...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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...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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...Before this practical at A level I had previously performed a gram staining of bacteria, although I had not observed them under a microscope before. I had studied about fungal structure and growth but had not observed them under the microscope before. I had learned about small motile aquatic organisms although methods for their observation were not included in my previous study. In this practical we began first by setting up our microscopes using standard operating procedures of practice. This involved following a series of steps in order to ensure correct working of the microscopes in the observation of our sample. For this practical we examined bacteria and fungi microscopically following a series of preparation and staining techniques...
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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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...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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... An environmental study was conducted at Westchester Community College to observe the different types of micro bacteria growing on campus. Several different locations on the second floor of the Science Building were sampled as well as samples that were collected from a living organism. The samples were given a nutrient source and incubated for a period of one week. The samples were then observed for growth; slides were created, stained and then studied to determine morphology of organisms. Materials - Sterile cotton swab - Water - Beaker - Slides - 1 Blood Agar plate and 2 Nutrient Agar plates - A Microscope - Emersion oil - Incubator - Refrigerator - Loop - Slide Holder (Clothes pin) - Bunsen burner - Flint - Gram Staining Agents (Crystal Violet, Iodine, Decolorizer, Safranin) - Wax pencil (for documentation) Procedures Collection of Environmental Samples - Sterile cotton swab was taken and wet with water - A 1” sample was taken of the specific locations listed in the table - The applicator was then placed back into the paper container for transport back to the lab. This was done to preserve the integrity of the specimen - The sample was then smeared onto one half of an agar plate. The two samples from the biological specimen were smeared onto a blood agar plate and the specimens taken from the environment were placed on a nutrient agar plate. - The cotton applicator was then disposed of The agar plates were placed in the incubator where...
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...Microbiology Overview Interpretation of preliminary microbiology data Gram-positive cocci Aerobic In clusters ● Coagulase (+): Staphylococcus aureus ● Coagulase (-): Staphylococcus lugdunensis and other coagulasenegative staphylococci In pairs/chains ● Optochin sensitive: Streptococcus pneumoniae ● Alpha-hemolytic: Viridans group Streptococcus, Enterococcus ● Beta-hemolytic: ○ Group A Strep (Streptococcus pyogenes) ○ Group B Strep (Streptococcus agalactiae) ○ Group C, D, G Strep Anaerobic: Peptostreptococcus spp. and many others Gram-positive rods Aerobic ● Large: Bacillus spp ● Cocco-bacillus: Listeria monocytogenes, Lactobacillus spp ● Small, pleomorphic: Corynebacterium spp ● Branching filaments: Nocardia spp, Streptomyces spp Gram-negative cocci Aerobic ● Diplococcus: Neisseria meningitidis, N. gonorrhoeae, Moraxella catarrhalis ● Cocco-bacillus: Haemophilus influenzae, Acinetobacter Anaerobic: Veillonella spp. Gram-negative rods Aerobic Lactose fermenting (Lactose positive): ● Enterobacter spp, Escherichia coli, Klebsiella spp ● Citrobacter spp*, Serratia spp* Non lactose-fermenting (Lactose negative): ● Oxidase (-): Acinetobacter spp, Burkholderia spp, E. coli, Proteus spp, Salmonella spp, Shigella spp, Serratia spp*, Stenotrophomonas maltophilia ● Oxidase (+): P. aeruginosa, Aeromonas spp. Anaerobic ● Large: Clostridium spp Anaerobic: Bacteroides spp, Fusobacterium spp, Prevotella spp. ● Small, pleomorphic: P. acnes, Actinomyces spp *Serratia and Citrobacter spp...
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...over a long period of time, an orderly succession of microorganism communities takes place. In this experiment, me and my partner will work together to observe the environmental conditions, and pattern of succession that takes place in plain whole milk, over a period of eight days. Lactobacillus and streptococcus both survive pasteurization. These two ferment lactose to lactic acid, thus dropping in pH level. This acidic environment causes casein to curdle up. We expect to see yeasts and fungi, because they grow well in acidic environment. METHOD We recorded pH using pH paper of day 0, day 1, day 2, day 4, day 6, and day 8. Than we observed the consistency, odor, and color of each. Than we made slides using the gram stain method for each day. Which is to take a thin smear on the slide, and then heat fix it. Than place crystal violet on it for a minute. Rinse with water. Than cover it with iodine for one minute. Rinse again with water. Than decolorize with alcohol with 10-20 seconds. Rinse again with water. Than cover it with safranin for two minutes. Then rinse with water and gently blot dry. After repeating this method for each day, place under...
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...there were only two bacteria’s growing. After isolating one of the bacterium a Gram stain was performed. The Gram test concluded that the bacterium was a Gram negative rod. This narrowed down the unknown to 5 possible answers: Escherichia coli, Klebesiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginesa. The first biochemical test performed was a Simmons Citrate, and after observation a negative result was concluded. This narrowed down the answers to Escherichia coli and Proteus vulgaris. The next biochemical test that was performed was a H2S test. After observation, the H2S test had a negative result, which pointed to Escherichia coli. A third test was done in order to confirm that Escherichia coli is the unknown. The Urea test also had a negative result, which confirmed that the unknown #1 is Escherichia coli. The second culture’s results were also found with the help of the unknown chart, handed out by the instructor. The unknown tube, #125, was isolated in order to grow two separate cultures. However, like stated earlier, a problem was faced after the isolation was incubated. The plate had contamination, which prevented further testing from happening. A second isolation was completed and when observed it confirmed that only two bacterium grew. After isolating the second bacteria, a Gram stain was performed, hoping to see a purple color, indicating a gram positive. Unfortunately, another issue arose. When observed,...
