Amoeba Under The Microscope – Staining And Fixing Techniques

Every living creature or organism consists of cells. Human beings have roughly 37 trillion cells! But in the world, there are many living creatures that consist of only a single cell.

These are called unicellular organisms or single cell organisms. A prime example of a single cell organism is an amoeba.  

Amoebas are ver tiny organisms, measuring between 250 and 750 microns. They can typically be found in bodies of fresh water, such as ponds, rivers, or lakes.

They move slowly around using their pseudopodia, which they also use to capture particles of food. Amoebas have also been known to enter the human body and cause diseases sometimes.

An amoeba is a single cell organism that can change its form by expanding or retracting its pseudopods. They also move in a crawling manner by the use of these pseudopods, which act like temporary false feet.

They don’t belong to any one taxonomic class, and they can be discovered in all major lineages from the eukaryotic species. 

Amoebas are what’s known as “eukaryotes”, meaning that its genetic material is organized and enclosed inside a nuclear membrane. The word “amoeba” is derived from the Greek word “amoibe”, which means “change”. 

Scientific Classification

As a part of the Kingdom Protista, amoebas are considered protozoans that only get their food from other organisms.

They are not classified as fungi, plants, or animals, but they are classified as eukaryotes because of the fact that they have a nucleus and organelles that are bound to their membranes. 

Here is a further explanation of amoebas’ taxonomic classification:

• Domain: Eukarya 
• Kingdom: Protozoa 
• Phylum: Protozoa 
• Subphylum: Sarcodina 
• Superclass: Rhizopoda
• Class: Lobosa
• Order: Amoebida 
• Families: Amoebidae, Entamoebidae, Acanthamobidae, Hartmannallidae

Amoeba Under The Microscope

As amoebas are unicellular organisms, they can only be viewed beneath a microscope. There are some techniques that can be used to better understand these creatures through observing them through a microscope.  

The easiest and most basic way of looking at amoebas is by simply viewing them under the microscope without staining. This is a simple technique for watching amoebas move around.  

The other technique for viewing amoebas is by staining and fixing them in order to have a better view of the organelles in addition to the structure of the microorganism. 

Plasma Membrane

The entire body of the amoeba is covered by a plasma membrane. This is what gives the body of the amoeba its shape, and it keeps all of the organelles within the cell.  It is a thin and elastic membrane that has the ability to produce pseudopodia. 

Cytoplasm

The cytoplasm of an amoeba can be categorized into two sections; the ectoplasm on the outside and the endoplasm on the inside of the amoeba.  

Ectoplasm

Ectoplasm exists right after the plasma membrane. It consists of a thick, contractile layer of cytoplasm which gives the amoeba its shape.  It also helps to form pseudopodia. 

Endoplasm

Endoplasm is a less viscous and granular section of the cytoplasm. This layer aids in producing pseudopodia, and it contains the organelles of the amoeba, in addition to performing most of the physiological functions. 

Pseudopodia

Pseudopodia are what sets amoebas apart from other protists. They are able to form on any part of the amoeba’s ectoplasm. They form by the cytoplasm pushing inwards or outwards, and they allow the amoeba to move around and eat particles of food. 

Vacuoles

Contractile Vacuole

The contractile vacuole allows the amoeba to excrete excess water in addition to carbon dioxide, allowing the amoeba to keep its osmotic pressure. 

Food Vacuole

The food vacuole surrounds the food particles that the amoeba consumes, allowing it to be stored, digested, and then excreted.  

Water Vacuole

This vacuole allows the amoeba to store water, maintaining its water balance. 

Nucleus

The amoeba’s nucleus holds its genetic material, which acts as a code or instructions for how to structure an amoeba and its behavior.

The nucleus of an amoeba is responsible for its reproduction, growth, and all of the cellular functions. The amount of nuclei within an amoeba is dependent on the particular species of amoeba. 

Other Organelles 

Other interesting organelles that exist within amoebas include the following:

• Golgi bodies
• Endoplasmic reticulum 
• Mitochondria

Where To Find Amoebas

Various species of amoeba can be discovered around the world. They are typically found within decaying vegetation or at the bottom of bodies of fresh water including ponds, lakes, slow moving rivers, ditches, and even springs. 

Simple Or Direct Method

Amoebas flourish in shallow ponds that are rich in organic matter.  

In order to effectively observe amoebas using a microscope, you need the following items:

• Water sample taken from a pond where there is a lot of organic matter
• Pondweed from the same pond 
• A petri dish 
• A compound microscope
• Water
• A dropper 

Process

This experiment requires you to examine a water sample from a pond in order to determine the species of microorganisms present. You can also perform a simple cultivation that increases the growth and number of amoebas. 

This is a relatively simple and straightforward process.  To cultivate amoebas, follow these steps:

• Put some pondweed into a petri dish, and add enough water to cover it.
• Put the petri dish in the dark for a few days, until a brown scum forms on the surface of the water.

