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 55-56
a. Review the Background.
b. Review terms and definitions
Basic Clinical Microbiology Lab Fall 2010
J. Hodgdon
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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
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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
produces organic
Fermentative
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 57-59 (skip OF-glucose questions);
questions 1-3; critical thinking 2-3.
G. Lab Practical Fermentation of Carbohydrates pages 61-53
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
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Purpose of carbohydrate fermentation tubes: to determine if an
organism ferments a carbohydrate or the peptone and if it produces
gas (N2 or CO2) 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
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“
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:
E. coli
ferments
glucose
large amt of acid produced, pH ↓
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
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VP Reaction:
VP reagents detect production of the acetoin.
ferments
E. coli
glucose
large amt of acid produced inhibit
E. coli growth; no acetoin produced.
ferments
E. aerog.
Glucose
Acids produced initially then these
converted to neutral acetoin.
Acetoin detected by
addition of VP1 and VP2
acetoin + VP1(alpha naphthol )+ VP2 (KOH) + O2 = 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
P. vulgaris
E.coli
E. aerog.
glucose, sucrose, lactose tubes only
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“
“
only
glucose, sucrose, lactose, MRVP tubes x2
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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 57-59 (skip OF-glucose); questions 13; 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
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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 O2. VP E. aerogenes pink is positive
and VP E.coli yellow is negative.
acetoin + alpha naphthol + KOH + O2 = 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
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