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BIO 175 Lab Practical 2
- Course
- BIO 175
- Institution
- Wake Technical Community College
This is a comprehensive and detailed lab practical on Different Media and Biochemical tests for Bio 175.
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Different Media and Biochemical TestsLab 9: Gram + Cocci: Staph Species Identification
● MSA: mannitol salt agar (7.5% NaCL, mannitol, phenol red)
○ Differential → detects mannitol fermentation by pH change detected by phenol
red
○ Selective → selects Staphylococcus species by high 7.5% salt
○ Positive result: If there was growth = Staph species, if it turned yellow = acid
fermentation of mannitol
○ Negative result: No growth
● SBA: Sheep’s blood agar
○ Non-selective
○ Differential → for RBC hemolysis
■ S. aureus produces toxin that lyses RBCs (beta), S. epidermidis cannot
lyse RBCs (gamma)
○ Positive result: Agar around colonies turned yellow = S. aureus
○ Negative result: Agar around colonies stayed red = S. epidermidis
● Novobiocin resistance/sensitivity
○ Positive result: ZOI around disk = sensitive
○ Negative result: no ZOI around disk = resistant
● Coagulase activity
○ Differential between S. aureus (coagulase +) and other coagulase - Staph
species
○ Positive result: clumping (white flecks in plasma) = S. aureus
○ Negative result: no clumping = other coagulase negative Staph species
Lab 10: Gram + cocci: Strep Species Identification
● Lancefield classification
○ Uses blood agar to classify Strep species:
■ Alpha = partial, green
■ Beta = complete, clear
■ Gamma = no hemolysis
○ Beta hemolytic strep: Group A and B
○ Alpha hemolytic strep: S. pneumoniae and S. salivarius
● Antibiotic sensitivity assays
○ Bacitracin: differentiates Beta hemolytic Strep
■ Sensitive = Group A (S. pyogenes)
■ Resistant = Group B (S. agalactiae)
○ Optochin: differentiates Alpha hemolytic Strep
■ Sensitive = S. pneumoniae
■ Resistant = S. salivarius
, ● Catalase Test
○ Positive result: immediate bubble formation = Staph (all species produce
catalase)
○ Negative result: No bubble formation (no catalase enzyme) = Strep (all strep
species)
● Rapid Strep Test
○ Positive result: Group A Strep (S. pyogenes) antibody-antigen complex = color
change/line on strip
○ Negative result: No GAS = no second line, only control line
Lab 11: Evaluation of an unknown GPC
● Catalase Test
○ Differentiates Strep (-) and Staph (+)
● Coagulase Test
○ Only if confirmed Staph species
○ S. aureus (+) or other Staph coagulase (-) species
Lab 12: Identification of Enteric ENR
● MacConkey (MAC)
○ Selective → uses bile salts and crystal violet dye (inhibits Gram + organisms)
○ Differential → for enteric GNRs by their ability to ferment lactose
○ Positive result: Growth (only gram -) plus lactose fermentation shown as fuschia
colonies
○ Negative result: No growth, no lactose fermentation (colorless)
● Triple Sugar Iron Agar (TSI)
○ Ability of organisms to ferment sugars present and produce hydrogen sulfide
○ Contains lactose, glucose (10x less), and sucrose: distinguish between species
that ferment glucose only and those that ferment all three
○ Ferrous sulfate → detection of hydrogen sulfide production
■ Iron salts react with H2S and form a black precipitate (at the bottom)
○ Phenol red → pH indicator for sugar fermentation
■ Yellow in acidic environments
■ Red in alkaline environments
○ Gas production: cracks or lifting of the agar
○ K= alkaline/neutral, A= acidic
● Simmon’s Citrate
○ Ability of organisms to utilize citrate as sole carbon source
○ Sodium citrate → only source of carbon
○ Ammonium salts → nitrogen
○ Bromothymol blue → pH indicator
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