I am a grad student working in S.cerevisiae. For the past month or so all of my liquid cultures have been contaminated and I am unable to determine how to prevent this issue for reoccuring so I can continue with my project. details of the growth and contamination are as follows.
Cells
Cells are S.cerevisiae By4741 derivitaves. They are grown either in SC-Leucine liquid media or YPD liquid media.
Media and Growth
The contaminant grows as small black clumps visible to the naked eye. The media is prepared and autoclaved, sugar solution (Glucose 50% or Raffinose 50%) is separately autoclaved and added to a final concentration of 2%. Cells are inoculated from a plate or a glycerol stock into 5mL of media and grown with shaking at 30 C.
Contaminant
In normal conditions liquid media becomes cloudy as the yeast grows. Since this issue began there have been large clumps of black substance that appears as the yeast grows. The media becomes cloudy as normal in addition to the black clumps (10-20 per 5mL tube). The clumps become larger an more pronounced as the cells are grown, appearing 16-24 hrs after inoculation and becoming more visible by 48hr. Attached is a picture of a 200mL culture grown for 48hrs with clumps visible as circled on the labelled image.
The contaminant is not present prior to cell growth and does not appear if sterile media+sugar is grown in the shaker next to the yeast.
Going back to old glycerol stock of base strains (>5 years old) the contamination appears in new liquid cultures from the stocks, although there was no sign of contamination when the stocks were made.
Microscopy of the cultures show yeast growing as normal, unless the black clumps are specifically pipetted out and loaded onto a slide. When a "clump" it appears as a block of cells but morphology is difficult to determine as they are so concentrated. A picture is attached where both the clump and free yeast cells are visible at 10000x magnification.
No contamination is visible when cells are grown on solid media, the yeast grow as expected with the typical colony morphology.
What has been considered
- Aseptic technique
The inoculation occurs in a fumehood with an open flame. The surrounding surfaces have been thoroughly cleaned with Ethanol and bleach. cells are inoculated with either a sterile pipette tip (autoclaved) a metal loop that has been flamed. While technique is the first answer for contamination, I have attempted all suggested improvements for technique and been shadowed by other lab members who can confirm that the technique is without issue. The contamination also occurs in yeast that they inoculate for me but not in their E. coli cultures (I am currently the only person growing yeast in the lab)
-Autoclave failure
The media and sugar solutions are autoclaved prior to use. standard autoclave testing (test cultures etc. has been done by the autoclave manager to confirm it is working. Contamination also appears in media prepared by another lab tech working in another building with a different autoclave.
-Contamination of individual glycerol stocks
While contaminated stocks would result in contaminated culture I have tested >10 stocks of 6 different strains prepared over the past 6 years including my own stock and those prepared by former students in the lab. I think it is unlikely sourced from any particular stock as it is in all cultures I have tried including ones that had not been opened since before the trouble began.
-Media and sugar solutions
The contamination appears in all liquid media that I have prepared over the past month. This includes combinations of SC-leu media or YPD media with glucose or raffinose in every possible combination. Media inoculated without sugar shows no growth of yeast and no contamination.
Overall
As it stands this is an issue with seemingly no solution. If it is environmental contamination getting into my media I have no idea how to prevent it moving forward. If anyone has any suggestions for removing this contamination moving forward (or even just an idea of what it is, as its morphology matches nothing I've ever encountered) I would greatly appreciate your advice.
I think you should filter-sterilize the sugar (glucose and raffinose) instead of autoclaving, You should prepare fresh sugar solution every time when required, don't use the old one and try to grow first on the agar plate and then from there, you pick a single colony for the culture. Also, autoclave the culture vessel in which you are setting up a culture. and keep yeast and bacterial culture work in separate hood for some time.
A few things to try while sorting it out.
1) Go through a mock inoculation of media without actually adding any yeast to a flask of your media. That way you can see if it is entering environmentally or not and tests all components except the yeast.
2) What happens when you streak on on solid agar for colonies?
3) Could your sterile hood be contaminated and blowing some spores or cells into your cultures as you try to inoculate?
4) Has the autoclave been calibrated recently?
5) yeast will actually grow fine on LB (just not quite as well as on yeast media). This has the advantage that everything was in the autoclave at one time so nothing is added. Or borrow from someone else.
6) Be sure glassware, pipettes etc are not the source of the problem.
- Badges
- Science topic
- Similar topics
- Computer Science
- Research Topics