...2014 ! ! ! ! Gram Staining Gram staining is the single most important and universally used staining technique in the laboratory. The name comes from it’s inventor Hans Christian Gram who developed the technique in 1882 (published 1884). Gram staining is almost always the first step in identifying a bacteria. The process is a complex and differential staining procedure with a series of staining and decolorization steps. Bacteria are differentiated by their cell wall composition. Gram positive cells have thick cell walls containing peptidoglycan which stain purple with Crystal Violet (or substitute). Gram negative cells with thin layers of peptidoglycan and high lipid content stain pink or red. The procedure is not used in Eukaryotic cells as they do not contain peptidoglycan. There are four basic steps to Gram staining a sample. The initial step is applying a primary stain to the heat fixed sample followed by Grams iodine as a mordant. The sample is rinsed with alcohol or acetone and finally counterstained with Safranin. The results, as mentioned above, yield purple (Gram positive) or pink/red (Gram negative) structures which differentiate the bacteria into classes. The process can also be indeterminate or variable which is beyond the scope of this paper. Gram Staining is a remarkable method of determining bacteria type and beginning to narrow down infectious etiology in medical application. In the proper context and setting, the use of Gram staining can have great implications...
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...Staining Microorganisms Microorganisms are often first observed by coloring the specimen with a dye to emphasize the structures for examination. In order to dye, (or stain ) the specimen, it is fixed, (attached to the microscope slide). ( Tortara, Funke, Case, 2013). To carry through this staining and fixing, the microorganisms are killed and attached to the slide. There are several staining techniques to accomplish this preparation. There are simple stains, differential stains, and special stains. The stain is cationic (basic) or anionic (acidic). Examples of cationic dyes are crystal violet, safranin , methylene blue and basic fuchsin. Examples of anionic dye are, acid fuschin, congo red and nigrosin. Simple stains are, as they are called, “simple”; they are an alcohol solution of a basic dye, and they will highlight the microorganism so the observer can examine the structures , arrangements of cells, and shape of the specimen. If needed, an additive mordant) can be added to the sample to thicken it, making it easier to see. Simple stains are the least expensive method of staining, and can be used with all microorganisms. These are effective because they stain a pure culture and do not have to be washed and re stained. Differential stains are the most widely used staining technique for examining bacteria, as the stains react differently with different cell typed. This method of staining uses the Gram stain and the acid-fast stain. Gram staining is the...
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...MLT1 Experiment 5/Task 6 Differential Staining There are many different ways to stain bacteria for viewing under a microscope. For the most part these are categorized as “either simple, nonspecific or differential (specific) (LabPaq, p.128). The most common staining method used is Gram staining. Gram staining is a differential or specific method of staining. Gram staining is a way to tell the difference between gram positive and gram negative bacteria. The cell wall of the gram positive bacteria is made up of several “layers of peptidoglygan,” and “techoic acids,” (LabPaq, p. 128). This peptidoglycan is what absorbs the color part of the crystal violet stain causing the gram positive bacterial cell to appear violet colored when viewed with a microscope. In contrast, the cell wall of the gram negative bacterial cell is not as thick as that of the gram positive cell wall. Peptidoglygans are on the inside of the cell rather than in outer layers. The outer part of the gram negative cell is made up of phospholipids and lipipoly-saccharides (Betsy and Keough, 2005). The outer cell wall does not hold onto the violet color of the crystal violet and appears pink in color when viewed under a microscope. The purpose of iodine staining is to aid the bacteria to keep the stain by creating an iodine-crystal violet mixture that will not dissolve. Iodine is also known as a mordant in this case. A mordant is usually an inorganic oxide that when mixed with...
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...organisms to handle and many ways to handle them. One of the oldest and most commonly used technique was gram staining. This technique was used to determine if bacteria is present. Then, it had to be determined if the organism was gram positive or gram negative. During the staining procedure there is a primary stain of crystal violet, the mordant gram iodine, a decolorizer acetone alcohol, and a counterstain of safranin. After the steps are completed the gram positive organism remains purple and the gram negative bacteria shows as a reddish-pinkish organism. Gram staining came about by Hans Christian Gram. He was trying to see cocci in the lungs of individuals who lost their lives to pneumonia. Once Gram gathered a collection of materials to complete a stain, he figured out a way to view the bacteria in the lungs. The stain of crystal violet retained to the people who were infected with pneumonia. He realized that his stain worked with envisioning various types of bacteria pertaining to “cocci of supportive arthritis following scarlet fever”. Because Gram finally discovered how to distinguish between a positive bacteria organism from a negative bacteria organism, many doctors are able to figure out ones illness at an early stage, once a doctor does so they might be able to use...
