núria feliubesora, marta orive camprubí, jaume lluís
TRANSCRIPT
INTRODUCTION
Núria Feliu Besora, Marta Orive Camprubí, Jaume Lluís Tartera, Benet Fité Luis
Mahou San Miguel, Innovation and Development Department, Lleida, Spain. [email protected]
INTRODUCTIONTraditionally, cell count and viability are manually analyzed under the microscope by using haemocytometer and appropriate staining reagents such as blue or methylene violet. This
method is laborious, time-consuming and affected by human error in counting and in colour distinction between live and dead cells. