Abstract
Gram staining is a laboratory technique used to differentiate gram negative and gram positive bacterial cells using light microscopy. The procedure was invented in 1884 by a bacteriologist, Christian Gram. Gram negative bacterial cells have a thin peptidoglycan wall within the cell membrane. During gram staining, these cells decolorize and the peptidoglycan wall maintains the red color of the counterstain. Gram positive bacterial cells do not have this peptidoglycan cell wall. Ultimately, these cells do not decolorize and instead maintain the purple color of the dye. Many infections in the body, such as a bloodstream infection, are caused by anaerobic gram negative bacilli. Therapy is effective but an unethical way to treat the infections. Many studies of these infections have been conducted in the past and these investigations will be continued for future diagnosis of causes, treatments, and preventions.
Literature review
Gram staining is a procedure used to distinguish between two different types of bacterial cells. Gram staining is reliant on the composition and structure of their cell walls. Light microscopy magnifies the samples of bacterial cells once the tissues are colored using an iodide solution. (Schleifer and Kandler, 1972)
In the past, detecting the presence of bacterial cells in tissues was very difficult because the traditional methods of staining actually colored the cells and the tissues, too. It was the Danish bacteriologist, Hans Christian Gram, who designed the method of gram staining used in modern research. At first, he attempted to design a method where he stained a particular bacteria, schizomycete, from tissue cells. Gram began his investigation by experimenting with pneumococci from victims of pneumonia in animal lungs. (Srinivasan, 2012)
During his work on the animal lung tissues, Gram used Ehrlich’s aniline-gentian violet, Lugol’s iodine, Bismarck brown as a counterstain to color the cells tissues (including their nuclei), and alcohol for decolorization. Gram observed that his method decolorized only some types of bacterial cells, causing them to retain the color of the brown counterstain. Unfortunately, he was not able to differentiate the bacterial cells that decolorized into the well-known gram positive and gram negative bacterial cell types. This connection was made initially in 1886 by Roux. According to Srinivasan (2012), Gram strived to achieve blue nuclei and brown cytoplasm in the sections of kidney which he stained with gentian violet and an iodine-potassium iodide solution.
Srinivasan (2012) presented a quote from Gram: “The experiments resulted from the accidental observation that aniline-gentian violet preparations of tissues, after treatment with iodine potassium iodide, are completely and quickly decolorized in alcohol.” Gram noticed how certain bacterial cells were unaffected by decolorization. Gram’s observations led to the development of the modern and useful gram staining procedure.
Gram’s staining procedure is currently recognized as an important contribution to biological science. It is a major focus specifically in bacteriology as well as classification and diagnosis. The ability to retain the dye during staining is a characteristic that few materials in nature have. Most gram negative cells are animal and plant cells. Yeasts, bacteria, and molds make up the majority of gram positive cells. Other characters are also acknowledged as gram positive, such as virus proteins, certain protozoa, cell nuclei, some protein form the ascarid gamete, and silk worm poliedes.
Kleanthous and Armitage (2015) supplied information concerning the bacterial cell envelope. The envelope is a complex and multilayered structure in bacterial cells. The envelope is the membrane that surrounds and helps in protecting the cell from the outside environment. It not only protects bacteria, but the bacterial envelope acts as a selectively permeable membrane in that it also allows selective nutrients to influx into the cell and waste products to leave the cell. There are two main groups of most bacteria, the gram negative and gram positive bacteria.
According to Kleanthous and Armitage (2015) the envelope of gram negative cells is composed of three major layers. These layers include a thin peptidoglycan wall, the cytoplasm or the inner membrane, and an outer membrane. Peptidoglycan is a substance composed of several reoccurring elements of the disaccharide N-acetyl glucosamine-N-acetyl muramic acid. The disaccharide is interlinked by many pentapeptide side chains. The peptidoglycan wall can be observed though a light microscope.
The outer membrane surrounds the peptidoglycan cell wall. The membrane contains lipopolysaccharide glycolipids, meaning it is the lipid bilayer. It is not a phospholipid bilayer; however, it does contain phospholipids. Lipopolysaccharide is a recognized molecule. This is because it is responsible for the endotoxic shock inside septicemia; gram negative bacteria causes the septic shock. The shock is an indicator of infection, hence the cause. Moreover, the human immune system is sensitive to lipopolysaccharide. The outer membrane is also composed of two classes of proteins, the lipoproteins and beta-barrel proteins. Lipoproteins have lipid moieties. They are connected to an amino terminal cysteine residue. Lipoproteins are not considered transmembrane proteins.
