Note: Come in the day prior to inoculate overnight cultures of your LAB's!
What are bacteriophage?
Bacteria, just like our cells, can be infected by viruses known as bacteriophage (or phage for short). Phage are mobile genetic elements that use the infected cell's machinery for protein and nucleic acid synthesis. Without a bacterium, a phage cannot replicate. For this reason, and like viruses, phage are not considered to be living things. You can read more about phage here. Phage are important players in mobilizing all sorts of genes between cells. For an interesting read on this check out this interesting paper.
Importantly, phage have two parts to their life cycle: they can exist as either lytic (where they are actively lysing their host cells) or lysogenic (where they are integrated into the genome and being passed on generation to generation). Phage that are lysogenic can be induced to become lytic under certain conditions. Could you think about what those conditions could be? What would make you want to abandon your host?
Today we will be using a drug, mitomycin C, to induce production of active phage particles from lysogenized phage in our LAB's. Your overnight cultures will serve as inocula for a larger culture of your LAB isolates. We will then add mitomycin C and measure the OD600 at 20 minute intervals. How do you think the OD600 will change if a lysogenized phage becomes lytic?
- Measure the OD600 of your overnight culture of LAB. You will do this two ways: either using the side-arm spec or by using a cuvette in the regular spec. You will use the cuvette method first, as it is more accurate for higher density cultures (like your overnight culture) but throughout your time course, you will use the side arm spec method.
regular spec use
1. Aseptically transfer 100 ul of your culture to a 1.5 ml microcentrifuge tube. Cap the tube and spin it at max speed for 1 minute.
2. Overturn the tube onto a paper towel to get rid of the media. The cells should stay as a pellet on the bottom of the tube.
3. Resuspend the sample in 1 mL of PBS by pipetting up and down and then pipette the sample into a labelled cuvette. Make up a blank cuvette as well with PBS alone.
4. Take the sample over to the spectrophotometer and measure the OD at 600 nm.
5. Remember - your sample is a 1:10 dilution of your original culture! Multiply the number you get from the spec by 10 to get the true OD600 / ml.
side-arm spec use
1. The side arm spectrophotometer takes a measurement of OD600 through the side arm of the flask. This makes it easy to measure the OD600 without contaminating your culture.
2. Insert the culture tube in the spectrophotometer. Take note of the measurement as it appears on the instrument.
Beginning the experiment: Mitomicin C treatment
Calculate how much of your overnight culture you will need to create an OD = 0.2 culture of your LAB's in 10 mL. You can use the calculation below:
10 mL x 0.2 OD = X mL x ## OD600 O/N culture
1. Aseptically add the volume necessary of your O/N culture to two 10 mL culture tubes containing fresh LAB liquid media. One of these you will label as your experimental tube and the other will be your control.
2. Aseptically add mitomycin C to your LAB experimental culture. Note the time in your lab notebook and set a timer for 20 minutes.
3. Measure the OD600 of your 10 mL cultures every 20 minutes for 2 hours. You'll fill in the table below:
Isolate Code Name
Plot the OD600 of both of your cultures (your experimental and your control) using excel. Did the mitomycin treatment result in a different rate of growth for your cultures? A drastic reduction in OD600 means that you successfully created phage particles from a lysogenized phage in your LAB genome. Now the question is: is your phage relevant to its environment? What is its host range? In the next lab, we will determine the specificity of your phage by testing it against important bacteria used in yogurt (Lactobacillus species).
if you did get phage particles, let's save them by placing your cultures in the fridge (4 C) until next time.