1. Phage identification and characterization
LECTURE 11:
Phage identification and characterization
Viro102:
Bacteriophages & Phage Therapy
3 Credit hours
Atta-ur-Rahman School of Applied Biosciences (ASAB)

2. 13 Bacteriophage families
Double stranded DNA,
Non-enveloped
Double stranded
DNA, Enveloped
SIRV 1, 2 P2
Rudiviridae
Myoviridae T2
Plasmaviridae
Fuselloviridae
SSV1
TTV1 λ
Tectiviridae
PRD1
Siphoviridae
Lipothrixviridae
PM2
P22
Corticoviridae
Podoviridae
Single stranded
RNA
Double stranded RNA
Single-stranded DNA
M13 & fd
Inoviridae
MS2
phi666
ΦX174
Leviviridae
Microviridae
Cystoviridae

3. Bacteriophage: ICTV Classification
Nucleic acid
Morphology
Family
Linear dsDNA
Non-enveloped, contractile tail
Myoviridae
Non-enveloped, long non-contractile tail
Siphoviridae
Non-enveloped, short non contractile tail
Podoviridae
Non-enveloped, isometric
Tectiviridae
Circular dsDNA
Corticoviridae
Enveloped, rod-shaped
Lipothrixvirida

4. Bacteriophage: ICTV Classification
Circular dsDNA
Enveloped, pleomorphic
Plasmaviridae
Linear dsDNA
Non-enveloped, rod-shaped
Rudiviridae
Non-enveloped, lemon-shaped
Fuselloviridae
Circular ssDNA
Non-enveloped, filamentous
Inoviridae
Non-enveloped, isometric
Microviridae
Linear ssRNA
Leviviridae
Segmented dsRNA
Enveloped, spherical
Cystoviridae

5. 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

6. 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.

7. 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

8. Electron Microscopy

9. Electron Microscopy

10. Bacteriophage host range
Phage host range can be done by using the following test method against different host
Spot test
Plaque assay
Streak Assay

11. Bacteriophage host range
Spotting can be used to provide a first approximation of the ability of a phage to lyse or just kill a bacterial strain. This is done such as during phage typing procedures or, at lower phage densities, as a means of phage titering that is less materials intensive than full plate-count assays. 
Spotting can be used as a
means of determining a
phage's host range.
Spot Test

12. Bacteriophage host range
Streak Assay

13. Plaque counting Unit (PFU)
virulent phage stock and a susceptible host cell culture.
10-fold dilutions of the phage stock are prepared.
The procedure requires the use of a Double-Layer Agar (DLA) technique also known as double agar overlay method, in which the hard agar serves as a base layer (to form gel), and a mixture of few phage particles (diluted stock) and a very large number of host cells in a soft agar forms the upper overlay.
When the plates are incubated, susceptible E. coli cells multiply rapidly and produce a lawn of confluent growth on the medium.
When one phage particle adsorbs to a susceptible cell, penetrates the cell, replicates and release new phage particles which infect other bacteria in the vicinity of the initial host cell.

14. The destroyed cells produce single circular, non turbid areas called plaques in the bacterial lawn, where there is no growth of bacteria.
Each plaque represents the lysis of a phage-infected bacterial culture and can be designated as a plaque-forming unit (PFU) and is used to quantitate the number of infective phage particles in the culture. 
Dyes that stain the living cells are frequently used to enhance the contrast between the plaques and the living cells

16. 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
To degrade bacterial DNA and RNA in the lysate, you will use a mix of nucleases (DNase I and RNase A). The capsid of the phage protects its DNA from the nucleases. However, if nucleases are present in later steps of the protocol, the phage DNA may become degraded.

17. 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

18. Phenol chloroform method

19. Phage Genome (Agarose Gel)
23130bp
9416bp
6557bp
4361bp
2322bp
2027bp 1 2 3 4
20-23 kb

20. 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.

21. 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.

22. Genome fingerprinting by RAPD analysis
The method used for randomly amplified polymorphic DNA (RAPD) analysis
Add primers and template DNA
PCR
Agarose gel

23. Phage A
Phage B

25. SDS-PAGE analysis of phage proteins.
Bacteriophage structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE).
Crude extract
Specific protein extraction

27. SDS-PAGE patterns of phage structural proteins
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.

28. Phage Burst Size, Latent period
801 LK1/ cell
24 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.

29. Thanks!! Measuring bacteriophage burst size
Does each phage-infected bacterial cell release the same number of phage or does the number vary from cell to cell?   If it varies, can we define the factors involved in this variation? 
To measure an average burst size lets perform this online experiment
Thanks!!