MMG Lab H

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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
What are hemolysins?: -proteins produced by some bacteria which act upon cell membranes to cuase lysis
What are the three results from BAP?: -alpha hemolysis: green halo
-beta hemolysis: clear, tan halo
-gamma hemolysis; no discoloration
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
How do you interpret the results of a BAP test?: -green halo= partial lysis of RBC
-clear halo= complete lysis of RBC
-no change= no lysis of RBC
(alpha, beta, gamma)
What is the Catalase test?: -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
How do you prepare a Catalase test?: -transfer a well isolated colony (or part of it) to clean glass slide using applicator
-add a drop of 3% H2O2
-watch for bubbling
Which bacteria is positive for catalase test? Negative?: -Staphylococcus aureus
-Enterococcus faecalis
How do you interpret results from a Catalase test?: -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!
What is catalase (the enzyme)?: -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
What is a Macconkey Agar plate?: -selective AND differential
-selective agents are crystal violet and bile salts
-these inhibit Gr + and fastidious Gr -
-Lactose is differential agent
How do you incubate a MAC plate?: -37 C, 24 hours
What is needed for efficient utilization of lactose in MAC plates?: -permease (facilitates entry of lactose into cell)
-beta galactosidase (when lactose is in cell, this breaks it down)
How is MAC plates innoculated?: -3 zone dilution/isolation method on agar plate
What species were originally meant to be differentiated on MAC plates?: -Salmonella
-Shigella
*normal fecal flora
How do you interpret the color results of a Macconkey Agar plate (MAC)?: -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
If you didn't have any growth on a MAC plate, what could this mean?: -Gr+ bacteria
-Fastidious Gr-
*plate selects against microbe
What is an m-Endo Agar plate?: -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
What are the selective agents in m-Endo Agar plates?: -sodium desoxycholate
-sodium lauryl sulfate
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
How does the red form in m-Endo plates?: -lactose fermenters produce intermediary aldehyd compounds that combine with the basic fuschin dye to produce red
How do you interpret the results of m-Endo tests?: -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
How do you prepare an m-Endo plate?: -filter 100ml of water and place filter on solid plate
How do you incubate m-Endo plates?: -37 C for 24 hours
How do you report results for m-Endo test?: -fecal coliforms, lactose fermenter
-lactose fermenter
-lactose nonfermenter
-microbe produces H2S
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!
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!
What are the ways a microbe can metabolize carbs?: -oxidation
-fermentation
Is glucose utilization aerobic or anaerobic? Fermentation?: -oxidation of glucose is aerobic
-fermentation is anaerobic
What do aerobic microbes do to their environment during oxidation of glucose?: -make it acidic
What is the indicator in O/F Leifson tests?: -bromthymol blue
How does bromthymol blue work?: -when neutral, it's green
-under alkaline conditions, it turns blue
-under acidic conditions it turns yellow
What is the relative concentration of peptone in O/F Leifson and why?: -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
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!
How do you prepare an O/F test?: -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
Describe what an O/F Leifson tube would look like for an oxidative microbe?: base: blue-green
open: yellow on top
closed: green
AKA: +/-
Describe what an O/F Leifson tube would look like for a fermentator?: base: blue-green
open: yellow
closed: yellow
AKA: +/+
Describe what an O/F Leifson tube would look like for an inert bacteria?: base: blue-green
open: green with or w/o blue top
closed: green
AKA: -/-
What is the pH indicator in O/F Glucose-Purple?: -bromcresol purple
Why is bromcresol purple used for Gr + bacteria and not Gr - bacteria?: -Gr + bacteria require more nutrients, so this allows them to grow
What are the results of an O/F purple test if the microbe is oxidative?: Base: purple
Open: yellow on top
Closed: purple
AKA: +/-
What are the results of an O/F purple test if the microbe is fermentative?: Base: purple
Open: yellow
Closed: yellow
AKA: +/+
What are the results of an O/F purple test if the microbe is inert?: Base: purple
Open: purple
Closed: purple
AKA: -/-
What is the purpose of the Oxidase test?: -to determine the presence of cytochrome oxidase
-first test used to identigy Gr - microbes
What is cytochrome oxidase used for in bacteria?: -aerobic respiration uses it to transfer electrons from cytochromes to oxygen (the terminal acceptor)
What is the artificial substrate that is oxidized from cytochrome oxidase?: -N,N,N,N tetramethyl-p-phenylenediamine (the oxidase reagent)
How do you perform an Oxidase test?: -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
What is the name of the technique we used in lab to perform the Oxidase test?: -Dry-slide oxidase technique
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!
