Essay: Gram Staining and Gram Reaction in Gram-negative Bacterial Cells

Abstract
Gram staining is a procedure which uses light microscopy. It is a method of differentiating gram negative bacterial cells and gram positive bacterial cells. The gram staining procedure was designed by Christian Gram in 1884. Gram negative bacterial cells contain a thin peptidoglycan wall in addition to the outer cell membrane. Gram positive bacterial cells do not have a peptidoglycan cell wall. Gram negative bacterial cells decolorize during the gram staining procedure and retain the red color of the counterstain. This is because of the peptidoglycan cell wall. Gram positive cells do not decolorize and ultimately retain the purple color of the dye. Anaerobic gram negative bacilli cause infections in many parts of the body. The symptoms include necrosis and the accumulation of pus which causes swelling. These infections can be treated fairly easily through surgical procedures and by taking. Studies of these infections have been carried out and more investigations and studies will continue to be conducted for future diagnosis of these infections and other conditions caused by anaerobic gram negative bacilli.
Literature Review
Gram staining is a method used to differentiate two different types of bacterial cells based on the structures and compositions of their cell walls. This staining is a light microscopy in which the procedure of gram staining distinguishes between gram positive and gram negative bacterial cell groups by a process of coloring the tissues red or violet using an iodide solution. (Bartholomew and Finkelstein 1954)
During the early days, it was very difficult to detect the presence of bacterial cells in tissues. This is because the methods of staining used back then actually colored the bacterial cells and the tissue equally. It was Christian Gram who developed the method of gram staining we now use. He first attempted to develop a procedure where he could differentially stain a certain bacteria called a schizomycete from tissue cells. He began this investigation by experimenting with pneumococci from the lungs of victims of pneumonia in animal lungs. (Bartholomew and Mittwer 1952).
Gram used Lugol’s iodine, Ehrlich’s aniline-gentian violet, alcohol for decolorization, and as a counterstain, Bismarck brown, during his work on the lung tissues. The Bismarck brown was used to color the tissues cells, including their nuclei. Gram noticed that his procedure decolorized some types of bacterial cells which made them take the color of the Bismarck brown counterstain. However, from this observation, he did not yet divide the bacterial cells which decolorized from those that did not into the gram-negative and gram-positive and gram negative cell types of bacteria well-known today. This connection was first made by Roux in 1886. (Bartholomew and Mittwer 1952)
By staining kidney sections with gentian violet and then an iodine-potassium iodide solution, Gram endeavored to obtain brown cytoplasm and blue nuclei in the kidney sections.
Bartholomew and Mittwer (1952) provided a quote from Gram, in Gram’s words, “The experiments resulted from the accidental observation that aniline-gentian violet preparations of tissues, after treatment with iodine potassium iodide, are completely and rapidly decolorized in alcohol.” Throughout his work, Gram observed how some bacterial cells were resistant to decolorization. His observations led to the development of the now commonly used and very useful gram-staining procedure.
Currently, Gram’s staining procedure is acknowledged as a major contribution to biological science, especially in bacteriology in addition to diagnosis and classification. The ability to hold the dye in the gram stain is a characteristic only a few materials of nature have. Most animal and plant cells are gram-negative. Gram positive cells include yeasts, bacteria, and molds. Other constituents like certain protozoa, virus protein, cell nuclei, poliedes of the silkworm, and some proteins from the ascarid gamete, are also recognized as gram-positive.
Silhavy, Kahne, and Walker (2010) provided information about the bacterial cell envelope. The bacterial cell envelope is a structure that is complex and multilayered. It is the membrane and other structures that surrounds and aids in protecting the organisms from their outside environments. The bacterial envelope not only protects bacteria, but also allows certain nutrients to come into the cell and waste products to go out of the cell through the selective membrane. For most bacteria, there are two groups of cell envelopes. These two groups include gram-negative bacteria, and gram-positive bacteria.
