...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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...Bone marrow examination | A Wright's stained bone marrow aspirate smear from a patient with leukemia. | Bone marrow examination refers to the pathologic analysis of samples of bone marrow obtained by bone marrow biopsy (often called a trephine biopsy) and bone marrow aspiration. A bone marrow aspiration should be performed as part of the same procedure. For patient safety and convenience, biopsies are usually performed on the posterior iliac crest. The biopsy specimen should measure at least 1.6 cm and, if it does not, consideration should be given to repeating the procedure, possibly on the contralateral iliac crest. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, lymphoma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells. While much information can be gleaned by testing the blood itself (drawn from a vein by phlebotomy), it is sometimes necessary to examine the source of the blood cells in the bone marrow to obtain more information on hematopoiesis; this is the role of bone marrow aspiration and biopsy. { A trephine (/trɨˈfaɪn/; from Latin trypan, meaning to bore) is a surgical instrument with a cylindrical blade. It can be of one of several dimensions and designs depending on what it is going to be used for. They may be specially designed for obtaining a cylindrically shaped core of bone that can be used for...
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...Process and Techniques of Finding Unknown J Abstract: As part of a focal study within Microbiology, many scientists have endeavored to identify the many species and types of microbes found in different environments. In an effort to categorize, understand and distinguish one microbe from another, scientists developed tests to show unique characteristics of microbes. This experiment enlists these tests, such as PCR, Simple and Gram staining, anaerobic growth tests, IMViC, Catalase, Oxidase, selective and differential media to identify an unknown microorganism. The Unknown organism studied was labeled “J” and found to be a gram negative, rod shaped bacteria that does not produce endospores. The selective and differential agars produced no growth on the MSA agar plate showing that the bacteria did not favor a salty environment of the Mannitol salts and showed an acidic by product in the selective and differential media of MacConkey’s Agar. The bacteria showed to metabolize sugars but did not produce any gaseous byproducts. After 16s rRNA was processed and run through a PCR, electricphoresis was used to run the RNA out on a gel in order to sequence the RNA which was then compared in a database. Furthermore, the Unknown J did not produce or metabolize starch. The bacteria did not react with the Citrate, KEY oxidase. When compared to other known bacteria tested, unknown J proved to be Escherichia coli. Introduction: Identifying bacterial causes for certain diseases that have...
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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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...Task 4 A. Describe the differences between gram-positive and gram-negative cell walls. The difference is the outer casing of the bacteria. Gram positive cell wall consists of a smooth and thick wall. A gram positive bacteria will have a thick layer of peptidoglycan (a sugar-protein shell) that the stain can penetrate and teichoic acids. In this case the lactobacillus and staphylococcus are gram positive. A gram negative cell wall is wavy and much thinner and has a couple of layers of peptidoglycan. This is enclosed by an outer membrane made of phospholipids other substances. The outer membrane prevents the initial stain from penetrating. The Escherichia coli has a gram negative cell walls dye (Levinson, 2014). B. Explain what causes gram-negative bacteria to stain pink. Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain procedure. The crystal violet dye is washed by acetone and counter stain stick on its cell wall. The cells appear pink because of the color of the counterstain (safranin) (Levinson, 2014). In this case, it applied to the Escherichia coli bacteria since the thinner peptidoglycan layer in their cell wall did not allow for the stain to retain. C. Explain what causes gram-positive bacteria to stain purple. The iodine binds the crystal violet stain in the cell wall preventing counter stain from sticking onto the wall...
