Microbiology Lab Review Questions with Certified Answers

Microbiology Lab Review Questions with Certified Answers Explain why the body tube of the microscope should not be lowered while you are looking through the ocular lens. to ensure that the slide or lens is not broken For what purpose would you adjust the Iris diaphragm? adjust the amount of light coming through the specimen For what purpose would you adjust the course-adjustment knob? to bring the specimen into view For what purpose would you adjust the fine adjustment knob? to bring the specimen into sharp focus For what purpose would you adjust the condenser? to direct the light from the light source into the lens system For what purpose would you adjust the mechanical stage? to adjust the position of the slide on the stage Describe the steps you would take to correct the problem while using the oil immersion lens: Inability to bring the specimen into sharp focus repeat the procedure for focusing under oil immersion and make sure to find the fine adjustment midpoint for each lens and not to put too little or too much oil Describe the steps you would take to correct the problem while using the oil immersion lens: Insufficient light while viewing the specimen raise the condenser and open the iris diaphragm Describe the steps you would take to correct the problem while using the oil immersion lens: Artifacts in the microscopic field clean the lens and slide with lens cleaner and paper Explain why the following step is essential during subculturing: Flaming the inoculating instrument prior to and after each inoculation prior: to prevent contamination of stock culture after: to prevent contamination of the laboratory table Explain why the following step is essential during subculturing: Holding the test tube caps i the hand as illustrated in Fig. 2.1 on pg. 14 to maintain their sterility. never set them down. Explain why the following step is essential during subculturing: cooling the inoculating instrument prior to obtaining the inoculum to prevent killing the cells with the heat Explain why the following step is essential during subculturing: Flaming the neck of the tubes immediately after uncapping and before recapping to kill any organisms that may be present on the neck Describe the purposes of the subculturing procedure. for transfer from one medium to another, preparation/maintenance of stock cultures. Explain why a straight inoculating needle is used to inoculate an agar deep tube. to make a straight line from the bottom of the tube to the top to maintain the redox potential of the medium There is a lack of orange-red pigmentation in some of the growth on your agar slant labeled S. marcescens. Does this necessarily indicate the presence of a contaminant? Explain. not necessarily, this organisms can produce variants in pigment and rate of pigment production. Upon observation of the nutrient agar slant culture, you strongly suspect that the culture is contaminated. outline the method you would follow to ascertain whether your suspicion is justified. make comparisons using gram stains and streak plates of each. Can a pure culture be prepared from a mixed-broth or mixed-agar-slant culture? yes, only after performing a streak or spread plate inoculation for isolation of discrete colonies Observation of a streak-plate culture shows more growth in Quadrant 4 than in Quadrant 3. Account for this observation. either the inoculating wire was repeatedly dragged through quadrant 3 or, moe likely, it entered Quadrant 1. Why is a needle used to isolate individual colonies from a spread plate or streak plate? it is thin enough to touch only one colony How can you determine if the colony that you chose to isolate is a pure culture? subculturing the isolate and gram staining Why are thick or dense smears less likely to provide a good smear preparation for microscopic evaluation? light passing through is diminished and cells are clumped making distinguishing morphology difficult Why is it essential that smears be air-dried? Why can't they be gently heated over a flame to speed us the drying process? air drying prevent the cells from shrinkage and distortion, thereby protecting their size and shape Why should you be careful not to overheat the smear during the heat-fixing process? excessive heating may distort the morphology, causing plasmolysis of the cell wall. Why do you thin the presence of grease or dirt on a glass slide will result in a poor smear preparation? (2-3 reasons) 1. may interfere with adherence to slide 2. artifacts may confuse the observer as to what is an organism and what is not Why are basic dyes more effective for bacterial staining than acidic dyes? basic dyes are positively charged which has an affinity for the negatively charged organism Can simple staining techniques be used to identify more than the morphological characteristics of microorganisms? Explain. no, simple staining can only be used to determine cell morphology. Any structural components are to small to be seen using a simple light microscope During the performance of the simple staining procedure, you failed to heat fix your E. Coli smear preparation. Upon microscopic examination, how would you expect this slide to differ from the correctly prepared slides? The organism would have been washed away during the staining process so little to no organisms could be observed. During a coffee break, your friend spills coffee on your lab coat and the fabric is discolored. Is this a true biological stain or simply a compound capable of imparting color? Explain your rationale. a coffee is