1. Lab #9

2. Review - pH Indicators pH Indicator Very acidic AcidicNeutralBasic Phenol red- pH <6.8 = yellow pH 6.9 - 8.0 = red pH >8.0 = magenta/ hot pink Litmus-pinkpurpleblue Bromocresol Purple -yellowburgundyviolet Methyl red pH <4.4 = red pH 5 - 6 = orange pH >6.3 = yellow - Memorize pH indicators and color reactions!

3. 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

4. Review Nitrate reduction test (pg. 81 – 83) Media: Nitrate broth  includes KNO 3 ETC KNO 3 KNO 2 + ATP Detect KNO 2 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

5. 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  CO 2 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

6. 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 H 2 S gas H 2 S 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

7. 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

8. 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

9. 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

10. Review Hydrolysis of Urea (pg. 91 – 93) Some microorganisms produce enzyme urease  breaks down urea into ammonia and CO 2 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

11. 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

12. 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

13. 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!

14. 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

15. Today’s Lab - DEMO Coagulase production (pg.117 – 118)

16. 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

17. Today’s Lab - DEMO Hemolysin production (pg.119 – 120) Alpha hemolysis Beta hemolysis Gamma hemolysis

18. 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

19. 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

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

21. 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

22. Hydrolysis of gelatin (pg. 69 – 70) Today’s Lab - DEMO Positive Negative

23. 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 + CO 2 gas (ACG): pink color, hard curd, and gas cracks Today’s Lab - DEMO

24. 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

25. 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

26. 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 Today’s Lab - DEMO