LECTURE 11:
Phage identification and
characterization
Viro102:
Bacteriophages & Phage Therapy
3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)
13 Bacteriophage families
Double stranded DNA,
Non-enveloped
P2
Double stranded
DNA, Enveloped
SIRV 1, 2
Rudiviridae
Myoviridae
T2
Fuselloviridae SSV1
λ
Tectiviridae
Plasmaviridae
TTV1
PRD1
Siphoviridae
Lipothrixviridae
P22
Corticoviridae
Podoviridae
Single-stranded DNA
Inoviridae
M13 & fd
PM2
Single
stranded
RNA
Double
stranded
RNA
phi666
MS2
Microviridae
ΦX174
Leviviridae
Cystoviridae
Bacteriophage: ICTV Classification
Family
Myoviridae
Morphology
Non-enveloped, contractile tail
Nucleic acid
Linear dsDNA
Siphoviridae
Non-enveloped, long non-contractile
tail
Linear dsDNA
Podoviridae
Non-enveloped, short non contractile
tail
Linear dsDNA
Tectiviridae
Corticoviridae
Non-enveloped, isometric
Non-enveloped, isometric
Lipothrixvirida Enveloped, rod-shaped
Linear dsDNA
Circular dsDNA
Linear dsDNA
Bacteriophage: ICTV Classification
Plasmaviridae Enveloped, pleomorphic
Circular dsDNA
Rudiviridae
Linear dsDNA
Non-enveloped, rod-shaped
Fuselloviridae Non-enveloped, lemon-shaped
Circular dsDNA
Inoviridae
Non-enveloped, filamentous
Circular ssDNA
Microviridae
Non-enveloped, isometric
Circular ssDNA
Leviviridae
Non-enveloped, isometric
Linear ssRNA
Cystoviridae
Enveloped, spherical
Segmented
dsRNA
Bacteriophage Characterization
• Electron microscopy (Morphological Studies)
• Bacteriophage DNA isolation
– RFLP
– Genome fingerprinting by RAPD analysis
• SDS-PAGE analysis of phage proteins
• Burst size
• Bacteriophage host range
Electron Microscopy
• Aliquots of a bacteriophage sample obtained
by ultracentrifugation were subjected to
electron microscopy for morphological
analysis.
• Purified phage particles were negatively
stained with 2% (wt/vol) uranyl acetate,
deposited on carbon-coated grids.
Isometric heads, visible collars, and shorter contractile tails
with terminal base plates, which are characteristics of
the family Myoviridae
Electron micrographs of L. fallax bacteriophages. (A) R01; (B) R03; (C) R05; (D) R09; (E) R12;
(F) R19. Bars
Electron Microscopy
Electron Microscopy
Bacteriophage host range
• Phage host range can be done by using the
following test method against different host
– Spot test
– Plaque assay
– Streak Assay
Spot Test
Bacteriophage host range
Streak Assay
Bacteriophage host range
Bacteriophage host range against its
own Evolved Host
Bacteriophage DNA isolation
• One hundred ml of phage lysate was
incubated for 1 h at 37°C after addition of
DNase I and RNase A(2 µg/ml).
• Incubated
• The phages were pellet down by
centrifugation
• phage pellets
Phenol Chloroform extraction
• The pellet obtained after centrifugation is
dissolved in 100 micro liter autoclaved H20.
• Treated with DNase I & incubated for 370C
• SDS, proteinase K
• Phenol chloroform extraction
Phenol chloroform method
Phage Genome (Agarose Gel)
23130bp
20-23 kb
9416bp
6557bp
4361bp
2322bp
2027bp
1
2
3
4
Genome fingerprinting by restriction
fragment length polymorphism (RFLP)
analysis
• Purified bacteriophage DNA samples were subjected
to restriction enzyme digestion
• with AluI, BamHI, EcoRI, HindIII, MboI, RsaI, and
Sau3AI
• The restriction digests were separated on a 0.8%
agarose gel and stained with ethidium bromide.
RFLP analysis of phage DNA: EcoRI digestion patterns of Myoviridae bacteriophages.
Lane M, 1-kb DNA ladder; lane 1, R03; lane 2, R05; lane 3, R12.
Genome fingerprinting by RAPD analysis
• The method used for randomly amplified
polymorphic DNA (RAPD) analysis
• Add primers and template DNA
• PCR
• Agarose gel
Phage A
Phage B
SDS-PAGE analysis of phage proteins.
• Bacteriophage structural proteins were
analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS
PAGE).
• Crude extract
• Specific protein extraction
SDS-PAGE patterns of phage structural proteins. Lanes M, molecular weight
markers; lane 1, Siphoviridae phage R01; lane 2, Myoviridae phage R03; lane 3,
Siphoviridae phage R09.
Phage Burst Size, Latent period
R
L
1400
P
Bacteriophage Count pfu/ml
1200
1000
800
801 LK1/ cell
600
24 min.
400
200
0
0
3
6
9
12
15
18
21
24
27
30
33
36
Time (min)
One step growth curve showing the latent period (24 min) and the average burst size (801 viral
particles per host cell). Latent time and burst size of phage LK1 were inferred from the curve
with a triphasic pattern. L: latent phase;R: rise phase; P: plateau phase.
Thanks!!