Ousman Jammeh

Lab 16

Lab report methods, results, and discussion

Materials and Methods

Isolate a Novel Phage from the Environment

A soil sample with a depth of 5 centimeters was collected. Characteristics of the

environment of soil sample were recorded: location, air temperature, GPS coordinate, soil

moisture, and defining features of site.

Enriched Isolations

A soil sample was obtained. Three to five milliliters of the soil sample (around 1 – 3

grams) was poured into the enrichment medium of the bioreactor. The entire contents (0.5

milliliters) of the M. foliorum from the red-capped tube was transferred into the bioreactor. The

lid on the bioreactor was secured tightly and labeled. The mixture was taken to the front bench to

be incubated (with shaking in room temperature at 250 r.p.m.) for one week to facilitate virus

replication.

About one milliliter of the enrichment was centrifuged for one minute in a microfuge

tube. The supernatant fluid was pipetted into another microfuge tube (without disturbing the

pellet). The centrifuged tube was discarded. Using a syringe filter (0.22 micrometer) and a three-

milliliter syringe, the supernatant was filtered into another microfuge tube.

The Spot Test and The Plaque Assay

 For the plaque assay, fifty microliters of filtrate were added to a half milliliter culture of

M. foliorum. The mixture was swirled and allowed to incubate on bench top for ten minutes,

allowing for infection of culture by any phage that could have been present in filtrate. After the

ten-minute incubation, twenty microliters of CaCl2 was added to a test tube containing molten

agar. Then, the solution was poured into the infected culture that was prepared in the first step.

The mixture was swirled and poured onto an agar plate.

For the spot test, twenty microliters of CaCl2 was added to a tube of molten agar. The

resulting solution was poured into a half milliliter culture of M. foliorum and gently swirled. The

resulting mixture was dispensed onto the solid agar in petri dish and allowed to solidify. After

solidification, five microliters of enrichment filtrate were applied to one side of plate. Then five

microliters of phage buffer were added to the other side. The phage buffer served as a negative

control.

Purify the Phage: The Plaque Streak Protocol

Two plaques were selected for streaking. Get two microcentrifuge tubes. One hundred

microliters of phage buffer were dispensed into two microcentrifuge tubes. A plaque was

touched with a wooden applicator and dipped into its respective tube. While the tip of the

applicator was submerged in phage buffer, it was twirled between fingers to mix the phage into

the buffer. A nutrient agar plate was obtained for the two plaques. The inoculation loop was

flamed until orange-hot. Then it was held outside of the flame for approximately forty-five

seconds to cool, to avoid destroying any phage in suspension. The inoculation loop was dipped

into phage suspension and streaked onto a plate. The inoculation loop could only be dipped into

the phage sample once (before the first streak). Separation of phage in successive streaks was

achieved by passing the loop twice through the previous streak. Twenty microliters of CaCl2 was
added to a tube of molten agar. The resulting solution was added to a half milliliter of M.

foliorum culture. The mixture was poured over the third streak in each plate. The plates were left

on the bench.

Results

Enriched Isolation

This experiment was performed to amplify phages from environmental samples by

providing favorable conditions for phage replication. The virus replication was inconclusive;

more tested are needed. The incubation was a success. The liquid was on top and the solid was

on the bottom.

Enriched Isolation Day 2

This experiment was performed to continue amplifying phages from environmental

samples by providing favorable conditions for phage replication. The enriched isolation was

unsuccessful. The petri dish did not return any phages, no plaque was visible. We did not obtain

a positive spot test; we did not isolate any phages.

Figure 1:Phage Enrichment

This is an enriched isolation plate. Whether phages are present is inconclusive; more tests
would have to be done
 Figure 2: Plaque Assay
This is a failed plaque assay. No phages are present.

Plaque Streaking

This experiment was performed to identify and purify a phage plaque. The plaque

streaking was unsuccessful. We were not able to purify the phage.

Figure 3:Plaque Streaking

This is a failed plaque assay. No phages were present.

Discussion

This experiment was undergone to isolate and characterize bacteriophages of M. foliorum

bacterium from soil. Bacteriophages are being explored for their potential as antibacterials,

alternative to antibiotics, vehicles for vaccine delivery, and diagnostic tools. They can be utilized

as biocontrol agents, vehicles for vaccine delivery, and detection of diseases causing bacteria

strains. (“Bacteriophages and Their Implications on Future Biotechnology: A Review”)

Enriched Isolation

Lab 2 went as expected; virus replication was inconclusive. There was no problem

putting the contents in the bioreactor. After incubation the liquid and solid separated as expected.

Lab 4 went as expected, despite having to start over; virus replication was inconclusive.

There was no problem putting the contents in the bioreactor. After incubation the liquid and solid

separated as expected.

Enriched Isolation Day 2

 Lab 3 went smoothly. We went through the procedure without any hiccups. All of the

steps were followed properly. The lab was unsuccessful; we didn’t acquire any phages. It could

have been due to the location where the soil sample was obtained. Maybe a more remote location

or soil that is from a dirtier area would have fostered better replication.

Lab 5 went as expected, as this procedure was already performed before. With that said,

the plaque assay was unsuccessful. This is could have been due to the location of the soil sample

collection. We might have needed to find out what type of environment is conducive to phage

growth and get soil from that area.

Plaque Streaking

Lab 6 went according to plan; the procedure was followed without any hiccups. Though,

there was a mix up with the storage of the agar plate, we eventually found our plate. With that

said, we didn’t get any phages on our plate. We probably needed to find a better area with soil

that is conducive for phage growth in order to have a successful experiment.

Lab 7 was unsuccessful. We didn’t get our agar plate back, due to it not solidifying

correctly. This was probably due to moving it before it solidified, consequently disturbing the

phage purification.
 References

Haq, Irshad Ul et al. (2012) “Bacteriophages and Their Implications on Future Biotechnology: A

Review,” Virology Journal, 9, 9.