I am culturing H. pylori in liquid medium and the growth is low, so I would like to do another test besides checking the absorbance after incubation with different concentrations of the tested drug.
Did you try by the commonly used E-test? What do you mean for "checking the absorbance"? Is it necessary for your purposes the culture in a liquid medium? Let me know, please.
Thank you for your comments. I am testing a new drug in a liquid medium. I have used several dilutions of the drug and have in parallel a culture of H. pylori without the drug that am I testing, and a culture with the drug that I am testing. This is being done in ELISA plates. Then the observance is read after 24h and 48h to check if the bacterial growth is lower when the drug is present. The problem is that H. pylori cultures never have big absorvances, so it is difficult to interpret the results. I do not a great amount of this new drug so preparing a solid culture medium with the new drug is difficult. Thank you for all your coming suggestions.
Some years ago I experienced a semiquantitative assessment of stool antigen of H. pylori. unfortunately, I have not a PDF version so I will attach the original word version with the hope it could be of help.
Dear Filipa, the method may be used even for semiquantitative detection of culrured bacteria. I attach the figure of our experiment and give you the reference.
Ierardi E, Margiotta M, Monno R, De Francesco V, Minenna MF, Burattini O, Faleo D, Panella C, Francavilla A, Cuomo R. A new semiquantitative method of quantifying Helicobacter pylori in antigen stools. J Clin Gastroenterol. 2002 Nov-Dec;35(5):375-8.
Are you using 96-well ELISA plates? And are your cultures static or do you have them shaking?
H. pylori is a particularly finicky grower in broth. Being a microaerophile, it does require some oxygen and is rather sensitive to changes in O2 and CO2. I find it is best grown under tissue-culture conditions (tissue culture cell incubator with a C02 level set to 10% for maximum growth) with modest shaking. When you reduce the surface area of your cultures by using low volume plates (or by growing static cultures), you are actually reducing the amount of oxygen that the bacteria are being exposed to, which in my experience leads to low growth, even when rich media is used. I have found that increasing well volume/size to 12 or 6 well plates results in more robust growth: you may even consider simply increasing the shaking force on your 96 well plates, just to ensure sufficient air exchange.
A 96 well assay isn't necessarily a problem if you are simply screening under low growth conditions (H. pylori, again in my experience, will eventually reach an OD600 of 1 in 96 well broth culture, though it may take several days), however, it will certainly bias your results. Low oxygen and gas exchange will result in a sluggish bacterial metabolism, which could either increase or decrease a given drug's effectiveness, depending on your target. Just keep this in mind.
Yes I am using 96-well ELISA plates without shaking for 48 hours. Thank you, I will try introducing shaking to see if I can resolve the problem.
You could add Resazurin (alamar blue) to the wells after the 24 hr or 48 hour incubation, then incubate for 3-4 hrs and read fluorescence.
See reference: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068456/
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Microorganisms
- Bacteria