not a permanent stain because it lacks the autochrome component and ionization cannot occur. Why can't methylene blue be used in place of nigrosin for negative staining? methylene blue is positively charged and you need a negatively charged stain as to not bind to the cell What are the practical advantages of negative staining? -does not kill the bacteria -no heat fixation so no distortion -bacteria that cannot be stained can be viewed Why doesn't nigrosin penetrate material cells? they are both negatively charged so they have no affinity for each other What are the advantages of differential staining procedures over the simple staining technique? simple staining uses a single dye and stains all cell components the same color. Differential staining uses two contrasting stains for separation of gram negative and gram positive bacteria what is the purpose of the following reagent in a differential staining procedure: Primary stain first stain and imparts color to all cells what is the purpose of the following reagent in a differential staining procedure: Mordant intensifies gram stain what is the purpose of the following reagent in a differential staining procedure: Decolorizing agent removes primary stains only from some cell types/structures what is the purpose of the following reagent in a differential staining procedure: Counterstain second stain of contrasting color absorbed only by the decolorized cells Why is it essential that the primary stain and the counterstain be of contrasting colors? it allows for differentiation between gram negative and gram positive cells Which is the most crucial step in the performance of the gram staining procedures? Explain. Decolorization, because it allows for the declaring of the cells that are gram negative so they can be counterstained. without it, all cells would be purple and distinctions could not be made Because of a snowstorm, your regular laboratory session was canceled and the Gram staining procedure was performed on cultures incubated for a longer period of time. Examination of the stained B. cereus slides revealed a great deal of color variability, ranging from an intense blue to shades of pink. Account for this result. as age of an organism increases, its ability to hold stain decreases Why must heat or surface-active agent be used with application of the primary stain during acid-fast staining? heat is required to soften the waxy cell wall components to facilitate the penetration of the primary stain into the cells Why is acid-alcohol rather than ethyl alcohol used as an decolorizing agent? to ensure that the primary stain is removed from the non-acid-fast organisms What is the specific diagnostic value of the acid-fast staining procedure? used to diagnose leprosy and tuberculosis (both Mycobacterium) Why is the application of heat or a surface-active agent not required during the application f the counterstain in acid-fast staining? the non-acid-fast cells readily accept the counterstain without the need for heat A child presents symptoms suggestive of tuberculosis, namely a respiratory infection with a productive cough. Microscopic examination of gastric washings reveals the presence of both acid-fast and non-acid-fast bacilli. Do you think the child has active tuberculosis? Yes, because it can be detected in the stomach because the acid-fast organisms would survive. Why is heat necessary in spore staining? because of the impervious nature of the protein spore coats, the stain-covered smear is heated to ensure (by softening) penetration of the stain into the spore Explain the function of water in spore staining. to remove primary stain without washing off the spores stain In the following case, indicate how your microscopic observations would differ from those observed when the slides were prepared correctly: You used acid-alcohol as the decolorizing agent the acid-alcohol would not decolorize the spore so the observations would be the same In the following case, indicate how your microscopic observations would differ from those observed when the slides were prepared correctly: You used safranin as the primary stain and malachite green as the counterstain Safranin cannot be washed out with water so both the spore and the cells would be red. In the following case, indicate how your microscopic observations would differ from those observed when the slides were prepared correctly: You did not apply heat during the application of the primary stain. the spores would not take up the stain so they would remain colorless Explain the medical significance of a capsule white blood cells won't kill the bacteria, increasing its virulence Explain the Function of copper sulfate in the procedure. It decolorizes the capsule while being absorbed into the capsule going it a light blue color in contrast to the deep purple color of the cell Fastidious definition: organism that has complex nutritional requirements Why did the most fastidious organism grow poorly in the chemically defined medium? chemically defined media contains little/no organic substance and fastidious organisms require growth supporting substances, enrichments, vitamins, or blood. Explain the advantages of using A readings rather than percent T as a means of estimating microbial growth. A is directly related to the amount of microbial growth and T is inversely related to microbial growth Explain the reason for the use of different medium blanks in adjusting get spectrophotometer prior to obtaining A readings. The medium itself will have an absorbency reading and the blank eliminates the reading of the