Microbiology Laboratory

MidTerms

Biochemical Reactions  Anaerobic breakdown of carbohydrates

 Detection of end-products
Purpose of Biochemical Tests o Acid
o Gas
 Determine the enzymes systems o Neutral Products
 Important in bacterial food acquisition
 Aids in identification of organisms Durham Fermentation Tube

Enzymes o Detects gas production: bubble

 Proteins
 Catalyze biochemical reactions
 Metabolic Reactions
A. Anabolic Reactions
 Simple compounds to complex compounds
 Requires energy
B. Catabolic Reactions
 Complex compounds to simple compounds
 Release energy
 Primarily used to identify bacteria
Two Methods of Identifying Bacteria
1. Detection of the End-Product
 If the END-PRODUCT is PRESENT,
the enzyme catalyzing reaction is
produced.
2. Detect the Substrate
 If the SUBSTRATE is still PRESENT,
the ENZYME is ABSENT.
General Biochemical Methods
I. Carbohydrate Utilization
inside the durham tube
o Detects acid production
Triple Sugar Iron
o Detects gas production: cracks at
the culture media or pulling away
o Detects pH changes: phenol red (pH
detector) red to yellow coloration
o Detects hydrogen sulfide: + black
coloration- ppt formed by reactions
of Fe, Pb, Bi and Sulfide.
II. Hydrolysis of Organic Compounds
A. Hydrolysis of Starch
 Require enzyme (amylase) to be hydrolyzed
 Checked by addition of iodine solution after
incubation.
 If the bacteria produce amylase to
hydrolyze starch, Iodine was added, then
there is no change because starch has been
hydrolyzed, it will produce a positive result.
If amylase is not produced and iodine is
added, it is a negative result.
Microbiology Laboratory
MidTerms

B. Hydrolysis of Protein
1. Casein
o Main protein is milk
o Enzyme: Casease
o Zone of clearing round the colony: positive
result for hydrolysis of casein
o No zone of clearing: negative result
2. Gelatin
o Gegraded by gelatinase
C. Hydrolysis of Fats
 Hydrolyzed by lipase
 Fatty acid + Glycerol
 Positive result using Tributylin Agaar: zone
of clearing around the colony
 Positive Result for Spirit Blue Agar: zone of
clearing
 Lipase is elaborated: positive result is
“irridescence”- play of colors around the
colonies.