Staining Techniques in Microbiology, Exams of Microbiology

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Microbiology 250 Lab Practical Exam Questions and Complete Solutions Graded A+ Denning [Date] [Course title]

brom cresol purple - Answer: indicator in MIO media that tests for decarboxylation of ornithine positive result is a lavender-purple color A hydrogen peroxide - Answer: H2O used to test for organism's possession of catalase copious amounts of bubbles are evidence for release of O2 as peroxide is broken down 2H2O2 + O iodine - Answer: indicates the presence or absence of starch agar - Answer: solidifying agent purified from marine algae colonial morphology - Answer: size, color, shape, elevation, consistency, form, margin, surface, optical characteristics should only be observed on isolated colonies YA10SC - Answer: yeast agar loaded with 10% sucrose (table sugar) encourages the growth of streptococcus organisms such as Streptococcus salivarius and Streptococcus mutans dental caries - Answer: result of bacterial fermentation of sugar, commonly sucrose

primary shapes of bacteria - Answer: rod - Bacillus sphere - coccus spiral - Spirillum parfocal - Answer: property of microscope which allows objectives to be changed without having to refocus refraction - Answer: the bending of light as it passes from one material to another immersion oil - Answer: addition of this minimizes the amount of light that is refracted or lost size of coccus cell - Answer: about 1 micrometer on 100x size of bacillus cell - Answer: about 4 micrometers on 100x size of spiral cell - Answer: about 15 micrometers on 100x size of average bacteria - Answer: 0.2 micrometers to 3.0 micrometers can reach a length of 10 micrometers arrangements of bacilli - Answer: singles, diplo, strepto, palisades arrangements of cocci - Answer: singles, diplo, strepto, tetrads, sarcinae, staphylo arrangements of spiral - Answer: vibrio, spirilla, spirochete (axial filament), stella, cubic, coccobacillus

how to prep an organism for staining - Answer: also known as a smear organism must be placed on a slide, spread out and fixed to the slide so it doesn't wash off heat fix stain composition - Answer: made from dyes and categorized as acidic, basic, or natural stain depends on which ion of the dye (cation or anion) carries the stain or color chromophore - Answer: color bearing ion basic stains - Answer: most stains used for microbiology color is carried on positively charged ion (cation) bacteria are slightly negatively charged so there is an attraction between the cationic chromophores and the organism simple stain - Answer: application of a single staining solution to make bacteria more visible quick and easy reveals basic information only - size, shape, arrangement dyes used in simple stain - Answer: carbol fuchsin - let sit for 15 seconds crystal violet - let sit for 1 minute

provides accurate view of cell and clearer picture of size, shape, and arrangement broth culture is a good choice to use gram stain - Answer: most widely used procedure for characterizing bacteria differential stain - positive and negative gram stain classifies gram-positive - Answer: occurs if organism retains primary or initial stain (crystal violet) and remains purple gram stain classifies gram-negative - Answer: occurs if primary stain is lost after an alcohol decolorization step because decolorized preparation will accept the pink counterstain (safranin) determining factor of gram stain - Answer: believed to be the cell wall crystal violet complexes with iodine and the chemical and structural nature of cell wall determines whether the complex will be washed out with alcohol or if it remains difficulties with gram stain - Answer: old cultures (more than 1-2 days) of gram+ may appear gram- due to loss of integrity of cell wall thick smears of gram- organisms may appear gram+ thin smears of gram+ may appear gram- some organisms due to their cell wall do not take up the dye under and over colorization may lead to erroneous results

