Remember the spec reading is 1/10 the Actual OD of the spores in your tube because you made a dilution. Multiply your Spec Readings by 10 to get the Actual OD. 1. Measurement Spec Reading Actual OD P. rubens spore suspension OD600 measurement P. crysogenum spore suspension OD600 measurement In order to normalize the amount of fungal spores you add to each culture, you will need to calculate the dilution of spores needed for a final OD of 0.05 in the final volume of 25mL. a. For the dilution use the calculation equation C1 x V1=C2 x V2 b. This can be re-written: (Actual OD in your 5ml tube) x (Volume you need in ml) = (0.05 final OD) x 25ml c. (Volume you need) = (0.05 x 25ml)/(Actual OD in your 5mL tube) d. Multiply answer by (1000l /1ml) to convert to l Mix the rubens spores by inverting the tube 3 times. “R” Pipet the volume from step d into one of the flasks containing 25ml LB. Label this flask with the contents, your group initials, and the date using tape. Repeat step 11 for the crysogenum spores. “C” Place both flasks on a shaker to be left for 72h or 3 full days at 25°C shaking at 100rpm. (You will have 1 of your 3 flasks of LB left over. Set it aside and keep it sterile. You will use it as a bacteria control in step 16.) Finally, we will use some of the remaining spores of each fungus to make a vertical stripe on LB agar plates. Label one LB agar plate rubens, the date, and your initials, repeat with crysogenum for the other plate. Dip a new sterile cotton swab into the tube. “R” Make one swipe across the center of the rubens LB agar plate. Repeat with the crysogenum plate from the tube. “C” Culturing Bacteria (DAY 3) Collect a patch of your bacteria from the plate using a sterile 200l pipette tip.
Remember the spec reading is 1/10 the Actual OD of the spores in your tube because you made a dilution. Multiply your Spec Readings by 10 to get the Actual OD. 1. Measurement Spec Reading Actual OD P. rubens spore suspension OD600 measurement P. crysogenum spore suspension OD600 measurement In order to normalize the amount of fungal spores you add to each culture, you will need to calculate the dilution of spores needed for a final OD of 0.05 in the final volume of 25mL. a. For the dilution use the calculation equation C1 x V1=C2 x V2 b. This can be re-written: (Actual OD in your 5ml tube) x (Volume you need in ml) = (0.05 final OD) x 25ml c. (Volume you need) = (0.05 x 25ml)/(Actual OD in your 5mL tube) d. Multiply answer by (1000l /1ml) to convert to l Mix the “R” rubens spores by inverting the tube 3 times. Pipet the volume from step d into one of the flasks containing 25ml LB. Label this flask with the contents, your group initials, and the date using tape. Repeat step 11 for the “C” crysogenum spores. Place both flasks on a shaker to be left for 72h or 3 full days at 25°C shaking at 100rpm. (You will have 1 of your 3 flasks of LB left over. Set it aside and keep it sterile. You will use it as a bacteria control in step 16.) Finally, we will use some of the remaining spores of each fungus to make a vertical stripe on LB agar plates. Label one LB agar plate rubens, the date, and your initials, repeat with crysogenum for the other plate. Dip a new sterile cotton swab into the “R” tube. Make one swipe across the center of the rubens LB agar plate. Repeat with the crysogenum plate from the “C” tube. Culturing Bacteria (DAY 3) Collect a patch of your bacteria from the plate using a sterile 200l pipette tip.
Penicillium%20Antibiotic%20Effect%20Class%20Protocol.docx