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...Diagnosis of an Infected Patient: Microbiology Paper BIO 212 Professor Fazely January 18, 2015 Professor Fazely Bio 212 18 January 2015 Diagnosis of an Infected Patient After collecting a sputum sample from a patient that is suspected or may be infected with a bacterium from one of the following genera: Escherichia, Mycoplasma or Bacillus; Each bacteria listed should be isolated by utilizing each of the various staining techniques. The best staining techniques to use is the Gram stain or the Acid- fast stain due to the fact that they both will provide a lot of information in detail regarding the bacteria being studied. It is very important to be observant of how each bacterium obtained reacts to each stain, and how the results obtained will lead us in a developing diagnosis. The first technique being used is the Gram stain. Gram stain is probably one of the most common used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of the two groups: either gram positive or gram negative bacteria. Bacteria prepared for the Gram stain is a heated fixed smear that is covered with a crystal violet. Because the purple stain impart its color to all cells. After a short period of time, the purple dye is then washed off, and the smear is then covered with iodine, a mordant. When the iodine is then washed off, both the gram-positive and gram-negative bacteria appear dark violet in color or...
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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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...Biochemical tests are often used to determine the identity of bacteria and differentiate between its species. There are numerous types of tests meant to detect key characteristics such as morphology, stain, motility, fermentation pathways used, oxygen requirements, enzymes present, and redox tests used. By conducting these tests with aseptic techniques, one can usually narrow down an unknown bacterium to its family; with the help of dichotomous keys and Bergey’s Manual of Determinative Bacteriology, particular genus and species can be identified. The first tests commonly conducted on a bacterium also give the broadest results. Initial tests employ differential staining and normally include gram, capsule, and endospore stains. All three indicate...
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...Discovering which bacteria are responsible for causing infection in a patient is a very helpful tool and allows for accuracy in providing appropriate antibiotic therapy. A technique utilized is called Gram staining. Gram staining is a type of differential staining method meaning that the stains will react differently depending on the bacteria present. This is a good starting point for identifying specific bacteria. In our scenario, Bacillus, Escherichia, or a Mycoplasma is expected to be found. Bacillus bacteria are rod-shaped and may appear in pairs or chains. They are characteristically Gram positive but may become Gram negative with age. Bacillus bacteria is aerobic in nature meaning that it uses oxygen. A unique feature of Bacillus is its...
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...Antibiotic Resistance and Gram Staining of Unknown Bacterial Samples Microbiology Lab July 1, 2013 Abstract: In this final laboratory assignment, each student was given an agar plate. Each plate was swiped with a pure culture bacteria sample. Where the sample came from and what kind of bacteria was unknown to the students. We were asked to observe the colony morphology on our plates and to perform the antibiotic sensitivity/resistance test. The plates were put into a 37° incubator oven. The next day the plates were taken out of the oven and we calculated the zone of inhibition. We then performed a gram stain of our bacteria and analyzed it under a microscope. We were then able to assess what kind of bacteria we had and if it was gram positive or negative. Introduction: Simple staining (the use of a single stain) allows a microbiologist to observe the morphology (shape) and arrangement of bacteria. In order to classify bacteria into different groups a differential staining procedure must be done. A differential stain involves the use of two or more stains. Depending on the components of the bacterial cell wall or outer layers, the bacteria will either retain the primary stain or have the primary stain removed in a decolorizing step and then retain the secondary stain. The gram stain is the most common differential stain used in the microbiology laboratory to categorize bacteria. The primary stain is the cationic dye Crystal violet and the secondary...
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...ruling them out one by one. I would start with Bacillus. They are rod shaped bacterium which form endospores. Their movement is propelled by flagella. Bacilli are gram positive, but because if forms endospores, I would do the Endospore staining. Endospores are resistant to stain and relatively uncommon in bacterial cells. You start with a heat fixed smear. Fixing a slide kills the microorganism. A thin film of the microorganism is put on the slid and allowed to dry. Once dried, it is then fixed by either being passed through the flame of a Bunson burner several times or covered with methyl alcohol for 1 minute. Malachite green is applied and the smear is heated to steaming for 5 minutes, helping the stain penetrate the endospore wall. It is then washed with water for 30 seconds. This removes the stain from the cell except for the endospore wall. Safranin is applied as a counterstain to stain portions of the cell other than the endospore. In the smear, the endospores appear green with the rest of the cell red or pink. If there no endospores in the smear, it would eliminate bacillus as a contributing factor. Escherichia, commonly called E-coli, are also rod shaped microorganisms, and are gram negative. A gram stain is a differential stain developed Danish bacteriologist Hans Christian Gram. You start with a heat fixed smear using the procedure stated above. After they are fixed, it is then covered with crystal violet (or basic purple dye). After a short time, the dye is...
