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141 Cards in this Set
- Front
- Back
What is a Blood Agar Plate?
|
-highly nutritious differential, but not selective, medium
-usually sheeps blood (5-10%) -tests for hemolysis (streptococci) *carbs inhibit hemolysin production |
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What are hemolysins?
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-proteins produced by some bacteria which act upon cell membranes to cuase lysis
|
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What are the three results from BAP?
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-alpha hemolysis: green halo
-beta hemolysis: clear, tan halo -gamma hemolysis; no discoloration |
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How do you prepare a BAP plate?
What do you incubate it at? |
-use zone 3 dilution/isolation; stab medium with loop (2 times in each zone)
-37 C for 24 hrs in 5% CO2 |
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How do you interpret the results of a BAP test?
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-green halo= partial lysis of RBC
-clear halo= complete lysis of RBC -no change= no lysis of RBC (alpha, beta, gamma) |
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What is the Catalase test?
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-usually first biochem test for Gr +
-tests for the presence of catalase which is found in most aerobic and fac organisms, but abssent in most anaerobes and microaerophiles |
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How do you prepare a Catalase test?
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-transfer a well isolated colony (or part of it) to clean glass slide using applicator
-add a drop of 3% H2O2 -watch for bubbling |
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Which bacteria is positive for catalase test?
Negative? |
-Staphylococcus aureus
-Enterococcus faecalis |
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How do you interpret results from a Catalase test?
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-lots of bubbling is positive which means the Gram positive bacteria has catalase
-few to no bubbling is negative *make sure you don't have RBC's on slide! |
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What is catalase (the enzyme)?
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-enzyme that catalyzes degredation of H2O2 to water and oxygen
-catalase is found in most aerobic and fac organisms, but abssent in most anaerobes and microaerophiles |
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What is a Macconkey Agar plate?
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-selective AND differential
-selective agents are crystal violet and bile salts -these inhibit Gr + and fastidious Gr - -Lactose is differential agent |
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How do you incubate a MAC plate?
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-37 C, 24 hours
|
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What is needed for efficient utilization of lactose in MAC plates?
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-permease (facilitates entry of lactose into cell)
-beta galactosidase (when lactose is in cell, this breaks it down) |
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How is MAC plates innoculated?
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-3 zone dilution/isolation method on agar plate
|
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What species were originally meant to be differentiated on MAC plates?
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-Salmonella
-Shigella *normal fecal flora |
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How do you interpret the color results of a Macconkey Agar plate (MAC)?
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-strong lactose fermenters: purplish-pink/red colonies and medium, medium also has precipitate
-weak lactose fermenter: pink colonies w/dark red center, medium purplish/pink/red NO precipitate -non-lactose fermenter: beige colonies and medium -non-lactose fermenter which is a pigment producer: red colonies, beige surrounding |
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If you didn't have any growth on a MAC plate, what could this mean?
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-Gr+ bacteria
-Fastidious Gr- *plate selects against microbe |
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What is an m-Endo Agar plate?
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-used for detection and enumeration of fecal coliforms by membrane filtration technique
-selective and differential -inhibits Gr+ and non-enteric GR- -lactose fermenters react with substance and forms color changes |
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What are the selective agents in m-Endo Agar plates?
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-sodium desoxycholate
-sodium lauryl sulfate |
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What is the differential agent in m-Endo Agar?
What are the two substances that detect by-products of metabolic processes? |
-lactose
-basic fuschin and sodium sulfite |
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How does the red form in m-Endo plates?
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-lactose fermenters produce intermediary aldehyd compounds that combine with the basic fuschin dye to produce red
|
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How do you interpret the results of m-Endo tests?
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-strong lactose fermenters (coliforms): dark red colonies, green metallic sheen
-Lactose fermenters: red to dark red colonies -Non-Lactose fermenters: light pink colonies (lactose not fermented) -Black colonies: H2S produced |
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How do you prepare an m-Endo plate?
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-filter 100ml of water and place filter on solid plate
|
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How do you incubate m-Endo plates?
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-37 C for 24 hours
|
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How do you report results for m-Endo test?
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-fecal coliforms, lactose fermenter
-lactose fermenter -lactose nonfermenter -microbe produces H2S |
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What is the purpose of the O/F Leifson test?
-What bacterium is this used for? |
-used to determine a microbes mode of utilization for a carbohydrate
-used only on Gr - rods! |
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What is the purpose of the O/F Glucose Purple?
-What bacteria are tested this way? |
-used to determine a microbes mode of utilization for a carbohydrate
-used only for Gr + bacteria! |
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What are the ways a microbe can metabolize carbs?
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-oxidation
-fermentation |
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Is glucose utilization aerobic or anaerobic?
