...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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...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, a spore test can be carried out. The presence of spores found with this test would indicate the presence of Bacillus, while no...
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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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...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 tests and bone studies, cutting holes in bones (i.e., the skull) or for cutting out a round piece of the cornea for eye surgery. A cylindrically shaped core of bone (or bone biopsy) obtained with a bone marrow trephine is usually examined in the histopathology department of a hospital under a microscope. It shows the pattern and cellularity of the bone marrow as it lay in the bone and is a...
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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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...that can distinguish them. There are also staining processes that can identify each. Let’s get started. The genera Bacillus is a gram positive rod shaped bacterium. When viewed under a microscope Bacillus can be seen as a single organism or in chains. It forms endospores that are resistant to heat, cold, radiation and disinfectants. Bacillus is also aerobic, it needs oxygen to grow. Bacillus can be the cause of infections including abcesses, wound, burn and ear infections, endocardistist, meningitis, ophthalmitis, osteomyelitis, peritonitis and respiratory and urinart tract infections. The genera Escherichia is a gram-negative rod shaped bacterium. It does not form endospores. They are anerobic and can survive with or without oxygen. Escherichia can normally be found in the lower intestines. This can be the cause of gastroenteritis and urinary tract infections. The genera Mycoplasma are also rod shaped, like Bacillus and Escherichia. Mycoplasma lack a cell wall which makes staining difficult. It is also so small it makes it undetectable with a normal light microscope. This can be the cause of lung infections and urogenital infections. Infections caused by Mycoplasma are not easily treated by antibiotics. A way to distinguish these genera is by staining. This is a procedure developed by Hans Christian Joachim Graham, a Danish bacteriologistis. This process became known as Gram’s stain or the Gram stain. Gram staining is the coloring of microorganisms with dye...
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...Bacteria can be found in plants and animals, in all habitats, on all surfaces and even in our bodies. To investigate the type bacteria existing in different locations, we took a culture from the inside of a mouth and the surface of a tabletop and let the bacteria grow in agar plates for one week. Separate agar plates were prepared for each of the two locations that also contained a small amount of two different cleaning solutions, Lysol and Green disinfectants, to observe which product worked better at killing bacteria. Both agar plates contained white and yellow bacteria growths. The bacteria in the agar plates was not evenly distributed throughout the surface, but rather was clustered in small areas. This was most like due to uneven distribution of the sample. It could also be attributed to the fact that bacteria grow in colonies. The plates with cultures from site 1 had slightly less bacteria coverage than the coverage from site 2. Although, this was really only the case for one of the cultures from site 2 while the other culture from the same site grew the same amount as the two cultures from site 1. It was observed that no bacteria grew on the portion of the agar nutrient where cleaning product was applied. It should be noted that the very minimal amount of bacteria that grew in the agar nutrient made it difficult to determine if this result was due to the effectiveness of the disinfectants. For this reason as well it is unclear which cleaning product out...
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...identification of an unknown bacterium. MEDIA LIST Unknown Bacterium #5 Mannitol Salt Agar Plate DNA Agar Plate Blood Agar Plate RESULTS/ DATA The Unknown #5 had the following morphology after Gram staining and observed under a brightfiled microscope: purple color, spherical shape and clustered like grape. After determining that it was a Gram positive staphylococcus, it was inoculated on a MSA plate, Blood Agar, DNA agar and Catalase test was also performed to help figure out the staphylococcus type. The Table below lists all of the biochemical tests, their purpose and results. TEST PURPOSE REAGENTS OBSERVATIONS RESULTS Gram Stain To determine the Gram reaction of the bacterium Crystal violet, Iodine, Alcohol, Safranin Purple grape cluster arrangement cocci Gram Positive coccus Mannitol Salt Agar To determine the if the bacterium can tolerate high saline levels and grow If the bacterium can ferment mannitol None There was a full growth and the mannitol turned to yellow (acidic) Positive Blood Agar To determine if the bacterium can hemolysis blood None Metallic gray, Creamy – golden color ß-hemolysis Positive DNA Agar To determine if bacterium has DNase and can hydrolyze DNA None Clearing around the bacterial growth Positive Catalase Test To determine the presence of the enzyme catalase (converts H2O2 to H2O and O2) Hydrogen peroxide Immediate bubbles Positive DISCUSSION/CONCLUSION After...
