Hi there! Brace yourself, this is going to be long winded and contains a year's worth of frustrations.
I culture moss, and for the last year, I have been fighting a gram negative bacterial invasion. I work in sterile laminar flow hoods, and we just had the hoods serviced and confirmed they are working properly, but anything I do comes out contaminated. We were fine for years, and then this all started July 2019, and has only gotten worse.
I've provided a list of everything I have tried so far, but to no avail. The last thing I can think to try is to add UV lights to the hoods, but I have been hearing conflicting opinions.
Everything I use comes directly from the autoclave, there is no storage in between use. It is all freshly sterilized.
To culture the moss, I first have to lay thin cellophane discs (that have been autoclaved on a 30 minute liquid cycle three times--it's just what we know works) onto the media. I use autoclaved and ethanol/flame sterilized forceps to lay the cellophanes. This never use to be an issue, but now probably 50-70% of the plates with cellophanes will be contaminated.
From there, we will homogenize the plant material in water and plate it on the cellophanes. I've been working with just water, no plant material, for the past few months to try and rule out the source. I've tried plating just water (autoclaved for 60 minutes), and plating water that has been homogenized. I've tried using different kinds of bottles for the water. Anything involving water comes out contaminated 95% of the time, and anything involving the homogenizer comes out contaminated 100% of the time.
In addition to these steps, I have done the following:
Everything is kept in one neat line at the very back of the hood
Bleached the homogenizer
Bleached the hoods
Constantly spray everything down with 70% EtOH
I wear a plastic hair net and a clean mask
At one point, I tried using a bleached pipet bulb instead of a Pipet Aid in case the Pipet Aid was the issue.
Ethanol all the way up my arms
I have run identical trials in both of our laminar flow hoods, with contamination resulting from both.
I move slowly in the hood to avoid air disturbance
I 100% ethanol/flame sterilize the forceps any time I do absolutely anything. Usually 2-5 times. About 10-15 times at the start of any day, and they're always freshly autoclaved.
Bleached/mopped out entire culture room and growth chambers.
My other lab mates are struggling as well, so I decided to come here for any advice.
I really don't know what else to try. Any input would be so appreciated. Thank you for taking the time to read this!
Kind regards,
Emilie-Kate
Did you check the air inflow of your lab for contamination? Airconditioners have been found to be a source of contamination.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating