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Boyd | Howard UniversityIsolation, Purification & Characterization of Bacteriophages

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Isolation, Purification & Characterization of

Bacteriophages

Chamille Boyd

Howard University


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Intro to PHAGES

What are bacteriophages?

Bacteriophages are viruses that attack bacteria. Their only objective is reproduction

which takes place with the help of their host bacteria. “Viruses cannot multiply through the

division of cells because they do not have cells (acellular). Instead, they seek a host cell in which

they replicate and assemble themselves using the metabolism and machinery of the host cell,”

(Everything about Bacteriophages). Phages are host specific viruses, meaning that each type of

phage can only successfully attack a certain type of bacteria. The phage’s reproductive cycle

begins when it uses its tail fibres to attach itself to its host. It then injects its DNA or RNA

(located in the capsid of the phage) into its host. Once injected, the DNA or RNA stops many of

the bacteria’s vital functions and forces it to replicate components of the phage virus – heads,

sheaths and tail fibres. These parts begin to come together, reproducing several phages. Once

enough phages are produced, they burst the bacteria – reaching the exterior and looking for more

bacteria to make their host.

The overall purpose of the PHAGES lab was to capture (get the phage from the

environment), tame (isolate/purify a single phage), dissect (analyze the phage’s physical and

molecular structure), analyze (elucidate the genomic properties), discover (identify novel

genomic traits), and share (disseminate data) their individual phages.

The first few classes were spent discussing bacteria and phages’ effect on them. Then

important, everyday lab concepts such as aseptic techniques, making top agar, plating bacteria,

and serial dilution were discussed and executed. A soil sample was collected from 30° 55’ 18” N

1’ 7” W – altitude 23 ft. for direct plating and an enrichment culture. Direct plating was

executed; no plaques were found. Enrichment was executed; plaques were found. Next, a spot


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assay was conducted to double check that found plaques were actual phages. If phages were

found in spot assay, streaking was performed to begin purification process. Streaking was

repeated until uniform plaque morphology was visible.

Materials and Methods

Aseptic Techniques

Materials: Flask of water 70% ethanol Paper towels

Bunsen burner Bottle of water

Procedure: Cleaned off work bench by removing all books, phones, writing utensils out of the way.

Sprayed generous amount of 70% ethanol onto wok bench and wiped down with paper towel. Let bench airdry. After bench is dry, used lighter to light Bunsen burner flame. Worked close to the flame while pipetting, taking off caps, and removing microcentrifuge tubes aseptically.

Plating Bacteria

Materials: Top agar Calcium chloride LB agar plates 7H9 broth Culture bacteria (U102)

Vortex mixer Pipette aid, pipettes,

micropipetter with tips Conicle tube

Procedures:Prepared bench (ethanol, supplies, and flame). Obtained one tube of melted top agar from

water bath. Used sterile pipettes and pipette aid to add 20mL 1x 7H9 broth to 20mL 2x top agar from water bath. Used micropipetter to add 400µL 100mM calcium chloride to 7H9 and 2x top agar solution to make final amount of “complete” 1x top agar. Used sterile pipettes and pipette aid, add 4.5mL of complete top agar to culture tube containing bacteria.Usedsame pipette as in the previous step, drew complete top agar and bacterium solution from culture tube and disperse into LB plates.

2-Fold Serial Dilution

Materials: 1000µl water 500µl colored water Microcentrifuge tubes and rack

Micropipetter and tips Vortex mixer Sharpie


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Procedures:Prepared bench (ethanol, supplies and flame). Filled 4 microcentifuge tubes with 250µl

water. Labeled each microcentrifuge tube 2−1, 2−2, 2−3 and 2− 4. Diluted the first tube (2−1) with 250µl colored water. Used pipette to move 250µl of 2−1 solution to 2−2 tube to dilute. Repeated steps for next two tubes. Took pictures for observation.


Materials: LB plates M. Smegmatis bacterium Conical phage buffer “Complete” top agar D29 (1.5mL)

Microcentrifuge tubes and rack Micropetter and tips Vortex mixer Sharpie

Procedures:Prepared bench (ethanol, supplies, and flame). Diluted D29 to 10−4using phage buffer ad

serial dilution techniques. Infected Mycobacterium Smegmatis (M. Smeg for short) with 10µl of dilution. Waited 15-30 minutes (infection period). Plated M. Smeg with complete top agar. Incubated at 30° Celsius for 48 hours.

