MCB 101 Exam 1 Spring 2019 9 9

MCB 101 Exam 1 –Spring 2019 9 – 9: 50 AM, Friday, Feb. 22 Place Sections - A, B, C & D in 180 Bevier Sections - E, F & G in 103 Mumford Covers: Experiments 1 - 8 pgs. 1 – 70 Format: Machine Graded Multiple Choice

MCB 101 On-Line Homework Assignments LON-CAPA: Pre-lab - 5 Due at 8: 00 AM Monday, 2/18/19 Covers: Biochemical Tests for Bacterial Identification Experiment 9 pgs. 71 – 89 LON-CAPA: Pre-lab - 6 Opens Today Due at 8: 00 AM Monday, 2/25/19 Covers: Bacterial Identification Experiments 10 & 11 pgs. 90 – 108 LON-CAPA: Quiz - 5 Opens Friday, 2/22/19 at 10: 00 AM Due: Friday 3/1/19 at 8: 30 AM Covers: Biochemical Tests for Bacterial Identification Experiment 9 pgs. 68 - 86 Aseptic Technique NO QUIZ OPENS TODAY, STUDY FOR THE EXAM NEXT WEEK.

MCB 101 Introductory Microbiology Laboratory Lecture 5 February 15, 2019 Today’s Topics -Experiment 8: Bacterial Enzymes and Adaptations to Specific Environments - Experiment 9: Biochemical Tests for Bacterial Identification Above: peritonitis Below: rotting apple

Expected Results for Experiment 7 7 A Growth Factor Requirements L-agar contains tryptone. This product is made from enzymatically digested milk proteins and therefore is a source of all 20 of the amino acids that are commonly found in proteins. M 9 is a defined, minimal medium. The only organic compound in M 9 is glucose. Medium LB M 9+T M 9+P S. marcescens DI + + WB 32 + + WB 34 + + -

7 B Citrate Utilization Serratia marcescens and Enterobacter aerogenes grow on Simmon’s citrate agar and turn the medium blue, Escherichia coli and Morganella morganii don’t grow on citrate agar.

Expected Results for Experiment 7 7 C Carbohydrate Fermentation Escherichia coli ferments glucose and should turn the tube that contains this sugar yellow. If there is no sugar present the bacteria can still grow by using the amino acids as a food source but they will produce basic waste products like ammonia or amines that cause the tube to turn blue or at least remain green.

7 D Cross Feeding Pigment Production by Non-pigmented strains of S. marcescens Both RWDE 2 and HLCW excrete compounds that allow MS 10 to make the red pigment. HLCW excretes a compound that allows RWDE 2 to make the red pigment.

Historically, auxotrophic mutants played an important role in helping biochemists to understand biosynthetic pathways.

This data was used to work out the last three steps of the arginine biosynthetic pathway by which the amino acid ornithine is converted to arginine.

Ex. 8 Bacterial Adaptation • To live, grow, and reproduce, microorganisms must be able to gather food from their environment and bring them into their cells. • Once they enter the cell, these nutrients must be converted into necessary cell components and/or used to obtain energy for cell functions. • Microorganisms must be able to adapt to changes in their environment. • Adaptation involves differences in metabolism.

Metabolism involves enzymes. • Enzymes are generally, globular proteins • Enzymes function as biological catalysts to speed up chemical reactions • Catalysts reduce the energy that must be supplied in order for molecules to start reacting with one another • Catalysts are not used up or changed by the chemical reactions they accelerate.

Characteristics of Enzymes • An enzyme contains an active site where the substrate binds. • Enzymes are usually present in relatively low concentrations since they are not used up or altered during their reactions. • Enzymes are highly specific for their substrate. Generally there is one specific enzyme for each specific chemical reaction.

