MCB 101 Final Exam 1 30 4 30

MCB 101 Final Exam: 1: 30 – 4: 30 PM Friday, 5/3/2019 sections A, B, C & D in 1024 Chem. Annex sections E, F & G in 100 Gregory Hall Conflict Final Exam: Contact KC if you need to take the conflict exam! Times: 8 – 11 am Fri. 5/3/2019 1: 30 – 4: 30 pm Mon. 5/6/2019 7 – 10 pm Mon. 5/6/2019 8 – 11 am Tue. 5/7/2019 What is the nature of your conflict? What is the best time for you to take the exam?

MCB 101 Introductory Microbiology Laboratory Lecture 12 April 26, 2019 - Experiment 18 Fermented Dairy Products - Experiment 19 The Nitrogen Cycle - Review for Exam 3 Exam – 3 (AKA: the Final Exam) is worth 100 points, & the format is similar to exams 1 and 2. Material Covered: Experiments 15 - 19, however, there will be questions that come from earlier material that is conceptually linked to materials in Experiments 15 – 19.

Second Chance at Lab Practical Parts C and D Part C - serial dilution and spread plate Maximum Score on the Repeat = 20 points (80%) You may try again if your score was < 14 points on the exam. Contact Dr. Chapman to sign up to take the Repeat. Times: Wednesday, May 1 at 8: 00 – 9: 00 AM Wednesday, May 1 at 10: 00 – 11: 00 AM Wednesday, May 1 at 3: 00 – 4: 00 PM Part D – dilution calculations Maximum Score on the Repeat = 16 points (80%) You may try again if your score was < 11 points on the exam. Contact Dr. Chapman to sign up to take the Repeat. Times: same as above

Yogurt Production - Lactic Acid Fermentation - Scald the milk. (Heat to 80 o. C, do not boil. ) This kills contaminating bacteria and drives off oxygen. (Fermentation is an anaerobic process. ) - Cool the milk to 65 C. - Inoculate the milk with lactic acid bacteria (use yogurt) S. thermophilus tolerates higher temperature and grows fastest at neutral p. H. L. bugaricus prefers a lower temperature (40 - 45 C) but can tolerate a lower p. H. - Incubate at 45 C for 16 - 24 hours The amount of lactic acid affects flavor and consistency of the yogurt. The temperature and p. H must be closely monitored in food industry. Stop the fermentation by refrigeration when the product is perfect. Add fruit or flavoring if desired.

Lactic Acid Bacteria Two types of bacteria are needed to make yogurt. - Streptococcus thermophilus - Lactobacillus bulgaricus We will use yogurt containing live active culture to inoculate the milk. Microbial Succession First, Streptococcus thermophilus (G+ cocci) ferments lactose to lactic acid. Acid inhibits growth of Streptococcus thermophilus when p. H goes below 5. 0. Second, Lactobacillus bulgaricus (G+ bacilli), because it can grow under more acidic conditions continues to produce lactic acid, lowering the p. H to ~ 4. 0. The acidic condition causes the milk protein casein to coagulate forming a semi- solid curd.

Experiment 18 C Making Cottage Cheese - Pasteurized milk is warmed to 30 o. C Add a culture of Lactococcus lactis and Lactococcus cremoris Incubate for 45 minutes at 30 o. C Add rennet Incubate at 30 o. C for about 6 hours or until a curd forms - Cut the curd into 1 inch cubes Gently warm the curds to drive out some of the whey Drain the curds using cheesecloth Add salt

The cheese curd is cut into smaller pieces and gently heated to drive out excess liquid (whey). Then the curds are drained and (usually) salted.

