Lab #9
Review - pH Indicators
Memorize pH indicators and color reactions!
pH
Indicator
Very
acidic
Acidic
Neutral
Basic
Phenol red
-
pH <6.8 =
yellow
pH 6.9 - 8.0 =
red
pH >8.0 =
magenta/ hot
pink
Litmus
-
pink
purple
blue
Bromocresol
Purple
-
yellow
burgundy
violet
Methyl red
pH <4.4 =
red
pH 5 - 6 =
orange
pH >6.3 =
yellow
-
Review
• Positive sugar fermentation reaction:
• A = acid production (yellow color change)
• A/G = acid production (yellow) and gas in Durham tube
• SA = slow reaction (orange color change)
• Negative sugar fermentation reaction:
• B = base production (pink/magenta color change)
• (-) = No change
• pH indicator: phenol red
Review
• Nitrate reduction test (pg. 81 – 83)
• Media: Nitrate broth  includes KNO3
• KNO3
ETC
KNO2 + ATP
• Detect KNO2 using reagents:
• Sulfanilic acid (SA), and
• Dimethyl-α-naphthlyamine (DAN)
• Procedure
• Take 1mL out from each culture
& pour into a clean test tube
• Add 3 drops of SA and 2 drops of DAN
• Positive result: Red/ magenta color
• Negative result: any other color
Negative
Positive
Review
• Production of decarboxylase (pg. 87 – 90)
• Enzyme decarboxylase removes carboxyl group from amino acids
• Media: Lysine, ornithine, or arginine amino acid + sugar +
bromocresol purple pH indicator + oil at top (anaerobic conditions)
• Positive result: If the amino acid
is decarboxylated  CO2 is produced 
pH become basic  purple
• Negative result:
• Sugar fermentation  acid production
 yellow color change
• No change
• Cover oil with thumb and then
observe results
Positive
Review
• SIM Reactions (pg. 95 – 99)
• SIM = Sulfide, Indole, and Motility (three tests in 1 tube!)
• 1) Production of Hydrogen Sulfide
• Some microorganisms can metabolize
amino acid cysteine (in media) and
produce H2S gas
• H2S gas reacts with iron sulfate (in media)
 black precipitate  positive result
• 2) Production of Indole
• Some microorganisms can metabolize
amino acid tryptophan (in media)
into indole (enzyme tryptophanase)
• Indole is detected by adding 10 drops
of Kovac’s reagent on top of the media
 red color change  positive result
Review
• SIM Reactions (pg. 95 – 99)
• SIM = Sulfide, Indole, and Motility (three tests in 1 tube!)
• 3) Motility
• Observe growth pattern
• Growth away from stab line = motility
Review
• MR-VP Reactions (pg. 101 – 104)
• Differentiate microorganisms on their fermentative end products
• Sugar glucose is added to media
• Each culture is split into two clean test tubes  pour in 1mL of
culture into each clean test tube
• Methyl Red (MR) Test
• Detects production of strong organic acids  pH lowered to 4.4
or less
• Add 10 drops of Methyl Red indicator
• Positive result: red color 
strong acid is present
• Negative result: yellow/ gold/ orange
color
Review
• MR-VP Reactions (pg. 101 – 104)
• Differentiate microorganisms on their fermentative end products
• Sugar glucose is added to media
• Each culture is split into two clean test tubes  pour in 1mL of
culture into each clean test tube
• Voges Proskauer (VP) Test
• Detects production of unusual alcohols  acetyl methyl carbinol
(AMC)
• Add 10 drops of α-naphthol and 10 drops
of KOH w/ creatine
• Let sit for 20min (do not shake tube)
• Positive result: a red ring on top
• Negative result: any other color ring
Review
• Hydrolysis of Urea (pg. 91 – 93)
• Some microorganisms produce enzyme urease  breaks down
urea into ammonia and CO2
• pH Indicator: Phenol red added to urea broth
• Ammonia is a strong base  media becomes basic
• Positive result: Hot pink color
• Negative result: yellow/ gold color
 indicates acidic conditions
Review
• Ammonium Phosphate Test (pg. 105 – 106)
• This test uses a minimal media with only one source of nitrogen
 ammonia
• Some microorganisms can metabolize ammonia and others cannot
• Media: ammonium phosphate, glucose, potassium chloride,
magnesium sulfate, and pH indicator bromocresol purple
• Positive result: ammonia is metabolized  media becomes acidic
 color change to yellow
• Negative result: ammonia is not metabolized  media remains
basic  color stays purple
Review
• Sodium Citrate Test (pg. 107 – 108)
• This test uses a minimal media with only one source of carbon
 citrate
• Some microorganisms can metabolize citrate and grow and others
cannot
• Positive result: growth in the media
• Negative result: no growth
Review – Mannitol Salt Agar
• Exp #24 (pg. 115) - Mannitol Salt Agar (MSA) plate
• Used for isolation and identification of Staphylococcus
species
• Contains mannitol (sugar), phenol red (pH indicator), and
7.5% NaCl (high salt concentration)
• Staphylococcus species will grow in high salt concentration
• Staphylococcus aureus is pathogenic
• Differentiated on ability to ferment
mannitol
• Mannitol fermentation  produces acid
 yellow color
• What do your plates look like?
Explain your results!