The gram-negative cell envelope is made of 3 main layers. According to Beveridge (1999), gram-negative bacterial cells have a thin peptidoglycan cell wall. Peptidoglycan is composed of several repeating elements of the disaccharide N-acetyl glucosamine-N-acetyl muramic acid. This disaccharide is cross-linked by pentapeptide side chains. The peptidoglycan can be seen through a light microscope. The peptidoglycan cell wall is surrounded by an outer membrane.
This outer membrane contains lipopolysaccharide, glycolipids; it is the lipid bilayer. It contains phospholipids, however, it is not a phospholipid bilayer. Lipopolysaccharide is a well-known molecule because is it responsible for the endotoxic shock inside septicemia. The shock is caused by gram-negative organisms. It is an indicator of infection, so the immune systems of humans is sensitized to LPS. The outer membrane also contains proteins. The proteins can be divided into two classes. These classes are lipoproteins and beta-barrel proteins. Lipoproteins have lipid moieties which are connected to an amino-terminal cysteine residue. These proteins are not transmembrane proteins.
The literature from Silhavy, Kahne, and Walker (2010) states that in order for E. coli to survive, the outer membrane is essential. In E. coli strains, the outer membrane only contains some enzymes. There is an enzyme that modifies lipopolysaccharide, a protease, and a phospholipase. The active sites of these enzymes face the exterior of the outer membrane.
It is true that gram-negative bacteria show more resistance to antibiotics than gram-positive bacteria.
The third layer is the cytoplasm or inner membrane. It contains the cytoplasm and the organelles of a bacterial cell. The organelles are the mitochondria, the smooth endoplasmic reticulum, and the rough endoplasmic reticulum. The inner membrane is where many functions of the cells are carried out because bacterial cells do not have the intracellular organelles of eukaryotic cells. The inner membrane is the phospholipid bilayer. In E. coli, the essential phospholipids are phosphatidyl ethanolamine and phosphatidyl glycerol.
Gram-positive bacterial cells lack the outer membrane consisting of lipopolysaccharide, but they have layers of the peptidoglycan cell wall. The peptidoglycan cell wall of gram-positive bacterial cells is much thicker than the peptidoglycan cell wall of gram-negative bacterial cells. The peptidoglycan layers contain teichoic acids, long anionic polymers.
The original procedure of gram staining uses Ehrlich’s aniline gentian violet, a decolorizer of absolute alcohol, and Bismarck brown as a counterstain. Ehrlich’s aniline gentian violet was an aqueous solution of iodide-potassium iodide. The modern method is fundamentally the same. There have been many modifications to the procedure which allow producing more reliable results. Some of these modifications were Hucker’s modification (75. 76, 77, 128), Burke’s modification (24), and the Kopeloff-Beerman modification (89) reviewed in Bartholomew and Mittwer (1952).
Today in the United States, the procedure used for gram staining consists of the following elements. The primary dye used is crystal violet (Bartholomew and Finkelstein 1954). This is because it is a more reproducible and reliable substance than the originally used gentian violet dye. To intensify the color of the dye, sodium bicarbonate can be added. Safranin is popular choice for counterstain. Ethyl alcohol is a popular decolorizer.
There are 3 stages involved in the gram staining process. First, the bacteria cells are stained with the crystal violet dye and a Gram’s iodide solution is applied to form a complex between the dye and iodine to cause it to be insoluble in water. Next, the ethyl alcohol decolorizer is added to the cell sample. This dehydrates the peptidoglycan layer, if there is one, which results in it shrinking and tightening. Lastly, the Safranin counterstain is applied, staining the cells red.
The application of the decolorizer is the most critical step of the procedure. One must be extremely careful not to apply to much or too little alcohol.
The peptidoglycan wall in Gram negative bacterial cells reacts with the dye and decolorizing agent, however the counterstain does not affect the peptidoglycan. The cells retain the reddish color of the crystal violet dye. Since gram positive bacterial cells do not have a peptidoglycan wall in addition to their cell membrane, they do not decolorize and the cells will not have the reddish appearance when viewed through a microscope. They will appear purple because they will retain the purple crystal violet dye.
