Koofers

Midterm Exam - Flashcards

Flashcard Deck Information

Class:BIO 3722 - Microbiology Laboratory
Subject:Biology
University:University of Texas - San Antonio
Term:Fall 2013
- of -
INCORRECT CORRECT
- INCORRECT     - CORRECT     - SKIPPED
Shuffle Remaining Cards Show Definitions First Take Quiz (NEW)
Hide Keyboard shortcuts
Next card
Previous card
Mark correct
Mark incorrect
Flip card
Start Over
Shuffle
      Mode:   CARDS LIST       ? pages   PRINT EXIT
TSA plate Tryptic Soy Agar
No selective or differential ingredients
Used to grow all types of bacteria
Used as a control against other tests
PEA Phenylethyl Alcohol Agar (blue line)
selective ingredient: phenylethyl alcohol
  • encourages Gram(+) and inhibits Gram(-)
Purpose: isolate Gram(+) bacteria

enterococci, streptococci, staphylococci 
CNA Columbia CNA Plate
Selective ingredients: Colistin & Naladixic acid; 5% sheep's blood
  • inhibits Gram(-) bacteria
Differential: Hemolysis ( α, β, γ )
  • Partial, complete, no hemolysis
Purpose: isolate Gram(+) bacteria

Enterococci, streptococci, staphylococci
MSA Mannitol Salt Agar
Selective ingredient: 7.5% NaCl 
  • S. aureus is a halophile
Differential: mannitol & phenol red
  • yellow growth - positive for halophile; mannitol fermented
  • red/pink growth - positive for halophile; nonfermenter
  • no growth - Gram(-)
Purpose: Grow & ID S. aureus
Generated by Koofers.com
MAC MacConkey
Selective ingredient: crystal violet & bile salts
  • inhibit growth of Gram(+) bacteria
Differential: lactose & neutral red
  • pink/red growth - organism ferments lactose, produces acid, coliform bacteria
  • colorless growth - no fermentation/acid, non-coliform
  • no growth - Gram(+)
Purpose: Isolate and differentiate; coliform and non-coliform
EMB Eosin Methylene Blue Agar 
Selective ingredient: dyes (methylene blue & Eosin Y)
  • inhibit growth of Gram(+) bacteria
Differential: lactose & dyes/pH indicator
  • growth is pink & mucoid - low acid production; possible coliform
  • growth is dark (w/ or w/o metallic sheen) - high acid production; probable coliform
  • growth is colorless - noncoliform, nonfermenter
Purpose: ID fecal coliform
HEA Hektoen Enteric Agar
selective ingredient: bile salts, bromthymol blue, acid fuchsin
  • inhibit growth of Gram(+) bacteria
differential: lactose, sucrose, salicin, bromthymol blue & acid fuchsin
  • pink/orange: fermentation; not Salmonella or Shigella
  • blue/green w/ black: no fermentation; sulfur reduction; pos. Sal
  • blue/green no black: no fermentation or reduction; pos. Shig/Sal
Purpose: ID Salmonella & Shigella
Na thiosulfate-->H2S + ferric ammonium citrate
MR-VP Methyl Red and Voges-Proskauer Tests
Broth is a combination medium for MR & VP tests. 
Differential: Glucose (fermentation)
MR: detects organisms capable of mixed acid fermentation, which overcomes phosphate buffer and lowers pH.
  • reagents: methyl red
  • red: positive result for mixed acid fermentation
VP: detects alkaline end product after oxidation of glucose
  • α-Napthol & KOH
  • red: positive result for acetoin production (alkalinity) 

Generated by Koofers.com
SIM Used for determination of sulfur reduction, indole production, motility

black: positive for sulfur reduction, H2S production
  • Na thiosulfate-->thiosulfate reductase-->H2S + ferric ammonium citrate
red after addition of Krovac's reagent: positive for indole production
  • tryptophan-->tryptophanase-->indole
radiating growth pattern: positive for motility
Citrate Test Purpose: differentiate members of Enterobacteriaceae; G(-) rods
Selective: citrate
  • only bacteria with the enzyme citrate-permease will grow
Differential: bromthymol blue
  • citrate-positive bacteria alkalinize the medium and turn it blue

