Microbiology Lab Midterm Test Questions – Flashcards
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| Endo Agar |
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| Used to detect fecal contamination in water and dairy Contains color indicators sodium sulfite and basic fuchsin, which also act as gram positive inhibitors |
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| Lactose Fermenters on Endo Agar |
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| They will appear red or pink and darken the medium This is due to a reaction with sodium sulfite with fermentation intermediate acetaldehyde |
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| Lactose Non Fermenters |
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| will produce colorless to slightly pink growth |
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| Metallic Sheen on Endo Agar |
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| is produced due to large amounts of acid from lactose fermenters like Escherichia coli and Klebsiella pneumoniae |
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| Eosin Methylene Blue Agar (EMB) |
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| Used for isolation of fecal coliforms Contains sugars to encourage growth of fecal coliforms Inhibit growth of Gram-positive organisms |
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| EMB agar under acidic conditions |
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| form into dark purple, a long with a green metallic sheen possibly |
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| Green metallic sheen on EMB agar |
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| indicates vigorous lactose or sucrose fermentation |
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| Pink Coloration on EMB plate |
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| means that there only was small amounts of acid production |
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| Non fermenters on EMB plate |
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| Non fermenters remain their normal color or take on coloration of the medium |
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| Hektoen Enteric Agar |
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| used to isolate and differentiate Salmonella and Shigella species from other gram negative enteric organisms |
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| Enteric |
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| gram negative rod shaped bacteria |
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| Enterics that produce acid from fermentation on Hektoen Enteric Agar |
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| Usually produce yellow to salmon pink colonies. |
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| Organisms that don't ferment Sugars on Hektoen Enteric Agar |
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| produce blue-green colonies, examples are Salmonella, Shigella, and Proteus Proteus and Salmonella specied that reduce sulfur to H2S form colonies containing a black precipitate. |
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| MacConkey Agar |
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| used to isolate and differentiate members of the Enterobaceriaceae based on the ability to ferment lactose Contains bile salts and crystal violet inhibit growth of Gram-positive bacteria |
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| Lactose fermenters vs. non fermenters on MacConkey Agar |
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| Neutral Red Dye is used and is colorless at pH over 6.8 and red at pH under 6.8 Acid accumulating from lactose fermenters turn a shade of red on the agar while the non fermenters remain their normal color or the color of the medium. Formulations without crystal violet allow growth of Eneterococus and some species of Staphylococcus, which ferment lactose and appear pink on the agar |
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| Mannitol Salt Agar (MSA) |
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| Used for isolation and differentation of pathogenic staphylococci, principally S. aureus Contains carbohydrate mannitol, NaCl, and phenol red the NaCl is used to be selective to staphylococci because they can live at high salinity. |
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| Pathogenic staphylcoccus v nonpathogenic staphylcoccus on MSA |
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| Pathogenic ones will turn the indicator yellow, forming a yellow halo around the growth. Non pathogenic ones will have good growth with no color change. |
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| Phenylethyl Alcohol Agar (PEA) |
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| used to isolate staphylococci and streptococci from specimens containing mixtures of bacterial flora. esp. containing E.coli or Proteus. It is a selective media that allows growth of gram positive organisms and stops or inhibits growth of most gram negative organisms. This is due to its active ingredient phenylethyl alcohol, which interferes with DNA synthesis in gram negative species |
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| Chromogen |
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| a colored molecule in the solvent of stain solutions. Chromophore is the portion of chromogen that gives it its color. It may have multiple chromophores, each adding intensity to the color |
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| Auxochrome |
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| the charged portion of a chomogen that allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell |
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| Basic stains |
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| are the result of the auxochrome becoming positively charged by picking up a hydrogen ion or losing a hydroxide ion. It then becomes attracted to the negative charges on the surface of most bacterial cells making them colored. Examples are methylene blue, cyrstal violet, and safranin |
