microbiology help TOT Zoo Add Page Insert Table Chart Text Shape Media Comment These questions will…

microbiology help

TOT Zoo Add Page Insert Table Chart Text Shape Media Comment These questions will serve in lieu of a lab report for Exercise 15, 16, and 17 You will find the answer to these questions in the background, procedure, results and interpretation sections of manual Exercise 15, 16, and 17, videos, Actions of Selective and Differential Media Chart, and the Principle/Theory article in homework section.) General Questions 1. What is the purpose (function) of selective media? (How does this test help identify organisms.) 2. What is the purpose (function) of differential media? (How does this test help identify organisms.) Questions about Mannitol Salt Agar (MSA) 3. Would this medium have the function of being selective for Staphylococci aureus, if it did not have sait in it? Why or why not? 4. Why would you not choose MSA to help you differentiate Gram negative organisms? (explain based on the media’s selective properties) 5. What is the agent (or chemical) and pH indicator in mannitol salt agar that makes the medium differential? How does that agent and indicator work? 6. On MSA, a) gram negative organisms display no inhibited growth. What notation (symbol) would you use to signify that on the Data About Bacteria chart? b) SE displays growth with no change of media around the growth (stays red). What type of organism is it? c) SA displays growth with yellow media all around it. What type of organism is it d) Both LL and BC display no inhibited growth. Why don’t they grow on MSA even though they are gram positive organisms? Questions about Eosin Methylene Blue Agar (EMB) 7. Is EMB media helpful to differentiate a) gram negative or b) gram positive organisms? 8. Name the differential agent included in EMB and describe how it differentiates between the three types of organisms (describe the results/appearance of each type of organism). MacBook Air 5 Mannitol Salt Agar CASE FILE In early summer, a festival was held in a community 50 miles south of Dallas, Texas. Within hours of attending. 98 people became with severe nausea, vomiting, and diarrhea. Seventeen of them sought medical attention. Most had recovered by the next day. Testing of various samples of food by the health department found evidence that toxin-producing Staphylococcus aureus may Source Janico Honey Care Centers be present in ham from one of the food vendors at the event. for Disease Control and Prevention You’ll be testing Sample M to help find out if S. aureus caused the symptoms in the ill people. LEARNING OUTCOMES At the completion of this exercise, students should be able to Understand the selective and differential functions of mannitol salt agar. * Interpret growth on a mannitol salt agar plate and determine the bacterium it represents describe the use of mannitol salt agar in the identification of Staphylococcus aureus. 9 Background of all the disease-causing bacteria, the Staphylococci are some of the most talented. Staphylococcus aureus is the most pathogenic of the staphylococci. It can infect the skin and many other tissues in the body. It can produce a wide range of harmful enzymes and toxins. Among these are enzymes and toxins that kill white blood cells, trigger a fibrin clot, or cause shock. As antibiotic-resistant strains of staphylococci, such as methicillin-resistant S. aureus (MRSA), and vancomycin-resistant S. aureus (VRSA), continue to spread, the infections that they cause are becoming increasingly difficult to treat. Even worse, S. aureus doesn’t even have to be inside of you to make you sick. It can release an enterotoxin as it grows in food that will make an individual sick within hours of eating. Mannitol salt agar is an example of a medium that is both selective (figure 15.1) and differential. This medium contains a high concentra- tion of salt (7.5% NaCl), which makes the medium selective by inhibiting the growth of most bacteria but allowing salt-tolerant (halophilic) 105 Med Sample General-purpose O n onselective medium All species grow) Selective medium One species grows) Figure 15.1 Punction of a selective medium. Staphylococel to grow. In addition, this medium contains the sugar mannitol and pH indicator phenol red. Disease causing staphylococci ferment the sugar mannitol. Mannitol fermenters release acid into the medium, which turns the phenol red in the medium around the colony yellow as the pH changes. Non disease causing staphylococci (like Staphy lococcus epidermidis) grow but do not change the medium’s color. The combination of mannitol and phenol red allows for differentiation between pathogenic