Gram's iodine - Answer: to observe the results on starch agar plate, the plate must be flooded with... 95% alcohol - Answer: decolorization step in the gram stain helps detect gram- result by "erasing" primary stain acid-fast stain - Answer: differential stain used to identify organisms of the genus Mycobacterium (specifically Mycobacterium tuberculosis) cell wall of Mycobacterium - Answer: bacteria contains high levels of lipid material (40-60%) in their cell walls, specifically mycolic acid waxy nature of this cell wall resists penetration by conventional staining techniques moist heat must be applied to penetrate the dye into the cells result of Mycobacterium accepting carbol fuchsin - Answer: will retain the red dye even when decolorized with acid alcohol 95% alcohol with 3% HCl added yields a positive acid-fast result result of methylene blue in acid-fast stain - Answer: after counter-staining with this dye acid-fast organisms remain red and non-acid-fast cells stain blue acid alcohol - Answer: 95% alcohol with 3% HCl added methylene blue - Answer: dye used as a counter-stain in acid-fast stain technique Mycobacterium characteristics - Answer: typically small branched or fragmented rods

vegetative cells release initial stain with washing and appear pink as a result of staining with safranin malachite green - Answer: In order for the stain to penetrate the impervious coat of the spore, which primary stain is steamed into the cell surface? endospores under microscope - Answer: bright green ovals either free or within a red vegetative cell detection of motility - Answer: precipitating dyes on flagella to increase their diameter presence of flagella, number, and location on bacterial cell can be determined flagella - Answer: bacteria that are motile possess fine hair-like appendages called... hanging drop - Answer: suspend the organism in a drop of water and determining microscopically if they possess true directional movement or Brownian movement Brownian movement - Answer: random jiggling and bumping movements observed after preparing a hanging drop stab line motility - Answer: inoculating organism into a tube of motility testing medium to determine if organism has migrated or spread from the line isolation by streak plate method - Answer: streaking a dilute sample from a mixed culture onto an agar plate in order to obtain single isolated colonies after appropriate incubation pure culture - Answer: all organisms in the culture are of the same species, derived from one cell cfu - Answer: colony forming unit colonies arose due to numerous divisions of a single original cell

colonial morphology size - Answer: record diameter of a well-isolated colony in millimeters measure colony from bottom of plate colonial morphology form - Answer: overall shape of colony circular, irregular, spindle, filamentous, rhizoid colonial morphology elevation - Answer: flat, raised, convex, domed, conical, umbonate colonial morphology margin - Answer: entire, undulate, lobate, filamentous colonial morphology colony surface - Answer: smooth, rough, rugose (wrinkled), contoured (irregular, smoothly undulating surface), granular (fine, medium, coarse) colonial morphology optical characteristics - Answer: opaque (doesn't allow light to pass through) translucent (allowing light to pass through without allowing complete visibility of objects seen through the colony) transparent (allowing complete visibility of objects seen through the colony opalescent (resembling the color of an opal), iridescent (exhibiting changing rainbow colors in reflected light) dull, shiny, glistening colony morphology pigmentation - Answer: note where the pigment diffuses into the medium around the colony or if it is restricted to the cells themselves

facultative anaerobe - Answer: organisms that grow under either aerobic or anaerobic conditions methods for attaining anaerobic culture - Answer: mechanical removal of oxygen chemical removal of oxygen use of reducing compounds mechanical removal of oxygen - Answer: mechanically pumping oxygen out of a closed container and replacing it with oxygen free gas (N2 or CO2) before placing culture in the container chemical removal of oxygen - Answer: combining oxygen in the system into some other compound so it is no longer available in the free state use of reducing compounds - Answer: preparing medium with a reducing agent added to lower oxygen content of the system medium deteriorates with time so a colored indicator like resazurin is added to detect presence of oxygen (sodium thioglycollate) sodium thioglycollate - Answer: found in reducing media, reduces the oxidation-reduction reduction potential for the medium Gaspak - Answer: in an anaerobic jar, the _____ is used to grow anaerobic organisms catalase test - Answer: based on the ability of the organism to break down toxic peroxides that may accumulate when growth occurs in the presence of oxygen if an organism can break down peroxides, it must possess this enzyme