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...M2D1: Microscopy and Differential Staining 1. What are the advantages and disadvantages of the different types of light and electron microscopes discussed in Chapter 3 that are used to study microorganisms? Focus your response in terms of the following parameters: o Range of magnification o Resolving ability o Sample preparation o Possible states of sample (e.g. whole organism, part of, living, non-living, etc Compound Light microscopes magnification is 2000X. Resolution of about 0.2μm. Can only see very small specimens and specimens are stained. Darkfield – used to study live microorganisms that cannot be stained or staining distorts the image or they are invisible using the normal light microscope. Phase-Contrast – in living microorganisms, this scope allows you to see detailed internal structures, plus you do not have to fix or stain the microbes. Differential Interference Contrast (DIC) – instead of one beam of light, 2 beams are used. Image looks almost 3-dimensional and is brightly colored. Fluorescence – used mainly as a diagnostic technique. Stained with fluorochromes and viewed with an ultraviolent light. Confocal – makes 3-dimensional images using a computer. Able to see entire cells and their components. Two-Photon – living cells can be seen up to 1mm (1000um) deep in tissues. Can also track, in real time, the activity of cells. Scanning Acoustic – living cells that are attached to cancer cells, artery plaque and biofilms can be seen through...
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...In 1884, Hans Christian Gram developed the Gram staining method when trying to discover the cause of pneumonia. While looking at lung tissues, he discovered that the staining method was adhering better to some bacteria than others. (Allen , Anderson , Nester , & Salm , 2016) This led to the discovery that two different types of bacteria were causing the pneumonia. By discovering that the staining method showed two different types of bacteria, the technique became the Gram Staining method. The technique allowed for the bacteria to either show results for gram positive or gram negative. To determine the different characteristics between Gram Positive and Gram-Negative, we start by using the differential stain that is the gram stain, The gram stain helps differentiate between bacteria’s by identify what color they would become. The Gram positive would stain blue or purple in color where the negative results would be pink to red in color. These colors allow scientist to categorize based off the cell wall of the bacteria. If a bacterium is determined to be a gram positive the cell wall will have a thicker layer of peptidoglycan outside the plasma membrane compared to the negative bacteria will have a thinner peptidoglycan layer between the plasma membrane...
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...Acid-Fast Staining Acid-fast stain is a differential stain. More so, it is a physical property of certain bacteria, particularly a bacteria’s resistance to decolorization by certain acids during staining procedures. Most bacteria are decolorized by the use of acid-alcohol. However, the families of Mycobacteriaceae, Nocardiceae, Gordoniaceae, Dietziaceae, and Tsukamurellacea are acid fast. In 1882 acid fast staining was developed by Paul Ehrlich and was later successfully modified by Ziehl And Neelsen. Ehrlich first discovered that Mycobacterium tuberculosis, which is the main causing agent of tuberculosis, retained the primary stain given even after washing it with an acid-alcohol mixture. Acid fastness in the property of certain bacteria (mycobacterium), their structures (spores) or protozoa forms; resist the decolorization by weak mineral acids following staining by the use of an intense dye. The most commonly known acid fast staining technique is the Ziehl-Neelsen technique. The cell walls of acid-fast organisms contain a wax like lipid called mycolic acid. As a result the cell wall is relatively impermeable to ordinary staining techniques. They can be stained by aniline dyes using drastic measures such as application of heat and phenol. The heat softens the wax in the cell wall, allowing the stain to enter, which is a basic fuchsin. The fuchsin dye is more soluble in phenol than in water or alcohol. Phenol in turn is more soluble in lipids or waxes, thus...
