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1 Basic Clinical Microbiology Lesson Plan Week 5 Carbohydrate Fermentation, MRVP Class 1 A. Take attendance. B. Review Lab Report questions from previous week. C. Instruct students to put on lab coats and gloves; clean their benches. D. Materials: Students work in pairs Students must use racks for working with and transporting all tubes 1 incinerator 1 inoculating loop Sharpie Lab tape for label 1 starch plate Grams Iodine 4 Glucose fermentation tubes 4 Lactose fermentation tubes 4 Sucrose fermentation tubes 4 MRVP tubes Stock cultures-1 set per bench (4 students share) Bacillus subtilis on nutrient agar slant Escherichia coli on nutrient agar slant Pseudomonas aeruginosa on nutrient agar slant Alcaligenes faecalis on nutrient agar slant Proteus vulgaris on nutrient agar slant Enterobacter aerogenes on nutrient agar slant E. Review of new bacteria in this lab: These organisms are Biosafety Level 2. Aseptic technique, gloves and lab coats are required. Alcaligenes faecalis a Gram negative rod, found in the environment (soil, water), associated with mixed infections of diabetic foot ulcers and infections of lower extremities. Enterobacter aerogenes-- a Gram negative rod, found in environment (soil, water, sewage), animals and dairy products; normal intestinal flora of humans; causes UTI s, wound infections, respiratory infections, bacteremia-just to name a few! F. Lab Practical Carbohydrate Catabolism pages a. Review the Background. b. Review terms and definitions Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 1 of 6

2 Catabolism a type of metabolism, the process of using enzymes to break down complex carbohydrates into simple carbohydrates; this process releases energy--atp. Carbohydrates large molecules containing carbon, hydrogen and oxygen. Polysaccharides complex carbohydrate; e.g. starch Monosaccharide simple carbohydrate; e.g. glucose Enzymes proteins that increase the rate of reactions; necessary for metabolism. Hydrolytic exoenzymes leave the cell, use water to break down the molecule to a smaller size, then transports that molecule back into the cell. Example amylase. Differential medium uses metabolic differences to distinguish and identify bacteria e.g. carbohydrate fermentation tubes Selective medium uses antibiotics, dyes or other chemicals to inhibit or induce the growth of bacteria e.g. MacConkey agar selective for GNRs. c. Starch hydrolysis test lab practical Purpose= to distinguish bacteria which can hydrolyze starch from bacteria that cannot hydrolyze starch. Starch is differential medium. Reaction: amylase Starch glucose 1) Label bottom of starch plate as below 3 sections. 2) Use a loop (and aseptic technique!) to inoculate Basillus subtilis, P. aeruginosa and E. coli onto each section. Draw on board. Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 2 of 6

3 3) Instruct student to stack plates; instructor tapes and labels plates, place in 35ºC incubator. 4) At class 2 students examine plates for growth and record in lab report; then pour small amount of Gram s iodine onto plate. 5) Examine and interpret results. The iodine will stain the starch blue; if starch was hydolized then the medium will not stain and appear clear. No stain= starch was hydrolyzed, organism produces amylase Stain= starch present, organism does not produce amylase. d. Oxidative and Fermentative Catabolism page 56 explain and show digital images Oxidative catabolism requires oxygen. Fermentative catabolism occurs with or without oxygen. Oxidative-fermentative test (OF Medium) purpose if to determine if organism metabolizes a carbohydrate oxidatively or fermentatively or not at all. They can only do one! Show digital image of Hugh-Leifson medium. A semi-solid deep medium with glucose and peptone. Bromthymol blue is the ph indicator. Reaction: Oxidative Glucose Fermentative produces organic acids which the ph and turns medium yellow bromthymol blue is yellow at ph < 6.0 blue at ph of >7.6 e. Lab report questions pages (skip OF-glucose questions); questions 1-3; critical thinking 2-3. G. Lab Practical Fermentation of Carbohydrates pages a. Review the Background. b. Review terms and definitions Fermentation a complex series of reactions and enzymes which occurs with or w/o oxygen. The end-products produced are dependent on the substrate, duration of enzymatic activity, temperature etc. Fermentation is a process incorporated into many methods for identifying bacteria. MRVP Methyl Red and Voges-Proskauer test determines if an organism produces a large amount of acid from fermentation of glucose. Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 3 of 6