Ex. – 8 Bacterial Enzymes and Adaptation Enzymes of Interest: Lipase: an exoenzyme for digesting fats Amylase: an exoenzyme for digesting starch Gelatinase: an exoenzyme for digesting protein

Lipase • Catalyzes breakdown of lipids – This includes fats (solid at room temperature), oils (liquid at room temperature), lecithins and sterols • Hydrolyzes fat to yield glycerol and fatty acids • Important for microbes that survive by digesting dead animal and plant material

Lipase Test • Egg yolk agar plate (naturally cloudy) • Inoculate with both organisms (one positive, one negative) • Incubate, let bacteria grow & make enzymes • Look for a clear zone around the bacterial growth (this indicates lipid breakdown)

Amylase is Needed to Digest Starch • Starch is a major form of stored carbohydrate in plants. • Composed of a mixture of two substances – amylose (10 -20%) – amylopectin (80 -90%). • Amylose molecules consist typically of 200 to 20, 000 glucose units. • Amylopectin molecules may contain up to two million glucose units.

Amylase Test Amylase (-) Amylase (+) Starch agar plate Inoculate with both organisms, incubate overnight Flood plate with iodine (reacts with starch - blue-brown color) Clear zone indicates starch breakdown (amylase +)

Gelatinase • Exoenzyme • Protease that degrades gelatin • Hydrolyzes peptide bonds • Allows microorganisms that produce it to break down gelatin into smaller polypeptides, and amino acids that can cross the cell membrane.

Gelatinase Test • Gelatin agar plate • Inoculate with test organisms Pseudomonas aeruginosa: positive for gelatinase Escherichia coli = negative for gelatin hydrolysis • Incubate at 37 o. C for 2 days • Flood the plate with ammonium sulfate (precipitates gelatin, makes plate appear cloudy) • Look for a clear zone around bacterial growth (indicates gelatin breakdown)

The O-F Glucose Test Oxidative vs. Fermentative Metabolism Oxidative metabolism (aerobic respiration) The terminal electron acceptor is O 2. Bacteria grow aerobically. Fermentative metabolism The terminal electron acceptor is an organic molecule. The bacteria can grow aerobically or anaerobically. This test uses a semisolid glucose Uninoculated Fermentative medium in a tube. (No growth) The medium contains a p. H indicator dye, bromthymol blue Two tubes of media are inoculated with each strain of bacteria tested. One tube is left open to the air. The other tube is kept anaerobic by addition of a layer of mineral oil. Oxidative (Respiration)

Metabolism • Respiration (+O 2 or some oxidized mineral) – More ATP can be made per sugar consumed – Complete oxidation (loss of e-) of compound – An external electron acceptor is required • Fermentation (Anaerobic) – Makes much less ATP than respiration – But there is less competition in anaerobic places – Partial oxidation of substrate (nutrient) – Internal electron acceptor (organic) – Acids are usually produced when bacteria ferment sugars

Experiment 8: Enzymes and Adaptations Procedures to Check the Results Amylase Test Flood the starch agar plate with iodine, the media will turn a dark color. The color is caused by a complex that forms between starch molecules and iodine. It may be brown, blue or black. A clear zone around the bacterial growth is a positive result. Lipase Test Hold the egg yolk agar plate up to window and look through it. The lipids in the egg yolk agar cause the medium to be cloudy. A clear zone around the bacterial growth is a positive result. Gelatinase Test Flood the gelatin agar plate with a saturated ammonium sulfate solution. This salt will cause proteins, like gelatin, to precipitate, turning the medium cloudy. (It may take a few minutes for the plate to turn cloudy. ) Dump the excess ammonium sulfate down the sink. Hold plate up and look through it. A clear zone around bacterial growth is a positive result. O-F glucose Test A yellow color indicates production of acid. Fermentative bacteria can turn the closed tube completely yellow.