Direct Plating vs. Enrichment

Materials Baffled flask Collection kit Bacterium U102 “Complete top agar Phage buffer LB plates

Soil sample Pipetter and tips Shaking incubator Vortex mixer Pipette aids and sterilogical pipettes 0.22mM filter

Procedures:Went out to find soil sample. Prepared bench (ethanol, supplies and flame). Weighed 1

gram of soil, pour into flask. Inserted 45mL of 7H9, 500µL CaCl², and 5mL bacterium U102 into flask with soil. Placed mixture into 30° shaking incubator. Added enough phage buffer to remaining soil to cover it (about 10mL). Incubated at room temp for 20-30 minutes. Pipetted 1mL supernatant into syringe and filter into fresh tube. Infected bacterium U102 with 50µL filterate. Incubated at room temp for 20-30 minutes. Plated, then incubated at 30°C for 48 hours. Transferred 7H9, CaCl², U102, and soil sample solution to conical tube. Centrifuged at 2000xg for 10 minutes. Transferred supernatant and filter sterilized enrichment sample. Diluted enrichment sample to the -4 using serial dilution technique. Added 50µL of sample to 0.5mL bacterial U102 and mixed by vortexing. Then let sit for 20-30 minutes Plateed samples, then inverted and incubated for 48 hours.

Spot Assay

Materials:

Enriched culture plate Micropipetter and tips


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Bacterium U102 (host) Top agar Sterile pipette tubes

Pipette aid Sharpie LB plates

ProceduresPrepared bench (ethanol, supplies, flame). Looked for plaques on enrichment culture

plates. Prepared complete top agar. Made bacterial lawn with U102 and let solidify completely. Prepared 6 tubes of 100µl phage buffer (label “a” – “d”, “+” and “-“). Picked a single plaque for each tube “a”- “d”. Used positive and negative controls for “+” and “-“ plates. Labeled LB plate by creating sections on the plate for each labeled tube. Spoted 5µl of putative phage and “+” and “-“ onto solidified plate in designated areas. Waited about 30 minutes to let the spot soak into bacterial lawn, then inverted and incubated plate at 37°C.

Plaque Streaking

Materials: Sterile pipettes Sterile wooden sticks U102 bacteria culture Culture tubes

Complete top agar LB plates Vortexer

ProcedurePrepared bench (ethanol, supplies and flame). Looked for plaques in spot assay plate.

Labeled new culture plates with sharpie (one plate per phage). Aseptically removed wooden stick from culture tube. With wooden stick, touched spot “a” from spot assay plate. On new plate, streaked spot “a” VERY LIGHTLY . Got new autoclaved wooden stick and streaked spot “a” once more on the SIDE moving from inside of the first streak to untouched space. Win new wooden stick, created a third streak, moving from inside second streak to untouched spaceLabeled area “x” at the end of the third streak. Repeated this for spots “b”, “c”, “d”, “+”, and “-“ on separate plates. Covered streaks with U102 and top agar, waited for solidification, inverted, and incubated at 30° C for 24 hours.

Experimental Results

Plating Bacteria

The positive control plate had much bacterial growth. The plate was smooth but cloudy because of the bacterial lawn it was beginning to grow. The negative control, on the other hand, had no growth at all. No clumps, no bacteria, just a clear plate.


The dilution was made visible through this experiment. The color in the 2−1 tube was much stronger than all of the other tubes hit the color in the 2−4 tube was weaker than the rest.

Chamille, 10/29/15,

??? this should be the title of the experiment


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The most plaques were found in the 10−1 dilution and the least were in the 10−4 dilution The most plaques were in the least diluted plates.

Direct Plating

There were NO visible plaques in any of the plates. This was very surprising. All of the plates (except for the negative control) were cloudy, so it was obvious that there were bacteria growing. But, there were not plaques whatsoever (except for on the positive control).

Enrichment

In the enrichment plates there were clearly visible plaques. The most isolated plaques appeared in the 10−3& 10−4plates. Too much top agar was put into the other plates (-2, -1, 0, ), and so there may have been plaques but they were not visible.

