Best tips for sterile phyto?

[DaJMasta]

As others have been talking about and as you seem to know, truly sterile is a very, very high bar which even labs struggle to maintain (thus the frequent microscope checks), so my focus has always been to minimize potential contamination vectors and then have redundant cultures. There are probably dozens of small optimizations you can make to your process that don't involve extra equipment to help minimize contamination.

Sterilize containers and airline tubing between runs
Make sure there is positive pressure running on the culture when it is active to keep out airborne contaminants
Make sure the inlet of your air pump (especially if sponge or cloth material) is clean/sterilized
Make sure to restart cultures regularly (running a longer time gives contaminants longer to take hold and start taking over, generally the phyto will outcompete contaminants in early stages of growth)
Sterilize the equipment used to measure out fertilizer
If the fertilizer pipette (or similar) touches an active culture vessel, immediately replace it with a new sterilized one
Wash your hands between handling different strain's culture vessels
Immediately cover an active vessel when not using it/starting to work with another
Don't overfill vessels to reduce chance of bubbling out and spatter
Keep fertilizer in a cool, dark place to minimize growth of contaminants
Make sure the method to check salinity of the fresh saltwater to start cultures doesn't cross contaminate (different pipette and fully dry refractometer in my case)
Physical separation between different strains on your growth rack reduces chance of random spatter, physical separation from your tanks or any zooplankton cultures further helps
Being far away from other vessels when transferring between another (reducing spatter risk)
Not keeping strains above each other (I don't do this, but a single row reduces contamination risk)

 

[salty joe]

I can't disagree with that but I have a routine that is simple and rock solid.

Wash hands, large cup hooks to hold airline while harvesting, spray bottle with rubbing alcohol to disinfect funnel and working surface.

I just set the syringes for the f2 and dechlor on a shelf beside their bottles, I don't bother disinfecting them.

I agree, multiple cultures is a must, the last crash I had was quite a while ago but it happens.

I use a HEPA filter from a vacuum cleaner on the inlet of the air pump.

I have a drill bit that fits exactly inside the rigid tubing that I use to ream out the tubing. Otherwise, crusty stuff will clog the things up after awhile.

 

[fryman]

I use a HEPA filter from a vacuum cleaner on the inlet of the air pump.

I do this too! I put my air pumps in vacuum cleaner bags, and this works quite well. Syringe filters are another option but they cut the airflow alot more than vacuum bags.

I have a drill bit that fits exactly inside the rigid tubing that I use to ream out the tubing. Otherwise, crusty stuff will clog the things up after awhile.

The "crusty stuff" is from abiotic precipitation. A healthy, fast-growing phyto culture has really high pH and so calcium carbonate forms anywhere wet, in particular on the airline. I recommend using an acidic cleaner like vinegar, CLR, or citric acid. I believe this is more important than bleach/IPA for the air line.

 

[DaJMasta]

FWIW I haven't had any precipitation issues in my cultures unless I had a contaminant algae that grew on the sides (didn't inhibit growth that substantially, but meant a bunch more cleaning and if the harvested algae sat unagitated it started to go bad.) I fill my used containers about 1/6th of the way with water, add a jigger of bleach, shake with the lid on (with airline in place), let sit, shake again, and rinse with freshwater then set to dry, and I haven't had any long term issues with airline clogging or precipitation of healthy cultures in my couple years of using the same equipment.

Also worth mentioning part of the reason I mention cleaning the filter is that even though they can trap things, things can also grow. I found a fungus growing in my inline air filters (syringe style) - they had trapped it, but then it grew and ended up sending spores through the other side after colonizing the filter. Those things are expensive enough to replace that I don't bother with filters now, and just occasionally sterilize the input for the air pump.