The Effect of Fertilizer Runoff on the Infectivity of T2 Bacteriophages

Luke Diorio-TothGrade 10Pittsburgh Central Catholic HS

Bacteriophages

Bacteriophages - Viruses that infect bacteria

Lytic Cycle

Phages’ Role in the Environment

Phages – may be very important to the environment

There are more phages on earth than all other life forms combined

Extremely ubiquitous

T2 Phage

T2 Phages demonstrate host specificity (E. Coli)

T2 phages were one of the most important models in the early history of molecular biology.

Escherichia coli

Escherichia coli (E. coli) – very common, found in intestinal tract of most mammals

There are many strains of E. coli, most are non-pathogenic Pathogenic strains can cause illness and death in humans Frequently studied in biology – ubiquitous, simple

structure, easily manipulated in the laboratory

E. Coli

Dangers of Fertilizers

Eutrophication - an increase in chemical nutrients (usually nitrogen or phosphorus) in an ecosystem.

Can lead to excess plant growth – accelerates degradation of ecosystem

Disrupts normal functions in the ecosystem Excessive fertilizer use – can be toxic to organisms. Contain very small amount of harmful chemicals –

magnified at high concentrations

Potomic river estuary

Chemical Breakdown of Fertilizer Total Nitrogen (N) = 10% 1.6% ammoniacal nitrogen 0.2% nitrate nitrogen 8.2% urea nitrogen Available Phosphate (P2O5) = 15% Soluble Potash (K2O) = 10% Iron (Fe) = 0.10% 0.10% chelated iron Manganese (Mn) = 0.05% 0.05% chelated manganese Zinc (Zn) = 0.05% 0.05% chelated zinc Derived from: urea, ammonium and potassium phosphates, potassium nitrate, iron EDTA, manganese EDTA and

Zinc EDTA

Purpose

The purpose of this experiment was to determine if excess fertilizer could affect the infection rates of ground and water dwelling bacteriophages.

Hypotheses

All tested concentrations of fertilizer will significantly affect T2 phage infectivity.

Fertilizer Exposure time will not have a significant effect on T2 phage infectivity.

Null – Fertilizer will not significantly affect T2 phage infectivity.

Materials E. Coli B culture T2 Bacteriophage culture Shultz Liquid Fertilizer Distilled water Top Agar 30 Petri Dishes poured with

LB agar 30 15mL Sterile Polystyrene

Conicals tubes 5 50mL Sterile Polystyrene

Conicals 10 Sterile Microtubes 1 Micropipette with Sterile

Tips Sterile Dilution Fluid (SDF) 1 Plastic Tub 1 Thermometer 1 Tube Rack 1 Macropipette with sterile

tips

1 Magnetic Stirrer 1 Fine Tip Permanent Marker Klett Spectrophotometer LB media Sterile water

LB Media (per 1 liter)

10g Tryptone (1%)

5g Yeast Extract (0.5%)

10g NaCl (1%)

2 mL 1N NaOH

SDF (per 1 liter)

100mM KH2PO4

100mM K2HPO4

10mM MgSO4

1mM NaCl

Top Agar (per 1 liter)

10g Tryptone (1%)

3g Peptone (0.3%)

5g NaCl (0.5%)

5g Agar (0.5%)

Procedure

1. A culture of T2 phages was prepared in a tube with a phage density of 107 phages/mL

2. The phage suspension was reduced to a density of 105 phages/mL by adding 0.1mL of the phage to 9.9mL of Sterile Dilution Fluid (SDF).

3. 0.01mL of the reduced phage was added to 9.99mL of SDF to create a T2 phage stock solution with a suspension of 104 phages/mL.

4. A culture of E. coli B was prepared in LB media.5. After 24 hours in the incubator, 1 mL of the culture was transferred to 50

mL of LB media in a sterile 250-mL sidearm flask.6. The flask was placed in a shaking water bath until the culture was in mid-

log phase (about 100-150 Klett units)7. Sterile plates poured with LB agar were pre-warmed in an incubator set at

37° C8. A stock solution of liquid fertilizer was prepared in a flask with a

concentration 10 times the manufacturers suggested concentration. The fertilizer solution was then sterile filtered into a 50mL sterile polystyrene conical.

Procedure (part 2)

1. 5 sterile microtubes were then placed in a rack and filled with the fertilizer stock, phage stock, and sterile water to create the desired concentrations:

2. The tubes were set aside for 30 min.3. Process was repeated with a second set of

microtubes.4. These tubes were set aside for 45 min.

