...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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...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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...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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...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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...distinct nucleus and occur singly or in chains or clusters and form distinct colonies. Bacteria are classified on the basis of many characteristics. Morphological and physiological features such as cell shape, motility, formation of spores and other distinguishable structures, and reaction to Gram stain is a good start in identifying bacteria. Other staining techniques such as Acid Fast stain are also useful in determining species. More important in identification of a genus and species of bacteria are biological tests, including the determination of the types of nutrients a cell can use, the products of its metabolism, and the response to specific chemicals. Other factors that can assist in identification of bacteria are their ecological habitats and more advanced methods such as genetic and molecular composition. Using various techniques one is able to distinguish and ultimately assign then genus and species of the unknown bacteria. Methods: Gram Staining: The Gram stain separates bacteria in two distinct classes and is also useful in distinguishing morphology. Through this technique one is able to identify bacteria as either gram positive or gram negative. The gram-negative bacteria have thin peptidoglycan layers that do not hold the primary stain, rather...
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...In this experiment we were able to use the technique of Gram staining to differentiate two types of bacteria that were unknown. Gram staining is a method that is used by many scientists in order to identify unknown bacteria’s via the unique physical and chemical characteristics of their cell walls. In a gram test a bacteria can either be gram positive or gram negative. The Gram-positive bacteria have an intricate set of amino sugars which we call peptidoglycan that form a thick cell wall around the plasma membrane. On the other hand, Gram-negative bacteria have an uncomplicated cell wall which thus has less peptidoglycan. The main source of identification within the Gram test is the fact that Gram-positive bacteria appear blue-purple while Gram-negative bacteria appear pink (Gram Stain). Bacteria can come in one of three different shapes; spirilla (spiral-shaped), cocci (round-shaped), and bacilli (rod-shaped). In order to be able to see these shapes a bacteria must be stained or dyed. I hypothesis that we will have one Gram-positive culture and one Gram-negative culture. (Lab Manual) In order to prepare this lab experiment we performed five steps. The first step in preparing this experiment was to heat fix a bacteria culture sample which in return stopped the sample from coming off the slide during the experiment. In order to perform this first step we first turned on a Bunsen burner and set the flames to a blue color which was used to sterilize an inoculating loop. Next, a...
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... And a Gram Stain Janet Myers 12:30p.m. Tuesday February 23 2016 Introduction: Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violet-iodine complex so that the dye cannot be removed easily. This step is commonly referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain...
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...on very tight. Next, the broth was incubated on a slant, at 37 °C. The slant had surface growth that was echinulate. There was also a smooth texture, with an accumulation of white growth on the bottom of the slant. There was not any apparent branching or dots. After that, a streak plate was inoculated. The streak plate had a round configuration of white clumps that was diffusing outward. The growth was slightly raised, with a convex shape. It was noted that there was also an abundance of growth. After observation, the media testing began. The first test was a gram stain. The gram stain showed purple cocci in chains. As a result, it was concluded that the bacterium was gram-positive, based on the fact that, gram-positive bacteria will stain purple due to their thick peptidoglycan layer (Figure 1). If the result had shown a red/pink bacterium that would prove that the bacterium was gram-negative. Gram-negative bacteria contain a thin peptidoglycan layer, with no teichoic acid, surrounded by an outer membrane. It was also determined that the bacterium had cocci morphology because it was circular. If it had a rod shape, it would have been bacilli morphology. The second test that was performed was a catalase test. The catalase test result was negative. There were not any bubbles, on neither the solid nor the broth. The lack of bubbles meant that H2O2 was not produced during aerobic respiration, and that it was not an oxidizing agent. Additionally, it meant that...
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...Clinical Microbiology Lab Final December 13, 2013 Table of content Gram Stain Technique……………………………………………………………………………………………… page 1 Culture Transfer Technique……………………………………………………………………………………… page 2 Acid-Fast Stain Technique………………………………………………………………………………………… page 3 The importance of the Gram Stain Technique to a physician……………………………………. page 4 The importance of varying shapes/colonies formation of bacteria……………………………. page 5 Spore Stain Technique………………………………………………………………………………………………. page 6 The Importance of incubation/protocol techniques…………………………………………………... page 7 The importance of various types of media for bacterial growth…………………………………. page 7 The importance of biochemical analysis in the microbial process……………………………… page 8 The importance of studying Clinical Microbiology and how the course will assist me in reaching my professional goals……………………….. page 9 Bibliography……………………………………………………………………………………………………………… page 10 Gram Stain Technique The Gram Stain is one of the most important differential stains used in bacteriology. (Cappuccino and Sherman, Microbiology A Laboratory Manual) Using the gram stain it is possible to determine purple gram-positive cells (S. aureus) from pink gram-negative cells (E. coli). The results of the Gram Stain make it possible to identify microorganisms by their shape, number and morphology. In a clinical setting these results can help in treatment by identifying the type of microorganism...
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...play a role in the ecology of life, by decomposing wastes, both natural and man-made, such as creating nitrogen fertilizer at the root zones of certain crops. Other several pathogens that can cause serious harm, even immediate death due to the diseases or disease causing products they produce. Overall, microorganisms play an important role in life. The purpose of this study was to identify the unknown bacterium using biochemical tests and various methods that had been learned from previous the microbiology laboratory class. Identifying the unknown bacterium was determined by separating and differentiating possible bacteria based on specific biochemical characteristics. The differential tests used to identify the unknown cultures were Gram staining, oxidase, indole test, urea test, and casein test. MATERIALS AND METHODS: The unknown bacteria were given out by the lab instructor. Each student chose their own unknown bacteria according to the number. All methods have been practiced to ensure proper procedure identifying bacteria have been applied to this unknown. Procedures were followed as stated in the course laboratory manual provided by the instructor, unless otherwise noted. Each test performed identified was used to determine the specifics and identify the unknown bacterium. All of the following tests were performed on this unknown on February 09, 2008. Some of the tests required a follow-up right after the next lab. The first procedure that needed to be accomplished...
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...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 a TSA slant today before leaving. Day 3...
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...so as to know how it can be treated, to knowing the correct microorganism to be used for making certain foods or antibiotics. This study was done by applying all the methods that have been learned so far in the microbiology laboratory class for the 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...
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...Microbiology Final exam Study reference 1. Gram Stain * Verify if bacteria are present or not. * Controls – positive (purple) – S.aureus * negative ( red/pink) – E.coli 2. Endospore Staine Positive controller – B. magneterium Green spore- Positive Pink (vegetative ) – Negative 3. Acid fast Positive control – M. smeagmatis Blue – negative Pink /red- positive 4. Motility Positive control - P.vulgaris A. non motile is negative test B. motile is a positive test 5. Carbohydrates fermentation (test for gram -) ( glucose , manitol, lactose) Positive control – ecoli (yellow) Red- no color change – negative test Yellow – color change – positive test 6. Mae Conkey’s Agar Ecoli – positive control Selective for negative gram stain Differential for organism that could ferment lactase Growth pink – positive No growth – negative 7. Gelatin Hydrolysis ( Gelatinase) Positive control – P.aeruginosa Liquid –positive test Solid - Negative test 8. Blood agar Positive control – S. aureus A. Betahemolysis B. alphahemolysis C. gammahemolysis 9. FTM *broth – O2 relationship with 10. MRVP (mix acid fermentation) MR- methyl red (PH lower than 4.4) Positive control – e.coli red color- positive test no red – negative test 11. Voges Proskauer ( acetone) –precursor for those who fermented butane Positive control- E. aerogenosa Red color – positive test No color...
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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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...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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