Lecture Activity 12 MCB 101 4/26/19 1) The temperature used for making cheese is 30 o. C, while yogurt is made at ~_____o. C. A. B. C. D. E. 0 o. C 4 o. C 22 o. C 45 o. C 63 o. C

MCB 101 Lecture 12 4/26/19 2. What two species of bacteria are used in cheese production? A. Escherichi coli & Staphylococcus aureus B. Salmonella typhi & Shigella dysenteriae C. Lactococcus lactis & Lactococcus cremoris D. Proteus vulgaris & Morganella morganii E. Lactobacillus bulgaricus & Streptococcus thermophilus

Experiment 19 Nitrogen Metabolism and Soil Bacteria

Exp. 19 a: The urease reaction is an example of mineralization, an organic compound is broken down and ammonia is released. We have looked at several other reactions that produce ammonia from organic compounds. Phenylalanine Deaminase Test, Indole Test, Cysteine Desulfhydrase Test (Kligler’s Test)

Trommsdorf Reagents are used to detect both the reduction of nitrate and the oxidation of ammonia.

The Chemistry of Trommsdorf Reagents Trommsdorf reagent II is 6 M sulfuric acid. It reacts with nitrite to produce nitrous acid. Nitrous acid can oxidize the iodide in Trommsdorf I to iodine. The iodine can form a blue colored complex with the starch, which is also in Trommsdorf I. Nitrate (NO 3 -) can react with Zinc metal (Zn 0). The zinc is oxidized to Zn+ while nitrate is reduced to nitrite (NO 2 -). Trommsdorf I Zinc Chloride Starch Zinc Iodide 20 g/l 4 g/l 2 g/l Trommsdorf II 6 M H 2 SO 4

Test for Reduction of Nitrate • Add Trommsdorf reagents, I and II (We will use these same reagents for Ex 19 c, Nitrification, but the production of a blue color means something different. ) – The immediate appearance of an inky blue color indicates presence of NITRITE (NO 2 -) – No color change could mean one of two possibilities, either: • No nitrate reduction took place or • Nitrate reduction took place, but the nitrite was further reduced to nitrogen gas (N 2) or nitrous oxide (N 2 O).

Exp. 19 b Nitrate Reduction Nitrate Broth has peptone, beef extract and nitrate. PBE doesn’t contain nitrate. Sterile nitrate broth does not turn blue when you add Trommsdorf reagents. But zinc reduces nitrate to nitrite and then you see the blue. Enterococcus strains don’t reduce nitrate. Nitrate reduction is anaerobic. Soil contains denitrifying bacteria. Most nitrate reducing bacteria use organic carbon sources such as sugars and proteins. Proteus reduces nitrate to nitrite , so you get blue color when you add Trommsdorf reagents. Pseudomonas reduces nitrate to nitrogen gas so you get no blue even after adding zinc.

Growth in PBE vs. Nitrate Broths PBE broth Peptone 5 g Beef extract 3 g Nitrate broth Peptone 5 g Beef extract 3 g KNO 3 1 g

OF Glucose Test to see if bacteria can grow using fermentative metabolism or if they need respiration O-F Glucose Test Results Alkaligenes faecalis does not make acid from glucose Enterobacter aerogenes is Fermentative Pseudomonas fluorescens is Oxidative • Ea = Enterobacter aerogenes • Pf = Pseudomonas fluorescens • Af = Alkaligenes faecalis

Ammonia broth contains very little organic carbon, a lot of ammonia, no nitrate or nitrite. Exp. 19 c Ammonia Oxidation Trommsdorf test of sterile ammonia broth gives no blue color, before or after zinc. Ammonia oxidizing bacteria live in the top layer of soil. Oxidation of ammonia requires oxygen. Aerobic culture shows oxidation of ammonia to nitrite. Anaerobic culture shows no color, even after adding zinc. Bacteria that make nitrite or nitrate from ammonia include: Nitrosococcus and Nitrobacter. Soil contains ammonia oxidizing bacteria. They are not the same as nitrate reducing bacteria.