Spot Assay

The preparation of the top agar made a problem with its solidification (perhaps the calcium chloride was left out). So, after the 24 hours of incubation, it was difficult to evaluate the plaques of the lack thereof. A second round of the spot assay was performed, with correct preparation of top agar, and the plaques were clearly visible in all of the sections except for the negative control section.

2-fold serial dilution using colored water. It was clearly visible that the -1 solution was

strongest while -4 solution was the weakest.

To the left is the -1 diluted plate and to the right is the -4 diluted plate.

The plates are in order from strongest to weakest (left to right)

On the top row are the tested plates. They are cloudy with the bacterial lawn but have no plaques. On the bottom left is the negative

control which is completely clear. And on the bottom right is the positive control which did

show a few plaques.


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Plaque Streaking (Round #1)

In this first round of streaking, plaques were seen on plates “a”, “b”, and “c” as well as on the positive control plate. The plaques are seen around the area of lowest concentration (the area labeled “x”). Plate D had nothing on it because it was accidentally plated without bacteria. The negative control had some kind of strange spreading taking place. It was never determined what this spreading might have been.

Since these pictures were taken in front of an open window instead of a solid colored surface, it’s a little hard to

make out the plaques from this streak.


Similar to what happened in the first round, there was an accident involving the plating of the bacteria. Only top agar was plated in ¾ of the plates that were being tested. There was one plate, though, that have bacteria and visible plaques – plate “c”. The phages in this plate were lytic and looked to be of the same two morphologies.


The second spot assay had 5 clearly visible plaques on spots “a”, “b”, “c”, “d”, & “+”. No plaques showed in the “-“spot.

The best plaques to pick from are the ones who are most isolated. In the close up picture of plate “c”, the plaque to the left of the “x” label is an ideal isolated plaque.

Chamille, 10/29/15,

Delete pictures


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This streaking round had obvious signs of multiple plaque morphologies. There were many different shapes and sizes. In plate “C1”, the phages looked more turbid or cloudy than they did in plates “C2” or “C3” where they were clear looking (lytic).


In this round there were still several different phage morphologies of all different shapes and sizes.


In this round of streaking, there were only two morphologies in my experimental plates. There were no plaques in my negative control and bout three morphologies in my positive control.


Round 6 still had multiple morphologies, but all were lytic. The positive control had multiple morphologies while the negative control had no plaques at all.

The more streaks that were performed, the closer the phages get to being isolated.

Morphology of a plaque includes shape and size. All of the plates from this round of streaking have

multiple morphologies.


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Discussion

Aseptic Techniques

Learning the aseptic techniques was one of the most important concepts to learn because these techniques are used literally every day in the lab. It is because of this that theplates haven’t experienced any contamination.

Plating Bacteria

Plating Bacteria is another concept that is used basically ever day in the lab as well as the preparation of top agar which was involved in the plating process. Once this concept was understood, questions started forming about the visibility of plaques.


This dilution was really just a practice run of dilution so that dilution was made clearly visible. Dilution is purposely weakening the effect of a substance. So, in this example, the color dye was weakened by the regular water.


This dilution was the first actual experience with phages. This experiment was another example of the visible effects of dilution, but in a way that was more useful for this lab.

Direct Plating

When comparing the results of direct plating to the results of enrichment plating, it’s clear that enrichment plating has much more effectiveness than direct plating does. This is because in direct plating, a short process, that phage does not have time to yield its putative plaques. This is why there were no phages present.

The plaques were becoming more and more uniforn.


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Enrichment

Enrichment plating is much more effective in yielding putative plaques. It does take much more time but this allots more time for the phages to yield their plaques. The enrichment process also required the use of new lab equipment that hadn’t been used before, such as the centrifuge and the 0.22mM filter.

Spot Assay

The spot assay is used to double check that what we see in our enrichment culture are actual phages. In doing so, there is no risk of testing something irrelevant to the experiment as a whole. Spot assay is the first step in the purification process.

Streaking

Streaking is the first step on the purification process. How streaking works is that it drags a high concentration of phages to a lower concentration of phages so that we get less and less each time. The end goal is to end up with one singular, uniform morphology phage (a purified phage).

Conclusion

From isolation through enrichment and spot assay, to purification though streaking and soon to be tittering, the phages and being examined and understood more every day. Every experiment is a step closer to the characterization and identification of these phages.

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