2. 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

3. 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

4. Blood agar as a differential medium
Beta-hemolysis
Blood agar as a differential medium
Alpha-hemolysis
No hemolysis (gamma-hemolysis)

5. Tests for fermentation and gas production
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

6. Phenotypic Methods: Direct Examination of Specimen
Direct observation of fresh or stained specimen
Stains most often used
Gram stain
acid-fast stain

7. 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

8. MacConkey Agar: Selective and Differential
Selects for Gram-negative and tells you if the bacterium ferments lactose

9. 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?
Tells you that this bacteria goes through ETC in presence of O2

10. Phenotypic Methods: Biochemical Testing
Unknown
microbe +
different
substrates
DNPG
ADH
LDC
ODC |
CIT |
H2S
URE
TDA
IND | VP | |
GEL |
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
Results (+/–) – – + + – – – – + – – + – – – – – – – –
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

11. Flowchart: We will use this to ID our MM!
Cocci
Gram (+)
Gram (–)
Catalase (+),
irregular clusters,
tetrads
Catalase (–),
pairs, chain
arrangement
Aerobic,
oxidase (+),
catalase (+)
Anaerobic,
oxidase (–),
catalase (–)
Streptococcus
Strictly
aerobic
Facultative
anaerobic
Neisseria
Branhamella
Moraxella
Veillonella
Micrococcus
Staphylococcus
Planococcus

12. 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

13. 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