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...four activities of fresh wet mount, direct staining using crystal violet, and indirect staining using both Congo Red and crystal violet. The fresh wet mount slides, as the name suggests, is bathed in some form of liquid, whether it is water or some kind of liquid that the organism itself came from. The microscopic images produced are clear or somewhat colorless, but the edges are distinct and we can see the shapes of the bacteria very well. Direct staining using crystal violet produces bluish or purplish colored bacteria and the shapes are well distinguished also Indirect staining using Congo Red is like the negative film version of direct staining. The bacteria or organism is left uncolored but it can be seen because of the reddish background that surrounds it. Since it leaves the inside of the bacteria untouched, we can see the inside of the cell wall. B. Discuss whether you were able to identify specific bacterial morphologies. Looking at the prepared slides on the Lab Manual, I was able to identify the various bacteria via their shapes. Cocci are round shaped bacteria, while bacilli look to me like medicine capsules or very small submarines. The slides with spiral bacteria seem to have multiple arms sticking out of a round center, kind of like the spokes of a bicycle wheel or a clock with multiple hands. The rest are variations on those shapes C. Explain the difference between direct and indirect staining. Direct staining is the technique of applying a dye that...
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...different activities. They include the fresh wet mount, direct staining using crystal violet and indirect staining using both Congo red, and crystal violet. A close examination of these examples showed that the wet mount slide examples were all the same in appearance. the cheek and yeast both appear to have some circular cells. the cheek smear was the most difficult to see. The direct staining images were very easy to make out the cells, as they turned the color of the crystal violet. The indirect staining, also known as the negative staining, used the Congo red dye and produced interesting results. The cheek smear looked like the cells were stained, rather than the background as it was explained in the experiment. It shows in the up close cheek smear, as the yeast smear didn't seem to retain the red color. It was however easier to make out the shapes of the cells. The plaque smear, retained some of the red dye but not in any sort of pattern in which to clearly be able to see cells. B. The bacterial morphologies, also known as the shape of the cells, were very easy to see with the crystal violet stain. The slides showed cocci and bacillus shaped cells. On the wet mount cheek smears it was hard to make out any shapes, but in the direct staining slides you could make out cocci shapes on the cheek and yeast smears. You could also make out some cocci on the indirectly stained yeast slide. C. Direct staining uses dyes that stain the cells, such as the crystal violet...
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...The staining test is a test for fiber identifycation. The test aims to identify four fiber contents of samples. It is to distinguish four fiber samples A, B, C and D from cotton, wool, polyester and nylon. The principle of that is to make use of small tuft of fiber samples and treated it with staining agent by dyeing it at short time. When different types of fibers are treated with staining agent, they will produce different coloration due to the differential of fibers construction and degree of staining agent uptake. The chemical composition, morphological structure and reativity of fibers differ from each other leading them to have particular affinity for specific staining agent. Test procedure i. At first, fiber sample A is taken for test. ii. Put the sample in the beaker with shirlastain A at 2 minues with tweezer and use glass rod to stir the fiber sample soaked into the staining agent thoroughly under room temperature.. iii. Take out the sample from the staining agent with tweezer and rinse it with cool water to wash out any excess staining agent from fiber surface. iv. Examine the fibres for staining and compare the color of the fiber sample with the samples. Observation of experiment i. During the experiment, when fiber sample A immersed into the staining agent, it is hard to soak thoroughly. While it keeps the better amount of staining agent after rinsing. Thus, it obtained the deepest color result. ii. When fiber sample B immersed into the staining agent...
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...Thin blood smears consist of a layer of blood that is one erythrocyte thick, it preserves erythrocyte shape and parasites, therefore is for suitable for species identification, as in the thick blood smear, the erythrocytes undergo lysis (Tek et al, 2010). To prepare the thin blood smear, a drop of blood is placed onto a pre-cleaned glass slide. Ensure there is an angle of 45° between the slide and the spreader, and place the spreader into the drop of blood, then smear the blood in a swift and steady movement across the slide. The smear needs to air dry before being fixed with absolute methanol. Once dry, the sample is stained with diluted Giemsa (1:20, vol/vol) for 20 minutes and washed by momentarily dipping the slide into a jar of buffered...