0x 0.1x 0.5x 1x 2x

Fertilizer stock 0 mL 0.01 mL 0.05 mL 0.1 mL 0.2 mLT2 stock 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mLSterile Water 0.9 mL 0.89 mL 0.85 mL 0.8mL 0.7 mL

Standard Overlay Technique for Phage Plating

1. A 45° hot water bath was prepared in a plastic tub.2. A tube rack was placed in the bath, and 3 (for each

concentration, 15 total) 15mL sterile polystyrene were placed in the rack.

3. 3 mL of liquefied top agar was added to each conical.

4. 0.1 mL of 30 min. phage suspension and 0.3 mL of mid-log phase E. coli B was added to each conical.

5. The tubes were then briefly vortexed and the contents poured onto pre-warmed LB agar plates.

6. This process was repeated with the 45. min phage.7. After setting, all plates were incubated at 37°C for

24 hours, and then the resulting plaques were counted.

▲Microtubes and pipettes

▲Hot water bath

◄Plaques

Plaque Counts

0x 0.1x 0.5x 1x 2x

Plate 1 111 128 145 175 123

Plate 2 115 135 152 167 128

Plate 3 109 125 153 173 119

Avg. 111.6667 129.3333 150 171.6667 123.3333

0x 0.1x 0.5x 1x 2x

Plate 1 138 151 165 178 131

Plate 2 142 149 168 185 133

Plate 3 143 155 171 181 129

Avg. 141 151.6667 168 181.3333 131

30 Minutes

45 Minutes

30 Minute Fertilizer Exposure

0

20

40

60

80

100

120

140

160

180

200

0x 0.1x 0.5x 1x 2x

Concentrations

Plaq

ue C

ount Plate 1

Plate 2

Plate 3

Avg.

45 Minute Fertilizer Exposure

0

20

40

60

80

100

120

140

160

180

200

0x 0.1x 0.5x 1x 2x

Concentrations

Plaq

ue C

ount Plate 1

Plate 2

Plate 3

Avg.

0

20

40

60

80

100

120

140

160

180

200

0x 0.1x 0.5x 1x 2x

Fertilizer Concentrations

Plaq

ue C

ount

30 Min.

45 Min.

P = 0.006868

P = 0.000238

P = 3.6E-05

P = 0.020654

P = 0.01025

P = 0.000306

P = 9.17E-05

P = 0.006417

The Effect of Fertilizer Exposure Time on T2 Phage Infectivity

P = 7.72E-08 P = 7.48E-09

Sig Sig Sig Sig Not Sig

Conclusions

All tested concentrations of fertilizer will significantly affect T2 phage infectivity. - Accepted

Fertilizer Exposure time will not have a significant effect on T2 phage infectivity. – Rejected, except for 2x fertilizer concentration

Null – Fertilizer will not significantly affect T2 phage infectivity. - Rejected

Conclusions (Limitations/Extensions) There was a lag between the time tubes were filled with top agar,

and when they were poured. During this time, the agar could have cooled, and started to congeal early, making counting plaques more difficult. This could be remedied by having a team of students working together to speed up operations.

Additional sets of microtubes will be prepared, and more plates will be poured to increase accuracy of the ANOVA.

Different phage and host will be tested.

New experiment will be designed to test if fertilizer affected the host, rather than the phage.

Testing longer times of exposure may reveal new findings

Testing higher concentrations may reveal new findings.

References

Dr. John Wilson, University of Pittsburgh, Biostatistician http://en.wikipedia.org/wiki/Fertilizer http://en.wikipedia.org/wiki/Eutrophication http://www.starnursery.com/show_details.php?

root=2&sub1=28&product=522&PHPSESSID=01be5cdfbfca1a322d7e21555fa62f8e

http://www.schultz.com/ http://www.oregontoxics.org/monk.html http://epa.gov/ http://en.wikipedia.org/wiki/Phage http://en.wikipedia.org/wiki/Biosphere http://en.wikipedia.org/wiki/Phage_ecology http://en.wikipedia.org/wiki/ANOVA http://www.des.state.nh.us/factsheets/bb/bb-3.htm http://www.ppath.cas.psu.edu/extension/plant_disease/

overfer.html http://www.usc.edu/CSSF/History/2004/Projects/J0921.pdf http://pubs.usgs.gov/of/1998/of98-805/lessons/chpt6/act2.htm