Objective: Experimental Setup of microbial Knockdown project.

Broader Context

This project will monitor the success of microbial knockdown under two seperate Antibiotic solutions being treated fro 7 days, and will also monitor the duration for which the microbiome stays reduced. Initial sample priming will involve the reduction of the microbiome through keeping the organisms in Seawater filtered with a .22 micron filtered, and feeding with Artemia that have had their microbiome knocked down through the use of antibiotics.

Protocol

prepare stock solutions for each of the individual antibiotics

Nalidixic acid (50mg/ml)

• 2.5ml MilliQ water
• 0.25 g nalidixic acid
• add 5M NaOH until the nalidixic acid dissolves
• bring the volume up to 5ml the pH should be about 11

Rifampicin (50mg/ml)

• 0.25g rifampicin (light sensitive wrap falcon tube in tinfoil)
• 5ml DMSO
• vortex to dissolve

Carbenicillin (50mg/ml)

• 0.25g carbenicillin
• 5ml millQ water
• vortex to dissolve

Chloramphenicol (50mg/ml)

• 2.5g chloramphenicol
• 50ml EtOH

Penicillin (50mg/ml)

• .25g penicillin
• 5ml MilliQ water
• vortex to dissolve

Streptomycin(50mg/ml)

• .25g streptomycin
• 5ml MilliQ water
• vortex to dissolve

Neomycin (50mg/ml)

• .25g neomycin
• 5ml MilliQ water
• vortex to dissolve

To make 500ml of ABS 1 (final concentration 50ug/ml) add:

• 498ml Filtered Artificial Seawater
• 500 ul nalidixic acid stock solution
• 500 ul chloramphenicol stock solution
• 500 ul carbenicillin stock solution
• 500 ul rifampicin stock solution

To make 500ml of ABS 2 (final concentration 100ug/ml) add:

• 498ml Filtered Artificial Seawater
• 1000 ul penicillin stock solution
• 1000 ul streptomycin stock solution
• 1000 ul neomycin stock solution
• 1000 ul rifampicin stock solution