Microscope Process

1. Drop some droplets of the pondwater sample into a microscope slide 
2. Cover the sample with a coverslip and put it on the stage of the microscope for viewing 
3. Start with low power, and gradually turn up the power until the sample can be observed 

Observation 

When viewed from afar, amoebas look like transparent jelly-like creatures that move slowly as they change their form. As they change their form, you can see long protruding projections that look like fingers,

Fixing And Staining Amoebas 

To stain and fix amoebas for closer observation, you will need the following items:

• Amoeba culture
• Bench-top MSE centrifuge 
• Neff’s saline, seawater
• Moist chamber 
• Cover slip 
• Sodium hydroxide 
• Distilled water
• Nissenbaum’s fixative 
• Lugol’s iodine 
• Formalin seawater 
• Carnoy’s fixative 
• Heidenhain’s iron Haematoxyli 

Process 

This method involves using a cultured amoeba sample, which is then concentrated with the use of a centrifuge at 3 Krpm for approximately 10 minutes. Once this has been done, follow these steps:

1. Use Neff’s Saline or another suitable solution to clean the pellet 
2. Allow the specimen to settle onto the coverslip inside a moist environment until the amoebas show morphology of locomotion

Fixing The Amoeba 

1. Once the coverslip has been settled, take some Nissenbaum’s fixative in a pipette and place it on the sample.  Let it sit for approximately five minutes. 
2. Clean up the sample with the use of acidified HgCl2 for approximately 7 minutes.
3. Next, clean the sample using 50%, 35%, and 15% ethanol for approximately 5 minutes each.
4. Finally, clean the sample with distilled water for approximately 5 minutes.

Other fixatives that may be used include seawater formalin, Lugol’s iodine, and Carnoy’s fixative.

Staining The Amoeba

By staining the amoeba, you are able to enhance the visibility of its mitotic figures, allowing you to observe them in more detail. There are a range of substances that can be used to stain the amoeba, including:

• Heidenhain’s iron haematoxylin 
• Kernechtrot (nuclear red)
• Modified Field’s stain
• Klein’s silver relief stain 

Staining using Heidenhain’s iron haematoxylin involves these steps:

1. Inoculate the sample using 2% ammonium ferric sulfate for approximately 1.5 hours.
2. Clean the sample using distilled water.
3. Inoculate the sample using 0.5% haematoxylin and 2% ammonium ferric sulfate for approximately 1.5 hours.
4. Clean the sample using regular tap water.

Once the amoeba has been stained, you may mount the slide onto the microscope stage for observation. 

Observation

When observing a stained amoeba beneath the microscope, you will be able to see small dark spots in the amoeba’s cytoplasm, which is where it has been stained lightly.

The downside of staining the amoeba is this process kills the amoeba, so that you will not be able to see the amoeba moving around by elongating and shortening its pseudopods.

Staining the amoeba, however, does increase the contrast thus allowing you to see the organelles of the cell more clearly.

Structure Of Amoeba

As mentioned previously, amoebas are eukaryotes, meaning that amoebas have cell membranes around their cytoplasm, and their DNA exists inside their central nucleus.

When observed beneath a microscope, these aspects of the amoeba are easily seen, especially with a stained sample. 

Some other organelles that you can see beneath a microscope are:

• Food vacuoles
• Cytoplasm
• Contractile vacuoles 

Pseudopodia

Simply put, pseudopodia are projections from the cytoplasm of the amoeba, allowing it to move around.

They are one of the most recognizable features of an amoeba, and the formation of the pseudopodia are based upon the flow of protoplasm. 

The pseudopodia allow the organism to contract in a way that pushes out cytoplasm, filling and thus expanding the pseudopod whilst pulling on adhesions located on the back of the cell. 

Studies have revealed how the amoeba projects its pseudopodia. This process follows these steps:

1. Pressure from the exterior gel (ectoplasm) causes the interior fluid (endoplasm) to be forced outwards in the cell.
2. Once the endoplasm reaches the membrane’s tip, the pressure forms a pseudopod in the endoplasm.
3. Then the endoplasm gets forced backwards to the ectoplasm and turns into a gel like substance, causing the new pseudopod to vanish.  This new gel that forms then convers to endoplasm and the process repeats itself.   The pressure moves to the membrane, forming another pseudopod. 

Amoebas also use their pseudopod to engulf particles of food, in addition to allowing them to move about.

This process involves the pseudopod surrounding the food particles whilst opening up a membrane, allowing the food particles to move inside the cell and into the amoeba’s food vacuole.  Once inside, enzymes digest the particles of food. 

How Amoebas Breathe

Much like humans, amoebas also require oxygen for all of the cellular processes to function. Amoebas, however, don’t have lungs. So how do they breathe?

Instead of breathing through a mouth or nose into lungs, an amoeba uses their plasma membrane to take in oxygen.

As amoebas generally live in wet environments, they can absorb dissolved oxygen through the amoeba’s plasma membrane. This process is called diffusion.  

The oxygen then moves to the mitochondria, which can be considered the cell’s “power plant”. The oxygen in addition to nutrition from food provides energy for the amoeba.

Carbon dioxide and other nitrogenous waste is then excreted through diffusion too.  

Conclusion

Amoebas are very amazing single celled organisms, and they are fascinating to study under a microscope. Even though they are relatively simple organisms, they have the ability to do some amazingly advanced functions such as moving, eating, and communicating.

They are also able to live in incredibly harsh environments.  We hope that we have shown you how amazing amoeba are, and we hope you find them as interesting as we do. 

https://www.youtube.com/watch?v=vlUjBOpdSI0