Lab 12 Goals and Objectives:

1
Lab 12 Goals and Objectives Exercise 42
Hydrolytic and Degradative Reactions Read
results some will require additional
reagents Exercise 43 Multiple Test Media No
Kligers Iron Agar or Litmus milk, No
inoculations for IMViC discuss next lab,
Inoculate SIM with straight stab for each
unknown Inoculate three separate controls per
pair using Proteus vulgaris, Escherichia
coli, Staphylococcus aureus Each pair needs 7
SIM stabs Exercise 72 Gram Negative Intestinal
Pathogens Perform according to revised
directions, supplement p. 59-60 You still need
to read the lab to know how the media
works! Each pair needs 3 MacConkey agar
plates 7 Russells Double Sugar slants streak
and stab! Control broth cultures Proteus
vulgaris (Mac) Escherichia coli (Mac,
RDS) Shigella flexnari (RDS) Pseudomonas
aeruginosa (RDS) PA will inoculate additional
controls for observation next lab Make new
streaks for isolation on BHIA Each pair needs 4
BHIA plates (new streaks for isolation)
2
Starch Agar Plate Inoculation method surface
streak with loop Contains the starches
amylopectin and amylose in agar to
solidify Additional reagents added iodine,
combines with starch to produce a blue-black
precipitate Discriminates organisms that can
produce amylase to hydrolyze amylopectin and
amylose into maltose, glucose and
dextrans Results Halo around the streak no
starch present, positive for amylase
production Blue-black precipitate up
to/under streak starch present, negative for
amylase production
_

3
Skim Milk Plate Inoculation method surface
streak with loop Contains fat free milk in agar
to solidify (plate appears opaque
white) Discriminates organisms that can produce
proteases to hydrolyze the white milk protein
casein into clear amino acids Results Halo
(clearing) around the streak no casein present,
positive for casein protease
production Opaque white up to/under streak
casein present, negative for casein protease
production
_

4
Spirit Blue Agar Plate Inoculation method
surface streak with loop Contains tributyrin
(triglycerides) in agar to solidify, Spirit Blue
pH indicator neutral pH pale blue, acidic pH
dark or bright blue Discriminates organisms
that can produce lipases to hydrolyze
triglycerides into glycerol and fatty acids
(acid pH). (Some lipases will completely
hydrolyze the tributyrin thus not producing acid
wastes but instead completely clearing the
visible lipids.) Results Dark blue
precipitate partial hydrolysis of
triglycerides, positive for lipase
production Halo around the streak (clearing of
lipids) complete hydrolysis of
triglycerides, positive for lipase production
(agar could be pale blue due to complete
clearing of all fatty acids or dark blue
because a few fatty acids remain) Pale blue
agar, no halo tributyrin present, negative for
lipase production
5
Results Dark blue precipitate partial
hydrolysis of triglycerides, positive for
lipase production Halo around the streak
(clearing of lipids) complete hydrolysis of
triglycerides, positive for lipase production
(agar could be pale blue due to complete
clearing of all fatty acids or dark blue
because a few fatty acids remain) Pale blue
agar, no halo tributyrin present, negative for
lipase production
_

6
_


acid
neutral with halo
7
Tryptone Broth Inoculation method loop
transfer Contains high amounts of
tryptophan Additional reagents added Kovacs
reagent (amyl alcohol, hydrochloric acid,
para-dimethylaminobenzaldehyde), reacts with
indole to produce a red product Discriminates
organisms that can produce tryptophanase to
hydrolyze the amino acid tryptophan into indole,
ammonia and pyruvic acid Results Red with
Kovacs cleavage of tryptophan into indole,
positive for tryptophanase production Colorless
no indole present, negative for tryptophanase
production
_

8
Urea Broth Inoculation method loop
transfer Contains yeast extract, urea, buffer,
Phenol red pH indicator acidic/neutral pH
yellow/orange, alkaline pH bright
pink Discriminates organisms that can produce
urease to hydrolyze urea into carbon dioxide and
ammonia Results Bright pink cleavage of urea
into ammonia, positive for urease
production Yellow/orange no ammonia
present, negative for urease production
_

_

9
Phenylalanine Slant Inoculation method surface
streak with loop Contains high amounts of
phenylalanine Additional reagents added 10
ferric chloride, reacts with phenylpyruvic acid
to form a green product Discriminates organisms
that can produce phenylalanine deaminase to
deaminate phenylalanine producing phenylpyruvic
acid and ammonia Results Green cleavage of
phenylalanine into phenylpyruvic acid, positive
for phenylalanine deaminase production Yellow
no phenylpyruvic acid present, negative for
phenylalanine deaminase production
_

10
Lab 12 Goals and Objectives Exercise 42
Hydrolytic and Degradative Reactions Read
results some will require additional
reagents Exercise 43 Multiple Test Media No
Kligers Iron Agar or Litmus milk, No
inoculations for IMViC discuss next lab,
Inoculate SIM with straight stab for each
unknown Inoculate three separate controls per
pair using Proteus vulgaris, Escherichia
coli, Staphylococcus aureus Each pair needs 7
SIM stabs Exercise 72 Gram Negative Intestinal
Pathogens Perform according to revised
directions, supplement p. 59-60 You still need
to read the lab to know how the media
works! Each pair needs 3 MacConkey agar
plates 7 Russells Double Sugar slants streak
and stab! Control broth cultures Proteus
vulgaris (Mac) Escherichia coli (Mac,
RDS) Shigella flexnari (RDS) Pseudomonas
aeruginosa (RDS) PA will inoculate additional
controls for observation next lab Make new
streaks for isolation on BHIA Each pair needs 4
BHIA plates (new streaks for isolation)