Citrate-->citrate permease-->pyruvate
           -->                           -->acetate
Nitrate Reduction Test Purpose: test for Gram(-) bacteria containing the enzyme nitrate reductase
Differential: 
  • gas production: denitrification has taken place
  • red color after addition of sulfanilic acid and α-naphthylamine means nitrate reduction to nitrite took place
  • red color after addition of zinc indicates no nitrate reduction took place (zinc artificially reduced)
  • no color change after reagents or zinc means the nitrate was reduced to a nongaseous nitrogenous compound
Starch Hydrolysis Test
(red line)
Purpose: Determine if organism can break down starch into glucose; organism produces α-amylase
Differential: Gram's Iodine reacts with starch. Any hydrolysis will be shown as a clear zone around the growth; amylase is present.
starch-->amylase-->maltose
          -->maltase--->glucose
Generated by Koofers.com
Urea Hydrolysis Test
white lid
Purpose: test for enteric bacteria which can metabolize urea
urea-->urease--> CO2 + H20 + NH3
Selective: urea
  • only rapid urease-positive organisms can alkalinize the medium; Proteus, Morganella, and Heliobacter
Differential: phenol red
  • pink: positive for rapid urease-positive organism
  • orange/yellow: negative
Casein Hydrolysis Test Purpose: Detect bacteria that produce casease and hydrolyze casein

Differential: Casein
  • clearing in agar - positive result - casease is present
  • no clearing - negative - casease is absent
Gelatin Hydrolysis Test Purpose: Determine organism's ability to produce gelatinase

Differential: Gelatin
  • liquid after 3 minutes on ice - gelatinase is present
Gelatin--> Gelatinase--> amino acids
Snyder Test Purpose: Determines susceptibility to dental cavities
Selective: Salt, Glucose
  • Inhibits Gram(-) organisms
  • fermentation
Differential: bromeresol green
  • color change from green to yellow indicates fermentation 
Generated by Koofers.com
Carb. Fermentation Test Purpose: differentiate members of Enterobacteriaceae from other Gram(-) rods

Differential: phenol red, durham tube
  • glucose/sucrose/lactose/mannitol fermentation - yellow color
  • peptone deamination - pink color
  • bubbles in durham tube - gas production

red cap = lactose; green cap = glucose; yellow cap = sucrose

BPA Sheep's Blood Agar
Differential: differentiation of bacteria based on ability to hemolyze RBCs
  • 5% sheep's blood
  • Hemolysins: Streptolysins: O-oxygen labile & S-oxygen stable
Purpose: Detect hemolytic ability of Gram(+) cocci
Catalase Test Purpose: Identify organisms that produce the enzyme catalase.

2 H2O2--> catalase--> 2 H2O + O2

bubbles after addition of H2O2 indicates the enzyme catalase is present
Oxidase Test Purpose: determine if an organism produces cytochrome c oxidase

dark color change on paper indicates cytochrome c oxidase is present
Generated by Koofers.com
Colony Characteristics 1. shape: round, etc.
2. margin: edges
3. elevation: look from side
4. texture: moist, dry, etc.
5. pigment: color
6. size: influenced by time
7. optical: shiny, translucent, etc. (not the same as pigment)
Gram stain Differentiate between Gram(+) and Gram(-) bacteria
  • Gram(+) - thick layer of PDG, no LPS layer
  • Gram(-) - thin layer of PDG, LPS outer layer
1st: Primary - crystal violet - colors ALL cells
2nd: Mordant - Gram's iodine - increases cell's affinity to the primary dye
3rd: Decolorize - 95% EtOH - protein dehydrator and lipid solvent; decolorizes susceptible cells (Gram(-))
4th: Counter - safranin - stains cells that lost color after 3rd step

Gram(+) = purple & Gram(-) = pink
Simple stain Basic stain: stains the bacterial cell
  • safranin, 1% crystal violet, methylene blue, carbol fuchsin
Three parts to every stain:
1. Solvent - typically benze
2. Chromophore - color
3. Auxochrome - charge

1&2 = Chromogen

Air dry and heat fix before stain.
Negative stain Acidic stain: stains the background rather than the bacterial cell
  • nigrosin
Do not air dry or heat fix. View immediately.
Generated by Koofers.com
Brightfield Background light, specimen dark, requires stain (which kills specimen)

