Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates.
The growth indicators 2,3,5-triphenyltetrazolium chloride (TTC), 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride (INT), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and resazurin were tested for their ability to indicate bacterial growth/growth inhibition. Two reading devices were evaluated and compared, a microplate spectrophotometer and a digital flatbed scanner. The bacteria used in the study were cultivated in 96-wells microplates and readings were made after 24 h. The scanned pictures were analysed with a software developed in-house to generate numerical values. It was found that resazurin was difficult to use since it shifts between three colours. MTT and TTC had a high correlation between the spectrophotometer data and the data from the scanned images. The reproducibility was similar for both reading devices. In no case was there a need to resuspend the pellets before reading. Both the XTT and INT showed lower correlations. It is concluded that bacterial growth/growth inhibition can be easily and reproducibly measured from microplate cultivations with a flatbed scanner or with a microplate spectrophotometer.
Gabrielson
Jenny
J
Microbiology and Tumorbiology Center, Karolinska Institutet, Box 280, 171 77 Stockholm, Sweden. jenny.gabrielson@mtc.ki.se
Hart
Mark
M
Jarelöv
Anna
A
Kühn
Inger
I
McKenzie
Douglas
D
Möllby
Roland
R
D001419:Bacteria / Q000254:growth & development*; D003470:Culture Media; D007202:Indicators and Reagents / Q000737:chemistry*; D008828:Microbiological Techniques; D010084:Oxidation-Reduction; D012815:Signal Processing, Computer-Assisted; D013053:Spectrophotometry / Q000295:instrumentation / Q000379:methods*; D013778:Tetrazolium Salts / Q000737:chemistry*
D003470:Culture Media; D007202:Indicators and Reagents; D013778:Tetrazolium Salts