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
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
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
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
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
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
