Colony forming unit (CFU): refers to individual colonies of bacteria, yeast or mold. A colony of bacteria or yeast refers to a mass of individual cells of the same organism, growing together. For molds, a colony is a group of hyphae (filaments) of the same mold growign together. CFUs are used as a measure of the number of microorganisms present in or on surface of a sample. To determine the number of CFUs, a sample is preapred and spread uniformaly on a surface of an agar plate and then incubated. The colonies that form are counted. 

CFU/mL=(# colonies)*dilution factor)/(volume plated in mL). So if you pipete 100 ul of the bacterial onto a plate with a 10 x 5 dilution, your CFU/mL = (400 * 10 to the 5th)(0.1 mL)=4*10 to the 8th) CFU/mL

Experiment; Determining CFUs (Legionella)

(1) spread your plates that have special agar for Lp growth on table. Do in triplicates for each sample.  Label the plates (time: eg. 24 hrs), (same #: eg., 1, 2…), (immature vs. mature DC, eg, i vs m), 

1 24 i LF only         2 24 i                1 24 PA only    2 24 PA only

1 24 i   LF only      2 24 i                1 24 PA only     2 24 PA only

1 24 i LF                2 24 i                1 24 PA only    2 24 PA only

(2) Stack plates under hood

1 24 10e1     1 24 PA only  ….

(3) Obtain polysterine glass tubes with plastic tops (kept in cabinet next to pcr). set up in groups as with plates in blue holder

(4) obtain 0.2% Saponin in water (lyses cells and releases bacterium from the cells). Use the small filter to refilter the saponin into a well plate

(5) add 100 ul to 100 ul of your samples. This will make [finial] saparin 0.1 %.   Time this for 10 min

Stop Reaction

(1) place HBSS into a 50 ml tube

(2) unscrew your polysterine tubes and place 800 ul of HBSS into each.

(3) after 10 min reaction time is up, transfer well of plate to polysterine tube (3 at a time, 100 ul)

(4) If need to you can do a dilution. Would put 800 ul of HBSS into 2nd set of polysterine tubes and take 100 ul of the 1st set and put into this set. This will be 10e2 dilution.

Streak Plates

(1) obtain bunsen burner (light one glass) and alcohol 95% ETOH.

(2) take sample polysterine tube, vortex and put 100 ul into plate

(3) light glass rod tip under burner, let cool slightly, steak.

(1) incubate plates for 3 -4 days to let bacterial grow.