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...Labset Two Worksheet 1. What is differential staining? How does it differ from simple staining? (2) Differential staining uses more than one chemical stain. It can help differentiate between different microorganisms, or different parts of the cell. The simple stain uses one stain and is used to see cell shape and size. 2. What are the differences between gram positive and gram negative cell walls? (2) A gram positive has a thick wall of peptidoglycan and stains purple. A gram negative has a membrane covering the peptidoglycan and doesn’t stain leaving it a pink color. 3. What is a mordant? What serves as a mordant in the gram stain protocol? (2) A mordant is a chemical that can deepen the reaction of the dye. Iodine is the mordant in the gram stain protocol. 4. Why do gram negative cells stain pink? Gram positive cells purple? (2) Gram positive cells have cell walls and can retain the dye. Gram negative cells do not have cell walls and cannot retain the dye. Cristal Violet is added to the sample and penetrates the cell walls, staining the cell in the gram positive. Gram negative will not retain color as there is no cell wall. A counterstain is added to make the cell visible and turns it a pink. 5. What is measured by the methyl red portion of the MR-VP test? (2) It measures acid in a bacterial broth. It turns red in pH under 4.4, yellow in pH over 6.2, and orange in between. 6. What is measured by the Voges-Proskauer...
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...Diagnosis of an Infected Patient: Microbiology Paper Bio 212 18 January 2015 Diagnosis of an Infected Patient After collecting a sputum sample from a patient that is suspected or may be infected with a bacterium from one of the following genera: Escherichia, Mycoplasma or Bacillus; Each bacteria listed should be isolated by utilizing each of the various staining techniques. The best staining techniques to use is the Gram stain or the Acid- fast stain due to the fact that they both will provide a lot of information in detail regarding the bacteria being studied. It is very important to be observant of how each bacterium obtained reacts to each stain, and how the results obtained will lead us in a developing diagnosis. The first technique being used is the Gram stain. Gram stain is probably one of the most common used staining procedures used in the field of microbiology. It is one of the differential stains that are used to characterize bacteria in one of the two groups: either gram positive or gram negative bacteria. Bacteria prepared for the Gram stain is a heated fixed smear that is covered with a crystal violet. Because the purple stain impart its color to all cells. After a short period of time, the purple dye is then washed off, and the smear is then covered with iodine, a mordant. When the iodine is then washed off, both the gram-positive and gram-negative bacteria appear dark violet in color or purple. The next, process is the slide is then washed with alcohol...
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...Name ___________________________________________(5 pts.) Chap 3 study Questions - You will learn and understand more if you complete these questions within 1 -2 days after lecture! Must use textbook or think it through yourself to answer some questions. What is the range of individual cell length in micrometers? 1) What do the following abbreviations stand for? m = mm= nm = 2) How many micrometers in a millimeter? How many millimeters in a meter? How many nanometers in a micrometer? To convert from one unit of measure to another, use an equivalency statement: Starting units (equivalency statement) = ending units 1) write the starting units with number 2) write the ending units 3) For the equivalency statement, put the ending units on top and the starting units at the bottom as shown below. This way the starting units cancel out and you are left with the ending units. Starting units (ending units/starting units) = ending units 4) Ask yourself which is the smaller unit: starting or ending? How many of the smaller units make up the larger unit? The unit that is smaller will be 1000 or 100 etc. The unit that is bigger will be 1. 5) Multiply to determine answer. Example: 6.8 m == ? nm Step 1 &2: 6.8 m (equivalency statement) = ____ nm Step 3: 6.8 m ( nm ) = _____ nm m Step 4: 6.8 m ( 1000 nm ) = _____ nm 1 m Step 5: 6800 nm 3) Convert the following (SHOW YOUR WORK to receive credit...
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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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