Biochemistry

Class Nine

Macromolecules

Macromolecules

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

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

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.

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

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

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

Endoenzymes

• Endoenzymes function within the cell for vital cellular metabolic reactions

a. Energy producing processesb. Fermentative processesc. Biosynthetic reactions

Note – Many of these processes produce byproducts that are useful in the identification and characterization of microbes

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

Results of starch hydrolysis

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

Negative test for starch hydrolysis

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.

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

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

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

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

Fermentation results

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

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

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

TSI results