The literature from Kleanthous and Armitage (2015) claims that the outer membrane is essential in order for E. coli to survive. The outer membrane contains only some enzymes in E. coli strains. The active sites of the enzymes protease and phospholipase face the exterior of the outer membrane. These enzymes modify liposaccharide.
The inner membrane, or the cytoplasm, contains the organelles of the bacterial cell, including the mitochondria, the smooth endoplasmic reticulum, and the rough endoplasmic reticulum. Bacterial cells do not have many of the intracellular organelles of eukaryotic cells. Therefore, the inner membrane is where many cellular functions are carried out. It is also the phospholipid bilayer. Phosphatidyl ethanolamine and phosphatidyl glycerol are the essential phospholipids in E. coli.
Bacterial cells that are gram positive lack the lipopolysaccharide outer membrane, but they do have a much thicker layer of the peptidoglycan cell wall than gram negative bacterial cells. The layers of peptidoglycan contain long anionic polymers called teichoic acids.
The traditional procedure of gram staining uses Bismarck brown as a counterstain and Ehrlich’s aniline gentian violet as a decolorizer of absolute alcohol. The current method of gram staining is essentially the same though there have been many alterations to the procedure which allow for more reliable results. Some of these alterations were the Kopeloff-Beerman modification, Hucker’s modification, and Burke’s modification reviewed in Srinivasan (2012).
In today’s society, specifically in the United States, the method used for gram staining entails the following elements. Crystal violet is the primary dye used (Schleifer and Kandler, 1972) because it is more reliable and reproducible substance than the gentian violet dye traditionally used. Sodium bicarbonate can be added to intensify the color of the dye. A modern popular choice of counterstain is safranin and a popular decolorizer is ethyl alcohol.
In the gram staining process, there are three stages. The bacteria cells are first stained with the crustal violet dye. A Gram’s iodide solution is then applied so that the dye and the iodine forms a complex which is insoluble in water. The ethyl alcohol is added to the sample of cells next. Adding the decolorizer shrinks and tightens the peptidoglycan layer by dehydrating it. Lastly, the cells are stained red by applying counterstain. The most critical step of gram staining is the application of the ethyl alcohol decolorizer. However, applying too much or too little alcohol is bad.
In gram negative cells, the peptidoglycan wall reacts with the decolorizing agent and the dye. Peptidoglycan is not affected by the counterstain, so the cells take on the red color of the crystal violet dye when viewed through a microscope. Gram positive bacterial cells no not decolorize and do not take on the red color because they do not have the outer membrane in addition to the peptidoglycan wall. Gram positive bacterial cells will absorb the crystal violet dye and appear purple instead.
Weinstein (2005) reviewed their study which showed that infections acquired in hospitals are associated with gram negative bacilli as the cause. The most common anaerobic gram negative bacilli is Bacteroides fragilis. It appears in many clinical infections. Bacteroides fragilis is also resistant to antimicrobial agents. It is proved that gram positive bacteria show less resistance to antibiotics than gram negative bacteria due to the presence of their outer membrane. This outer membrane is semipermeable, and it does not allow certain drugs, including antibiotics, from entering the cell due to their structures and compositions. There are many types of antibiotics, each with a different function in terminating the spread and influence of bacteria in bacterial infections. Antibiotics inhibit bacterial cell wall synthesis, bacterial protein synthesis, and bacterial DNA synthesis.
The Ethical Issue
Gram negative bacilli have been proved to be the leading causes of infections. Specifically, Enterobacteriaceae and Psuedomonas aeruginosa are the leading causes of blood stream infections, or bacteremia (Kang, 2005). A bloodstream infection is an invasion of the bloodstream by bacteria. This can occur through an open cut or a surgical procedure at a time when the immune system is suppressed. Symptoms of the condition may include fever, nausea, confusion, and swelling caused by an accumulation of pus. Bacteremia can lead to septic shock in some cases which is life threatening. This infection is diagnosed by a culture of the victim’s blood for bacteria. Treatment of a most infections consists of the combination of antibiotics, antimicrobial therapy, and sometimes surgery. Unfortunately, most of the time bacteremia is caused by antibiotic-resistant bacteria, so antimicrobial therapy is the primary treatment for a bloodstream infection.
