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Survey of Microbial Diseases
•How to identify bacteria in patient specimens or
in samples from nature? Or the MM project;)
-
phenotypic: considers macroscopic and
microscopic morphology, physiology, and
biochemistry
-
immunologic: serological analysis
-
genotypic: genetic techniques
increasingly being used as a sole resource
for identifying bacteria
•Data from these methods can provide a unique
profile for any bacterium
Survey of Microbial Diseases:
Phenotypic Methods
Physiological/Biochemical Characteristics
•Traditional mainstay of bacterial identification
•Enzyme production and other biochemical properties are
reliable ways to ID microbes
•Dozens of diagnostic tests exist for determining the
presence of specific enzymes and to assess nutritional and
metabolic activities:
- fermentation of sugars
- capacity to digest complex polymers
- production of gas
- sensitivity to antibiotics
- nutrient sources
Beta-hemolysis
Blood agar as a differential
medium
Alpha-hemolysis
No hemolysis
(gamma-hemolysis)
Survey of Microbial Diseases:
Phenotypic Methods
Tests for
fermentation and gas
production
Durham tube
(inverted tube
to trap gas)
No fermentation
Acid fermentation
with gas
Phenotypic Methods: Direct Examination
of Specimen
•Direct observation of
fresh or stained specimen
•Stains most often used
- Gram stain
- acid-fast stain
Survey of Microbial Diseases:
Phenotypic Methods
• Isolation Media and Morphological Testing
- Selective media: encourage the growth of
only the suspected pathogen
- Differential media: used to identify definitive
characteristics and fermentation patterns
MacConkey Agar: Selective and Differential
Selects for Gram-negative and tells you if the bacterium
ferments lactose
Phenotypic Methods: Biochemical Testing
• Physiological reactions:
indirect evidence of
enzymes present in a
species. If bacteria tests +
for cytochrome c oxidase
what does that tell you?
Phenotypic Methods: Biochemical Testing
Unknown
microbe +
different
substrates
Results (+/–)
DNPG ADH LDC
–
–
+
ODC |CIT | H2S
+
–
–
URE TDA IND
–
–
+
|VP| |GEL | GLU
–
–
+
MAN INO SOR
–
–
–
RHA SAC MEL
–
–
–
AMY ARA
–
• Enzyme-mediated metabolic reactions often
visualized by a color change
- microbe is cultured in a medium with a special
substrate, then tested for a particular end
product
- microbial expression of the enzyme is made
visible by a colored dye
–
Flowchart: We will use this to ID our MM!
Cocci
Gram (+)
Gram (–)
Catalase (–),
pairs, chain
arrangement
Catalase (+),
irregular clusters,
tetrads
Aerobic,
oxidase (+),
catalase (+)
Anaerobic,
oxidase (–),
catalase (–)
Streptococcus
Strictly
aerobic
Micrococcus
Facultative
anaerobic
Staphylococcus
Planococcus
Neisseria
Branhamella
Moraxella
Veillonella
Phenotypic Methods: Phage Typing
- Testing for sensitivity to various
phage groups
- a lawn of bacterial cells is
inoculated onto agar, mapped
off into blocks, and phage are
exposed to each block
- cleared areas corresponding to
lysed cells indicate sensitivity
to that phage
- Ex. S. aureus Phage Group I vs.
Group II
Determining Clinical Significance of Cultures
•Important to rapidly determine if an isolate from a specimen is
clinically important or if it is merely a contaminant or normal
biota
-
a few colonies of E. coli in a urine sample can indicate
normal biota, but several hundred can mean an active
infection
-
a single colony of a true pathogen such as
Mycobacterium tuberculosis in a sputum culture, or an
opportunist in a sterile site, is highly suggestive of
disease
-
repeated isolation of a relatively pure culture of any
microorganism can mean it is an agent of disease