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Lab Practical 3 Review Exercise 34 (Antimicrobial Sensitivity Testing) 1. Be able to define the following: a). Antibiotic: Antimicrobials that are produced by microorganisms that inhibit or kill other microorganisms. b). Sensitive: When a microorganism is very susceptible to a particular agent. c). Intermediate: When a microorganism is somewhat susceptible to a particular agent. d). Resistant: When a microorganism is minimally, or not susceptible at all, to a particular agent. e). Zone of inhibition: The area on an agar plate where growth of a control organism is prevented by an antibiotic usually placed on the agar surface. If the test organism is susceptible to the antibiotic, it will not grow where the antibiotic is. 2. The Kirby-­‐Bauer Test: The most widely used antibiotic susceptibility test in determining what treatment of antibiotics should be used when treating an infection. This method relies on the inhibition of bacterial growth measured under standard conditions. For this test, a culture medium (specifically the Mueller-­‐Hinton agar) is uniformly and aseptically inoculated with the test organism and then filter paper discs, which are impregnated with a specific concentration of a particular antibiotic, are placed on the medium. The organism will grow on the agar plate while the antibiotic “works” to inhibit the growth. If the organism is susceptible to a specific antibiotic, there will be no growth around the disc containing the antibiotic. Thus, a “zone of inhibition” can be observed and measured to determine the susceptibility to an antibiotic for that particular organism. The measurement is compared to the criteria set by the National Committee for Clinical Laboratory Studies (NCCLS). Based on the criteria, the organism can be classified as being Resistant (R), Intermediate (I) or Susceptible/Sensitive (S). 3. Understand that different antibiotics are used against Gram positive and Gram negative cells: -­‐ This is due to the difference in the cell membrane/wall of these bacteria. Gram positive bacteria have a thick peptidoglycan cell wall not surrounded by anything. Gram negative bacteria have an outer lipopolysaccharide membrane surrounding a thin peptidoglycan cell wall. 4. Be prepared to measure the zone of inhibition in mm for several antibiotics and determine which ones are most, or least, effective against a particular bacterial infection: -­‐ The smallest inhibition zones (in the first figure) are the least susceptible to the antibiotic in the disk. -­‐ When measuring the zone of inhibition, one measures the full diameter including the disk (see figures 2 & 3). 5. Know that antibiotics are made by both fungal and bacterial organisms as a means of defense and competition (antagonism). -­‐ Clostridium and Streptomyces are bacteria that produce antibiotics. -­‐ Penicillium and Cephalosporium are fungi that produce antibiotics. Exercise 35 (Effects of Disinfectants and Antiseptics) 1. Be able to define the following: a). Antibiosis: An association between organisms that is injurious to one of them. b). Antiseptic: Substances such as alcohol or betadine that inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue. c). Disinfectant: Chemical agents that are applied to inanimate objects such as floors, walls, and tabletops to kill vegetative microbes. d). Confluent growth: Continuous bacterial growth covering all or part of an agar plate in which the bacteria colonies are not discrete. e). Sterilization: To destroy all microbial life, including endospores. 2. Be prepared to measure the diameter of the zone of inhibition for a given antiseptic or disinfectant in mm: 3. Understand that a given disinfectant and antiseptic will have varying effects against different microorganisms: -­‐ This is due to the vastly different modes of action (of agents), chemical properties of various agents, and the susceptibility level of different microbes to these agents. 4. Have an understanding of why the disk diffusion method of testing the effectiveness of antiseptics and disinfectants is not ideal: -­‐ This method is less effective for anaerobic, fastidious, or slow-­‐growing bacteria. -­‐ Depends on rate of diffusion, evaporation, and other factors that are difficult to control Exercise 62 (Temperature) 1. Be able to define the following: A). Thermal Death Time (TDT): How long it takes to kill a specific bacterium at a specific temperature. B). Thermal Death Point (TDP): The temperature at which an organism is killed, or a virus inactivated, in 10 minutes. 