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
What is Plate count agar?: -a general nutritional medium that is designed to grow microbes used to dilute environments
Why do PCA plates work instead of TSA?: -TSA plates (and others) actually contain too many nutrients for dilute microbes to thrive
What is PCA plating most often used for?: -used to determine the number of microorganisms in food and water samples
What is Thioglycollate medium used for?: -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
What serves as the reducing agent in Thioglycollate medium and what does this do?: -sodium thioglycollate keeps the Eh of medium low and electron rich
What is agar in thioglycollate medium used for?: -retard the penetration of oxygen into the depths of medium helping bottom stay reduced
What is the energy source in thioglycollate medium?: -glucose
What is the carbon source in thioglycollate medium?: -glucose
What are two types of redox indicators used in Thioglycollate medium?: -Mehtylene Blue
-Resazurin
What would aerobic microbes look like in a thyoglycollate medium?: -growth in top part of tube only (depth depends on oxygen penetration and microbe oxygen requirements)
What would microaerophilic microbes look like in thyoglycollate medium?: -growth slightly below surface (depth depends on oxygen penetration and microbe oxygen requirements)
What would facultative microbes look like in thyoglycollate media?: -growth throughout the tube because they don't need oxygen
What would anaerobic growth look like?: -growth at very bottom of tube
*strict anaerobes still won't be able to grow in this environment because of minute oxygen amounts
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
What other plate test does this serve as the base for?: -Blood Agar Plate
What peptones are used in TSA plates?: -enzymatic hydrolytic product of casein
-pancreatic digest of soy meal
Why is TSA such a good growth promotor?: -because of the nutritional characteristics of the peptones used
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
How much of a sample do you transfer to a PCA plate?: -0.1 ml with a hockey stick
What tool should be used for inocculating thyoglycollate medium?: -sterile, plastic loop
-stab all the way to bottom
How do you incubate O/F glucose tests?: -37 C for 48 hours or longer
How do you incubate thioglycollate media?: -37 C for 24-48 hours
How do you incurbate TSA or TSA slant?: -37 C for 24 hours (or at other appropriate temp and time for plate)
What color is Bromcersol Purple in neutral pH? Acidic? Alkaline?: -light purple
-yellow
-light purple
What color is Bromthymol Blue in netural pH? Acidic? Alkaline?: -green
-yellow
-blue
What is Neutral Reds color in neutral pH? Acidic? Alkaline?: -pink-purple
-red-purple
-beige-tan
What is the color indicator in an MAC plate?: -Neutral Red
What is the KF Strep test?: -Kenner Fecal Streptococcus Agar
-selctive AND differential
-uses membrane filtration technique
What is the selective agent in KF Strep test?: -sodium azide
What is the differential agent in KF Strep test?: -triphenyl tetrazolium chloride
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)
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
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
How do you prepare a KF Strep test?: -filter 100 ml water through filter and put on agar plate
How do you incubate KF Strep tests?: -incubate 37 C for 24 hours
What do the colonies look like in a KF Strep test when TTC is reduced?: -dark intense red
-fecal streptococci
What do the colonies look like in a KF Strep test when TTC is NOT reduced?: -beige, pale red, or orange
-non fecal streptococcie
What is a CAP designed to grow?: -fastidious Gr negative
*can also grow wide assortment of other microorganisms
How do you incubate a CAP?: -37 degrees C
-5% CO2 atmostphere
-24 hours
*compare to BAP after!