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| Negative Stain |
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| used to determine the morphology and cellular morphology and cellular arrangement in bacteria that are too delicate to withstand heat fixing. It is also most helpful for accurate size determination because it produces minimal cell shrinkage |
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| How negative stains work |
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| the chromogen in the dye solution is acidic and carries a negative charge. The negative charge on the bacterial surface repels the negatively charged chromagen, so the cell remains unstained against a colored background |
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| Cell Morphology |
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| Main: cocci-spheres bacilli- rodss spirilla- spirals Variations: Vibrios- curved rods coccobacilli- short rods spirochetes- flexible spirals |
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| Cell Arrangement |
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| diplococcus or diplobacilli- single division where daughter cells remain attached Streptococcus or streptobacilli- remain in same plane and remain attached to form chains of variable length tetrad- there are divisions at two perpendicular planes. Sarcina- divide in three perpendicular planes to produce a cube arrangement Staphylococcus- staphylococci divide in omre than three planes to produce a characteristic grapelike cluster of cells |
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| Gram Stain |
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| used to distinguish between gram pos and gram neg. The primary stain is crystal violet which stains both gram pos and gram neg bacteria. Gram's iodine is added as a mordant to enhance the crystal violet stain Decolorization occurs with the use of alcohol or acetone, only Gram negative cells are decolorized, gram positive cells are not. Safranin is used as a counterstain so that the gram negative cells can be colorized. |
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| Why gram negative becomes decolorized? |
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| The higher lipid content in the outer membrane of gram negative cells gets extracted from the alcohol, making the wall more porous and incapable of retaining the dye Gram positive cells have thicker peptidoglycan and greater degree of cross linking that allows the stain to stay on more effectively. Leaving alcohol on too long will over decolorize causing reddish gram positive, or not leaving it on enough will under colorize causing purple gram negative cells. |
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| Endospore Stain |
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| used as a differential stain to detect presence and location of spores in bacterial cells |
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| Schaeffer Fulton Endospore Stain method |
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| Malachite green is the primary stain, which is forced into the spore by steaming the bacteria. Because the malachite green is water soluble it has a low affinity for cellular material, so the vegetative cells and mother cells can be decolorized with water, and counterstained with Safranin. All cells will show up pink or red, and the spores will be green. The bacteria is not spore forming unless you can find the spores inside the cell |
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| Flagella stain |
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| allows for direct observation of flagella. |
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| Types of flagella |
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| Monotrichous- has a single flagellum and is said to be polar Amphitrichous- flagella at both ends of cell Lophotrichous- tufts of flagella at the end of the cell Peritrichous- flagella emerging from the entire cell surface |
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| Wet Mount vs. Hanging Drop |
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| They are both used to test for motility Both at low light because no stain is used and most bacteria are transparent. For wet mount it dries faster. Hanging drop allows for longer observation |
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| Brownian motion |
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| motion created by collisions with water molecules, does not show true motility in bacteria. They seem to vibrate in place |
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| Bacitracin Susceptibility Test |
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| used to differentiate between beta-hemolytic group A streptococci from other beta-hemolytic steptococci. Similar test can be done with other antibiotics like the Noviobiocin test we did. The antibiotic diffuses into the agar and inhibits growth of susceptible bacteria |
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| Blood Agar |
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| used for isolation and cultivation of many types of fastidious bacteria. Also distinguishes between hemolytic charcteristics esp. for streptococcus, enterococcus, and aerococcus. Several gram positive cocci species produce exotoxins called hemolysins able to destroy RBCs and hemoglobin |
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| Different types of hemolysins |
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| Beta-hemolysis- is complete destruction of RBCs and hemoglobin, and results in clearing medium around the colonies Alpha-hemolysis is partial destruction of RBCs and produces a greenish decoloration of the agar around the colonies Gamma-hemolysis is actually non-hemolysis and appears as simple growth with no change to the medium |
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| Catalase Test |