Ex. 8 Enzymes and Adaptations Expected Results • Amylase Test Escherichia coli is – Bacillus subtilis is + • Lipase Test Escherichia coli is – Bacillus subtilis is + • Gelatinase Test Escherichia coli is – Pseudomonas is + • O-F glucose Test Escherichia coli is F Pseudomonas is O • Ea = Enterobacter aerogenes • Pf = Pseudomonas fluorescens • Af = Alkaligenes faecalis

Bacteria produce enzymes needed for survival in an ecological niche. Escherichia coli grows in the mammalian large intestine. It produces few hydrolytic exoenzymes because the host animal makes digestive enzymes. E. coli can grow fermentatively because the intestines are an anaerobic environment. Bacillus subtilis grows on plant matter. It produces amylase and lipase to digest organic matter. (B. subtilis, AKA “hay bacillus”) Pseudomonas aeruginosa grows on rotting plant and animal matter and can cause skin infections. It makes proteases such as gelatinase and is an obligate

Ex. 9 Biochemical Tests • Each organism has unique metabolic characteristics based on their habitat • Biochemical tests use differences to discriminate between bacteria • Used to identify bacteria – Can use energy source or not? – Enzymatic ability – Waste products produced (p. H change)

ENTEROBACTERIACEAE • Many are pathogenic and they are often seen in clinical microbiology. • All are Gram negative rods. • All ferment glucose anaerobically • They are often associated with intestinal infections • Some can cause meningitis, bacillary dysentery, typhoid, fever and food poisoning • Different species can cause similar symptoms, so biochemical tests are crucial for identification, diagnosis, and treatment of infection. Genera in the Family Enterobacteriaceae • • • ESCHERICHIA SHIGELLA EDWARDSIELLA SALMONELLA CITROBACTER KLEBSIELLA ENTEROBACTER SERRATIA PROTEUS MORGANELLA PROVIDENCIA YERSINIA

Typical Biochemical Tests Used For Identifying Bacteria Carbohydrate Fermentations - the broth contains a rich mix of amino acids and vitamins and one type of sugar - the broth usually contains a p. H indicator dye - the bacteria must be able to grow in the broth for the test to be valid - a positive result for fermentation = acid production = lower p. H Production of Specific Waste Products - large amounts of acid (methyl red test) - 2, 3 -butanediol (Voges-Proskaur test) - gas (CO 2, H 2) (Durham tube) - H 2 S (iron agar) Degradation of Specific Amino Acids - tryptophan indole - phenylalanine phenylpyruvate - lysine cadaverine - ornithine putrescene

General Hints About Biochemical Tests • For most tests, if the bacteria don’t grow you do not have a valid test, it’s not negative! • Incubation Time & Temperature Matter – You can get misleading results if you read the test too early or let it incubate too long. – Results can change with temperature change. • Compare to controls – Positive – Negative

Sugar Fermentations • Glucose, Sucrose and Lactose • p. H indicator - BTB – Fermentation = acid production – Hydrolysis of amino acids = alkaline waste • Look for net reaction of acid and base • Gas Production, (CO 2 & H 2) – Durham tube, look for bubble Brom-thymol blue Acid p. H 6. 8 Base

Results of Sugar Fermentations - A/G, acid and gas produced (yellow, with bubble in the Durham tube) - A, acid produced (yellow, yellow no bubble) - (A), some acid produced (lime green) - 0+, no acid or gas produced, but there is growth (the bacteria use the peptone) (green and cloudy) - 0+/B, no acid or gas but there is growth on peptones alkaline (blue and cloudy) - 0 -, no acid or gas; (green, clear) clear no growth = no test

Methyl Red/Voges-Proskauer • Distinguishes between coliform bacteria – E. coli (fecal contamination) – Enterobacter (non-fecal contamination) • Coliforms – G-, facultative anaerobes, ferment lactose – Indicator of possible presence of pathogens (diseasecausing microorganism) – Indicator of possible fecal contamination – Present in soil, water, plants and mammalian intestines – Don’t always cause disease themselves, but they are an indication that disease-causing organisms could be present, especially if confirmed fecal coliform

