Objects that can’t be seen by the naked or unaided eye may need a microscope, so a researcher or scientist can observe them accurately. That process is called microscopy, and it has allowed the discovery of many things.
You may view a wide variety of samples under a microscope, but some may need staining to see them correctly. It will provide a contrast that will make some structures discernible. It will make some parts, especially in a cell, change their color to set them apart from others. You may apply stains on a wide variety of samples.
In-Vivo
In-vivo staining includes a living matter. It means that you are going to stain it as it is alive and observe it accordingly. These include cells, tissues, and others. Many kinds of dyes will work best with the in-vivo method.
In-Vitro
The in-vitro staining technique involves a non-living biological matter. It also has stains that are suited for the process.
Preparation of Stains:
Hematoxylin
For examining biological tissues, Haematoxylin will work and turn nuclei blue. To make one, you will need to dilute 10 grams of crystal Haematoxylin and 500 mL of water in 70 to 80 degrees centigrade. Then, in another container, you have to mix 20 grams of alum with 500 mL of water at the same temperature.
Next, you will combine the two mixtures and add another gram of the Haematoxylin crystal. Keep the mixture away from sunlight and store it in a translucent flask with a paper towel cover, to allow air circulation, for one week. This is when it will have its early maturation stage. After that, you have to transfer it to a dark flask with a tight and secure cover and store it in a dark place for three weeks. This is for its late maturation.
Eosin
To turn the cytoplasm and other parts of the cell you need to observe, pink, you will require Eosin. To prepare one, you have to dilute 10 grams of Eosin crystals in 1000 mL of water at 70 to 80 degrees centigrade. Store it in a dark flask, and you may use it at once.
Techniques and Procedures:
For generally any sample, you may need to rehydrate it and use Haematoxylin for 20 to 40 minutes. Then, for 3 minutes, wash the sample in tap water and let it turn blue. Use 70% ethanol with 1% of HCL to get rid of the excess stain, and wash it again in tap water. Dye it with Eosin for 1o minutes and wash again with tap water for 1 to 5 minutes. Dehydrate it and mount it on a rack.
Papanicolaou Staining
Pap-staining or pap smear is used to inspect cells that were from body fluids. Some examples are lymphocytes and plasma cells. For this technique, you will need light green SF yellowish, Haematoxylin, Orange G, and Eosin Y. To do this, slides must first be fixed in acetic fixative for 15 minutes, in absolute alcohol for 2 minutes, in 70% alcohol for another 2 minutes, and in 50% alcohol for another 2 minutes.
Rinse it in tap water for 2 minutes and then put it in Haematoxylin for 4 minutes. Rinse it again and use acid alcohol for 5 seconds. Blue inside tap water then dehydrates it twice using absolute alcohol. Use Orange G for ten seconds that rinse twice in absolute alcohol. For two minutes, use E.A 50 then use absolute alcohol twice. Clear it with xylene three times and mount it again for another three times.
Acid and Basic Fuchsin Technique
For plasma staining, acid fuchsin dye is mostly used. It is magenta red and is an acid. On the other hand, basic fuchsin is magenta and is used for staining the nucleus.
The method is also called “acid-fast staining.” To prepare the dye, you only need to mix two solutions. The first solution is made of 0.2 grams of basic fuchsin and 10 mL of 95% ethanol, while the second solution is composed of 5 grams phenol and 90mL distilled water.
For this process, you may use Methylene blue as a counterstain and alcohol to decolorize. You will be able to identify acid-fast cells because they will remain red or pink, while non-acid-fast will remain blue.
Wright’s Stain
To make a metachromatic stain, you will need a specially treated Methylene blue dye and Eosin. To avoid poor staining, you will need a buffer of 6.4 to 6.7. The acid in the stain will color the basic components red, while the basic dye will stain the acidic components blue. To prepare this dye, you will need 1 gram of Wright’s stain powder to be mixed with 400mL of methyl alcohol.
Add some ethanol and mix well until it dissolves completely. You may use a warm water bath at 37 degrees centigrade to aid in dissolving the powder. Store it in a dark place and at room temperature. Take particular care because this is flammable and toxic, so make sure to label the container and keep it somewhere safe.
Gram Staining
One of the most common staining techniques is gram staining. Bacterial cell walls display different colors when stained, this helps in identifying if they are gram-positive or gram-negative. In this method, you will need crystal violet and iodine. Gram-positive cell walls get attracted to crystal violet, while iodine is the mordant. You may use ethyl alcohol to easily wash the stain off.
Learning to stain is basic in microscopy. It will help you learn the different parts and structures of cells. You will also know how to identify some cells from other cells. If you want to dive deeper into microscopy, then this is a must to learn.
In doing microscopy, make sure that you have the proper materials and equipment. Wear proper attire when conducting experiments and work in a clean or sterile environment to avoid contamination. That will help you protect your materials and samples. Practice and master staining techniques for better experiments in the future.
