Growth Curves for Yeasts and Molds - (Apr/26/2020 )

Hi all,

 

I recently attempted to Create growth curves for Candida Albicans and Penicillium rubens and have failed miserably, I'd like to share with you my protocol to see maybe I'm missing something:

• I grew starter cultures in YM broth over a few nights(yeast culture was 2-3 days old, mold culture was 5-6 days old). I grew both cultures in a semi sealed sterile erlemmayer at room temperature placed on a rotating orbital shaker(spins in a cyrcle) at about 65 rpms. - I saw massive growth in both starter cultures.
• I considered the grown starter cultures as 7 log (10,000,000 CFU/ml) concentration and I diluted both starters x4 and x5 and infected new samples (clean ym broth in erlenmeyer) at what I learned is considered 3 log and 2 log growth concentration.
• I placed the 4 samples(2 for each fungi, at 2 different infection levels) on the same rotating shaker and started mesuring time - sampeling each bottle into 2 agar plates (my agar is oxytetracycline - glucose yeast extract agar) at 2 different dilutions (2 log and 3 log).
• I sampeled the yeast samples every 3.5 hours, and diluted it another +1 ratio(1:10) each time considereing 3.5 hours is the average growth time for yeasts, for 7 times total.I sampeled the mold samples every 6 hours, and diluted it another +1 ratio(1:10) each time considereing 6 hours is the average growth time for molds , for 10 times total.
• All plates were left for incubation in room temerature for 3 days (yeasts) and 5 days(molds).

Non of my mold plates showed any growth at all, and my yeast plates showed very random growth, showing some growth over the first few time points, then dropping to very low growth(with some plates showing 0 colonies) before going back to show some growth toward the end of the time points.

 

Please, if anyone has experience with growth curves or with growing these organisms, I'd appriciate any assistance you can offer.

 

 

 

Unrelated to your question - but why are you using antibiotic-amended plating media?   

 

No idea re. problem with Candida albicans.  Might try incubating at 35C and use YM agar.

 

Penicillium rubens is a mycelial fungus, plate counts are not useful as the cellular units do not separate.  That should be evident to you.

Ziv on Sun Apr 26 13:36:28 2020 said:

Hi all,

 

I recently attempted to Create growth curves for Candida Albicans and Penicillium rubens and have failed miserably, I'd like to share with you my protocol to see maybe I'm missing something:

• I grew starter cultures in YM broth over a few nights(yeast culture was 2-3 days old, mold culture was 5-6 days old). I grew both cultures in a semi sealed sterile erlemmayer at room temperature placed on a rotating orbital shaker(spins in a cyrcle) at about 65 rpms. - I saw massive growth in both starter cultures.
• I considered the grown starter cultures as 7 log (10,000,000 CFU/ml) concentration and I diluted both starters x4 and x5 and infected new samples (clean ym broth in erlenmeyer) at what I learned is considered 3 log and 2 log growth concentration.
• I placed the 4 samples(2 for each fungi, at 2 different infection levels) on the same rotating shaker and started mesuring time - sampeling each bottle into 2 agar plates (my agar is oxytetracycline - glucose yeast extract agar) at 2 different dilutions (2 log and 3 log).
• I sampeled the yeast samples every 3.5 hours, and diluted it another +1 ratio(1:10) each time considereing 3.5 hours is the average growth time for yeasts, for 7 times total.I sampeled the mold samples every 6 hours, and diluted it another +1 ratio(1:10) each time considereing 6 hours is the average growth time for molds , for 10 times total.
• All plates were left for incubation in room temerature for 3 days (yeasts) and 5 days(molds).

Non of my mold plates showed any growth at all, and my yeast plates showed very random growth, showing some growth over the first few time points, then dropping to very low growth(with some plates showing 0 colonies) before going back to show some growth toward the end of the time points.

 

Please, if anyone has experience with growth curves or with growing these organisms, I'd appriciate any assistance you can offer.

 

 

 

 

Room temperature for C. albicans is too low for this experiment.

Not sure exactly where you got this protocol, but it is a bit weird.

3,5 hours is the average growth? At room temperature?

Phil Geis on Thu May 7 09:53:00 2020 said:

Unrelated to your question - but why are you using antibiotic-amended plating media?   

 

No idea re. problem with Candida albicans.  Might try incubating at 35C and use YM agar.

 

Penicillium rubens is a mycelial fungus, plate counts are not useful as the cellular units do not separate.  That should be evident to you.

 

Thanks for getting back to me.  I'm using Antibiotic amended plates for selection purposes of course(lower competition with bacteria), hence the lower incubation temp(room instead of 35C) From what I understand the ideal growth temp for these types of organisms is even lower then room temp, and is best done at 19C even.