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...Discussion: The purpose of this experiment is to identify an unknown organism over three weeks by performing lab exercises that were previously performed. The unknown #16 was chosen for this experiment. During the first week, The next step in determining this unknown was to perform the streak plate method for isolation of colonies of a 24-hour period in a 37o C incubator. The unknown organism grew on the media; observed white in color, raised with a smooth margin, shiny colonies on the TSA agar plate. The next step in identifying this unknown #16 was to steak for isolation on plated media; then incubated at 37o C for 24 hours and the stores at 4o C. The plates used for isolation were; Triple Salt Agar (TSA), Mannitol Salt Agar (MSA), Phenyl Ethyl Alcohol Agar (PEA), Eosin Methylene Alcohol (EMB), MacConkey (Mac), and a TSI slant. As seen in the observations in part II some growth was recognized. The TSA plate was streaked for isolation of the unknown. While observing, there was growth, white in color. The next plate was the MacConkey streaked plate. The colonies on the Mac plate were green or blackish in color with flat smooth colonies in every section of the plate. For the MSA plate there was no growth observed on the plate. The EMB plate had very little growth,, and interpreted as G/NLF as well. The Blood Agar plate was brownish/greenish growth with a raised margin. The PEA agar also had very little growth, white colonies that were flat and smooth; it was interpreted as...
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...thick b. How would a colony attain this shape? Equal divisions in two planes Procedure 24.6 Question 4: a. Where are the bacteria? Are they between cells or inside cells? Inside the cells b. Why is this relationship between a plant and bacterium called a mutualism? Mutualism is any relationship between individuals of different species where both individuals benefit. For some plants their relationship is mutual with certain bacterium as it benefits both species. In other cases one may harm the other in which case the relationship is not mutualism c. How does Rhizobium benefit from this association? Nutrients from the host d. How does the host plant benefit from this association? Nitrogen supply from the bacterium Gram Stain Question 3: a. Which type of bacteria is...
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...animal glue, etc.) to achieve the adhesion between the particles and consequently to be applied as paint film on the dry ground. The pigment must be dispersed or grinded as evenly as possible in the binding medium to take full advantage of the properties of both the pigment and the binder.[8] The typical stratigraphic structure of the Egyptian wall paintings is usually made of multiple layers. The plastering procedure was to initially smooth an irregular rock surface with one or more of plaster layers. The first layer is the coarse, so called ‘arriccio’, applied directly to the wall. This layer consists of siliceous aggregates, and the matrix binder is mainly of gypsum or lime. The second intermediate layer is the fine plaster called ‘intonaco’, which consists of gypsum or lime and quartz crystals of small dimensions. The third layer in the top is the ground layer for the application of pigments. This layer is based mainly on pure lime, gypsum or a mixture of both of them. In some cases, mainly in temples, only a single layer of gypsum was used as a preparatory layer for pigments. Egyptian blue was the first synthetic pigment ever produced by man; it is considered a great technology development in the ancient Egypt, as it appeared in Egypt during the third millennium BC. [9] This pigment consists of the mineral cuprorivaite (CaCuSi4O10), a blue tabular crystal about 15–30 mm in length, residual silica (quartz and/or tridymite) and an amorphous silica-rich phase. This pigment was...
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...different tests are performed for the diagnosis of different disease .they are used different techniques for the detection of presence of microorganism which are causing harmful disease Different test performed are • Test performed by ELISA technique a. Hepatitis B,C , b. Rheheumatoid arthritis (RF FACTOR ) c. Treponemapallidiumhaemagglutination test (TPHA) • Test performed by latex test a. CRP ( C reactive protein test ) b. ASO(anti - streptolysin o ) • Flocculation method Veneral disease research laboratory (VDRL) is the flocculation test. It is simple rapid convenient and economical procedure for serologic testing for syphilis • Culture a. Pus culture b. CSF or body fluid culture c. Blood culture • Media preparation • Staining • Sensitivity •...
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...Discussion a) Gram Staining Escherichia coli is a Gram-negative rod- shaped bacterium. Initially the bacterium is stained purple by the primary stain, crystal violet. The alcohol which is used as a decolourising agent washes away the outer membrane and allows the crystal violet complex to wash out from the thin peptidoglycan, making the cell colourless. When the counterstain, safranin is applied, they appear pink. Staphylococcus aureus is a Gram-positive bacterium and appear in cocci grape-like clusters. They have a very thick peptidoglycan layer that can retain the purple stain from crystal violet. The decolourising solution has no effect on this bacterium besides a slight drying effect on the wall, The pink counterstain does bind the cells but it is not seen because covered by a darker purple colour primary stain. Bacillus subtilis are Gram-positive rod shaped bacteria that appear as small chains or single cells. They have thick peptidoglycan layer that can retain the purple stain from crystal violet. Saccharomyces cerevisiae are circular which occur singly or in a group. They do not respond to gram staining due to their cell wall that consists of glucan and mannoproteins. They usually bind to the original dye, look blue but they are not Gram-positive. Thus, they are Gram non-reactive. Water isolates found in rod shaped and they are Gram-negative bacteria that stains pink after safranin applied due to the thin peptidoglycan layer. b) Acid fast staining Mycobacterium...
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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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