How to Isolate Single-Celled Organisms: Techniques and Methods

Single-celled organisms, such as bacteria, protozoa, and archaea, are fascinating subjects in biological research. These tiny organisms are capable of carrying out all necessary life processes, including reproduction, nutrition, and environmental sensing. Isolating a single-celled organism from a collection is a critical step in understanding their behavior and function. While it might seem simple to isolate a single cell by visually inspecting a tiny bit under a microscope, the practical process involves more intricate techniques.

Traditional Method: Visual Inspection and Microscopy

The most straightforward method would be to use tools like a lancet or a razor blade to cut off a very small piece of the organism and observe it under a microscope. However, this method is extremely tedious and inefficient, making it impractical for large-scale isolations. A more effective approach is to disperse the cell mass into a single-cell suspension.

Enzymatic Dispersal for Animal Cells

The first step is to take a small piece of tissue and macerate it, using enzymes to break down the cell-to-cell bonds. For animal cells, a proteolytic enzyme like trypsin is commonly used. This enzyme helps to separate individual cells from a tissue mass, ensuring that each cell is isolated and viable for further analysis.

Enzymatic Dispersal for Plant Cells

For plant cells, the process is similar but involves the use of a different cocktail of enzymes. Enzymes such as cellulase and pectinase are used to digest the cell walls, particularly the pectin middle lamella, which holds cells together. This process forms a suspension of single plant cells, groups of cells, and cell debris. The next step is to purify the cells using techniques like centrifugation or plastic sieves and then dilute the suspension to enhance the probability of obtaining a single cell.

Plasmolyzing and Protoplast Preparation for Plant Cells

Before the cell walls are completely digested, plant cells are often plasmolyzed. This means that the cell contents are made to shrink and detach from the cell wall, making the cells less prone to damage during the digestion process. Once the cell walls are removed, plant cells become protoplasts, which are essential for certain types of cellular experiments. If complete cells with cell walls are needed, a different approach can be taken. Establishing a callus culture and shaking it mechanically can be an effective method.

Callus Culture and Enzymatic Dispersal

A callus culture involves cultivating a mass of plant cells on a sterile nutrient medium. These cells usually grow quickly and form a soft, gel-like tissue that can be easily separated into individual cells. By transferring the callus to a liquid medium and shaking it continuously, cells can be separated mechanically. This approach avoids the need for expensive enzymatic digestion, but it takes significantly more time to achieve the desired result.

Isolating single-celled organisms is a fundamental technique in biology, and the choices between enzymatic and mechanical methods depend on the specific requirements of the experiment. Understanding these processes is crucial for advancing our knowledge in fields ranging from microbiology to genetic engineering.