Today we'll be testing the specificity of the phage you isolated last time we met. We has grown up overnight cultures of important organisms used in yogurt making. The question is, are your phage able to infect these alternative hosts? We will test this by using the double agar method. You will first filter out your host from last week to isolate the phage. These phage will be mixed with a small amount of the cultures provide by sherly, plus calcium chloride and molten agar. They will be poured over a hardened layer of agar. The bacteria grow within the agar and the phage, if able to lyse the host, produce circular plaques in the agar - basically wherever bacteria are missing. For cool pictures of bacterial plaques look here.
1. Filter your 10 mL culture presumed to include bacteriophage using a syringe and a syringe filter (0.2 um). You can do this by first suctioning up 5 mL into the syringe aseptically. With the plunger in the syringe, screw the 0.2 um filter into the end of the syringe. Put the tip of the syringe/filter into a 15 mL falcon and depress the plunger slowly. The drops that make it through the filter should be phage particles.
2. For each important host to test, label a 1.5 ml tube and place 250 ul of the overnight culture in the tube.
3. Take 250 ul of your phage solution and add it to each 1.5 ml tube.
4. Add 10 mM final calcium chloride to the tube. Do this by adding 5 ul of 1 M CaCl to the tube
5. Finally, add 2.5 mL of molten M17 agar (46C) and quickly mix by inverting before pouring it over the top of a M17 agar plate. You may nutate the plate in order to achieve a regular layer of agar.
6. As soon as the agar hardens, place your dish upside down and in the 37C room.
Check on your phage plaques daily and note any that form in your lab notebook. We can also take pictures of your plaques on Friday. A cool website about bacteriophage in LABs can be found here.