Lab 11 Goals and Objectives:

1
Lab 11 Goals and Objectives Exercise 41
Oxidation and Fermentation Tests Read results
some tubes will require additional reagents
(located at stations around the room) Do
controls first so you have something to compare
to! Exercise 42 Hydrolytic and Degradative
Reactions Set up according to Fig 42.1 except
both of your two unknowns well separated on
each type of plate and control on a different
plate (one control plate per pair) One set of
controls per pair using broth cultures Bacillus
subtilis Staphylococcus aureus Escherichia
coli Proteus vulgaris Each pair needs 3
starch plates 5 urea broths (replaces urea slant)
3 skim milk plates 5 phenylalanine slants 3
spirit blue plates 5 tryptone broths Save
streak plates of unknowns for use next class
2
Durham Sugar Tube Fermentation (Glucose, Lactose,
Mannitol) Inoculation method loop
transfer Contains single carbohydrate peptone
broth with durham tube for gas collection,
Phenol red pH indicator alkaline pH red,
acidic pH yellow Discriminates the ability
to ferment a single carbohydrate (glucose,
lactose, or mannitol) into acid products (e.g.
pyruvic acid) or acid plus gas Results Red
inert, negative for fermentation of specified
carbohydrate Yellow positive for
fermentation of carbohydrate to acid
products Yellow with bubble positive for
fermentation of carbohydrate to acid gas
3
Acid plus gas
Acid
Negative
4
MR-VP Medium Methyl Red Test Inoculation
method loop transfer Contains peptone,
glucose, and buffer (buffer will neutralize weak
acids so only strong stable acids will be
detected by methyl red) Additional reagents
added methyl red pH indicator acid pH red,
neutral or alkaline pH yellow Distinguishes
ability to catabolize glucose into stable mixed
acids (lactic, acetic, and formic acids) in the
mixed acid pathway Results Red positive for
mixed acid formation Yellow negative for
mixed acid formation
_

5
MR-VP Medium Voges-Proskauer Test Inoculation
method loop transfer Contains peptone and
glucose Additional reagents added Barritts A
(alpha napthol) and Barritts B (KOH) (will
react with acetoin to produce a red product,
alone produce a copper colored product)
Distinguishes the ability to catabolize glucose
into the neutral end product butanediol (the
oxidized product is acetoin) in the butylene
glycol pathway Results Red positive for
acetoin and thus for 2,3-butanediol
production Yellow/Orange no acetoin,
negative for 2,3-butanediol production
_
_


6
Simmons Citrate Agar Inoculation method streak
and stab slant with needle Contains citrate as
sole carbon source, ammonium salts as sole
nitrogen source, bromthymol blue pH indicator
neutral pH green, alkaline prussian
blue Discriminates organisms that can produce
citrase to metabolize citrate into oxaloacetate
and pyruvate. These organisms are forced to
utilize ammonium salts as the nitrogen source
producing alkaline ammonia waste. Results
Prussian blue slant and or butt positive for
citrase production Green negative
for citrase production
_

7
Oxidase Test Discriminates organisms that can
produce cytochrome oxidase which catalyzes the
transfer of electrons from reduced cytochrome c
in the electron transport chain to molecular
oxygen. Test uses NNNN-tetramethyl-p-phenylenedi
amine (Oxidase Reagent) as an artificial
electron acceptor when oxidized it is colorless,
when reduced it turns purple
Look for color change on the bacteria, not on
the cotton swab! (The reagent will turn light
purple when exposed to oxygen in the air)
8
Catalase Test Discriminates aerobic organisms
that produce catalase to degrade hydrogen
peroxide into water and oxygen
_

http//ftp.ccccd.edu/dcain/CCCCD20Micro/Catalase.
jpg
9
12 Possible Unknowns
Gram Positive
Gram Negative
Gelatinase
Gelatinase -
Gelatinase
Gelatinase -
Catalase
Catalase -
Pseudomonas aeruginosa
Bacillus subtilis
10
Nitrate Reduction Broth Inoculation method loop
transfer Contains beef extract, peptone, KNO3
as nitrate source, durham tube for gas
collection Additional reagents added sulfanilic
acid (reagent A), dimethyl-alpha- naphthylamine
(reagent B), (together form a complex with
nitrite creating a red product), zinc (reduces
nitrate to nitrite allowing reaction with
reagent A and B) Discriminates organisms that
can produce nitrate reductases to utilize
nitrate as a final electron acceptor resulting
in the production of either nitrite (partial
reduction) or N2O or N2 gas (complete
reduction). Results Red with reagents A and
B positive for nitrate to nitrite
reduction Red only after zinc
negative for nitrate reduction, negative
for nitrate reductases Clear with gas
positive for complete reduction of nitrate
to nitrogen gas
11
A B nitrite red Zinc converts nitrate to
nitrite
sulfanilic acid (reagent A) dimethyl-alpha-napht
hylamine (reagent B)
add zinc to negative tubes
Nitrate to nitrite
Complete reduction
No reduction
12
Fig. 42.1
Unknowns
Separate Plates!
Control
13
Lab 11 Goals and Objectives Exercise 41
Oxidation and Fermentation Tests Read results
some tubes will require additional reagents
(located at stations around the room) Do
controls first so you have something to compare
to! Exercise 42 Hydrolytic and Degradative
Reactions Set up according to Fig 42.1 except
both of your two unknowns well separated on
each type of plate and control on a different
plate (one control plate per pair) One set of
controls per pair using broth cultures Bacillus
subtilis Staphylococcus aureus Escherichia
coli Proteus vulgaris Each pair needs 3
starch plates 5 urea broths (replaces urea slant)
3 skim milk plates 5 phenylalanine slants 3
spirit blue plates 5 tryptone broths Save
streak plates of unknowns for use next class