Lab #8

Review of Lab #7 - pH Indicators

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 -

Memorize pH indicators and color reactions!

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

ETC• KNO3 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

PositiveNegative

• 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 of Lab #7

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 dropsof 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