 

Why would you do it in 35C? And why would YM agar be better then the one I'm using?(I've succesfuly grown both yeasts and molds on it before - just not with this protocol).

pito on Thu May 7 17:38:49 2020 said:

 

Ziv on Sun Apr 26 13:36:28 2020 said:

Hi all,

 

I recently attempted to Create growth curves for Candida Albicans and Penicillium rubens and have failed miserably, I'd like to share with you my protocol to see maybe I'm missing something:

• I grew starter cultures in YM broth over a few nights(yeast culture was 2-3 days old, mold culture was 5-6 days old). I grew both cultures in a semi sealed sterile erlemmayer at room temperature placed on a rotating orbital shaker(spins in a cyrcle) at about 65 rpms. - I saw massive growth in both starter cultures.
• I considered the grown starter cultures as 7 log (10,000,000 CFU/ml) concentration and I diluted both starters x4 and x5 and infected new samples (clean ym broth in erlenmeyer) at what I learned is considered 3 log and 2 log growth concentration.
• I placed the 4 samples(2 for each fungi, at 2 different infection levels) on the same rotating shaker and started mesuring time - sampeling each bottle into 2 agar plates (my agar is oxytetracycline - glucose yeast extract agar) at 2 different dilutions (2 log and 3 log).
• I sampeled the yeast samples every 3.5 hours, and diluted it another +1 ratio(1:10) each time considereing 3.5 hours is the average growth time for yeasts, for 7 times total.I sampeled the mold samples every 6 hours, and diluted it another +1 ratio(1:10) each time considereing 6 hours is the average growth time for molds , for 10 times total.
• All plates were left for incubation in room temerature for 3 days (yeasts) and 5 days(molds).

Non of my mold plates showed any growth at all, and my yeast plates showed very random growth, showing some growth over the first few time points, then dropping to very low growth(with some plates showing 0 colonies) before going back to show some growth toward the end of the time points.

 

Please, if anyone has experience with growth curves or with growing these organisms, I'd appriciate any assistance you can offer.

 

 

 

 

Room temperature for C. albicans is too low for this experiment.

Not sure exactly where you got this protocol, but it is a bit weird.

3,5 hours is the average growth? At room temperature?

 

 

Thanks for getting back to me.

 

Why is room temp too low? I've had success growing C.albicans at it multiple times in the past, why would this experiment be different? wouldn't the lower temp just prolong my waiting time for plate results but in turn provide better selection conditions? 

 

The protocol is one I've used in the past to formulate growth curves for bacteria, L.Mono and Salmonella in particular, so the template is the same, with different growth and incubation time variables added. - if you have any insights into what I'm doing wrong, and you could provide an explanation as well, I will greatly appreciate it.

You're working with pure cultures - there is no reason to use antibiotic-amended agar.  If you have contamination problems, you need to address technique.  Fungi can be sensitive to some antibiotics (used for bacteria) - at high concentrations, they can inhibit mitochondrial function.

 

YM because you have not been successful with the agar medium you're using now, and it supported growth of your inoculum.  If this protocol has worked for C. albicans before - suggest you do some QC for your plating medium

 

These are fungi, not bacteria.  Not sure where you got 19C - that is clearly false.  I'd use ~35C for Candida albicans (some report opt even greater temps https://www.ncbi.nlm.nih.gov/pubmed/6762776-).  3.5 hours is a waste of medium.   Consider a growth curve of 24 hours +/- for Candida albicans - sample at 6 hours.  Think generation time is ~3 hours - within standard deviation plate counts.

 

Suggest room temp for Penicillium rubens (25-28C - file:///C:/Users/phila/Downloads/9179.pdf).   But waste no more time with it..  Working with mycelial fungi requires different expertise.  Do you understand why serial dilution/plate counts of mycelial fungi is not useful?  

Phil Geis on Sun May 10 10:19:25 2020 said:

You're working with pure cultures - there is no reason to use antibiotic-amended agar.  If you have contamination problems, you need to address technique.  Fungi can be sensitive to some antibiotics (used for bacteria) - at high concentrations, they can inhibit mitochondrial function.

 

YM because you have not been successful with the agar medium you're using now, and it supported growth of your inoculum.  If this protocol has worked for C. albicans before - suggest you do some QC for your plating medium

 

These are fungi, not bacteria.  Not sure where you got 19C - that is clearly false.  I'd use ~35C for Candida albicans (some report opt even greater temps https://www.ncbi.nlm.nih.gov/pubmed/6762776-).  3.5 hours is a waste of medium.   Consider a growth curve of 24 hours +/- for Candida albicans - sample at 6 hours.  Think generation time is ~3 hours - within standard deviation plate counts.

 

Suggest room temp for Penicillium rubens (25-28C - file:///C:/Users/phila/Downloads/9179.pdf).   But waste no more time with it..  Working with mycelial fungi requires different expertise.  Do you understand why serial dilution/plate counts of mycelial fungi is not useful?  

