BIOCHEMICAL TESTS PART ONE Differentiation of organisms based on their ability to break down complex Macromolecules in to simpler nutritional constituents

Protein Starch Fat

Macromolecules are polymers of monomeric subsunits. Hydrolysis reactions cleave polymers into monomers by adding water.

Protein (Amino Acids) Starch (Sugars) Fat (Triglycerides And other lipids) Macromolecules and their (Monomeric Subunits)

Starch: a polymer of sugars The hydrolysis reaction is the reverse of the dehydration reaction Muscles store sugar as glycogen.

STARCH TEST Differentiates bacteria based on their ability to hydrolyze starch with the extra cellular enzyme amylase Starch (a polysaccharide) is to large to pass through the bacterial cell wall; to be of metabolic value to the bacteria, starch must be split into smaller fragments or individual glucose molecules

Procedure: Divide one starch plate into thirds and inoculate with E.coli, Bacillus cereus, and P.aeruginosa E.coli B.cereus P.aeruginosa

Results: Starch and its sugar subunits are clear in the medium. Iodine is used to detect the presence or absence of starch in the vicinity around the bacteria Iodine reacts with starch and produces a blue-black color. After addition of iodine to the plate media, any microbial starch hydrolysis by the exoenzyme amylase reveals a clear zone around the bacterial growth

Lipid Hydrolysis Test Used to identify bacteria capable of producing the exoenzyme lipase Triglycerides (a possible bacterial carbon and energy source) are too large to enter the bacterial cell; bacteria that produce and secrete lipase hydrolyze triglycerides into glycerol and 3 fatty acid chains

Triglyceride Hydrolysis by Lipase

Procedure: Divide one tributyrin agar plate into thirds and inoculate with E.coli, Bacillus cereus, and P.aeruginosa E.coli B.cereusP.aeruginosa

Results: Tributyrin agar plates contain the triglyceride tributyrin and are initially opaque; lipase-positive organisms will exhibit a clear zone around their growth—tributyrin has been hydrolyzed

Casein Hydrolysis Test Identifies bacteria capable of hydrolyzing casein (protein) with the enzyme casease Casein is the protein that gives milk it’s color. To be utilized by certain bacteria, casein must be broken down in to its smaller subunits, amino acids

Procedure: Divide one skim milk agar plate into thirds and inoculate with E.coli, Bacillus cereus, and P.aeruginosa E.coli B.cereusP.aeruginosa

Results: Bacteria that secrete the proteolytic exoenzyme casease hydrolyze milk protein thus creating a zone of clearing around the bacterial growth

Gelatin Hydrolysis Test for the ability of an organism to produce the exoenzyme gelatinase which digests and liquefies gelatin Gelatin is a protein derived from collagen, a connective tissue found in vertebrates Gelatin is too large to enter the bacterial cell; however its amino acids my be used as an energy source or built back up into bacterial protein

Procedure: 3 Nutrient gelatin tubes are stab inoculated with the following organisms then incubated: E. coli B.cereus P.aeruginosa

Results: Since nutrient gelatin melts at 28°C, care must be taken to distinguish between organisms capable of producing gelatinase and gelatin tubes that are affected by incubator temperature After incubation, tubes should be refrigerated; tubes inoculated with gelatinase positive organisms remain liquid after refrigeration

After refrigeration, E.coli is gelatinase negative and P.aeruginosa is gleatinase positive

Carbohydrate Fermentation (Phenol Red Carbohydrate Broth) Differential test to detect the ability of an organism to ferment various carbohydrates Phenol Red is the pH indicator (yellow below 6.8 and red above 7.4) A Durham tube (inverted small tube) is used to trap any gas produced from the fermentation of various sugars

Procedure: Obtain and label the following tubes: 4 Phenol Red Glucose Broth Tubes 4 Phenol Red Sucrose Broth Tubes 4 Phenol Red Lactose Broth Tubes Inoculate the above tubes with the following organisms: E.coli, P.vulgaris, A.faecalis, S.aureus

Results of Carbohydrate Fermentation Tests:

TRIPLE SUGAR IRON TEST (TSI) Used to differentiate among the different groups of Enterobacteriaceae based on their ability to ferment glucose, lactose and/or sucrose Also differentiates between groups capable of reducing sulfur to hydrogen sulfide gas (H 2 S)

Procedure: Medium contains: 1% LactosePhenol Red 1% SucroseSodium thiosulfate 0.1% Glucose Obtain 6 TSI agar slants; inoculate with the following organisms: P.aeruginosa, E.coli, P.vulgaris, C.freundii, P.mirabilis, A.faecalis Medium is inoculated by stab and streak

Results: Red slant/Red butt = no fermentation Red slant/Yellow butt = only glucose fermentation Yellow slant/yellow butt = lactose and/or sucrose fermentation Dark color: Hydrogen Sulfide produced