Class Nine

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

• 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

• 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.

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

• 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

• 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

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

• 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

• 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 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

• 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

• 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

• 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

• Alkaline slant and acid butt( with or without gas)
• Acid slant or acid butt( with or without gas)
• Alkaline slant and alkaline butt