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Biochemistry

Biochemistry. Class Nine. Macromolecules. Macromolecules. Microorganisms and their Identification. Determination of pathogens responsible for infectious diseases

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Biochemistry

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  1. Biochemistry Class Nine

  2. Macromolecules

  3. Macromolecules

  4. Microorganisms and their Identification • Determination of pathogens responsible for infectious diseases • Selection of microbes that are important for the development of new pharmaceuticals, critical to industrial processes, and important for their environmental impact • Isolation of organisms critical to the preparation of foods such as cheeses • Comparison for purposes of classification

  5. Microorganisms and biochemistry • Microbes possess a unique capacity for biochemical reactions • Despite the fact that for the most part their genes are located one circular chromosome, their biochemistry is quite complex • Recently the complexities of the biochemical processes has led to a new classification for microorganisms

  6. Gram Positive and Gram Negative • The organization of genes as well as the functionality of the genes differs in gram positive and gram negative bacteria • The construction of the cell wall is only one of the major differences • Many of the cellular reactions differ. Some are specific for gram negative and some for gram positive.

  7. Exoenzymes • Enzymes are catalysts in biochemical reactions • All cellular reactions require enzymes • Exoenzymes act on substances outside of the cell. • Large, complex molecules can be digested outside of the cell and then undergo transport into the cell

  8. Hydrolytic enzymes • Hydrolytic enzymes catalyze the breakdown of large molecules like lipids, carbohydrates, and proteins into their smaller subunits • Hydrolytic enzymes break bonds between the subunits to produce smaller molecules

  9. Hydrolytic enzymes and the Identification of Microbes • As hydrolytic enzymes are released in media( solid) they exert an influence on the molecules in the media itself • The effects then can be used to assist in the identification of microbes

  10. Endoenzymes • Endoenzymes function within the cell for vital cellular metabolic reactions • Energy producing processes • Fermentative processes • Biosynthetic reactions Note – Many of these processes produce byproducts that are useful in the identification and characterization of microbes

  11. Starch hydrolysis( Experiment 22) • Starch is a polysaccharide composed of subunits of the sugar glucose • The glucose molecules are connected by bonds ( glycosidic) • The enzyme amylase results in the breakdown of starch • In a media supplemented with starch - it is possible to assess microbial activity for this process

  12. Results of starch hydrolysis

  13. Interpretation of results • Starch + Iodine = a black or purple color • A positive test for starch hydrolysis is a clear zone around the bacteria • A negative test is the production of the purple or black color over the surface of the agar

  14. Negative test for starch hydrolysis

  15. Gelatin hydrolysis • Gelatin is a protein produced by the hydrolysis of collagen. Below the temperatures of 25oC the gelatin will remain solid with its gel like properties • Above the temperature of 25oC, the gelatin will become liquid.

  16. Gelatin hydrolysis • Some microbes are capable of producing the enzyme gelatinase. • Gelatinase has the ability to hydrolyze the bonds connecting the amino acids in the protein gelatin • This causes the liquefaction of gelatin or the change of the gel to a liquid • Once this process has occurred even low temperatures will not restore the gel

  17. Carbohydrate metabolism • Microbes use carbohydrates( sugars) differently. • These differences may be the result of whether the microbes prefer aerobic or anaerobic processes. • Facultative anaerobes are more flexible in their ability to use biochemical pathways

  18. Fermentation ( Experiment 23) • Fermentation involves the metabolism of sugars and alcohols to produce a variety of acidic products such as lactic acid, formic acid, and acetic acid. • Gases are also produced by fermentative processes such as CO2 and H2 • Please refer to the chart on page 152

  19. Fermentation tubes • Contain broths with the sugars, glucose, lactose, and sucrose • The culture tubes contain a small tube for the collection of gases( Durham tube) • Figure – 23.4

  20. Fermentation results

  21. pH indicator • The pH indicator is red in neutral pH • It is yellow at slightly acidic pH of 6.8 or lower • Results are reported as • + for fermentation( Acid produced) • + for gas

  22. TSI – Triple Sugar Iron • Designed to differentiate between the members of the Enterobacteriaceae • The identification of microbes is based upon subtle differences in the fermentation processes of the sugars, glucose, lactose, and sucrose. • The acid base indicator – phenol red is also incorporated into this agar

  23. Result analysis • Alkaline slant and acid butt( with or without gas) • Acid slant or acid butt( with or without gas) • Alkaline slant and alkaline butt

  24. TSI results

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