Test 2 Flashcards
(20 cards)
Aerotolerance
Obligate Strict Anaerobic
Obligate Strict Aerobic
Facultative Anaerobic
Ability to live with or without Oxygen
Grows on bottom of tube
Grows on top of tube
Grows throughout the tube
Thioglycollate Medium
Contains:
- Sodium thioglycollate
- L-Cystine (Reduces oxygen to water)
- Resazurin (Indicator: Pink - Pressance of O2, Yellow - No O2)
Psychrophile
Mesophile
Thermophil
0-20 C (Common in oceans, cold areas)
20-40 C (most bacterial & Pathogens in humans)
40-65 C (Hot Springs)
Anaerobic Jar
Cultures:
- CO2 enriched
- Microaerophilics
- Anaerobics
Gas generator pack
Sodium Bicarbonate
Sodium Borohydride
H20
Palladium - Catalyzes reaction b/t H & free O2
Indicator strip (methylene blue)
- White: Removal of free O2 = Anaerobic condition
- Blue: Oxidized
Eosin Methylene Blue (EMB)
Measures
- Presence of Coliforms = fecal = possible pathogens
- Lactose fermentation determination
Metallic Green/Dark Purple (E. coli)
- Acid production
- Ferments lactose or sucrose
- Possible Coliform
Pink (E. aerogenes)
- Little acid production
- Ferments lactose only
- Probable Coliform
No Color/Agar color
- No fermentation
Dyes Inhibits Gram + growth. Selective for Gram -
Contains
- Peptone
- Lactose
- Sucrose
- Eosin Y dyes
- Methylene Blue dyes
MacConkey Agar
Contains
- Lactose
- Bile
- salt
- Crystal Violet dyes
- Neutral red dyes (pH indicator)
Bile & Crystal violet inhibits Gram +
Selective for Gram -
Colors
- Red = pH less than 6.8 = produces acid from lactose = coliform
- Colorless - pH greater than 6.8 = no lactose fermentation = noncoliform
Mannitol Salt Agar (MSA)
Isolates Staphylococus aures
NaCl favors staphylococci growth. Inhibits others.
Contains
- 7.5% NaCl
- pH indicator Phenol Red
- Carbohydrate mannitol
Colors
- Yellow - Less than pH 6.8 = produces acid = Staphylococcus aureus
- Red - pH 7.4~8.4 = No fermentation = Staphyloccus but not S. aureus
- Pink - Greater than pH 8.4 = No fermentation = Staphyloccus but not S. aureus
Differential Media
Selective Media
Can grow more than one organisum and distinguishes one organism from another in a plate
Can only promote one growth and inhibits the growth of another in a plate
Oxidation-Fermentation Test (O-F Glucose)
Determines if bacteria performs Oxidation or Fermentation of specific sugars
Contains
One tube with mineral oil (sealed-keeps O2 out)
One tube w/o oil (Unsealed-keeps O2)
Colors
Sealed - Unsealed
**Yellow - Yellow: ** Oxidative & Fermented (O-F) or Fermented (F)
Yellow (Top) - Yellow (Top): Oxidation w/ slow fermentation (O-F) or Slow fermentation (F)
Green/Blue - Some Yellow: Oxidation (O)
**Green/Blue - Green/Blue: ** No sugar metabolized, Organism is nonsaaccharolytic (N)
Usable sugars
- Glucose
- Sucrose
- Fructose
- Lactose
Phenol Red Broth
Fermentation test
Determines Acid and Gas production
Durham Tube: Checks for gas production (A/G), (A/-), (-/-), (K)
Colors
Yellow - Acid Production from fermentation (A/G, or A/-)
Red - No Fermentation (-/-)
Pink - Degradation of peptone, Alkaline (K)
Methyl Red & Voges Proskauer Tests
(MR-VP)
MR test: detects organism performing mixed acid fermentation
Red: pH 4.4 (+) Mixed acid fermentation
Yellow: pH 6.2 (-) None
VP Test: Ferments glucose -> acetoin/2,3 butanediol -> diacetyl -> guanidine nuclei -> Red -> (+)
Red: (+) 2.3 butanediol is fermented, acetonin produced
No color chage: (-) None
After incubation steps
- transfer 1ml of broth to 2 separate tubes
- Add 3 drops of Methyl Red into one tube (Read immediately)
- Add 15 drops of Reagent A and 5 drops of Reagent B into other tube (Read in 60 mins. 10 min intervals)
Catalase Test
Electron Transport Chain: capable of accepting/donating electrons in aerobes and facultative anaerobes
Favoprotein: Carrier molecule of ETC. Transfer electrons directly into O2 producting 2 highly potent toxins-Superoxide radical & H2O2
Superoxide dismutase: Enzyme that breaks down toxin and converts to Hydrogen peroxide
Catalase: Converts Hydrogen Peroxide into H20 & gaseous O2.