Lecture Activity 12 MCB 101 4/26/19 3) What type of media is used to test for denitrifying bacteria. A. B. C. D. E. Ammonia Broth TYE – lactose Agar Urea Broth Potato Dextrose Agar Nitrate Broth

Lecture Activity 12 MCB 101 4/26/19 4) A water sample is tested for coliform bacteria by spreading samples on standard Petri dish cultures of EMB agar. After incubation, the dark colored coliform colonies are counted. A 0. 10 ml aliquot of a 1 x 10 -2 dilution of the sample gives 90 coliform colonies. What is the concentration of coliform bacteria in this sample of water? A. 9. 0 x 101 D. 9. 0 x 104 B. 9. 0 x 102 E. 9. 0 x 105 C. 9. 0 x 103

Review Questions 1) A water sample is tested for coliform bacteria by spreading samples on standard Petri dish cultures of Mac. Conkey lactose agar. After incubation, the dark red colored coliform colonies are counted. A 0. 10 ml aliquot of a 1 x 10 -4 dilution of the sample gives 90 coliform colonies. What is the concentration of coliform bacteria in this sample of water? A. 9. 0 x 101 D. 9. 0 x 104 B. 9. 0 x 102 E. 9. 0 x 105 C. 9. 0 x 103 2) In Experiment 15: Milk Pasteurization, we used 3 M Petrifilm cultures to count the bacteria in the diluted milk samples. What volume of liquid is used to inoculate a 3 M Petrifilm culture? A. 0. 1 ml B. 0. 3 ml C. 1. 0 ml D. 3. 0 ml E. 10. 0 ml

6) What is the term for microorganisms that can grow and cause food spoilage in the refrigerator? A. acidophillic C. thermophillic E. thermoduric B. psychrophillic D. mesophillic 7) What type of microorganism is Penicillium? A. Fungus D. Archaea B. Bacteria E. Virus C. Protozoa 8) The food preservative sodium nitrite is: A. an organic acid. B. a phenol derivative. C. a fat soluble antioxidant. D. an inhibitor of pyruvate-ferrodoxin oxidoreductase. E. produced as a waste product of microbial fermentation of sugar.

10) How do propionate and benzoate prevent microbial growth? A. They attack proteins, prosthetic groups, sugars and nucleic acids B. They inhibit pyruvate-ferrodoxin oxidoreductase and ferrodoxin. C. They inhibit ripening enzymes that are naturally found in food. D. They denature protein destroying the cytoplasmic membrane E. They increase the proton concentration of the cytoplasm in microbes. 11) Which one of the following is not classified as a disinfectant? A. alcohol B. bleach C. phenol D. tetracycline E. hydrogen peroxide 12) An antiseptic is: A. an antimicrobial substance used on an operating table. B. an antimicrobial substance that is used on the skin. C. an antimicrobial substance added to a food or beverage D. a drug derived from a natural product that is used fight microorganisms within a patient's body. E. a drug that is a completely man-made chemical used to fight microorganisms within a patient’s body.

19) Yogurt production primarily involves: A. the fermentation of a sugar to produce lactic acid. B. hydrolysis of protein to produce short peptides and free amino acids. C. putrifying bacteria. D. boiling the milk for 30 minutes. E. bacteria that grow best in the refrigerator. 20) Which two of the following bacteria are used to make yogurt? i. iii. iv. Lactobacillus bulgaricus Pseudomonas aeruginosa Staphylococcus aureus Streptococcus thermophilus

22) Which one of the following statements about the nitrogen cycle is false? A. B. C. D. E. Nitrosococcus bacteria produce energy by oxidizing NH 3 to nitrite. The oxidation of ammonia to nitrate is called denitrification. Some Pseudomonas bacteria can reduce nitrates to nitrogen gas. The enzyme urease breaks down urea to ammonia and CO 2. The breakdown of amino acids and other nitrogen-containing organic compounds to release ammonia is called mineralization. 25) Which one of the following statements about soil bacteria and the Nitrogen Cycle is false? A. B. C. D. E. Denitrification is a form of anaerobic respiration. Mammals excrete excess nitrogen primarily in the form of ammonia. The main carbon source in ammonia broth (Ex. 19 C) is carbonate. The carbon source in nitrate broth (Ex. 19 b) is organic compounds. The only organisms that can convert elemental nitrogen (N 2) to organic forms are certain prokaryotes. F. Nitrification is an aerobic process also called ammonia oxidation. G. Nitrification is the conversion of ammonia to nitrate.