PMID- 12378609
OWN - NLM
STAT- MEDLINE
DCOM- 20030519
LR  - 20191210
IS  - 0006-3592 (Print)
IS  - 0006-3592 (Linking)
VI  - 80
IP  - 6
DP  - 2002 Dec 20
TI  - Accurate and rapid viability assessment of Trichoderma harzianum using 
      fluorescence-based digital image analysis.
PG  - 677-84
AB  - Fluorescence microscopy and image analysis were evaluated in order to assess the 
      viability of Trichoderma harzianum, an economically important filamentous fungus. 
      After the evaluation of the two most commonly used fluorochromes, acridine orange 
      (AO) and fluorescein diacetate (FDA) as metabolic indicator stains, AO gave 
      ambiguous results and therefore FDA was chosen. The lower stability at room 
      temperature and fast fluorescence intensity decay (50% after only 30 s of 
      illumination in UV light) could be overcome by the use of a digital image 
      acquisition system including frame grabber and a video camera. Fresh (live) 
      fungal hyphae emitted bright green fluorescence when stained with this dye (7.5 
      microg/L), whereas a total absence of fluorescence was observed when using 
      sterilized (dead) fungal cells. Fresh cells were subjected to different lethal 
      and sublethal treatments and the percentage of FDA stained fluorescent hyphae was 
      then measured over the total hyphal area (% of FDA-stained area) by image 
      analysis. At the same time, samples were cultivated in shake flasks in order to 
      correlate this % of FDA-stained area with its growth rate, a functional indicator 
      of viability. The linear correlation (r = 0.979) was: growth rate (g/L x h) = 
      2.25 x 10(-3) (% of FDA-stained area). This method was used to evaluate the 
      viability of the fungus under two different fermentation conditions in a 10-L 
      bioreactor. Estimated viable biomass during fermentation was strongly influenced 
      by the process conditions. The use of FDA, with computer-aided quantitative image 
      analysis, has made it possible to rapidly and reliably quantify the viability of 
      T. harzianum.
CI  - Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 677-684, 2002.
FAU - Hassan, Mainul
AU  - Hassan M
AD  - Departamento de Bioingenieria, Instituto de Biotecnologia, Universidad Nacional 
      Autonoma de Mexico, Cuernavaca, 62250 Morelos, Mexico.
FAU - Corkidi, Gabriel
AU  - Corkidi G
FAU - Galindo, Enrique
AU  - Galindo E
FAU - Flores, Celia
AU  - Flores C
FAU - Serrano-Carreon, Leobardo
AU  - Serrano-Carreon L
LA  - eng
PT  - Comparative Study
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Biotechnol Bioeng
JT  - Biotechnology and bioengineering
JID - 7502021
RN  - 0 (Fluoresceins)
RN  - F30N4O6XVV (Acridine Orange)
RN  - YL39R93PRE (diacetylfluorescein)
SB  - IM
MH  - Acridine Orange
MH  - *Biomass
MH  - Bioreactors/*microbiology
MH  - Cell Division/physiology
MH  - Cells, Cultured
MH  - Colony Count, Microbial/*methods
MH  - Electromagnetic Fields/adverse effects
MH  - Fluoresceins
MH  - Image Enhancement/methods
MH  - Microscopy, Fluorescence/*methods
MH  - Quality Control
MH  - Sensitivity and Specificity
MH  - Statistics as Topic
MH  - Temperature
MH  - Trichoderma/*cytology/physiology/radiation effects
EDAT- 2002/10/16 04:00
MHDA- 2003/05/20 05:00
CRDT- 2002/10/16 04:00
PHST- 2002/10/16 04:00 [pubmed]
PHST- 2003/05/20 05:00 [medline]
PHST- 2002/10/16 04:00 [entrez]
AID - 10.1002/bit.10423 [doi]
PST - ppublish
SO  - Biotechnol Bioeng. 2002 Dec 20;80(6):677-84. doi: 10.1002/bit.10423.