mediums absorbency, only taking the growths absorbency Why are complex media preferable to chemically defined media for routine cultivation of microorganisms? most microorganisms nutritional growth requirements are encompassed in complex media and the specific nutritional needs of the organism are not needed Would you expect a heterotrophic organism to grow in an inorganic synthetic medium? Explain. No, heterotrophic organisms require the use of organic carbon sources and other organic chemicals which inorganic synthetic medium does not provide A soil is found to grow poorly in a basic artificial medium. You suspect a vitamin supplement is required: What supplement would you used to enrich the medium to support and maintain the growth of the organisms? Explain. yeast extract, because it has all the B Vitamins Outline the procedure you would follow to determine the specific vitamins required by the organism to produce a more abundant growth you should perform a vitamin assay by growing the organism on various plates each lacking a certain vitamin. Whichever does not grow is the essential vitamin. What is the specific selective and/or differential purpose of the following media: Phenylethyl alcohol agar phenyl ethyl alcohol partially inhibits growth of gram-negative organisms, selecting for gram-positive organisms What is the specific selective and/or differential purpose of the following media: Crystal violet agar crystal violet inhibits growth of gram positive organisms, selecting for gram-negative organisms What is the specific selective and/or differential purpose of the following media: 7.5% sodium chloride agar selection and differentiation of of halophilic (salt loving) organisms and members of the staphylococcus genus What is the specific selective and/or differential purpose of the following media: Mannitol salt agar high salt concentration inhibits growth of all organisms except for halophiles, selecting for halophiles What is the specific selective and/or differential purpose of the following media: MacConkey agar selective for gram negative bacteria and the lactose serves to differentiate between lactose fermenters and nonfermenters on their ability to produce acid. What is the specific selective and/or differential purpose of the following media: Eosin-methylene blue agar Eosin-methylene blue is selective for E. Coli (shiny green layer). also partially inhibits growth of gram-positive organisms. What is the specific selective and/or differential purpose of the following media: Blood agar blood serves to support the growth of fastidious organisms and to differentiate microorganisms, particularly streptococcal special, on the basis of their hemolytic activities Why are crystal violet agar and 7.5% sodium chloride agar considered selective media? they inhibit the growth of all organisms except a certain classification. -Crystal violet: selects for gram-negative -7.5% selects for halophiles and staphylococcus A patient exhibits a boil on his neck. You, as a microbiology technician, are asked to identify the causative organism and determine whether it is pathogenic. Describe the procedure that you would follow to make this determination. a boil is usually a staphylococcal or streptococcal bacteria so plating the organism on mannitol salt agar would allow for the identification because only staphylococcus will grow. Distinguish between respiration and fermentation. cellular respiration is biooxidative process that occurs aerobically, with molecular oxygen serving as the final electron acceptor, or anaerobically with an inorganic ion acting as a final electron acceptor. Fermentation is a bioxidation that utilizes an organic compound as the final electron acceptor. Do all microorganisms use pyruvic acid in the same way? Explain. No, some use it as a final electron acceptor, and others use it as a stepping stone into the revs cycle for further ATP production. Describe a pathway used for the degradation of carbohydrates by strict anaerobes the Embden-Meyerhof glycolytic pathway to pyretic acid with limited ATP production. the pyruvate is then further metabolized through germinative pathways. From you experimental data, you know that P. aeruginosa did not utilize any of the carbohydrates in the test media. In view of this, how do these organisms generate energy to sustain their viability? they hydrolyze proteins to amino acids that then enter the revs cycle for generation of ATP. Clostridium perfingens, an obligate anaerobe, is capable of utilizing the carbohydrates released from injured tissues as an energy source. During the infectious process, large amounts of gas accumulate in the infected tissues. Would you expect this gas to be CO2? Explain. no, because they utilize the carbohydrates anaerobically so no CO2 would be produced. what is the purpose of the TSI test? to distinguish enterobacteria from other gram negative bacteria and to differentiate the different types of enterobacteriaceae. Explain why the TSI medium contains a lower concentration of glucose than of lactose and sucrose. this allows for detection of the use of glucose only Explain the purpose of the phenol red in the medium This allows fro detect of acidic end products from carbohydrate fermentation Explain the purpose of thiosulfate in the medium thiosulfate act a substrate for hydrogen sulfide (H2S) production Explain why the test observations must be made between 18 and 24 hours after inoculation. to prevent the breakdown of proteins in the medium which would result in the formation of end product that would obscure the observation results