E. Puchkov
colonies of 0.3 mm in diameter and higher. Automatic counting was based on the “top hat” thresholding.
“Manual” counting was accomplished visually using virtual mouse-controlled “markers” with automated
counting of the marked colonies.
2.3. Microscopy and Digital Photography
The cells were concentrated by centrifugation up to ca. 5 × 108 cells/ml and supplemented with 3,8-d ia mino-
5-et hyl -6 -phenylphenanthridinium bromide (ethidium bromide, Eth) (Sigma) at a concentration of 50 μM and
4,6-diamidino-2-phenylindole, dilactate (DAPI) (Serva) at a concentration of 15 μM. Upon 5 - 10 min of in-
cuba tion at 25˚C ± 0.5˚C, 6 μl aliquots of the cell suspension were placed onto specimen slides, covered with
cover glass, and sealed with nail polish to prevent evaporation. Observations were started ca. 20 min after
sealing, when most cells had stuck to the slide’s glass.
Fluoresce microscopy and color digital photography were carried out on a ML-2B fluorescence microscope
(LOMO, Russia) equipped with a Sony DSC-V3 digital camera [8]. By applying appropriate filter sets, the fol-
lowing combinations of λex/λem were used: in the fluorescence mode—360 nm/>400 nm; in the light transmis-
sion mode—520 nm/>400 nm.
2.4. Computer Data Treatment
The color digital images of the fluorescing cells were processed by the Adobe Photoshop v. 8.0 (Adobe Systems
Inc., USA). Quantitative image analysis was made, in case of colony counting, using original KOMPANKOL-
M1 software, and in case of microscopic cell counting, using ImageJ 1.42 software (National Institute of Health,
USA, http://rsb.info .nih. gov/ij). Other quantitative treatments were made by MS Excel 2003 and OriginPro 7.5
(OriginLab, USA) software.
3. Results and Discussion
3.1. Computer-Aided Colony Counti ng
The “manua l” and automated KOMPANKOL-M1 colony counting methods in case of conventional spread- in-
oculation onto the entire area of a Petri dish were compared (Figure 3). The data indicated that, within the error of
determination, both methods gave close results. However, it was noticed that automatic counting systematically
gave some underestimation. This was the consequence of the presence of some fraction of merged colonies.
Unfortunately, the “thre s ho ld ” adjustments could not completely “resol ve” single colonies in comparatively large
aggre ga te s.
The Miles and Misra procedure [6] of inoculation of the small volume samples upon serial dilution onto a
single Petri dish significantly lessen labor consumption in assessing of the colony-forming capacity. However,
visual identification and counting of small colonies is practically impossible. Therefore, this procedure is nor-
mally used for estimating quantitatively the colony forming ability only to the order of magnitude (or the degree of
dilution) by evaluating of the colony growth presence/absence in the section of the corresponding dilution
(Figure 2(B)).
KOMPANKOL-M1 is capable of getting images of single colonies outgrown upon Miles and Misra [6] in-
oculation (Figure 2 (C)). Comparison of the “manua l” and automated colony counting methods for this protocol
of inoculation and the conventional one revealed good agreement of all counts (Figure 4). This opens new po-
tential applications of the Miles and Misra protocol of inoculation by assessing survival rates within the percent
scale. For example, there is need in assessing survival rate in the percent scale for evaluating efficacy of micro-
bial preservation procedures. Figure 5 illustrates application of the developed computer-aided approach for as-
sessing survival rates of three yeast species after freezing and freeze-drying.
3.2. Computer-Aided Fluorescence Microscopy Viability Assessment
Fluorescence of DAPI and Eth cation is known to markedly increase upon binding to nucleic acids. On the other
hand, cell membranes of intact yeast cells are well permeable for DAPI and impermeable for Eth. This is a basis
for revealing by fluorescence microscopy of the yeast cells with compromised membranes (stained red by Eth)
upon double staining with DAPI an Eth (Figure 6(B)). Using “RGB -split” option of the Image J software digital