Expert: Florence M Rollwagen - 3/31/2009

Question
QUESTION: Dear Florence,
I am a student, doing my 4th semester project. I study (M. Sc) biotechnology in India.We're working with Candida albicans, and are trying to do the confirmatory procedures. We inoculated it on Corn Meal agar with tween80, with a long coverslip over the streaks. I have never observed cultures directly on a petri plate before this, so could you tell me how to observe the chlamydospores? am i supposed to remove the lid of the petri plate? because i really can't see anything clearly by inverting the plate and observing under the microscope. i've tried the 10x and 40x objectives. also, could you tell me what is the best method to inoculate on corn meal agar? should we dig into the agar, as some papers suggest? os is just streaking a line and covering it with a coverslip sufficient?
I was really, really happy to stumble upon this website!

ANSWER: Sorry, Windy, this is something I've never done.  However, here is a good link you might try:

http://www.bd.com/ds/technicalCenter/inserts/Corn_Meal_Agar.pdf

Hope this helps!

FM Rollwagen, PhD

---------- FOLLOW-UP ----------

QUESTION: Thank you very much, Florence. that was superfast!
Those two pages were the very pages i used for my procedure, but I really don't know how to see organisms on a petri plate directly. I have couple more queries. Can you tell me if we really need to use double strength medium while testing for MICs?('coz we add equal amounts of the drug-in-water solution and growth medium). I have been seeing that a 2X solution of Mueller- Hinton broth often produces a precipitate in the tubes, on incubation.
I'd also like to know which one of the following is a better substrate for inducing heavy sporulaton in filamentous fungi like Mucor and Trichophyton -- barley, or bread? trichophyton hardly seems to sporulate on barley( at least, not visibly)..
Thank you very much for replying. Hope i can borrow from  your experience if i get any more nagging doubts during my project!

Windy

Answer
Hi again, Windy:  As I said, I'm not an expert on this subject.  But I'll give you the information I have.

I was under the impression that you could observe the chlamydospores directly on the plate.  They will be bigger than bacteria, so you should have no problem.  Try turning the light down a little, sometimes the image can be washed out by too much light.  The image on the website looks to be about 40-60X.  You won't be able to get oil in this kind of prep.

As for the other questions, we make up the media 2X so that when we dilute it for the first tube it's correct strength.  You shouldn't be incubating the 2X solution directly, it has to be diluted 1:1 with the drug.

Can't answer the question on media.  Perhaps try the net.  Often the culture media companies have good tutorials on their websites.  Here's the link for BD and trichophyton agar:
http://search.bd.com/query.html?col=all&qt=trichophyton

Hope this helps.

FM Rollwagen, PhD