Lab 12 : Microbial Metabolism II
I.
Lipid Hydrolysis
a. Lipids (fats) can be a carbon source
b. First step done by a lipase enzyme
A. Hydrolyzes a fat molecule into it’s components,
glycerol and fatty acids
B. Fatty acids then get further catabolized and used
for energy
c. Trybutyrin agar contains fats (looks opaque)
A. When fats are broken down the media becomes
more clear
II.
Citrate Utilization
a. Citrate is another potential source of carbon for bacteria
b. Utilization occurs though citrase or citrate-permease
c. Catabolism of citrate results in alkaline products
through the converstion of ammonium dihydrogen
phosphate to ammonia
d. Change in pH detected by bromothymol blue
A. Green @ neutral pH, blue in basic, yellow in
acid
B. Good at detecting vary slight changes in pH
around 7
III.
Indole Production
a. A very common biochemical precursor (benzyne ring
fused to a pyyrole)
b. Comes from the amino acid tryptophan
A. Need tryptophanase (first step) to break it
down into indole
c. Chemical indicator is known as Kovac’s reagent
A. Turns red in the presence of indole
IV. Urea Hydrolysis
a. Breakdown of urea into two molecules of ammonia is
catalyzed by urease enzyme
b. pH indicator, Phenol Red, is used to detect basic
ammonia
A. Basic = red/pink; Acidic = yellow
V.
Gelatin Liqufaction
a. Gelatin can be a source of protein for bacteria but it’s
molecules are too large to be taken up by the cells
A. So bacteria can produce gelatinases to break
down the gelatin into more accessible parts
B. Mercuric chloride causes gelatin to precipitate
… but if the gelatin is already broken down
there’s nothing to precipitate
C. Zones of clearing show gelatinase activity
D. MERCURIC CHLORIDE IS TOXIC – BE
CAREFUL
VI. Lysine Decarboxylase
a. Decarboxylation is removal of a carboxylic acid
functional group
b. Lysine decarboxylase removes the –COOH from
lysine forming a basic product
A. But it only does this under acidic conditions
1. So we induce fermentation by removing
oxygen thus leading to acid production
B. But lysine decarboxylation results in alkaline
products so …
C. pH indicator used is bromocresol purple so pH
goes from:
1. Neutral = Purple to
2. Acidic = Yellow then back to purple
VII. Phenylalanine Deamination
a. Deamination is the removal of an amine group forming
an acidic compound
A. In this case phenylalanine deaminase turns
Phenylalanine into phenylpyruvic acid
b. PPA is detected by Ferric Cloride which turns green in
the presence of phenylpyruvic acid indicating the
presence of phenylalanine deaminase
VIII. Bile Esculin
a. Two tests in one : 1) Can is grow in a high bile
concentration and 2) Does it utilize esculin
b. Bile is an intestinal secretion from the liver (stored in
the gall bladder) used as a selective agent
c. Esculin is a glycoside that can be hydrolyzed to release
glucose and esculetin
d. Esculetin reacts with ferric citrate in media to
precipitate into a brown / black compound
For quiz know all SUBSTRATES, ENZYMES, AND
INDICATORS