Lab #8
Review of Lab #7 - 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 of Lab #7
• 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
Review of Lab #7
• 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 of Lab #7
• 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
Lab #7 Results
• 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
Lab #7 Results
• 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
Lab #7 Results
• 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
Lab #7 Results
• 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
Today’s Lab – DEMO
• 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
Today’s Lab – DEMO
• 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
Today’s Lab – DEMO
• 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
Today’s Lab
• Experiment #24 (pg. 115)
• Mannitol Salt Agar (MSA) plate
• Use a sterile cotton swab to obtain a throat or nose sample
• Streak the first quadrant of an MSA plate using the cotton
swab
• Then use your sterilized loop to streak the remaining three
quadrants  streak for isolated colonies
• Place plates in plastic container  incubator
Water Analysis Project and Lab Report
• Pg. 131 – 138
• Test for presence of coliform (E. coli)  G- rod bacteria
found in the GI tract
• Perform three tests (Pg. 136):
• Presumptive
• Confirmed
• Completed
• Procedure for presumptive test
• Obtain 3 water samples A, B, and C
• Inoculate 1mL of each into a separate Lactose broth tubes
• Make sure tubes are properly labeled
• Incubate the tubes
• Read results in 24-48 hrs/ next lab
Water Analysis Project and Lab Report
• Presumptive test results (table on pg. 133)
• Positive: Acid and gas production = “a/g”
(color change to yellow and gas bubble in Durham tube)
• Use positive tube to streak an EMB (Eosin Methylene Blue)
plate  Confirmed test
• Confirmed test results
• Positive: Metallic green colonies
• Obtain a single colony from the positive streak plate and inoculate it
into a lactose broth  Completed test
• Today: Completed test results
• Positive: Acid and gas produced
• Indicates the presence of coliform bacteria  fecal contamination!
• Record your results and class results