Fermentation? |
-oxidation of glucose is aerobic
-fermentation is anaerobic |
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What do aerobic microbes do to their environment during oxidation of glucose?
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-make it acidic
|
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What is the indicator in O/F Leifson tests?
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-bromthymol blue
|
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How does bromthymol blue work?
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-when neutral, it's green
-under alkaline conditions, it turns blue -under acidic conditions it turns yellow |
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What is the relative concentration of peptone in O/F Leifson and why?
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-Low so that false negatives aren't found: microbes that can't use the carb will use peptones as carbon source. If enough are used, surface will turn blue
|
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What is the purpose of the first test tube in an O/F test?
What makes this test valid? |
-it lacks carbohydrate (glucose) so microbes have nothing to oxidize/ferment, thus staying green/purple
-the first tube must contain growth but should not change color! |
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How do you prepare an O/F test?
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-collect three test tubes: one base tube, two glucose tubes
-stab each to bottom of tube with microbe innoculation -seal ONE tube with 4mm oil |
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Describe what an O/F Leifson tube would look like for an oxidative microbe?
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base: blue-green
open: yellow on top closed: green AKA: +/- |
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Describe what an O/F Leifson tube would look like for a fermentator?
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base: blue-green
open: yellow closed: yellow AKA: +/+ |
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Describe what an O/F Leifson tube would look like for an inert bacteria?
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base: blue-green
open: green with or w/o blue top closed: green AKA: -/- |
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What is the pH indicator in O/F Glucose-Purple?
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-bromcresol purple
|
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Why is bromcresol purple used for Gr + bacteria and not Gr - bacteria?
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-Gr + bacteria require more nutrients, so this allows them to grow
|
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What are the results of an O/F purple test if the microbe is oxidative?
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Base: purple
Open: yellow on top Closed: purple AKA: +/- |
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What are the results of an O/F purple test if the microbe is fermentative?
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Base: purple
Open: yellow Closed: yellow AKA: +/+ |
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What are the results of an O/F purple test if the microbe is inert?
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Base: purple
Open: purple Closed: purple AKA: -/- |
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What is the purpose of the Oxidase test?
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-to determine the presence of cytochrome oxidase
-first test used to identigy Gr - microbes |
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What is cytochrome oxidase used for in bacteria?
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-aerobic respiration uses it to transfer electrons from cytochromes to oxygen (the terminal acceptor)
|
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What is the artificial substrate that is oxidized from cytochrome oxidase?
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-N,N,N,N tetramethyl-p-phenylenediamine (the oxidase reagent)
|
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How do you perform an Oxidase test?
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-take a small sample of microbe colony using applicator stick or fired hematocrit tube
-place colony by touching a test square (1 of 4) -REUSE and do NOT throw out |
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What is the name of the technique we used in lab to perform the Oxidase test?
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-Dry-slide oxidase technique
|
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How do you interpret a positive Oxidase test?
How long does this take? |
-dot of microbe turns a blue/purple color
-microbe is + for producing cytochrome "c" *must occur within 20 seconds! |
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How do you interpret a negative Oxidase test?
Why do false negatives occur? |
-card doesn't change color
-microbe - for producing cytochrome "c" *many reasons, especially if stored in refridgerator |
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What is Plate count agar?
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-a general nutritional medium that is designed to grow microbes used to dilute environments
|
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Why do PCA plates work instead of TSA?
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-TSA plates (and others) actually contain too many nutrients for dilute microbes to thrive
|
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What is PCA plating most often used for?
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-used to determine the number of microorganisms in food and water samples
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What is Thioglycollate medium used for?
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-growing wide variety of microbes
-may be used to determine the oxygen requirements or sensitivity of a microorganism -can also contain redox indicators to show presence of oxygen |
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What serves as the reducing agent in Thioglycollate medium and what does this do?
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-sodium thioglycollate keeps the Eh of medium low and electron rich
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What is agar in thioglycollate medium used for?
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-retard the penetration of oxygen into the depths of medium helping bottom stay reduced
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What is the energy source in thioglycollate medium?
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-glucose
|
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What is the carbon source in thioglycollate medium?
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-glucose
|
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What are two types of redox indicators used in Thioglycollate medium?
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-Mehtylene Blue
-Resazurin |
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What would aerobic microbes look like in a thyoglycollate medium?
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-growth in top part of tube only (depth depends on oxygen penetration and microbe oxygen requirements)
|
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What would microaerophilic microbes look like in thyoglycollate medium?
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-growth slightly below surface (depth depends on oxygen penetration and microbe oxygen requirements)
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What would facultative microbes look like in thyoglycollate media?
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-growth throughout the tube because they don't need oxygen
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What would anaerobic growth look like?