Today’s Lab - DEMO
• Coagulase production (pg.117 – 118)
• Detect enzyme coagulase – causes blood plasma to clot  clots
fibrinogen to form fibrin
• Staphylococcus aureus produce enzyme coagulase  confirms the
presence of pathogenic S. aurues
• Plasma contains clotting factors  naturally forms a clot  to
prevent this anticoagulant (EDTA) is added to rabbit plasma
Procedure:
• Positive colonies from MSA plate are inoculated into a tube of rabbit
plasma  incubate at 37°C for 2-3 days
• Tilt tubes and observe for clotting
• Positive result (coagulase): clotting of plasma
• Negative result (no coagulase): no clotting of plasma
Today’s Lab - DEMO
• Coagulase production (pg.117 – 118)
Today’s Lab - DEMO
• Hemolysin production (pg.119 – 120)
• Staphylococcus species and Streptococcus species produce
hemolysins (enzymes) that allow them to breakdown blood cells to
obtain nutrition
• Bacteria are streaked onto blood agar plates (5% sheep blood)
• Three hemolysis patterns:
• 1) Alpha hemolysis (α): partial breakdown of blood cells  colonies
have a greenish, murky zones around them
• Example: Streptococcus pneumoniae
• 2) Beta hemolysis (β): complete breakdown of blood cells  colonies
have a clear zone around them (no blood)
• Example: Streptococcus pyogenes (causes strep throat)
• 3) Gamma hemolysis (γ) – blood cells are not broken down 
colonies grow on the surface of the plate but no zones appear
around the colonies
• Example: Enterococcus faecalis
Today’s Lab - DEMO
• Hemolysin production (pg.119 – 120)
Alpha hemolysis
Beta hemolysis
Gamma hemolysis
Today’s Lab - DEMO
• Latex agglutination (pg. 123 – 126)
• Detect if a Staphylococcus organism produces coagulase and/or
protein A
• Direct vs. indirect test
• Direct – detect the antigen (coagulase/ protein A) – perform in lab
• Indirect – detect the antibodies
• Antibody + Antigen (Coagulase/ protein A) = clumps
• Latex beads are coated with antibodies for coagulase and/or
protein A
• Use disposable cards provided and follow instructions on pg. 123124
• Positive result: presence of clumps
• Negative result: no clumps present
Today’s Lab - DEMO
• Hydrolysis of Starch (pg. 67 – 68)
• Detect enzyme amylase – breaks down starch into sugar
amylase
Starch
(polysaccharide)
Maltose
(disaccharide)
• Not all bacteria produce this enzyme
• Procedure:
• Inoculate bacteria onto starch agar plate  incubate
• Open lid of plate  pour a thin film of iodine onto incubated plate 
wait 10 mins
• Starch reacts with iodine  dark purple/ brown color
• Positive result (amylase): no starch  no color change 
“clear zone”
• Negative result (no amylase): color change to dark purple/ brown
Today’s Lab - DEMO
• Hydrolysis of Starch (pg. 67 – 68)
Negative
Positive
Today’s Lab - DEMO
• Hydrolysis of gelatin (pg. 69 – 70)
• Detect enzyme gelatinase – breaks down gelatin
gelatinase
Gelatin
(solid)
Amino acids
(liquid)
• Not all bacteria produce gelatinase
• Procedure:
• Using an inoculating needle, stab the gelatin deep
• Incubate tubes at room temp – 3 to 7 days
• Refrigerate tubes for ~5minutes and then read results
• Positive result (gelatinase): no gelatin present  media inside
tube is liquid
• Negative result (no gelatinase): gelatin still present  media
remains solid
Today’s Lab - DEMO
• Hydrolysis of gelatin (pg. 69 – 70)
Positive
Negative
Today’s Lab - DEMO
• Litmus milk (pg. 75 – 80)
• Media with whole milk + litmus pH indicator
• Many different reactions:
• Fermentation: milk sugar lactose is
fermented by bacteria  acid production
• 1) Acid only (A): litmus turns pink
• 2) Acid + hard curd (protein) (AC):
acid coagulates milk proteins 
pink color and hard curd
• 3) Acid + hard curd + CO2 gas (ACG):
pink color, hard curd, and gas cracks
Today’s Lab - DEMO
• Litmus milk (pg. 75 – 80)
• Media with whole milk + litmus pH indicator
• Many different reactions:
• Alkalinization (B): milk protein casein is
partially broken down  alkaline end
products (polypeptides and amines) 
pH increases  litmus changes color
to blue (blue milk)
Today’s Lab - DEMO
• Litmus milk (pg. 75 – 80)
• Media with whole milk + litmus pH indicator
• Many different reactions:
• Peptonization (P): complete breakdown of milk
proteins
• No proteins = no colloids = milk becomes
translucent/ clear
• Breakdown of proteins  alkaline end
products  pH increases  litmus changes
color to purple
• Peptonization + rennet curd (PR) 
some organisms produce enzyme renin
 clots milk  translucent and soft curd
Today’s Lab - DEMO
• Litmus milk (pg. 75 – 80)
• Media with whole milk + litmus pH indicator
• Many different reactions:
• Reduction (R): anaerobic respiration occurs  litmus is reduced
(picks up Hydrogens)  litmus loses its colors and becomes
whitish
• Reduction can occur alongside:
• Fermentation: AR
• Peptonization: PR or PRC
• Alkalinization: BR
• Fermentation, alkalization, and
peptonization do not occur together