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| used to identify organisms that produce the enzyme catalase. Most often used to differentiate between catalase positive Micrococcaceae and catalase negative Streptococcaceae. Catalase turns hydrogen peroxide into water and gaseous oxygen. When hydrogen peroxide is dropped on a species, if it begins to bubble (O2 formation) the test is positive for catalase |
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| Coagulase test |
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| used to differentiate Staphylococcus aureus from other Gram-positive cocci. S. aureus is an oppurtunistic pathogen that is highly resistant to normal immune response and antimicrobial agents. Part of its resistance is due to production of the coagulase enzyme. Coagulase can be either bound or free. We used the Tube test which test the presence of both by using rabbit plasma treated with anticoagulant. If the plasma thickens or form threads within 24 hours of the inoculation then the test is positive. |
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| Litmus Milk Medium |
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| used to differentiate members within the genus Clostridium. Differentiates Eneterobacteriaceae from other gram-negative bacilli based on enterics' ability to reduce litmus. |
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| Lactose Fermentation |
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| acidifies the medium and turns the litmus pink. This acid reaction begins with the splitting of the disaccharide into the monosaccharides glucose and galactose by the enzyme beta-galactosidase. Accumulating acid may form an acid clot. |
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| Litmus Mile Reaction results |
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| Pink Color- acid reaction, by splitting of disaccharide into monosaccharides glucose and galactose. PInk and solid- acid clot, from accumulating acid Fissures in clot- gas Clot broken apart- stormy fermentation White color (lower portion of medium)- reduction of litmus Semisolid and not pink; clear to gray fluid at top- Curd, from caseases in bacteria that coagulate casein. Clarification of medium; loss of "body"- Digestion of peptone; peptonization Blue medium or blue band at top- alkaline reaction |
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| Novobiocin Susceptibility Test |
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| used to differentiate coagulase-negative staphylococci. usually to identifey novobiocin-resistant staphylococcus saprophyticus. Most other staphylococcus are susceptible to novobiocin. |
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| Oxidase test |
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| Used to identify bacteria containing the respiratory enzyme cytochrome oxidase, which is responsible for the transfer of electrons to oxygen, reducing it to water. Can identify the oxidase positive Neisseria. It can also differentiate between oxidase negative Enterobacteriaceae from oxidase positive Psuedomonadeaceae A positive test comes out purple or dark blue a negative test has no color change |
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| Pseudomonas aeruginosa |
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| Negative Stain- small, clustered bacillus Gram stain- negative EMB Plate- Purple PEA Plate- limited growth MSA Plate- no growth MacConkey Plate- Yellow growth |
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| Enterococcus faecalis |
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| Negative Stain- cocci,random Catalase- negative Bact. O2- facultative anaerobe Bact. temp- similar in both incubator and room temp Hemolysis- alpha |
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| Staphylococcus citreus |
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| Motility- observed Gram Stain- positive EMB- no growth PEA- whitish/small growth MSA- yellowish growth MacConkey- no growth |
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| Proteus vulgaris |
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| Motility- motility observed Gram- negative Temp- incubator showed small growth, should grow around 23 degrees C |
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| Bacillus cereus |
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| Gram positive Endospore stain- spore forming EMB- slight growth PEA- white growth MSA- small growth, yellow MacConkey- no growth NA plate- yellow growth |
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| Streptococcus agalctine |
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| Gram positive Hemolysis- beta Litmus- on going |
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| Escherichia coli |
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| Gram negative EMB Plate- green/metallic sheen PEA growth- low growth MSA- no growth MacConkey- reddish growth |
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| Micrococcus luteus |
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| Endospore stain- none found Catalase- positive Bact. O2- obligate anaerobe |
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| Micrococcus roseus |
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| Gram positive Oxidase- positive Bact. temp- a lot of growth in incubator, yellow/green growth at RT |
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| Enterobacter cloacae |
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| Gram negative Bact. O2- obligate aerobe |
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| Serratia marscesens |
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| Gram negative Bact. temp- incubator showed a lot of growth, RT showed some |
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| Staphylococcus aureus |
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| Coagulase- positive Novobiocin- highly succeptible |
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| Staphylococcus epidermis |
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| coagulase- negative |
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| Staphylococcus saprophyticus |
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| Novobiocin- no sensitivity |
