3. Results
3.1. Comparison of effectiveness and timein
manual and automatic counting
The biggest disadvantage of manual yeast cell counting with a
haemocytometer is that the process is very time consuming and
inefficient in batch processing; thus, the first assessment index of the
automatic counting procedure is its efficiency. Manual and automatic
counting were used simultaneously to analyse the same yeast samples in
this study regardless of whether the samples were in simple or complex
backgrounds. According to the principle of manual haemocytometer
counting, in a complex background, it took an average of at least 1-2
minutes to count cells in one middle square, which was further divided
into 16 smaller squares, and the total time for one sample was
approximately 5-10 minutes by trained observers. To reduce errors, the
counting process was repeated three times, which made the total analysis
time for each sample approximately 15-30 minutes, and the total counting
time for 12 samples was at least 2 hours.
In contrast, when yeast cells were
counted automatically, a trained operator took approximately 5-10
minutes to take 36 images (3 images per sample included 400 small
squares that needed to be counted), 2-5 minutes to set the parameters,
and 1-2 minutes for the automatic PC calculation. Therefore, automatic
counting in this study took only 2 to 5 percent of the time required for
manual counting, which greatly improved the efficiency of the experiment
(Figure 3A). These processing times can be further improved after the
first operation for samples in the same backgrounds due to the use of
the reproducible “Yeast Counter” macro, which made the average
counting time for one sample 2-5 minutes. These results demonstrated the
high efficiency of yeast cell counting with
ilastik and ImageJ in a complex
background.
3.2.Higher
accuracy exhibited by the automatic method
Subjective factors can play a role in the yeast cell counting process
with a haemocytometer; for example, some cells can be half-in/half-out
of the square being counted, and it is difficult to be consistent
throughout manual counting. Moreover, for budding yeast cells, the
standard of sub-cells with different sizes that can be counted as a
single individual varies by different observers, and the same observer
would also obtain different statistical results. As the software
standard can be unified by the operator, we speculated that the
automatic yeast cell counting method would be more accurate than manual
counting. Thus, the spotting test was applied in this study. Briefly, 5
out of 12 yeast samples in complex backgrounds or not were taken and
diluted to the same concentration and then subjected to 10-fold
dilutions. As the number of yeast cells is theoretically consistent at
the same dilution, it is obvious that the results of manual counting
exhibited a greater deviation between samples (Figure 2A and 2C), while
those of automatic counting showed better consistency (Figure 2B and 2D)
whether the background was complex or not. We also calculated the CFUs
(colony-forming units) of the last spot on YPD media, which were
anticipated to be 10 in theory according to the formula for
concentration. The results revealed that the average value of the CFUs
in automatic counting was 7.8, while the value for manual counting was
1.8 (Figure 3B), demonstrating the higher accuracy of the automatic
method.