Marcie the Bacteriophage:Hannah Street, Hunter Stoltz

Biochemistry and Biotechnology, Minnesota State University Moorhead, 1104 7th Avenue South, Moorhead, MN 56563

Introduction:Bacteriophage or phage for short is a virus that infects bacteria. To infect

bacteria a phage injects its DNA into the bacteria. There are two common

phage life cycles. A lysogenic cycle is when a phage injects it genome into the

bacteria and it joins with the bacteria’s genome, and a lytic cycle is when a

phage injects its genome into the bacteria and creates new phages and then

lyses or bursts the cell.

A phage consists of a head, tail, and tail fibers. The head of a phage contains

DNA or RNA and is enclosed in a protein coat. Siphoviridae is a group of

phages with long, flexible, and non-contractile tails. Myoviridae is a group of

phages with contractile tails. Podoviridae is a group of phages with short non-

contractile tails.

Phages are important to learn and study in the world of medicine specifically

against multidrug-resistant bacterial infections. Phage therapy may be used as

an alternative or supplement to antibiotic treatments (Lim, 2017). We still

know so little about phages and every bit of research helps.

Our objective was to isolate, purify, and amplify a phage that infects

Microbacterium foliorum.

Acknowledgments:We would like to thank the Howard Hughes Medical Institute and the Hatfull

Lab at the University of Pittsburgh for providing bacteria, support, and training

materials.

Methods:

Purification:❖ Picking a Plaque: Retrieve phage particles from a plaque to create a lysate.

❖ Serial Dilution: Using serial dilution to the 8th step to create a High Titer

Lysate.

❖ Spot Titer: Using Lysate created create a spot titer to determine the

concentration of the produced lysate.

Results:

Isolation:❖ Collect an environmental sample

❖ Direct Isolation: Use a liquid media to extract phages from the environmental

sample.

❖ Plaque Assay: Plate the mixture of top agar, bacteria, and sample to detect

the presence of phages on the bacterial lawn.

❖ Picking a Plaque: Retrieve phage particles from a plaque to create a lysate.

Amplification:❖ Spot Titer: Using spot titer plate to determine concentration of lysate.

❖ Webbed Plates: Once concentration is known to be above 1x10^9 pfu/mL or

around create webbed plates to produce further high titer lysates.

❖ Flood Plate: Flood the webbed plate with phage buffer to produce a high titer

lysate.

Figure 1. This was the first plate with

phage on it. Circles are marking air

bubbles.

Figure 2. This is an undiluted plate

where you can see the plaques clearly.

The plaques are a defined circle but

there is some cloudiness outside of the

circle.

Figure 3. This plate was a spot titer

test. There was contamination (the

lighter colored spots), but we were

still able to calculate the titer by

counting three plaques on the 10^-8

dilution. We calculated the titer to be

1x10^11 pfu/mL.

Figure 4. This was an electron

microscope picture taken with a scale

of 0.5 µm. This picture shows

multiple phages.

Figure 5. This is an electron

microscope picture taken with a scale

of 100 nm. This shows a close up of

Marcie. We estimate the tail to be

170 nm and the overall size of Marcie

is estimated to be 210 nm.

Sources:

Lin D, Koskella B, Lin H. 2017. Phage therapy: An alternative to antibiotics

in the age of multi-drug resistance. World Journal of Gastrointestinal

Pharmacology and Therapeutics 8(3): 162-173.

Microscopy:❖ Collect High Titer Lysate: From webbed plates produced collect high titer

lysate for electron microscope imaging.

❖ Electron Microscope: Phage lysate was collected and given to lab to give us

our Electron microscope images of phage.

Discussion:

Marcie was found in a soil sample near First Silver Lake in Minnesota

(46.30913, -95.738483). This sample was collected by our professor.

When first discovered, the plaques were small but easily visible. The

plaques morphology was somewhat cloudy. This could mean that Marcie is

lysogenic.

We calculated our concentration by performing spot titer tests. Most of our

lysates we collected were considered to be high titer lysate because it was

greater then 5x10^9 pfu/mL. We calculated one titer to be 1x10^6 but this

was an educated guess because after the 10^-3 dilution the plaques

suddenly stopped. We think there was an error made when performing our

serial dilutions.

We had issues with contamination. At first we believed there was

contamination in the bacterial stock, but contamination still occurred after

the bacterial stock was replaced. We are still unsure why contamination

was happening because it only affected high titer plates.

We had electron microscope pictures taken of Marcie at NDSU. These

pictures allowed us to see Marcie. Marcie’s tail is estimated to be 170 nm

and the overall size to be 210 nm. We determined that Marcie was a

siphoviridae because of the long flexible tail.

In conclusion, Marcie was a successful phage that survived throughout our

research. Marcie was entered into the phage database along with other

phages that have been discovered.

Future Plans:

Due to unforeseen circumstances our research had to be cut short. Future

plans for research involving Marcie include DNA extraction and DNA

characterization. Furthermore research into what bacteria Marcie is able to

attack will be conducted. Using bacteria that is antibacterial resistant Marcie

might be able to combat the bacteria that is resistant to antibiotics.