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-growth at very bottom of tube
*strict anaerobes still won't be able to grow in this environment because of minute oxygen amounts |
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What is TSA?
What is it used for? |
-trypticase soy agar
-a general nutritional medium which supports growth of wide variety of microorganisms -frequently used for phenotypic testing such as temp tolerance/preferance, pigment production, and endospore production |
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What other plate test does this serve as the base for?
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-Blood Agar Plate
|
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What peptones are used in TSA plates?
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-enzymatic hydrolytic product of casein
-pancreatic digest of soy meal |
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Why is TSA such a good growth promotor?
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-because of the nutritional characteristics of the peptones used
|
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What is TSB?
What is it used for? |
-Trypticase soy broth
-same as TSA but no agar -used for base medium for: +blood cultures +antimicrobial sensitivity testing +determining Gram reaction +morphology +true spacial arrangement +etc |
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How much of a sample do you transfer to a PCA plate?
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-0.1 ml with a hockey stick
|
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What tool should be used for inocculating thyoglycollate medium?
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-sterile, plastic loop
-stab all the way to bottom |
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How do you incubate O/F glucose tests?
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-37 C for 48 hours or longer
|
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How do you incubate thioglycollate media?
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-37 C for 24-48 hours
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How do you incurbate TSA or TSA slant?
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-37 C for 24 hours (or at other appropriate temp and time for plate)
|
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What color is Bromcersol Purple in neutral pH?
Acidic? Alkaline? |
-light purple
-yellow -light purple |
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What color is Bromthymol Blue in netural pH?
Acidic? Alkaline? |
-green
-yellow -blue |
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What is Neutral Reds color in neutral pH?
Acidic? Alkaline? |
-pink-purple
-red-purple -beige-tan |
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What is the color indicator in an MAC plate?
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-Neutral Red
|
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What is the KF Strep test?
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-Kenner Fecal Streptococcus Agar
-selctive AND differential -uses membrane filtration technique |
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What is the selective agent in KF Strep test?
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-sodium azide
|
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What is the differential agent in KF Strep test?
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-triphenyl tetrazolium chloride
|
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How does sodium azide work as a selective agent in KF Strep tests?
What does it NOT select against? |
-chelator that binds heme iron in cytochromes (therefor stopping electron transfer for respiration)
-inhibits most microbes except Streptococcus and Enterococcus (no heme or respiration) |
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How do you interpret results of KF Strep test?
|
-no growth: microbes NOT Strep or Entero
-growth: likelihood of strep or entero -Yellow colonies: streptococcal microbes but NOT fecal -Red colonies: fecal streptococci or enterococci |
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How does TTC work in KF Strep tests?
What does TTC stand for again? |
-triphenyl tetrazolium chloride
-fecal streptococci reduce it resulting in bright red (maroon) colonies |
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How do you prepare a KF Strep test?
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-filter 100 ml water through filter and put on agar plate
|
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How do you incubate KF Strep tests?
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-incubate 37 C for 24 hours
|
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What do the colonies look like in a KF Strep test when TTC is reduced?
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-dark intense red
-fecal streptococci |
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What do the colonies look like in a KF Strep test when TTC is NOT reduced?
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-beige, pale red, or orange
-non fecal streptococcie |
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What is a CAP designed to grow?
|
-fastidious Gr negative
*can also grow wide assortment of other microorganisms |
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How do you incubate a CAP?
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-37 degrees C
-5% CO2 atmostphere -24 hours *compare to BAP after! |
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What is important about the CAP plate?
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-when compared to BAP, similar colonies exist. HOWEVER, CAP can grow Haemophilus or Neisseria which are potentially pathogenic
|
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What is the green discoloration in CAP plates?
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-due to reduction of hemoglobin to its methemoglobin form
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What does the CAP contain?
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-hemoglobin (X factor)
-NAD+ (V factor) |
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What are the colonies found on CAP plates and what do they look like (results interpretation)?
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-flat grey colonies not on BAP could be Hemophilus sp.
-Conves, yellowish tan colonies not found on BAP could be Neisseria sp. |
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What does the Coagulase test indicate?
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-the ability of a microbe to clot plasma using coagulase
|
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What does the coagulase test differentiate (genus)?
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-Staphylococcus
|
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What are the two types of coagulase tests?
|
-slide
-tube |
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What is benefiicial about using coagulase testing?
|
-most inexpensive
-accurate -single criterion to differentiate pathogenic from non-pathogenic staph |
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What does the coagulase slide test indicate?
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-indicates presence of bound coagulase and referred to as the "clumping factor"
|
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What does the coagulase tube test indicate?
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-measures free coagulase
|
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What are the overall results to a slide test?