 

Thank you for answering.

 

I understand what you're saying regarding antibiotic amended agar, but I've had sucess multiple times growing both strains of Candida Albicans and Penicillium Rubens that I've been using for the growth curve protocol, I'll take what you said into consideration when I order new agar, but for now I don't see why that would be the cause for my growth curve protocol to fail(Correct me if I'm wrong).

 

Regarding Candida Albicans sampeling time during growth curves protocol, you say sample every 6 hours instead of 3.5 that I'm doing now? why is 3.5 hours false if the generation time is 3 hours? I'd like to sample every 1 multiplication of the population, not 2 - every 6 hours the pupulation theoratically devides twice. Again, correct me if I'm wrong on any of this.

 

Regarding your final remark about Penicillium rubens - I understand why plate count is not usful in the case of mold(Impossible to seperate colonies), but I still think that it's still usful to assess growth via precentege of plate covered(or at least that's what I'm expected to do through out this project). Regardless of that and more important - Why is serial dilution not useful for these type of fungi?

 

Thanks in advance.

 

Ziv

3.5 isn't false - just not very useful. It's discretionary and not very revealing as it isn't far enough from plate count accuracy to be mean much vs 6 hour.

 

It's useless to chase plate coverage with a mycelial fungus.   You'll chase mycelial fragmentation, spore production, "microcycle" and other phenomena irrelevant to cell multiplication."Why is serial dilution not useful for these type of fungi?"  Please consider what do you think you're counting and it's relevance to biomass?

 

Back to your original problem - poor growth in plating media.  Have you performed QC for your plating medium?

Ziv on Sun May 10 08:49:07 2020 said:

 

pito on Thu May 7 17:38:49 2020 said:

 

Ziv on Sun Apr 26 13:36:28 2020 said:

Hi all,

 

I recently attempted to Create growth curves for Candida Albicans and Penicillium rubens and have failed miserably, I'd like to share with you my protocol to see maybe I'm missing something:

• I grew starter cultures in YM broth over a few nights(yeast culture was 2-3 days old, mold culture was 5-6 days old). I grew both cultures in a semi sealed sterile erlemmayer at room temperature placed on a rotating orbital shaker(spins in a cyrcle) at about 65 rpms. - I saw massive growth in both starter cultures.
• I considered the grown starter cultures as 7 log (10,000,000 CFU/ml) concentration and I diluted both starters x4 and x5 and infected new samples (clean ym broth in erlenmeyer) at what I learned is considered 3 log and 2 log growth concentration.
• I placed the 4 samples(2 for each fungi, at 2 different infection levels) on the same rotating shaker and started mesuring time - sampeling each bottle into 2 agar plates (my agar is oxytetracycline - glucose yeast extract agar) at 2 different dilutions (2 log and 3 log).
• I sampeled the yeast samples every 3.5 hours, and diluted it another +1 ratio(1:10) each time considereing 3.5 hours is the average growth time for yeasts, for 7 times total.I sampeled the mold samples every 6 hours, and diluted it another +1 ratio(1:10) each time considereing 6 hours is the average growth time for molds , for 10 times total.
• All plates were left for incubation in room temerature for 3 days (yeasts) and 5 days(molds).

Non of my mold plates showed any growth at all, and my yeast plates showed very random growth, showing some growth over the first few time points, then dropping to very low growth(with some plates showing 0 colonies) before going back to show some growth toward the end of the time points.

 

Please, if anyone has experience with growth curves or with growing these organisms, I'd appriciate any assistance you can offer.

 

 

 

 

Room temperature for C. albicans is too low for this experiment.

Not sure exactly where you got this protocol, but it is a bit weird.

3,5 hours is the average growth? At room temperature?

 

 

Thanks for getting back to me.

 

Why is room temp too low? I've had success growing C.albicans at it multiple times in the past, why would this experiment be different? wouldn't the lower temp just prolong my waiting time for plate results but in turn provide better selection conditions? 

 

The protocol is one I've used in the past to formulate growth curves for bacteria, L.Mono and Salmonella in particular, so the template is the same, with different growth and incubation time variables added. - if you have any insights into what I'm doing wrong, and you could provide an explanation as well, I will greatly appreciate it.

 

 

 

I am not saying you can't grow C. albicans at such low tempratures, what I am saying is that doing a growth curve at those temperatures is difficult.

 

As Phil Geis already mentioned: it is a fungus , 35°C is more appropriate and lets not forget: it is a human commensal. It is rarely found outside.

I am pretty sure you are even working with a strain that was collected from a  patient. Environmental C. albicans strains are rarely used in labs.

 

Also what Phil mentioned: not sure why you use plates with antibiotics.