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...Diagnosis of an Infected Patient Infection is the invasion and growth of microorganisms such as bacteria, viruses, and parasites that are not normally present within the body. A prokaryotic cell is a simple cell that does not have a nucleus. One of the most common types of prokaryotic cells is a bacterium. Bacteria are differentiated by many factors including shape, chemical composition, nutritional requirements, biochemical activities, and sources of energy (Tortora 76). A patient with an infection in the upper respiratory system will need to have a sputum sample sent to the lab for further evaluation to determine the cause in order to accurately treat the infection. While many microorganisms can be the cause of infection, this essay will focus on the following genera: Bacillus, Escherichia, and Mycoplasma as the cause of the patient’s infection. Bacillus is a rod-shaped, endospore-forming, facultatively anaerobic and gram-positive bacterium (Tortora G2). The gram stain is fundamental to identify the characteristics of bacteria as this process differentiates organisms according to the cell wall structure. Gram-positive cells have a thick cell wall layer and will stain blue to purple. The Gram stain process requires four steps which include applying a primary stain, usually crystal violet, to a heat-fixed smear, then adding a mordant, usually Iodine, followed by rapid decolorization with alcohol or acetone and finally counterstaining with safranin (Hussey). At...
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...Diagnoses of an Infected Patient By: Reathea Felder Microbiology Excelsior college | | When given a sputum sample it is important to take in consideration the type of bacteria that could be present, causing illness in the patient. There are hundreds of different types of bacteria, but I will only focus on how to detect three of them: Bacillus, Escherichia, and Mycoplasm. I will also discuss how to identify the genera causing the patients infection by describing the different staining procedure protocols. The term bacillus has two different meanings. The lowercase bacillus means bacterial shape while the italicized bacillus refers to a specific genus. Bacillus cells are usually single rods that may look like straws that often form long twisted cells (p. 78). They are typically rods that produce endospores. Bacillus is a gram-positive cell that may at times have gram negative cells. In a gram positive cell, there are many layers of a thick, rigid structure known as peptidoglycan. They also have teichoic acids which are made primarily of alcohol and phosphate. This acid plays a role in cellular growth and prevention of cell wall breakdown. One of the most well known bacteria in microbiology, Escherichia can be characterized as rod shaped having short hair like appendages. Escherichia is a gram negative...
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...Gram Staining: Gram positive and Gram negative bacteria One of the most important cytological features of bacteria is their reaction to a simple procedure called, after its discoverer, the Gram stain. The cell wall of bacteria shows a characteristic reaction to the gram stain devised by C. Gram. Gram Staining Procedure Gram staining procedure Source: deenahere How Gram Stain works? Gram Staining Principle: Step by step procedure with explanation * Gram Staining Principle: Step by step procedure with explanation Reagent Preparation / Step by step procedure& Principle. The gram staining procedure involves staining cells with the dye crystal violet and all the bacteria will be stained blue. The bacteria are then treated with an iodine solution and decolourized with alcohol. Those bacteria which retain stain are known as Gram positive and those which do not retain the stain are termed as Gram negative. This is the fundamental difference between gram positive and gram negative bacteria. Gram positive Gram negative Difference between Gram Positive and Gram Negative Bacterial Cell wall: Bacterial Cell wall Character | Gram positive | Gram negative | Number of layers | One | Two | Thickness | Thick(20-80nm) | Thin (8-10 nm) | Outer membrane | Absent | Present | Periplasmic space | Present in some | Present in all | Chemical composition | Petidoglycan, Teichoic acid and lipotechoic acid | Lipopolysaccharide, lipoporteins and peptodoglycan | ...
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...Strain-Testing to Determine Genera Class Name University/School Name Cytoplasm is generally transparent, making it difficult to study under a microscope. Various methods of staining microbes are used to differentiate them and draw attention to their distinguishing features. In the case of a patient whose symptoms are consistent with an infection of the Bacillus, Escherichia and Mycoplasma genera, a sputum sample can be analyzed with staining procedures to narrow down possibilities to one genera. All tests begin by applying a sample to a slide and allowing it to dry. Most tests then require that the culture be passed over a flame to kill the cells and allow them to accept dyes. Some tests don’t require heat fixing due to the damage it causes to fine structures. The Bacillus, Escherichia and Mycoplasma genera can be distinguished by their distinct features anatomically as well as their reactions to various solutions. The acid-fast test, spore test, flagella test, and the negative strain technique, taken together, will eliminate two of the possibilities and leave one. Mycoplasma can be distinguished from other genera using the acid-fast test. In this process, sputum is applied to a slide and an initial bright red dye is applied to the sample via heat or through a solvent. The dye is then rinsed off with an acid-alcohol solution; Mycoplasma cells retain the red dye after being washed, while Escherichia and Bacillus cells would take on a blue stain. If Mycoplasma is ruled out...
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