4 Purpose of carbohydrate fermentation tubes: to determine if an organism ferments a carbohydrate or the peptone and if it produces gas (N 2 or CO 2 ) in the process. Reactions in the Carbohydrate fermentation tube: Carbohydrate fermentation acid produced, ph, gas? acetoin (neutral) produced, ph to neutral, gas? ph indicator phenol red which is red at >7.0 yellow at <6.8 **an orange color indicates neutral ph Reactions in MRVP tubes two tests acid prod and acetoin prod. : MRVP broth contains glucose, peptone and methyl red ph indicator MR Reaction: ph indicator Methyl Red is red at <4.4 yellow at >6.2 Large amount of acid production from glucose fermentation, the acid limits the growth of the E.coli and its ability to ferment peptone: ferments E. coli large amt of acid produced, ph glucose so when MR reagent added color of tube is red Large amount of acid production from glucose fermentation, E. aerogenes able to survive in acidic environment some acids converted to neutral acetoin which increases the ph. ferments glucose E. aerog. acid produced initially then the acetoin produced the ph to about 6.0 so when MR reagent added the color is yellow. Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 4 of 6

5 VP Reaction: VP reagents detect production of the acetoin. ferments E. coli large amt of acid produced inhibit glucose E. coli growth; no acetoin produced. ferments E. aerog. Acids produced initially then these Glucose converted to neutral acetoin. Acetoin detected by addition of VP1 and VP2 acetoin + VP1(alpha naphthol )+ VP2 (KOH) + O 2 = pink color c. Label the 4 sets of glucose, lactose and sucrose fermentation tubes with the substrate, the organism name (A, faecalis, P. vulgaris, E. coli, and E. aerogenes), the date and your initials. Repeat as above for MRVP tubes except use E. coli and E. aerogenes only. A. faecalis glucose, sucrose, lactose tubes only P. vulgaris only E.coli glucose, sucrose, lactose, MRVP tubes x2 E. aerog. d. Use aseptic technique to inoculate each set of tubes. e. When all tubes and plates inoculated, students bring to instructor who places tubes in 1 large rack labeled with the section. Tape the stacked plates together and label with the section. Place in 35ºC incubator. f. Lab reports: Carbohydrate catabolism pages (skip OF-glucose); questions 1-3; critical 2-3 Fermentation pages 65-67; questions 1-3, 6; critical thinking 1-4 Class 2 Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 5 of 6

6 H. Take attendance. I. Clean benches, put on lab coats and gloves. J. Materials for Class 2: Methy Red and Vogues Proskauer Reagent (4 students share) Empty large test tubes 4 per 2 students Plastic Transfer pipettes 2 per 2 students K. Process MRVP tubes first because VP reaction takes up to 30 minutes. a. Label 2 large test tubes E. coli MR and E.coli VP b. Label 2 large test tubes E. aerogenes MR and E. aerogenes VP c. Use pipette to aseptically transfer MRVP broth to labeled tubes volume to transfer is 1 inch of liquid in each large tube. d. Add 5 drops MR reagent to MR tubes result is almost immediate; E.coli is red and E. aerogenes is yellow. e. Add 12 drops VP1(alpha naphthol) and 3 drops VP2 (40%KOH) to VP tubes. Shake tubes and leave the caps off. VP reaction is oxidative reduction reaction and needs O 2. VP E. aerogenes pink is positive and VP E.coli yellow is negative. acetoin + alpha naphthol + KOH + O 2 = pink color f. Instructor can set up 1 set of MR and VP controls for whole class. L. Students may work in groups to complete lab questions. M. Turn in lab reports to instructor labs may be removed from manual. N. Instructor corrects reports and returns to student before or at next class. Basic Clinical Microbiology Lab Fall 2010 J. Hodgdon Page 6 of 6

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