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EasyPhage Lab—Basic Instructions for Teachers

Timing is crucial in this lab. You must have three consecutive class days to perform the experiment. This lab cannot go over a weekend.

Checklist(provided in the EasyPhage kit from Scientific Methods, Inc.):

___ 1. Sterile medium EasyPhage EP-10 (10 bottles)

Store at 4 °C.For best results, warm to 35 °C or at least room temperature before use.

___ 2. Sterile pretreated Petri dishes (10)

Store at room temperature.

___ 3. E. coli Famp bacterial cells on an agar slant (nonpathogenic)

Store at 4 °C. Warm to 24-35 °C before use.

___ 4. MS2 (male specific) coliphage virus in a tiny vial in a small, dark bag (nonpathogenic)

Store at 4 °C.

___ 5. Bacterial stain, sterile in a tiny vial in a small, dark bag

Store at 4 °C.

___ 6. Microbiological loop, sterile, plastic, disposable, in a plastic bag

Store at room temperature.

___ 7. Trypticase Soy Broth (TSB) in large centrifuge tube

Store at 4 °C. For best results, warm to 35 °C or at least room temperature before use.

___ 8. Micropipets, sterile (2)

Store at room temperature.

Checklist (provided by the teacher):

___ 1. Sterile water ___ 3. Sterile, individually wrapped plastic pipets

___ 2. 35 °C incubator___ 4. Parafilm to seal Petri dishes

Timing: Timing is crucial in this lab. You must have three consecutive class days to perform the experiment. This lab cannot go over a weekend.

  • Day 1: TransferE. coli from the agar slant into the TSB tube using the sterile plastic loop. Let it grow overnight. A fresh, 24-hour culture of E. coli is required. The bacteria must be actively dividing for the virus to infect the bacteria, so the lab will not work with an older culture.
  • Day 2: Conduct the lab. Inoculate media bottles with E. coli, phage, and stain. Pour the media into the pre-treated plates and allow them to harden for 1 hour. Incubate plates overnight in a 35 °C incubator. Plaques will not form at room temperature.
  • Day 3: Read results within 24 hours! The plates must not stay in the incubator longer than 24 hours. E. coli and phage will continue to grow if the plates incubate longer. This will cause plaques to enlarge until they overlap and are no longer distinct. The indicator dye will also fade over time.
  • Students should be able to see “holes” in the colored medium on the Petri dishes. These empty spots are called plaques. Each plaque indicates a place where one virus infected a single bacterium. This virus then replicated and released hundreds or thousands of new viral particles to infect new bacterial cells. This leaves an empty spot.

Aseptic(Sterile) Technique:

  • Do not touch any of the lab materials with your bare hands. Wear gloves throughout the investigation to protect yourself from contamination.
  • Lab gloves are not sterile. Avoid touching sterile items even while wearing gloves.
  • When inoculating the TSB tube with E. coli, the loop is sterile until you take it out of the bag. Only touch the handle. Do not let the loop touch anything but the bacteria and the broth.
  • Do not allow the lower surfaces of vial lids to touch anything. When you open a vial place its lid upside down on the counter. Doing otherwise may contaminate the contents of the vial.
  • Do not breathe or cough into open containers (sterile water, vials, E. coli culture tube, or the Petri dish). Air can carry contaminants.
  • Do not leave the Petri dish lid open longer than necessary. Dust from the air will fall onto the surface and contaminate the culture. Open the plate with one hand and slowly pour the contents of the bottle onto the surface with the other hand. Quickly replace the lid.
  • Do not reuse any items used in this lab. Soak contaminated materials in a 10% bleach solution overnight then discard in the trash the next day.

Procedure:

Day 1: CultureE. coli overnight.

1. Allow E. coli agar slant to warm to 24-35 °C before use. For best results, warm TSB broth tube to 35 °C or at least room temperature before use.

2. Wear gloves and use proper sterile technique. Remove the sterile plastic loop from the plastic bag. Only touch the handle. Do not allow the loop to touch any surface.

3. Carefully open the agar slant of E. coli. Gently touch the loop onto the bacterial growth. A small amount of visible bacteria on the loop is enough.

4. Open the TSB tube carefully and dip the loop into the broth. Swirl it around a bit without touching the rim. Close the tube and let it incubate upright overnight (18-24 hours) at 24-35 °C.

5. Use this broth culture of E. coli to complete the lab. Coliphages (viruses that infect E. coli) will only infect bacteria that are in an active phase of growth. Older cultures of E. coli will not work.

Bacteria: Escherichia coli bacterial cells must be actively growing for the virus to infect them. The bacteria are the host and the virus is the pathogen. In the stage of bacterial growth called the log or exponentialphase, bacteria have enough nutrients to grow very quickly. The entire population can actually double every 20 minutes! This is the E. coli generation time. In humans, a generation takes about 25 years.

Day 2: Conduct the lab.

1. For best results, warm EasyPhage bottles and TSB broth tube to 35 °C or at least room temperature before use. Mix by gently swirling the bottle to avoid bubbles. (Cold medium will take longer to solidify. Medium may form layers during cold storage.)

2. Each student (or small student group) should receive:

(1) EasyPhage bottle

(2) Pretreated plate

Make sure that students label the bottom of the plate with name, date and identification by writing along the outer edge of the bottom with a marker. Tell your students not to write in the center of the plate to avoid obscuring any plaques that form.

3. Using aseptic (sterile) technique, add 6 mL of sterile water to each EasyPhagebottle with a sterile plastic pipet.

4. Using aseptic (sterile) technique, add 70 µL (orone drop) of bacterial stain to each EasyPhagebottle with a sterile plastic micropipet.

5. Using aseptic (sterile) technique, add100 µL (or one drop) of MS2 coliphage to each EasyPhagebottle with a sterile plastic micropipet.

Viruses, also called phages, are often grouped by the host they infect. Bacteriophages only infect bacteria and coliphages only infect E. coli bacteria. Every major group of organisms has a specific group of phages that infects it.

6. Using aseptic (sterile) technique, add 300 µL (orthree drops) of the E. coli overnight culture to each EasyPhagebottle with a sterile plastic micropipet.

7. The EasyPhage bottles now contain water, stain, MS2 coliphage and E. coli.Swirl the bottle very gently to mix reagents without introducing bubbles.

8. Pour contents onto the bottom of apretreated Petri dish(from kit). While resting the Petri dish on the table, swirl it gently so that any bubbles that form move to the edge of the plate. Allow the plate to harden for 1 hour. Incubate overnight (18-24 hours)in 35 °C incubator.

Day 3: Read the results within 24 hours!

Students should be able to see “holes” in the colored medium on the Petri dishes. These empty spots are called plaques. Each plaque indicates a place where one virus infected a single bacterium. This virus then replicated and released hundreds or thousands of new viral particles to infect new neighboring bacterial cells, killing the bacteria in the process. This leaves an empty spot. (The stain will only color live bacteria.)

You can perform this activity as an inquiry lab or as a demonstration of viral plaques.