Lab #9

Review - pH Indicators

pH Indicator

Very acidic

Acidic Neutral Basic

Phenol red -pH <6.8 =

yellowpH 6.9 - 8.0 =

red

pH >8.0 = magenta/ hot

pink

Litmus - pink purple blue

Bromocresol Purple

- yellow burgundy violet

Methyl redpH <4.4 =

redpH 5 - 6 =

orangepH >6.3 =

yellow-

Memorize pH indicators and color reactions!

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

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

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 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

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

• 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. 123-

124• Positive result: presence of clumps• Negative result: no clumps present

Today’s Lab - DEMO

Today’s Lab - DEMO• Hydrolysis of Starch (pg. 67 – 68)

• Detect enzyme amylase – breaks down starch into sugar

amylase Starch Maltose

(polysaccharide) (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)

PositiveNegative

• Hydrolysis of gelatin (pg. 69 – 70)• Detect enzyme gelatinase – breaks down gelatin

gelatinase Gelatin Amino acids

(solid) (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)

Today’s Lab - DEMO

Positive

Negative

• 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 ispartially broken down alkaline end products (polypeptides and amines) pH increases litmus changes colorto 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 changescolor 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, andpeptonization do not occur together

Today’s Lab - DEMO