and nonpathogenic staphylococci _ Tips for Success Make short streaks, so that the color zones from adjacent samples don’t merge and make it hard to read your results. Incubating plates for more than 24 hours may cause zones around the colonies to become too large and interfere with the results. Removing and/or refrigerating plates may be needed. Organisms (in Broths) Staphylococcus aureus Staphylococcus epidermidis Escherichia coli Sample M E Materials (per Team of Two or Four) 1 mannitol salt agar plate Inoculating loop Bunsen burner or microincinerator (Optional) 1 tryptic soy agar (TSA) nlate Illlllllllll en Figure 15.2 Marking and inoculating the plate. Steven D Procedure Period 1 1. Use your marker to divide the plate into quarters (mark the bottom of the plate as shown in figure 15.2); label each quarter with the name of the organism that will be inoculated there. As an optional activity, the organisms can also be inoculated onto a control TSA plate, which is not selective 2. Make a short streak of each organism. 3. Put the plate lid side down in the incubator. Period 2 1. Observe the growth on your plate and record the results in your lab report Results and Interpretation Mannitol salt plates can be read against either a dark or light background. First, determine if the organism in each area grew. Second, did the color of the medium around the growth change! Figure 15. Ja to e shows possible results. An organism can grow and produce acid, grow and not produce acid, or not grow at all. Figure 153 Growth on mannitol salt agar. (a) Pathogenic Staphylococcus aureus growing on mannitol salt anar fermenting mannitol, producing acid, and changing the color of the pH indicator. (b) Staphylococcus amidar 08 epidermidis rowing but not fermenting mannitol and not producing acid. (c) Lack of growth due to inhibition hu the nhibition by the high level of salt in medium Steven D. Obenauf 107 VLULU Microbes: Media 1b Eosin Methylene Blue Agar CASE FILE Two water samples have arrived in a testing lab. One is from a group of homeowners with homes on the same street. sharing a common well A new resident of the street is concerned about water quality. The second is from individuals concerned about the safety of the water in a river near their homes. Drinking water is not sterile. It contains bacteria. The key Source: CDC/Peggy S. Hayes & thing to know is what type. Normally, the bacteria found in Elizabeth H. White. MS water supplies are harmless. Finding a type of bacteria known as coliform bacteria, and in particular. Escherichia coll, can be a cause for concern Livestock, inadequate wastewater treatment plants, leaky septic systems, landfills, and stormwater runoff are common sources of fecal contamination of water supplies, including rivers, lakes, and wells. While water used for swimming or boating (“recreational water”) is allowed to have relatively small amounts of E. col (the EPA standard for primary contact water is 235 E. coll per 100 milliers in a single sample) in addition to the other bacteria found in that water, the standard for drinking water is zero. So if any E. col are detected in drinking water, it fails the standard. The phrase “one is too many applies here. The eosin methylene blue (EMB) agar that you will use in this exercise is used as part of the process of confirmatory testing for E. coll in water. LEARNING OUTCOMES At the completion of this exercise, students should be able to . understand the selective and differential functions of EMB agar. examine the various colors of growth on an EMB agar plate and determine the category of bacteria they represent. Background Water can be a dangerous thing. A wide variety of serious microbial dis- eases can be transmitted by contact with or ingestion of water. These include cholera, shigellosis, amoebiasis, giardiasis, schistosomiasis, hepatitis A. and viral gastroenteritis. Coliform bacteria can live in the environment or the intestines of many animals (including us). Fecal coliform bacteria such as Escherichia coll live primarily or exclusively within the intestines of warm blooded and cold- blooded animals. Therefore, the presence of these bacteria in water or food 111 may indicate possible fecal contamination. When safety, we usually do not look diety for all the borne pathogens such as Gordis or hepatitis A test for the presence and quantity of fecalcom The as indicators of the presence of other harmful micon that pose significant threat to humans. In c ise 19. Tate we will perform a coolsed test for quantitating bac but it provides iele information that can be used to ident The higher the level of calcolo persent in a wa the greater the risk