What is important about the CAP plate?: -when compared to BAP, similar colonies exist. HOWEVER, CAP can grow Haemophilus or Neisseria which are potentially pathogenic
What is the green discoloration in CAP plates?: -due to reduction of hemoglobin to its methemoglobin form
What does the CAP contain?: -hemoglobin (X factor)
-NAD+ (V factor)
What are the colonies found on CAP plates and what do they look like (results interpretation)?: -flat grey colonies not on BAP could be Hemophilus sp.
-Conves, yellowish tan colonies not found on BAP could be Neisseria sp.
What does the Coagulase test indicate?: -the ability of a microbe to clot plasma using coagulase
What does the coagulase test differentiate (genus)?: -Staphylococcus
What are the two types of coagulase tests?: -slide
-tube
What is benefiicial about using coagulase testing?: -most inexpensive
-accurate
-single criterion to differentiate pathogenic from non-pathogenic staph
What does the coagulase slide test indicate?: -indicates presence of bound coagulase and referred to as the "clumping factor"
What does the coagulase tube test indicate?: -measures free coagulase
What are the overall results to a slide test? Tube test?: -invalid, positive, negative
-positive
-negative
Why is it important to watch a coagulase tube test as it incubates?: -some pathogenic staph make fibrinolysin which breaks clots back down again
What does CNA stand for?: -Colistin Nalidixic Acid Blood Agar Plates
What is a CNA plate used for?: -to select for Gram positive microbes
-to test hemolysis
*prevents some bacillus
What is the selective medium in CNA plates? Differential?: -colistin and nalidixic acid
-red blood cells (no pH indicator like most plates!)
How does Colistin work?: -targets cell membranes of Gram negative microbes
-blocks DNA synthesis of Gram neg microbes
How do you incubate a CNA plate?: -37 C
-5% CO2
-24 hours
What are McFarland Standards?: -barium sulfate, nephelometer (turbidity) standards
What are McFarland standards used for?: -mimicking density of various conc. of bacteria in broth
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
What are the components of a McFarland standard?: -1% aqueous barium chloride
-1% sulfuric acid
What are the nutrient sources in a Minimal Glucose plate?: -glucose
-nitrogen
-salt
-phosphorous
What are MG plates used to grow?: -prototrophs
What are prototrophs?: -microbes that can synthesize needed nutrients
What can be added to a MG plate for further classification?: -Streptomycin
-Histidine
-Tryptophan
What is the purpose of a MS plate?: -to isolate and differentiate among oral strept
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
What is inhibited on an MS plate?: -most gram positive other than streptococcus
-most gram negative rods
What do SAB plates stand for?: -Sabouraud Dextrose Agar, Emmons
What grows on SAB's?: -fungi
-take longer so medium is deeper to reduce drying
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
What is the new concentrations of on a SAB plate?: -2% glucose
-pH of 6.9
What is the purpose of a Tomato Juice Agar plate?: -isolation and identification of C. albicans and Lactobacillus
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
What do Lactobacillus look like on TJ agar? -Candida albicans?: -pinpoint colonies
-creamy, opaque colonies about 1-2 mm in diameter
What is Vogel-Johnson Agar used for?: -isolating Staphylococcus aureus
What are the components of a VJ plate?: -tellurite, lithium chloride, glycine, mannitol, phenol red
What is the pH indicator in a VJA?: -phenol red
What are the selective agents in VJA?: -tellurite
-lithium chloride
-high conc. glycine
What are the differentials of VJA?: -tellurite
-mannitol
What are the results for VJA tests?: -salt/tellurite tolerance
-tellurite reduction
-mannitol fermentor
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
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

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