Tube test? |
-invalid, positive, negative
-positive -negative |
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Why is it important to watch a coagulase tube test as it incubates?
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-some pathogenic staph make fibrinolysin which breaks clots back down again
|
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What does CNA stand for?
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-Colistin Nalidixic Acid Blood Agar Plates
|
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What is a CNA plate used for?
|
-to select for Gram positive microbes
-to test hemolysis *prevents some bacillus |
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What is the selective medium in CNA plates?
Differential? |
-colistin and nalidixic acid
-red blood cells (no pH indicator like most plates!) |
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How does Colistin work?
|
-targets cell membranes of Gram negative microbes
-blocks DNA synthesis of Gram neg microbes |
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How do you incubate a CNA plate?
|
-37 C
-5% CO2 -24 hours |
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What are McFarland Standards?
|
-barium sulfate, nephelometer (turbidity) standards
|
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What are McFarland standards used for?
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-mimicking density of various conc. of bacteria in broth
|
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What are the standards used in lab for McFarland standards?
|
-0.5 = 1.5 x 10^8
-1.0 = 3 x 10^8 -2.0 = 6 x 10^8 -3.0 = 9 x 10^8 |
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What are the components of a McFarland standard?
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-1% aqueous barium chloride
-1% sulfuric acid |
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What are the nutrient sources in a Minimal Glucose plate?
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-glucose
-nitrogen -salt -phosphorous |
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What are MG plates used to grow?
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-prototrophs
|
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What are prototrophs?
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-microbes that can synthesize needed nutrients
|
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What can be added to a MG plate for further classification?
|
-Streptomycin
-Histidine -Tryptophan |
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What is the purpose of a MS plate?
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-to isolate and differentiate among oral strept
|
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What is the selective and differential agents in an MS plate?
|
-crystal violet and potassium tellurite
-differentials are a combo effect of trypan blue, tellurite, glucose and sucrose |
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What is inhibited on an MS plate?
|
-most gram positive other than streptococcus
-most gram negative rods |
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What do SAB plates stand for?
|
-Sabouraud Dextrose Agar, Emmons
|
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What grows on SAB's?
|
-fungi
-take longer so medium is deeper to reduce drying |
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What was the original concentrations of a SAB plate?
*What grew on this? |
-pH 5.6
-4% glucose *allowed for many fungi especially dermatophytes and not much bacteria |
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What is the new concentrations of on a SAB plate?
|
-2% glucose
-pH of 6.9 |
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What is the purpose of a Tomato Juice Agar plate?
|
-isolation and identification of C. albicans and Lactobacillus
|
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What is the selective agent in TJ agar?
|
-the tomato juice!
*caused from low pH of medium (6.1) which inhibits a wide variety of microflora |
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What do Lactobacillus look like on TJ agar?
-Candida albicans? |
-pinpoint colonies
-creamy, opaque colonies about 1-2 mm in diameter |
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What is Vogel-Johnson Agar used for?
|
-isolating Staphylococcus aureus
|
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What are the components of a VJ plate?
|
-tellurite, lithium chloride, glycine, mannitol, phenol red
|
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What is the pH indicator in a VJA?
|
-phenol red
|
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What are the selective agents in VJA?
|
-tellurite
-lithium chloride -high conc. glycine |
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What are the differentials of VJA?
|
-tellurite
-mannitol |
|
What are the results for VJA tests?
|
-salt/tellurite tolerance
-tellurite reduction -mannitol fermentor |
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What grows black on VJA's?
|
-tellurite reducers that are salt tolerant
*such as S. aureus |
|
What does a positive tejjurite reduction look like in VJA plates?
Negative? |
-very black colonies
-white to light grey colonies |
|
What does a positve mannitol fermenter look like on VJA?
Negative? |
-medium around colonies is yellow
-medium around colonies is orange or bright pink |
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How do you incubate VJA plates?
|
-37 C
-24 hours |
|
What media contains Bromcresol Purple?
|
-6.5% NaCl (SF)
-MIO -O/F Purple |
|
What media contains Bromthymol Blue?
|
-Citrate (Simmons)
-O/F Leifson's |
|
What media contains Neutral Red?
|
-MAC
|
|
What media contains Phenol Red?
|
-VJA
-TSI |
|
What color is neutral Bromcresol purple?
Bromthymol Blue? Neutral Red? Phenol Red? |
-purple (light purple in OF)
-Green -Pink-Purple -Orange (redish in VJA) |
|
What color is acidic Bromcresol purple?
Bromthymol Blue? Neutral Red? Phenol Red? |
-Yellow
-Yellow -Red-Purple -Yellow |
|
What color is alkaline Bromcresol purple?
Bromthymol Blue? Neutral Red? Phenol Red? |
-purple
-blue -beige-tan -red |