that something hare will also be there E colis a particularly good indicator because it is not typically found living in oor wat its presence is more strongly comed with fecal contamination than other bacte A standard qualitative test for determining water way is the fecal coliformis test. Since color bacteria ferment the Lactose, the first presumptive) step in the fiecal coliforms General purpose looks for lactose-femmenting on in the sample nonderential medium that step All species have a similar is positive, a second confirmatory) test looks more specifically appearance) Dermedium for fecal coliform bacteria One of these confirmatory tests is the Ale species grow bulmay use of easi mehe le A ber of other media in Show contractions) addition to FNB C bet t o the presence of E Figure 16.1 Punction of a differential Eosin mechiene Mari e of medium that is both selective and differential thigure 16.1). The two dves together cosin and methylene blue cake the s um lective by boting the growth of a positive bacteria but allowing um gatves includ medium which allows for differentiation between sonlatos fermente, lactose fermentes, and heavy lactose e s pecially useful for identity ing fecal colestorm bacteria. The production d e fermenting lactose will trigger a pink to me ane color change that will distinguish coliform bacteria from other and Som the colors _ Tips for Success The way to read EMB agar places is dit om blood MacConkey or mannitol salt agar planes EMS plane best read with a dark background underneath the do not hold them up and look through them or you will not see the colors of the colonies will Organisms (in Broths) Sample P (water) or student water sample Escherichia coli Enterobacter aerogenes Pseudomonas aeruginosa Materials (per Team of Two or Four) 1 EMB agar plate Inoculating loop Bunsen burner and striker Procedure 1. Interested students should get a sample bottle or container to obtain water from a river, canal lake, or other freshwater source Period 1 1. Use your marker to divide the plate into quarters (mark the bottom of the plate as shown in figure 16.2); label each Figure 16.2 (a) Uninoculated plate and (b) pattern for marking and inoculating the plate. Steven D. Obenauf 112 er with the name of the organism or simple that will be each organism as shown in the diagram or with a single inoculated there 2. Streak each organism as sho straight streak 3. Put the plate lid side down in the incubator Period 2 serve and record the growth on your plate. Look at the color of the growth or colonies on the plate not the medium (figure 10.3). Figure 16.3 Growth on the plate Steven D. Obenauf Results and Interpretation Read your plates against a dark background (for example, your lab bench top) you may need to lift the lid bit. Gram-positive bacteria will grow Poorly on this medium or not at all (figure 16.da). Gram-negative bac teria that are not coliforms will grow well but are usically colorless (figure 16.4b). Coliform bacteria (lactose fermenters) will grow well and the colonies will be pink with purple in the center (figure 16.4c). Rapid or strong lactose fermenters will grow well and will appear dark purple with a distinct metallic green color (figure 16.4d). Figure 16.4 Growth on EMB agar. (a) Gram-positive organism growing poorly due to inhibition by methylene blue in medium () Gram-negative non-lactose fermenter. (c) Coliform lactose fermenter with mucoid pink and purple growth d Ecold with green sheen caused by rapid lactose fermentation Steven. Obend 113 MacConkey Agar CASE FILE A 43-year-old woman with severe head trauma and lacerations from a car accident was transported to the intensive care unit. She received one unit of packed red blood cells and required Intubation and mechanical ventilation. On her third day at the hospital, the patient was still unresponsive. Her temperature rose to 39.3 C and pulse oximetry indicated worsening gas Source: Centers for Disease exchange. A chest radiograph revealed a right-side infiltrate, Control and Prevention (CDC) and analysis of a blood sample showed an elevated white blood cell count. Staining of a bronchoalveolar lavage was positive for the presence of gram-negative bacteria and negative for fungi. Treatment with IV antibiotics was initiated, and the infection- control nurse and the respiratory therapy supervisor were notified. LEARNING OUTCOMES At the completion of this exercise, students should be able to explain how MacConkey agar is both selective and differential Interpret the various colors of growth on a MacConkey agar plate and determine the category of bacteria they represent. explain when and how MacConkey agar would be used in