**Used in this class**
Darkfield Dark background, light specimen
Flourescent Need antibody to target with dye; antibody-antogen reaction
Phase-contrast stain not needed
Generated by Koofers.com
Electron Microscope Transmission & Scanning (3D image)
Resolving Power ability to distinguish between two objects
  • Light microscope: 0.2 mm
  • Electron microscope: 2 mm
Par Focal technique that allows the use of only the fine focus when increasing magnification.
Generated by Koofers.com

List View: Terms & Definitions

  Hide All 31 Print
 
Front
Back
 TSA plateTryptic Soy Agar
No selective or differential ingredients
Used to grow all types of bacteria
Used as a control against other tests
 PEAPhenylethyl Alcohol Agar (blue line)
selective ingredient: phenylethyl alcohol
  • encourages Gram(+) and inhibits Gram(-)
Purpose: isolate Gram(+) bacteria

enterococci, streptococci, staphylococci 
 CNAColumbia CNA Plate
Selective ingredients: Colistin & Naladixic acid; 5% sheep's blood
  • inhibits Gram(-) bacteria
Differential: Hemolysis ( α, β, γ )
  • Partial, complete, no hemolysis
Purpose: isolate Gram(+) bacteria

Enterococci, streptococci, staphylococci
 MSAMannitol Salt Agar
Selective ingredient: 7.5% NaCl 
  • S. aureus is a halophile
Differential: mannitol & phenol red
  • yellow growth - positive for halophile; mannitol fermented
  • red/pink growth - positive for halophile; nonfermenter
  • no growth - Gram(-)
Purpose: Grow & ID S. aureus
 MACMacConkey
Selective ingredient: crystal violet & bile salts
  • inhibit growth of Gram(+) bacteria
Differential: lactose & neutral red
  • pink/red growth - organism ferments lactose, produces acid, coliform bacteria
  • colorless growth - no fermentation/acid, non-coliform
  • no growth - Gram(+)
Purpose: Isolate and differentiate; coliform and non-coliform
 EMBEosin Methylene Blue Agar 
Selective ingredient: dyes (methylene blue & Eosin Y)
  • inhibit growth of Gram(+) bacteria
Differential: lactose & dyes/pH indicator
  • growth is pink & mucoid - low acid production; possible coliform
  • growth is dark (w/ or w/o metallic sheen) - high acid production; probable coliform
  • growth is colorless - noncoliform, nonfermenter
Purpose: ID fecal coliform
 HEAHektoen Enteric Agar
selective ingredient: bile salts, bromthymol blue, acid fuchsin
  • inhibit growth of Gram(+) bacteria
differential: lactose, sucrose, salicin, bromthymol blue & acid fuchsin
  • pink/orange: fermentation; not Salmonella or Shigella
  • blue/green w/ black: no fermentation; sulfur reduction; pos. Sal
  • blue/green no black: no fermentation or reduction; pos. Shig/Sal
Purpose: ID Salmonella & Shigella
Na thiosulfate-->H2S + ferric ammonium citrate
 MR-VPMethyl Red and Voges-Proskauer Tests
Broth is a combination medium for MR & VP tests. 
Differential: Glucose (fermentation)
MR: detects organisms capable of mixed acid fermentation, which overcomes phosphate buffer and lowers pH.
  • reagents: methyl red
  • red: positive result for mixed acid fermentation
VP: detects alkaline end product after oxidation of glucose
  • α-Napthol & KOH
  • red: positive result for acetoin production (alkalinity) 

 SIMUsed for determination of sulfur reduction, indole production, motility

black: positive for sulfur reduction, H2S production
  • Na thiosulfate-->thiosulfate reductase-->H2S + ferric ammonium citrate
red after addition of Krovac's reagent: positive for indole production
  • tryptophan-->tryptophanase-->indole
radiating growth pattern: positive for motility
 Citrate TestPurpose: differentiate members of Enterobacteriaceae; G(-) rods
Selective: citrate
  • only bacteria with the enzyme citrate-permease will grow
Differential: bromthymol blue
  • citrate-positive bacteria alkalinize the medium and turn it blue

