. Now, rewash the sample with tap water and stain it with Safranin. Next, destain the specimen with 70% alcohol, 90% alcohol, and absolute alcohol.
The Bottom Line
Among the many types of microscope slides, permanent slides are ideal for preserving samples. These samples can be kept for a long time in solidifying media, eliminating the need to prepare slides frequently. This article provided a complete guide on how to make permanent slides of insects and other specimens. The steps include killing, fixing, hardening, staining, dehydrating, clearing, and mounting the sample. Use an adequate fixing agent and mounting medium for long-lasting slides.
How to make permanent slides using permount?
You can also use permount when preparing permanent slides by passing the specimen through 70%, 80%, 90%, 95%, and 100% ethanol. Then, soak the sample in xylene after pasting through the ethanol.
How to make permanent slides in botany?
Preparing permanent slides in botany includes dehydration, clearing, and mounting the samples on the slide for later viewing. You might also need to stain the samples to observe intricate details easily.
How to make permanent slides using water samples?
Making a permanent slide using water samples requires killing and fixing them before staining, dehydrating, and clearing the specimen. Use a water-based mounting medium for water samples to ensure compatibility.