Therapeutic treatment of bacteremia comes at some costs. Kang (2005) examined an investigation of 1045 patients with infections caused by various bacteremia, 499 of which identified with pneumoniae and 183 with Enterobacter. 47% of the patients with the Enterobacter bacteremia as well as 74 other patients with other bacteremia were infected with strains that were antibiotic-resistant. Of these 286 patients, only 135 of them received the proper treatment including antibiotics and therapy.
The therapeutic treatment following the diagnosis of bloodstream infections does not conform to ethical principles of science. Kang (2005) states in his scientific journal that the specific therapy threatens the lives and health of patients with bacterial bloodstream infections. This is proved through his examination of the study conducted with the 1045 patients. He identifies the therapy as “inappropriate” in the context that the patients received antibiotics that were inactive against the pathogens causing their infections. The effects of the antimicrobial therapy on the survival of the patients was evaluated.
Medical ethics is the application of morals and values to clinical situations in the practice of medicine. As stated in the Principles of Medical Ethics, physicians are expected to be dedicated to delivering proficient medical care with respect and compassion for human rights. Physicians are also expected to consider their patients’ best interests when it comes to treating their condition in a situation other than an emergency (American Medical Association, 2016). In Kang’s review, he mentions the clinicians’ failure to comply by these Principles in that the patients were treated with therapy which gave a 38.4% mortality rate for the 286 patients with the gram negative bacilli caused bloodstream infection.
Future Directions and Conclusions
Without Christian Gram’s dedication and contribution to biological science, the advancements made in present medical research and the pharmaceutical industry helping with the discovery and diagnosis of gram negative bacteria could not be possible today. Today’s research gives doctors and clinics the capacity and ability to identify and treat many life threatening infections of the human body.
Weinstein (2005) discussed anaerobic gram negative bacilli and reviewed that they are common in many parts of the body including mucous coating. There were over two dozen known genera of gram negative bacilli in 1996. Gram negative bacilli act like secondary pathogens and are more commonly involved in infections than any other anaerobes. Many gram negative bacilli are resistant to antibiotics. Some gram negative bacilli are pleomorphic and have the ability to change their shape or size as an adaptation to environmental factors. Others have a certain morphology. Motility, having organic properties, and having flagella are physical characteristics of anaerobic gram negative bacteria.
Numerous studies researching anaerobic gram negative bacilli have been carried out. Weinstein (2005) concluded that infections acquired in hospitals during surgery and other various procedures caused by gram negative nosocomial bacilli are showing an increase in resistance to many drugs. These diseases and infections are beginning to lack treatment options and are expected to present a serious health concern for the public in the future. More studies will be conducted in the future about the diagnosis and treatments of infections and conditions caused by gram negative bacilli.
Literature Cited
American Medical Association. Principles of medical ethics of the American Medical Association. American Medical Association Press, 2016. http://www.ama assn.org/ama/pub/physician-resources/medical-ethics.page?
Kang, Cheol-In, et al. “Bloodstream infections caused by antibiotic-resistant gram-negative bacilli: risk factors for mortality and impact of inappropriate initial antimicrobial therapy on outcome.” Antimicrobial agents and chemotherapy 49.2 (2005): 760-766. http://aac.asm.org/content/49/2/760.full.pdf+html.
Kleanthous, Colin, and Judith P. Armitage. “The Bacterial Cell Envelope.” Philosophical Transactions of the Royal Society B: Biological Sciences 370.1679 (2015): 20150019. PMC. Web. 29 Mar. 2016. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632596/#.
Paterson, David L. “Resistance in gram-negative bacteria: Enterobacteriaceae.” The American journal of medicine 119.6 (2006): S20-S28.
Schleifer, K H, and O Kandler. “Peptidoglycan Types of Bacterial Cell Walls and Their Taxonomic Implications.” Bacteriological Reviews 36.4 (1972): 407–477. Print. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC408328/.
Srinivasan, Usha et al. “Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies.” Ed. Patrick M. Schlievert. PLoS ONE 7.10 (2012): e42898. PMC. Web. 29 Mar. 2016. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469605/.
Weinstein, Robert A., et al. “Overview of nosocomial infections caused by gram-negative bacilli.” Clinical infectious diseases 41.6 (2005): 848-854. http://cid.oxfordjournals.org/content/41/6/848.full.pdf+html.