Citrate-->citrate permease-->pyruvate
           -->                           -->acetate
 Nitrate Reduction TestPurpose: test for Gram(-) bacteria containing the enzyme nitrate reductase
Differential: 
  • gas production: denitrification has taken place
  • red color after addition of sulfanilic acid and α-naphthylamine means nitrate reduction to nitrite took place
  • red color after addition of zinc indicates no nitrate reduction took place (zinc artificially reduced)
  • no color change after reagents or zinc means the nitrate was reduced to a nongaseous nitrogenous compound
 Starch Hydrolysis Test
(red line)
Purpose: Determine if organism can break down starch into glucose; organism produces α-amylase
Differential: Gram's Iodine reacts with starch. Any hydrolysis will be shown as a clear zone around the growth; amylase is present.
starch-->amylase-->maltose
          -->maltase--->glucose
 Urea Hydrolysis Test
white lid
Purpose: test for enteric bacteria which can metabolize urea
urea-->urease--> CO2 + H20 + NH3
Selective: urea
  • only rapid urease-positive organisms can alkalinize the medium; Proteus, Morganella, and Heliobacter
Differential: phenol red
  • pink: positive for rapid urease-positive organism
  • orange/yellow: negative
 Casein Hydrolysis TestPurpose: Detect bacteria that produce casease and hydrolyze casein

Differential: Casein
  • clearing in agar - positive result - casease is present
  • no clearing - negative - casease is absent
 Gelatin Hydrolysis TestPurpose: Determine organism's ability to produce gelatinase

Differential: Gelatin
  • liquid after 3 minutes on ice - gelatinase is present
Gelatin--> Gelatinase--> amino acids
 Snyder TestPurpose: Determines susceptibility to dental cavities
Selective: Salt, Glucose
  • Inhibits Gram(-) organisms
  • fermentation
Differential: bromeresol green
  • color change from green to yellow indicates fermentation 
 Carb. Fermentation TestPurpose: differentiate members of Enterobacteriaceae from other Gram(-) rods

Differential: phenol red, durham tube
  • glucose/sucrose/lactose/mannitol fermentation - yellow color
  • peptone deamination - pink color
  • bubbles in durham tube - gas production

red cap = lactose; green cap = glucose; yellow cap = sucrose

 BPASheep's Blood Agar
Differential: differentiation of bacteria based on ability to hemolyze RBCs
  • 5% sheep's blood
  • Hemolysins: Streptolysins: O-oxygen labile & S-oxygen stable
Purpose: Detect hemolytic ability of Gram(+) cocci
 Catalase TestPurpose: Identify organisms that produce the enzyme catalase.

2 H2O2--> catalase--> 2 H2O + O2

bubbles after addition of H2O2 indicates the enzyme catalase is present
 Oxidase TestPurpose: determine if an organism produces cytochrome c oxidase

dark color change on paper indicates cytochrome c oxidase is present
 Colony Characteristics1. shape: round, etc.
2. margin: edges
3. elevation: look from side
4. texture: moist, dry, etc.
5. pigment: color
6. size: influenced by time
7. optical: shiny, translucent, etc. (not the same as pigment)
 Gram stainDifferentiate between Gram(+) and Gram(-) bacteria
  • Gram(+) - thick layer of PDG, no LPS layer
  • Gram(-) - thin layer of PDG, LPS outer layer
1st: Primary - crystal violet - colors ALL cells
2nd: Mordant - Gram's iodine - increases cell's affinity to the primary dye
3rd: Decolorize - 95% EtOH - protein dehydrator and lipid solvent; decolorizes susceptible cells (Gram(-))
4th: Counter - safranin - stains cells that lost color after 3rd step

Gram(+) = purple & Gram(-) = pink
 Simple stainBasic stain: stains the bacterial cell
  • safranin, 1% crystal violet, methylene blue, carbol fuchsin
Three parts to every stain:
1. Solvent - typically benze
2. Chromophore - color
3. Auxochrome - charge

1&2 = Chromogen

Air dry and heat fix before stain.
 Negative stainAcidic stain: stains the background rather than the bacterial cell
  • nigrosin
Do not air dry or heat fix. View immediately.
 BrightfieldBackground light, specimen dark, requires stain (which kills specimen)

**Used in this class**
 DarkfieldDark background, light specimen
 FlourescentNeed antibody to target with dye; antibody-antogen reaction
 Phase-contraststain not needed
 Electron MicroscopeTransmission & Scanning (3D image)
 Resolving Powerability to distinguish between two objects
  • Light microscope: 0.2 mm
  • Electron microscope: 2 mm
 Par Focaltechnique that allows the use of only the fine focus when increasing magnification.
36, "/var/app/current/tmp/"