Methyl Red Test • Looks for LARGE quantities of acids (Mixed Acid Fermentation Pathway) • MR/VP broth contains glucose • Methyl Red is a p. H indicator, added after growth. Acid color = Red, Red Base color = Yellow p. Ka ~ 5. 4 • E. coli produces a lot of acidic end products from glucose fermentation – Results in a p. H of less than 4. 5 (Red) Red • Enterobacter produces acids, but also some nonacidic compounds such as: acetoin, 2 -3 butanediol and ethyl alcohol – Results in a p. H of less than 6. 5 but more than 5. 0 (Yellow or Orange) Orange

Voges-Proskauer Test • Checks for use of Butanediol Pathway – Excretion of butanediol into medium – Acetoin = intermediate in pathway • Add VP I = alpha-naphthol – Oxidizing agent • Add VP II = KOH & creatine – KOH = oxidizing agent – Creatine = acetoin indicator (color change) • Shake vigorously to introduce oxygen for oxidation back to acetoin – Acetoin + creatine = pinkish red color

MRVP • The Voges-Proskauer test is complementary to the Methyl Red Test – If MR+, VP usually -, mixed acid pathway – If MR-, VP usually +, butanediol pathway – If MR-, VP- glucose cannot be fermented, (these bacteria probably grow on the peptones) MR test: - + VP test: uninoc. - + -

Phenylalanine Deaminase • Catalyzes removal of amino group from phenylalanine - oxidative deamination – Reaction needs oxygen so this agar is NOT stabbed. • Produces ammonia and phenylpyruvic acid, which turns green in presence of Ferric Chloride • Differentiates between – Proteus, Morganella and Providencia – Other Enterobacteriacae

Phenylalanine Deaminase Results Proteus: Can be a cause of opportunistic nosocomial infections Other Urinary tract infections Neg (-) Pos (+) Green

Lysine Decarboxylase • Lysine decarboxylase removes the carboxyl group from the amino acid lysine, producing cadaverine. • The broth contains a mixture of lysine and glucose (5: 1) All Enterobacteriaceae ferment glucose and produce acid, so the medium turns Yellow. If the bacteria can make Lysine Decarboxylase, they start to do so when the medium turns acidic and they run out of glucose, then you get CO 2 and cadaverine, which is alkaline, so the medium turns Purple Lysine decarboxylase is involved in breaking down an amino acid. Rot that includes a lot of protein degradation is called putrifaction.

Indole Production from Tryptophan • Tests for production of the enzyme tryptophanase • breakdown of tryptophan releases indole • Isoamyl alcohol extracts indole • p-dimethylaminobenzaldehyde reacts with indole to make a red color • The indole test is used to distinguish between Salmonella and Shigella

Hydrogen Sulfide Production: Kligler’s Iron Agar • • Cysteine and Methionine contain sulfur. Some bacteria degrade these amino acids and make H 2 S reacts with iron to form Fe. S, a black precipitate. Kligler’s iron agar also contains Glucose and Lactose plus a p. H indicator and can be used to test for fermentation of those sugars. C = uninoculated control 3 & 5 show the black precipitate characteristic of H 2 S production 1 is - for sugar fermentation 2 is + glucose but - for lactose fermentation 4 & 4 A show gas production

Catalase - distinguishes between Staphylococcus (+) Streptococcus (-) 2 H 2 O 2 + 2 H 2 O Aerobic microorganisms like Staphylococcus, which is an inhabitant of the skin, tend to make catalase while many anaerobes and microaerophiles like Streptococcus do not.

Looking Ahead The tests from Experiments 8 and 9 are used in clinical labs to identify bacteria. In Experiments 10 and 11 we will use or these tests to identify an unknown culture. BUT, before doing biochemical tests, you have to have a PURE CULTURE. Observations of basic traits, like the Gram stain reaction can reveal the BROAD group to which the bacterium belongs. Biochemical tests can reveal differences between related species due to differences in the enzymes they possess.