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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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... The following lab (Rubisco Western Blot) procedure was done over a course of three weeks. During Week 1, gels were prepared, plant extracts were produced and the Bradford Assay was completed. First 2 identical gels were poured. This was done by assembling 2 sets of glass plates, inserting combs between the plates, and using a permanent marker to mark a line 1 cm below the teeth of the comb. The comb was then removed. Vinyl Gloves were put on for protection from acrylamide due to the toxicity of the liquid for of the substance. In a smaller flask, the components were mixed for the resolving gel in said order: 5.0 ml deionized water, 6.0 ml 30% acrylamide mix, 3.75 ml 1.5 M Tris (pH 8.8), 0.15 ml 10% SDS 0.15 ml, 10% APS 6 μl TEMED. Polymerization began after the TEMED was added. The mixture was then swirled using a plastic Pasteur pipet, immediately after the gel was then poured up to the line drawn earlier. Using a p1000 micropipette, the gel was overlayed with a thin layer of DI water. Said steps were repeated for the second gel. The mixture was left at room temperature for 30 minutes allowing the mixture to completely polymerize. Any excess acrylamide in the mixing vessel was allowed to solidify before being thrown away. The top of the resolving gel was then rinsed with DI water and blot dried with a paper tower. Vinyl gloves were put back on for the next step. Using the same flask (cleaned) mentioned before, the following components of the stacking gel were mixed in said...
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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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...by conducting a series of tests in an attempt to isolate our particular bacteria. Daily Notes Day 1 – Chose unknown bacteria #70, which is a broth culture. It is cloudy and yellow in color, with sediment gathered at the bottom. It has a strong odor, and I question whether I have smelled it before. The first test I’m doing is a Gram stain. Doing 3 slides – all air dried and heat fixed. Also streaking 2 plates today: TSA plate, and 2nd plate will be based on the results of the Gram stain. Either: MSA= isolates S. aureus, inhibits G-, EMB= fecal coliforms, G- growth (selective isolation G- rods), MAC= promotes G- growth. I am worried that I won’t be able to achieve isolation of separate colonies from the broth culture when I streak the plates. Viewed results of Gram stain with microscope under 100x oil immersion. Results of 1st Gram stain were inconclusive, so staining again with another slide. Many more cells on this slide. 2nd Gram stain = G- bacilli (rods), confirmed with Claudia. Decide to streak 2nd plate = MAC since I believe I have a G- bacteria. Day 2 – Achieved isolation on both TSA and MAC agar’s. Definite growth on MAC means I do have a G- bacteria. Will now perform a 2nd Gram stain from TSA plate to confirm Gram status; confirmed that it is G- . After looking at the chart, decided to try to narrow down options by doing an oxidase test. There are 6 oxidase positive bacteria, and 8 oxidase negative bacteria. Oxidase test results = negative. Inoculated...
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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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...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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...Agar plate e.g trypticase soy agar (TSA) an already prepared pure culture Agar plate e.g trypticase soy agar (TSA) an already prepared pure culture Streak or pour plate method can be used to obtain pure culture Streak or pour plate method can be used to obtain pure culture UNKNOWN UNKNOWN Has a thin peptidoglycan layer but much thicker then G + and don’t take up crystal violet stain used in gram staining method instead pink/red gram negative Has a thin peptidoglycan layer but much thicker then G + and don’t take up crystal violet stain used in gram staining method instead pink/red gram negative Salmonella typhimurium can cause typhoid fever Salmonella typhimurium can cause typhoid fever Klebsiella pneumonia can cause pneumonia, meningitis Klebsiella pneumonia can cause pneumonia, meningitis Positive test indicates carbohydrates fermentation giving a yellow acidic color with bubbles in tube Positive test indicates carbohydrates fermentation giving a yellow acidic color with bubbles in tube No reaction with color remain purple No reaction with color remain purple Negative result remains the same nothing forms Negative result remains the same nothing forms Staphylococcus epidermidis Staphylococcus epidermidis Staphylococcus (aureus, schleiferi, intermedius) Staphylococcus (aureus, schleiferi, intermedius) Ability to clot plasma (coagulase) if positive they clump together forming lumpy look like gel Ability to clot plasma (coagulase) if positive they...
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