Lab 12 Goals and Objectives:
Exercise 40: Hydrolytic and Degradative Reactions
Read results: some will require additional reagents
Exercise 41: Multiple Test Media
No Kliger’s 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 71: Gram Negative Intestinal Pathogens
Perform according to revised directions, supplement pg
You still need to read the lab to know how the media works!
Each pair needs:
3 MacConkey agar plates
7 Russell’s 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)
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
+
_
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
+
_
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
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
+
_
+
+
acid
neutral with halo
_
Tryptone Broth
Inoculation method: loop transfer
Contains: high amounts of tryptophan
Additional reagents added: Kovac’s 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 Kovac’s = cleavage of tryptophan into indole, positive for
tryptophanase production
Colorless = no indole present, negative for tryptophanase production
+
_
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
+
_
+
_
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
+
_
Lab 12 Goals and Objectives:
Exercise 40: Hydrolytic and Degradative Reactions
Read results: some will require additional reagents
Exercise 41: Multiple Test Media
No Kliger’s 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 71: Gram Negative Intestinal Pathogens
Perform according to revised directions, supplement pg
You still need to read the lab to know how the media works!
Each pair needs:
3 MacConkey agar plates
7 Russell’s 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)