positive catalase test - Answer: if H2O2 (peroxide) is placed on a slide and copious amounts of bubbles are produced evidence is release of oxygen as the peroxide is broken down enriched media - Answer: composed of a nutrient base to which rich nutrients such as blood serum or plant or animal tissue extracts have been added will enable even fastidious heterotrophs to grow selective media - Answer: have certain chemical substances incorporated into the agar or specifically lacking from the agar that selectively inhibit growth of one specific group of bacteria without inhibiting the growth of other types differential media - Answer: have certain reagents or chemicals incorporated into the agar that may result in apparent and observable differences in the appearance of the growth of one bacterial type as compared to others control media - Answer: nutrient agar plate is provided to use as a comparison of typical morphology and growth rates of the organisms nutrient agar - Answer: serves as a control media most organisms grow well on this media helps to study typical growth rates and morphology PEA - Answer: phenylethyl alcohol agar no pH indicator selective

no change EMB - Answer: eosin methylene blue selects for gram- bacteria inhibits gram positive organisms bile salts dyes - eosin and methylene blue differential for fermentation of lactose if positive colonies appear dark, metallic green, or pink large amounts of acid: green metallic sheen on colony surface less acid: pink coloration if negative: colonies appear colorless Escherichia coli: dark with green metallic sheen enterobacter aerongenes: dark centered colonies/look like fish eyes useful for water testing-differential for lactose coliforms

lactose fermentation on EMB plate - Answer: if this occurs acid is produced and colonies turn dark purple as acid acts upon dyes coliforms - Answer: gram- rods that are facultative anaerobes and ferment lactose to acid and gas, non endospore forming most frequently used indicator organism MacConkey - Answer: selects for gram negative bacteria bile salts and crystal violet both inhibit gram+ bacteria lactose is the only carbohydrate in the media selective and differential differentiates lactose fermenters MacConkey indicating acid production - Answer: pH indicator neutral red below pH 6.8 is red lactose fermenting bacteria on MacConkey media - Answer: bacteria appear pink non-lactose fermenting bacteria appear colorless mannitol salt - Answer: selective and differential media contains 7.5% NaCl

will glow under UV light pGLO system - Answer: illustrates bacterial transformation, use of an antibiotic selective medium to identify transformed cells operon as a mechanism of microbial genetic regulation bacterial transformation - Answer: process by which competent bacterial cells pick up DNA from the environment and make use of the gene it carries only occurs naturally in certain genera (ex. Pneumococcus) can occur artificially competent cells useful for genetic engineering green fluorescent protein - Answer: GFP responsible for bioluminescence in the jellyfish Aequorea victoria fluoresces more than the natural vision indicator of gene transcription and cell transformation in our experiment competent E. coli cells - Answer: introduce GFP gene into these and use the ability of the cell to fluoresce as visual evidence of successful transformation and subsequent gene expression operons - Answer: structural and functional genetic units of prokaryotes carries genes for enzymes in the same metabolic pathway

each minimally contains a promoter site and two or more structure genes promoter site - Answer: component of operon for binding RNA polymerase structural genes - Answer: component of operon coding for enzymes in the same metabolic pathway arabinose operon - Answer: consists of promoter Pbad and 3 structural genes araB, araA, and araD araB, araA, araD - Answer: code for enzymes used in arabinose digestion in vivo arabinose - Answer: enzymes are needed only when the substrate is present araC - Answer: DNA binding protein attaches to the promoter of arabinose operon and acts like a switch arabinose binds to a receptor on this and causes it to change to a shape that allows the DNA binding to occur operons with genes for catabolic enzymes - Answer: transcribed only when a specific substrate is present (ex. sugar arabinose) if substrate is not present RNA polymerase is unable to bind to the promoter and begin transcription of the genes what occurs when arabinose binds to a receptor on araC - Answer: transcription of the structural genes occurs, enzymes are produced, and arabinose is catabolized pGLO plasmid - Answer: has been engineered to contain the arabinose promoter and the gene for GFP instead of the genes for arabinose catabolism