Another main difference between protoplasts and spheroplasts is the number of membranes present. Protoplasts are bounded by a single membrane while spheroplasts have two - an inner membrane and an outer membrane.
Protoplasts and Spheroplasts: How are they formed?
The formation of bacterial protoplasts and spheroplasts can be induced in the laboratory. Since cell walls are composed of a variety of polysaccharides, viable protoplasts can be prepared by weakening the primary stress-bearing layer of the cell wall (peptidoglycan) using the appropriate enzymes. To accomplishing this purpose, researchers may either use mechanical or enzymatic methods.
Mechanical Method
In the past, protoplasts were isolated using mechanical methods which were labor-intensive, tedious and inefficient. Additionally, these methods were restricted to certain tissues with vacuolated cells and produced extremely low yields and barely viable protoplasts. For these reasons, most researchers now prefer the enzymatic method, except in cases where the enzymes can have a damaging effect on the resulting protoplasts.
Enzymatic Method
For plant cells, cellulase, pectinase, and xylanase can be used to break down the cell walls while lysozome (+EDTA) can be used to produce protoplasts from gram-positive bacteria. For fungal cells, using chitinase will do the trick. Similarly, spheroplasts can be prepared from gram-negative bacteria using procedures similar to those used in preparing protoplasts.
Upon the degradation of the peptidoglycan, the bacteria loses all its ability to control its response to the differences in the ionic concentration between its internal and external environment, so it becomes extremely sensitive to osmotic stress during and after the digestion of the cell wall.
For this reason, it should be stored in isotonic solution to prevent the plasma membrane from rupturing or shriveling. Maintaining the optimal ionic balance will also ensure the successful transformation of the bacteria into the desired form.
Protoplasts and spheroplasts can also occur naturally. In this case, they are referred to as L-forms (also L-phase bacteria, L-phase variants and cell wall-deficient (CWD) bacteria). Bacillus, Clostridium, Haemophilus, Pseudomonas, Staphylococcus and Vibrio are known to produce L-forms in nature.
Due to the absence of a defining cell wall, the morphology of L-form bacteria is very much different from the strain from which it was derived. Interestingly, while L-forms can be derived from both gram-positive and gram-negative bacteria, it is always detected as gram-negative when subjected to a gram stain test.
Some Useful Applications
Protoplasts and spheroplasts are valuable research tools and can be used in a wide range of applications.
Protoplasts
Spheroplasts
Photo by Mnolf
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