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Bacterial classification
 Wall structure
 Gram +
 Staphylococcus, Streptococcus, Clostridium,
Bacillus
 Gram  Enteric, respiratory and others
Bacteria
 Acid-fast
 Mycobacterium
 Wall-less
 Mycoplasma
 Unusual
 Obligate intracellular
 Rickettsia, Chlamydia
G+
G-
Bacterial classification
 Cell morphology
 Shapes



Rod
Cocci
Spiral
 Associations




Bacteria
Individual
DiploStaphyloStrepto-
G+
Rod Cocci
G-
Rod Cocci Spiral
Bacterial classification
 Growth characteristics
 Oxygen requirement



Bacteria
Aerobic
Anaerobic
 Microaerophilic, aerotolerant
Facultative
 Spore formation
G+
Rod Cocci
G-
Rod Cocci Spiral
 Intracellular/extracellular
 Fastidious/non-fastidious
+ spore -
+
O2
Classification & Diagnosis
 Type of colonies
 Appearance

Color, shape, size and smoothness
 On differential media

Blood, MacConkey, EMB
 On selective media

MacConkey, Thayer-Martin
Classification & Diagnosis
 Metabolism
 Utilization of specific substrates


Lactose (Sal/Shi/Yer/)Citrate (E. coli-/Klebsiella+)
 Production of certain end products

Fermentation end products
 Acid (acetate, propionic acid, butyric acid etc.)
 Acetoin
 Alcohol
 Amine
 H2 S
Classification & Diagnosis
 Specialized tests
 Immunological


O-, H- & K-Ag (serotype)
Precipitation, agglutination
 Specialized enzymes




Catalase--- Staph+. vs. Strep-.
Coagulase---S. aureus+ vs. S. epidermidisOxidase---Neisseria gonorrhoea+
Urease---Proteus+, Helicobacter+
 Phage typing
 Fatty acid profile
Conventional diagnosis methods
Molecular diagnosis
 Ribotyping
 Restriction fragment length polymorphism
(RFLP)
 DNA hybridization
 PCR, RT-PCR and RAPD
 Nucleic acid sequence analysis
Ribotyping
 involves the fingerprinting of genomic
DNA restriction
fragments By digesting the genes with a specific restriction
enzyme,
 fragments of different lengths are generated. By performing
a Gel electrophoresis with the digested samples, the
fragments can be visualised as lines on the gel, where larger
fragments are close to the start of the gel, and smaller
fragments further down.
Ribotyping of bacteria
 Ribotyping is a method that can identify and classify bacteria
based upon differences in rRNA. It generates a highly
reproducible and precise fingerprint that can be used to
classify bacteria from the genus through and beyond the
species level.
 DNA is extracted from a colony of bacteria and then
restricted into discrete-sized fragments.
 The DNA is then transferred to a membrane and probed with
a region of the rRNA operon to reveal the pattern of rRNA
genes. The pattern is recorded, digitized and stored in a
database.
 It is variations that exist among bacteria in both the position
and intensity of rRNA bands that can be used for their
classification and identification. Databases for Listeria (80
pattern types), Salmonella (97 pattern types), Escherichia (65
pattern types) and Staphylococcus(252 pattern types) have
been established.
RFLP
GGATCC
CCTAGG
In situ
Hybridization
PCR RT-PCR
Random amplified polymorphic
DNA (RAPD)
 RAPD is a modification of the PCR in which a single,
short and arbitrary oligonucleotide primer
 able to anneal and prime at multiple locations
throughout the genome,
 produce a spectrum of amplification products that are
characteristics of the template DNA
Molecular diagnosis
 Reduce reliance on
culture
 Faster
 More sensitive
 More definitive
 More discriminating
 Techniques adaptable to
all pathogens
 Technically demanding
 Relatively expensive
 Can be too sensitive
 Provides no information
if results are negative
Bioterrorism
 Pathogen detection
 Fast and accurate
 Mobile
 Inexpensive
Differentiating Staphylococci from Streptococci
 Gram stain and morphology
 Both Gram +
 Staphylococci: bunched cocci
 Streptococci: chained cocci (S. pneumoniae form
diplococcus)
 Enzyme tests
 Staphylococci: catalase +
 Streptococci: catalase -
 Growth
 Staph.: large colonies , some hemolytic
 Strep.: small colonies , many hemolytic (a or b)
Differentiating the Gram- bacteria
 Cocci
 Neisseria
 Rods
 Type of disease they cause
 Enteric Gram- rods
 Curved
 Vibrio, Campylobacter, Helicobacter
 Spiral Gram- organisms
 Spirochetes
Gram negative
Curved rods
Straight rods
Lactose+
Citrate+
Klebsiella
CitrateE. coli
Lactose-
H2S+
Salmonella
Campy blood agar
42oC+ 25oC-
TCBS agar
Yellow
Oxidase+
Campylobacter
Vibrio
H2SShigella
Thiosulfate-citrate-bile salts-sucrose agar
Bacteria
Gram+
Cocci
Staph.
Gram-
Rod
Strep.
Non-spore
Fil
Rod
Spiral
Spore
Acid Fast
Rod
Cocci
Treponema
Borrelia
Leptospira
+O2 -O2
+/-O2
-O2
Curve
Resp.
Bordetella.
H. influenzae
Legionella
Other
Vibrio
Campylobacter
Helicobacter
Zoo
Yersinia
Pasteurella
Brucella
Francisella
Streptobacillus
Wall
Less
Rickettsia Mycoplasma
Coxiella
Erlichia
Chlamydia
Neisseria
Moraxella
Straight
+O2
M.t.
Intra
Cellular
H. ducreyi
Gardnerella
Calymmatobacterium