2. Be able to interpret a plate and determine the thermal death temperature based on growth or no growth. 3. Understand why a difference in temperature tolerance is observed between spore formers and non-­‐spore formers: -­‐ Spore forming bacteria will be more resistant to heat than non-­‐spore formers. Endospores have a much lower water content than vegetative cells. As a result, their macromolecules are less susceptible to denaturation. -­‐ Spore formers include the genera Clostridium and Bacillus. 4. Know which temperature range contains the organisms that are most often pathogenic to humans and which contains the organisms often responsible for food spoilage: -­‐ Mesophiles, responsible for human pathogenicity, live in a temperature range of 20°C to 45°C (68°F to 113°F). Psychrotrophs are bacteria capable of growth at temperatures at or less than 7°C (44.6°F) and can be responsible for food spoilage. 5. Be able to define sterilization standards in the food industry and their ultimate goal: -­‐ The ultimate goal of the food industry is to eliminate or reduce microorganisms in food to a level that is safe for consumption. Pasteurization of milk is required in order to reduce the number of vegetative microbes for safe consumption as well as to provide a longer shelf life. More drastic measures, such as high heat or radiation (sterilization), must be taken in order to kill endospores. Safety standards from various federal or state agencies are usually expressed as a maximum allowable cells or CFU per 100 ml. Exercise 19 (Standard Plate Count) 1. Be able to define: -­‐ CFU (Colony Forming Unit): Used to determine the number of viable bacterial cells in a sample per mL. -­‐ TFTC (Too Few To Count): Plates on which fewer than 30 colonies are growing. -­‐ TNTC (Too Numerous to Count): Plates on which more than 300 colonies are growing. 2. Be prepared to perform a standard plate count with or without a colony counter: 3. Be prepared to calculate dilution factors and from that information calculate cells per mL in the original solution from a given plate count. 4. Know the purpose and procedure behind a serial dilution: -­‐ This procedure is used to identify the number of viable microbes in a fixed amount of a liquid. Serial dilution involves repeatedly mixing known amounts of source culture with (sterilized) liquid. 1 ml added to 9 ml gives a 10-­‐fold dilution; 1 ml added to 99 ml gives a 100-­‐fold dilution. When fixed amounts of this dilution series are inoculated onto an appropriate agar and incubated, different numbers of colonies will be obtained. By working back from an easily counted plate and using the appropriate dilution factor, the number of microorganisms in the original source culture can be calculated. 5. Understand the purpose behind performing replicate plates: -­‐ Greater accuracy can be obtained by counting two or three plates and averaging the counts. 6. Be able to explain the purpose and function behind a spread plate technique: -­‐ The standard plate count method is very widely used to estimate the number of bacteria in environmental samples, drinking water, and various juices and food. This is used to determine the safety of where you swim and what you eat and drink. Safety standards from various federal or state agencies are usually expressed as a minimum allowable cells or CFU per 100 ml. An advantage of the surface plate count method over directly counting microbes under the microscope is that both living and dead cells look the same under a microscope, but only living cells will grow on a plate. Therefore, plate counts provide a better indicator of disease risk. 7. Know how to use a micropipettor: 8. Know what number of colonies on a given plate is considered countable: -­‐ Plates containing between 30 and 300 colonies are considered to be valid and countable. Exercise 27 (Aerotolerance) 1. Be prepared to define: -­‐ Reducing media: Contains reducing agents that deplete oxygen in order to grow obligate anaerobes, since they are unable to grow in the presence of oxygen. -­‐ Aerobic respiration: A form of cellular respiration that requires oxygen in order to generate energy (ATP). -­‐ Anaerobic respiration: A form of cellular respiration using electron acceptors other than oxygen to generate energy (ATP). -­‐ Fermentation: A metabolic process that converts (catabolizes) sugar to acids, gases and/or alcohol. 