Biochemistry

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Biochemistry

• Class Nine

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Macromolecules
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Macromolecules
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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

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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

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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.

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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

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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

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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

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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

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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

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Results of starch hydrolysis
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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

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Negative test for starch hydrolysis
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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.

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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

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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

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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

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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

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Fermentation results
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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

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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

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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

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TSI results
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