1. The Story of “Phernando”
the Bacteriophage
By
Christopher Harness
Victoria Torres
University of Detroit Mercy 2015

2. Background (Bacteriophages)
Harness-Torres 2
 Viruses known to
infect/replicate in bacteria
 Caudovirales phage consist of:
 Siphoviruses
 Myovirus
 Podoviridae
 Uses horizontal gene transfer
towards hosts
 Are found in almost every
environmenthttp://www.biologyexams4u.com/2012/1
1/bacteriophages.html#.VldNBvmrTIU

3. Harness-Torres 3
Overview

4. Finding the Phage
Harness-Torres 4
Collected by C.H.
Date 9/6/2015 at 10:36 AM
Sample Type Soil
Depth 13.1763 cm or 5 3/16 in
Air Temp 25 Celsius
General Location Moravian Park, Sterling Hts, MI 48312
GPS Reading 42.545778 N 82.975505 W
Description Damp, dark, shaded area
Moravian Park

5.  Purpose: To allow the phage from the soil sample to
reproduce in Mycobacterium smegmatis
 Process:
 Preparing an enrichment culture with the sample +
various formulas
 Performing serial dilutions with the culture /plating them
with top agar and M. smeg
 Result: 8 serial dilution plates successfully created
(10 – 10 , Neg)
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Soil Enrichment
0 -6

6. Spot Plating
• All spot samples taken from 10 Enrichment Plate
• B and C contained the most phage samples
Harness-Torres 6
-1

7. Streak Plating
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 Phage chosen from Spot
C were streaked
 Three streaks were
performed (Streak 3 not
shown)
 Method used to help
isolate and purify plaques
for titer assays

8.  Purpose: Further purify
phage sample
 First titer taken from
isolated plaque (Streak
2)
 Further titering taken
from “10/20” plaque
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Titer Assays

9.  Titer Assay 2 had both
lysogenic (A) and lytic (B)
plaque morphologies
 Further Titers:
 A consisted of hybrid
population (lytic-
lysogenic)
 B had two separate as
before
Harness-Torres 9
Problem Solving

10.  MTL created from 10−3
flood plate of Assay 2A
 Spot Plate: Series of
dilutions of MTL (100 to
10-10)
 Formula (MTL):

# 𝑝𝑓𝑢
5 𝜇𝐿
×
1000 𝜇𝐿
1 𝑚𝐿
× 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
Harness-Torres 10
Medium Titer Lysate (MTL) Stocks
Conentration: 1.2 × 108 𝑝𝑓𝑢
𝑚𝐿
(6 pfu on 10−5
)

11.  MTL Amount Equation:

𝜋 𝑝𝑙𝑎𝑡𝑒 𝑟 2
𝜋 𝑝𝑙𝑎𝑞𝑢𝑒 𝑟 2 /𝑀𝑇𝐿 𝐶𝑜𝑛.
 Result:
 7.0 × 10−3
𝜇𝐿 𝑜𝑓 𝑀𝑇𝐿
 Four, 5X flooded
webplates would create
the HTL
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High Titer Lysate (HTL)
Plate Factor Amount of MTL (𝝁𝑳)
1/4x 17.5x10-4
1/2x 35.0x10-4
1x 7.0x10-3
2x 14.0x10-3
4x 28.0x10-3
5x 35.0x10-3

12.  HTL Concentration: 3.4x108 pfu/mL (17 pfu on 10−6)
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HTL Plating

13. DNA Restriction Analysis
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 Purpose: Isolate/Purify
Phage DNA for restriction
enzymes
 DNA sample taken from
1 mL of 3.4 × 108 𝑝𝑓𝑢
𝑚𝐿
of
HTL concentration
 DNA Concertation: 51.5
𝑛𝑔
𝜇𝐿
 Restriction Enzymes
Used:
 BamHI
 ClaI
 EcoRI
 HaeIII
 (NOTE): All DNA was
combined with ddH2O
and 6X Dye

14. Gel Electrophoresis
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 Gels help identify the DNA
sequence of phage
 Placements show number of
fragments made by the
restriction enzyme cuts
 HaeIII had the highest
distance from the well
 The DNA may have been
denatured
Ladder BamHI ClaI EcoRINeg HaeIII

15. TEM Imaging
• Photos taken by the curtesy of Wayne State University
Harness-Torres 15

16. Harness-Torres 16
Phernando’s Story
Moravian
Park 𝟏𝟎−𝟏
Plate
Enrichment
Spot Plate
Spot C
Streaks 1 – 2
Assays 1 – 2(A) Assay 1 – 2
𝟏𝟎−𝟑
Flood
MTL
𝟏. 𝟐 × 𝟏𝟎 𝟖
𝒑𝒇𝒖
𝒎𝑳
𝟕. 𝟎 × 𝟏𝟎−𝟑
𝝁𝑳 𝒐𝒇 𝑴𝑻𝑳
(4)
5X Plates
HTL
3.4× 𝟏𝟎 𝟖 𝒑𝒇𝒖
𝒎𝑳
DNA: 51.5
𝒏𝒈
𝝁𝑳
Gel
Electrophoresis
TEM Imaging

17.  Abedon, Stephen T et al. “Phage Treatment of Human
Infections.” Bacteriophage 1.2 (2011): 66–85. PMC. Web. 30 Nov.
2015. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278644/>.
 BioExams4U. “Bacteriophage Structure.” Biology Exams 4 U.
2015. Image. 26 November 2015.
<http://www.biologyexams4u.com/2012/11/bacteriophages.html#
.VlvicPmrTIV>.
 Conant, Stephanie, and Jack Thompson. “SEA Phages.”
University of Detroit Mercy. 2015. PowerPoint. 29 November 2015.
Harness-Torres 17
Works Cited

18. QUESTIONS?