Future Directions and Conclusions
Finegold (1996) reviewed anaerobic gram-negative bacilli are very common throughout the body. In 1996, there were over two dozen known genera of gram-negative bacilli. Anaerobic gram-negative bacilli are features of the mucous coating throughout the body. Many times, they act as secondary pathogens. These gram-negative bacilli are involved in infections most commonly than any other anaerobes. Some are antibiotic-resistant.
These gram-negative bacilli can have a characteristic morphology, however others are pleomorphic, meaning the bacteria have the ability to modify their size or shape as an adaptation to environmental changes. Anaerobic gram-negative bacteria have many physical characteristics such as motility, having flagella, and organic and volatile fatty acid products.
Anaerobic gram-negative bacilli invade the mucous as pathogens, a microorganism that causes disease in a body. Ultimately, they cause inflections. These inflections can be anywhere in the body; the most common types of infections are dental and oral, bone infections, female genital tract and skin, intra-abdominal and pleuropulmonary. Anaerobic gram-negative bacilli can also cause periodontal disease and colon cancer. Some of these bacilli, for example, Fusobacterium, Prevotella, Bacteroides, and Porphyromonas, produce enzymes such as proteinases, deoxyribonuclease (DNase), collagenase, and heparinase. These enzymes can help bacterial organisms to penetrate tissues and cause inflections after surgery or some other trauma, or even at the habitation of a tumor.
Evidence of anaerobic infection includes the formation of a swollen are within a body tissue, containing a buildup of pus, foul-smelling discharge, necrosis (the death of all or most cells in a tissue or an organ), and septic thrombophlebitis. Septic thrombophlebitis is a condition in which bodily extremities become tender and swollen.
In order to treat this infection, the abscesses must be drained through surgical procedures and the necrotic tissue must be debrided. In addition, the patient must be given antimicrobial medication such as combinations of amoxicillin, ticaricillin, ampicillin, or piperacillin with beta-lactamase inhibitors, or chloramphenicol, imipenem, or metronidazole. The best way to reduce this infection is by avoiding situations which would cause the redox potential of tissues to decrease and also by preventing the anaerobes from getting into the host tissues.
The most common of all anaerobes is Bacteroides fragilis. This is because of its recurrent show ups in clinical infections and also because it is resistant to antimicrobial agents. It is a gram-negative bacillus and is most of the encapsulated. The structure of B fragilis is similar to the structure of other gram-negative bacteria.
There have been numerous studies of anaerobic gram-negative bacilli, and it was concluded in Finegold (1996) that the Bacteroides fragilis endotoxin has little to no lipid A , heptose, or 2-ketodeoxyocctanate composition. An endotoxin is something present in a bacterial cells and is harmful to the body. It is responsible for many diseases. In the future, there will more studies conducted about the diagnosis of these infections and other treatments for the conditions.
Literature Cited
Bartholomew, J. W., & Finkelstein, H. (1954). CRYSTAL VIOLET BINDING CAPACITY AND THE GRAM REACTION OF BACTERIAL CELLS. Journal of Bacteriology, , 67(6), 689–691. http://jb.asm.org/content/67/6/689.full.pdf+html
Bartholomew, J. W., & Mittwer, T. (1952). THE GRAM STAIN. Bacteriological Reviews, 16(1), 1-29. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC180726/
Beveridge, T. J. (1999). Structures of Gram-Negative Cell Walls and Their Derived Membrane Vesicles. Journal of Bacteriology, 181(16), 4725–4733. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC93954/
Finegold SM. Anaerobic Gram-Negative Bacilli. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 20. Available from: http://www.ncbi.nlm.nih.gov/books/NBK8438
Silhavy, T. J., Kahne, D., & Walker, S. (2010). The Bacterial Cell Envelope. Cold Spring Harbor Perspectives in Biology, 2(5), a000414. http://doi.org/10.1101/cshperspect.a000414
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