a clinical setting Background A hospital can be a dangerous place to stay. Nosocomial, or hospital- acquired, infections present a significant risk to patients and cause well over a million illnesses and around 100,000 deaths each year. Urinary tract Infections, blood infections, and pneumonias are the three most commonly seen categories of nosocomial infections. A type of hospital-acquired pneu- monia seen in patients on mechanical ventilators is known as ventilator- associated pneumonia (VAP). The most commonly seen causes of VAP include the gram-positive cocci Streptococcus and Staphylococcus (including MRSA), Gram-negative causes include several coliform bacteria: Klebsiella. Enterobacter and Escherichia coli as well as non-coliform bacteria such as Pseudomonas Acinetobacter, and Serratia. The fungus Candida is also a possibility. MacConkey agar is an example of a medium that is both selective and differential. Bile salts and the dye crystal violet make the medium selective 117 INT Figure 171 Marking and Inoculating the plate Divide the plate into gudants and then streak each urant with one of the organisms by inhibiting the growth of gram positive bacten but satives (including coliforms) to grow. In addition, this medium wingman the sugar lactose and the pH indicator neutralne which allows for dat ferentiation between non-lactose fermenters such as Parade lactose fermenters such as Kits and Eschericht. It is especially Identifying enteric bacteria Acid is produced and released into the me during the fermentation of lactose in response to the lower pH, neutral red turns from pink to red. The color change distinguishes Lactose fermenting coliform bacteria from other gram negative bacteria Colonies of lactose fermenters will appear pink Non lactose fermenting gram negative organ isms will utilise peptone in the medium for growth. Their colonies will be clear or uncolored There are versions of MacConkey agar with different chemical makeups that are used to identify crococcus or inhibit Pro Tips for Success . If you are doing this exercise at the same time as other one such as manitol sales or EMB make sure to label your places as you pick them up. They are similar in color Organisms (in Broths) Sample 1 (CU patient sample) Staphylococcus epidermis Escherichia coli Figure 17.2 Growth on EMB and MacConkey agar EMB agar is shown on the left and MacCoy agar is shown on the right. The top quadrant on each plate was inoculated with ICU patient sample the left quadrant on each plate was Inoculated with E call the bottom quadrant Od Bach place was inculaced with Poocomor rust and the night quadrant with Staphylococcus auren Gram-positive organisms grow poorly on these media due to Inhibition toy bile sults and crystal violet. Gram-negative non lactose- fermenting bacteria grows colorless colonies. Coliform bacteria form pink colonies. Susannarra Materials (per Team of Two or Four) . 1 MacConkey war plate Inoculating loop . I nutrient agar plate (optional) Bunsen burner and striker Procedure Period 1 1. Use your marker to divide the plate into fourths mark the bottom of the place as shown in figure 17.1) label each quarter with the name of the organism or simple that will be inoculated there. 2. Streik each organism as shown in the diagram or with a single straight streak 3. Put the plate lid side down in the incubator Period 2 1. Observe and record the growth on your plate. 2. Look at the color of the growth or colonies on the plate, and the medium around them (figures 17.2 and 17.3). 3. Gram positive bacteria will grow poorly on this medium or not at all (figure 17.2, right quadrant). 4. Gram-negative bacteria that are not coliforms will grow well but are basically colorless (figure 17.2. bottom quadrant) 5. Coliform bacteria (Lactose fermenters such as E. coli and Enterobacter) will grow well and the colonies will be pink or red (figure 17.2. top and left quadrants). 6. Record your results. Figure 17.3 A comparison of the growth of Ecolon MacConkey and EMS agars. E colis a gram-negative Lactose fermenting coliform. When growing on MacConkey agar eft). Eco produces a pink colory and causes pink dye to be deposited in the agar Surrounding the colony When growing on EMB agar (right) colonies of E collexhibit a metallic green color. Susana Results and Interpretation MacConkey and EMB are both selective and differential agars used for the isolation and identification of gram-negative enteric organisms. Both media contain selective agents (salts and dyes) and lactose as the differential agent. See figure 173 for a comparison of the two 118 Purpose The developentot lalos tund the bacterial s that the organism is a path