Bubbles indicate: catalase positive cutures
Add 1ml of H2O2 and check for bubbles
Oxidase Test
Identifies bacterias containing respiratory enzyme (cytochrome c oxidase)
Cytochrome C oxidase - Carrier molecule in Electron transport chains that is responsible for transfering electrons to O2 & reducing H2O
Tetramethyl-p-phenylenediamine - Detects bacteria that produces cytochrome C oxidase
Chromogenic Reducing Agent - chemical that changes or produces color as they become oxidized.
Results (within 20 secs)
Dark Blue/Purple = (+) oxidized: cytochrome C oxidase is present.
No Color Change = (-)
Nitrate Reduction Test
Presence of (mostly Gram -) nitrate reductase reduces Nitrate to Nitrite
Anaerobic respiration involves reduction of inorganic molecule other than O2
Nitrate reductase - Single step reduction (Nitrate-Nitrite)
Denitrification - Multi step reduction
Steps after Incubation
- First, check for denitrificaiton via Phenol Red test. No further test required if (A-/G+)
- Other wise, add sulfanilic acid (Nitrate reagent A) & Naphthylamine (Nitrate reagent B)
Colors after reagent
Red: (+) Nitrate reduction
No change: (-) no reduction or reduced to something else. Add zinc powder
Colors after Zinc powder
Red: (-) no Nitrate reduction
No Chnage: (+) Nitrate reduced to nongaseous nitrogenous compounds
Citrate Test
Use of Citrate as sole Carbon source in Enterobacteriase
Color
Green: pH 6.9 (-)
Blue: pH 7.6 (+) Citrate utilized
No color change w/ growth: (+) Citrate utilized
No color change w/o growth: (-)
Phenylalanine Deaminase Test
Ability to remove amine group NH2
Green: (+) for phenylalanine deaminase & phenylpyruvic acid
Yellow (-)
After Incubation
Add few drops of 12% Ferric Chloride solution into each tube. Color fades quickly so read immediately
Starch Hydrolysis
Organism that produces & secretes extracellular enzymes: alpha-amylase & Oligo 1,6 glucosidase
Starch (Polysaccharide) is too big for bacterias. They need to break down (hydrolyze) into single glucose units.
Steps after incubation
Stain starch plate wit Gram Iodine turning it Blue/Dk Brown
Results
Clearing with Halo around growth: (+) amylase present
No Clearing: (-)
Urea Hydrolysis
Urea, product of decarboxlation of amino acids
Urease - Enzyme in bacteria that hydrolyzes to ammonia & CO2
Use Phenol Red
Yeloow/Orange: Less than pH 8.4 (-) for urease
Red/Pink: Greater than pH 8.4 (+) for urease
Gelatin Hydrolysis Test
Presence of gelatinase enzyeme using Nutrient Gelatin
Gelatin protein is derived from collagen
Positive organism secretes gelatinase which liquifies medium,
Results
Gelatin is liquid: (+) for gelatinase
Gelatin is Solid: (-) for gelatinase
SIM Medium
Sulfur reduction, Indole production & Motility
Determines 3 bacterial activities
- Sulfur Reduction - reduce to H2S by Cysteine desulfurase (catalyzes Cysteine to Pyruvate) & Thiosulfate reductase (catalyzes sulfur at end of ETC)
- Indole Production - presence of tryptophan. Hydrolyzes to pyruvate, ammonia and indole)
- Motility - movement reduces of agar concentration
Results
Sulfer reduction
Black: (+) sulfur reduction, H2S produced
No Black: (-)
Indole
Red on top: (+) tryptophan is broken down to indole and pyruate
No color change: (-)
Motility
Growth radiating outward: (+) motility
No growth radiating: (-) Nonmotile
Steps after incubation
First, examine for Motility and Sulfer reduction
Add 2-3mm of Kovac’s reagent to each tube. Check for red layer on top (Indole)