2. Be prepared to identify an organism as strict (obligate) aerobe, strict (obligate) anaerobe, facultative anaerobe, microaerophile, or aerotolerant anaerobe based on its growth in fluid thioglycollate broth: 3. Be prepared to identify an organism as a strict (obligate) aerobe, strict (obligate) anaerobe, or facultative when grown on Brewer plates under different conditions: -­‐ Obligate anaerobes will grow on this agar in anaerobic conditions, obligate aerobes will not. Conversely, obligate anaerobes will not grow on this agar if placed in an aerobic environment and obligate aerobes will. Facultative organisms can grow under both conditions. *4. Know that the Clostridium genus are obligate anaerobes and B. subtilis is an obligate aerobe. 5. Understand the purpose and function of the following: -­‐ Fluid Thioglycollate Tubes: Thioglycollate broth (Fluid Thioglycollate Medium) is a medium designed to test the aerotolerance of bacteria. It contains the dye resazurin, which is an indicator for the presence of oxygen. In the presence of oxygen the dye becomes pink. Since the oxygen tension is always higher near the surface of the medium, the medium will be pink at the top and colorless in the middle and bottom. The medium also contains a small amount of agar, which helps to localize the organism and favors anaerobiasis in the bottom of the tube. Obligate anaerobes will only grow in the lower areas of the tube. Microaerophiles will grow in a thin layer below the richly-­‐oxygenated layer. Aerotolerant anaerobes can grow throughout the medium but will primarily grow in the middle of the tube, between the oxygen-­‐rich and oxygen-­‐free zones. Facultative anaerobes will grow throughout the tube as well but will a little more growth near the top where it is oxygen-­‐rich. -­‐ (Gas Pack) Anaerobic Jar: Provides an oxygen-­‐free incubation environment for petri plates of anaerobic agar. Hydrogen is generated in the jar, which removes the oxygen by forming water. Palladium pellets catalyze the reaction at room temperature. The generation of hydrogen is achieved by adding water to a plastic envelope of chemicals. CO2 is also produced, which is a requirement for the growth of many fastidious bacteria. A Methylene blue strip is placed in the jar to be sure an anaerobic condition exists. Methylene blue is blue in the presence of oxygen but is colorless in an anaerobic environment. When the oxygen is converted to water and condensation forms on the side of the jar, the indicator strip will turn from blue to white. -­‐ Brewer Plates: (anaerobic agar) This is a medium that is excellent for cultivating anaerobic bacteria in petri dishes. It contains thioglycollate (reducing agent) and resazurin (an oxidation/reduction indicator). For strict anaerobic growth, it is essential that plates be incubated in an oxygen-­‐free environment as seen above in the anaerobic jar. 6. Know the different atmospheric conditions needed by the following organisms: -­‐ Obligate aerobe: Must grow in oxygen because their metabolism requires it. -­‐ Obligate anaerobe: Cannot tolerate oxygen and must be grown under conditions in which oxygen is completely eliminated. -­‐ Facultative aerobe (anaerobe): These bacteria grow very well in the presence of oxygen but also have the capacity to grow in anaerobic conditions when oxygen is not available. -­‐ Microaerophile: These bacteria prefer to grow in low oxygen concentrations of 2-­‐10% as opposed to 20% found in the atmosphere. 7. Be able to name the causative anaerobic organism behind the following: -­‐ Botulism – Clostridium botulinum -­‐ Gas gangrene – Clostridium perfringens -­‐ Tetanus – Clostridium tetani Exercise 24 (Killing by UV light) 1. Be able to differentiate between the following: a). Ionizing radiation: -­‐ Gamma rays, x-­‐rays, high energy electron beams. -­‐ creates free radicals, which are toxic and can damage DNA. -­‐ can sterilize. -­‐ penetration capabilities. b). Nonionizing radiation: -­‐ Ultraviolet (UV) radiation. -­‐ damages DNA; cause pyrimidine dimers. -­‐ cannot penetrate; only effective on surfaces, in air, and in water. 