1. Lab #8

2. Review of Lab #7 - 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 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

4. Review of Lab #7 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 of Lab #7

6. 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 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. 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

8. 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

9. 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

10. Today’s Lab – DEMO 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. 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

12. 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

13. 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

14. 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

15. 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