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

  2. Fat Protein Starch

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

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

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

  6. 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

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

  8. 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

  9. 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

  10. Triglyceride Hydrolysis by Lipase

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

  12. 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

  13. 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

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

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

  16. Gelatin Hydrolysis • Test for the ability of an organism to produce the exoenzyme gelatinasewhich 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

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

  18. 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

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

  20. 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

  21. 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

  22. Results of Carbohydrate Fermentation Tests:

  23. 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 (H2S)

  24. Procedure: • Medium contains: 1% Lactose Phenol Red 1% Sucrose Sodium 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

  25. 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