2. UV light damages DNA by causing the formation of thymine or cytosine dimers (bonds between two adjacent pyrimidines in the DNA strand). These can lead to mutations. Exposure to large amounts of UV light will cause more damage than cell systems can repair, leading to cell death. UV light, however, is limited as a sterilizing method because of its poor penetrating ability. Because of this, it is mainly used to kill microbes on surfaces, in air, or in water. 3. Understand why differences in survival occur between spore formers and non-­‐spore formers: -­‐ Spore forming microorganism are generally more resistant to methods of destruction because the endospores they produce are metabolically inactive, extremely well protected and are capable of withstanding prolonged periods of unfavorable conditions. *The figure above shows the white bacteria on the left, Bacillus cereus (spore former), to be more resistant than its non-­‐spore forming partner on the right. Exercise 4 (Microbial Phototrophs: Algae and Cyanobacteria) 1. Know the basic cell structure of cyanobacteria: -­‐ Prokaryotic; no nucleus or chloroplasts; have chlorophyll. 2. Be prepared to identify and name the following: -­‐ Anabaena: Filaments of round cells. -­‐ Oscillatoria: Filaments of disc-­‐shaped cells. 3. Know the basic cell structure of algae: -­‐ Eukaryotes; contain a nucleus and chloroplasts. *4. Marine algae produce majority of atmospheric oxygen. 5. Be prepared to identify and name the following: -­‐ Spirogyra: Filamentous green algae; spiral chloroplasts. -­‐ Chlamydomonas: Unicellular oval green algae; two flagella. -­‐ Diatoms: Rigid, silica, cell wall; usually unicellular; near bilateral symmetry; geometric shapes; common phytoplankton. -­‐ Dinoflagellates: Unicellular; flagella; cellulose plates (thecae); can cause algal blooms and release toxins (red tide); can bioluminesce (appear to be glowing). bioluminescence red tide Exercise 6 (Fungi) 1. Be familiar with some of the beneficial roles of fungi: -­‐ Bread, cheese, mushrooms, alcohol, antibiotics, decomposition. 2. Know the cellular characteristics of organisms in kingdom fungi: -­‐ Eukaryotes; chitin cell wall; usually multicellular; heterotrophs – usually saprobes, feed on dead or decaying organic matter. 3. Know what is meant by dimorphic growth: dimorphism literally means occurring in two forms: In fungi, it is usually used to describe the ability to exist as a filamentous (mycelial) form, or as a unicellular yeast form and to switch between these growth habits. 4. Be prepared to identify the various structures of fungi to include: -­‐ Hypha [septate(left) and non-­‐septate(right)]: -­‐ Mycelium (the vegetative part of a fungus, consisting of a mass of branching, thread-­‐like hyphae): 5. Know the feeding strategy of fungi and have an understanding of their importance in recycling nutrients: -­‐ Fungi are usually saprobes, which means they feed on dead, decaying organic matter. They do this by secreting digestive enzymes into their surrounding environment. These enzymes break down or digest macromolecules. These smaller digested components can then diffuse more quickly across the membrane. Their ability to decompose complex macromolecules like lignin and cellulose (plant cell wall components) and chitin (insect skeletal material) makes the fungi important in recycling nutrients within ecosystems. 6. Know the different reproductive methods of each of the fungal organisms mentioned: -­‐ Rhizopus stolonifer: Reproduce by forming asexual and sexual spores. -­‐ Penicillium: Reproduces asexually using spores. -­‐ Aspergillus: Reproduce both sexually and asexually. -­‐ Candida albicans: Reproduce asexually through budding. -­‐ Saccharomyces cerevisiae: Usually reproduces asexually by budding, but can reproduce sexually (by sporulation). 7. Be prepared to differentiate between Candida albicans and Saccharomyces cerevisiae: C. albicans: -­‐ normal (resident) human biota, but -­‐ can cause disease in humans (yeast infection, thrush). -­‐ displays dimorphic growth. -­‐ is not used to make beer. S. cerevisiae: -­‐ can be used to make beer and in baking (Brewer’s/baker’s yeast). -­‐ unicellular; does not display dimorphic growth. 8. Be prepared to identify the following organisms, their spores, and be able to name their phylum: -­‐ Rhizopus stolonifer: (Black bread mold) Phylum: Zygomycota -­‐ Penicillium: (Common bread mold) Phylum: Ascomycota -­‐ Aspergillus: (mold, often cause mildew) Phylum: Ascomycota. -­‐ Candida albicans: (oral thrush, yeast infection; dimorphic) Phylum: Ascomycota -­‐ Saccharomyces cerevisiae: (brewer’s/baker’s yeast; unicellular) Phylum: Ascomycota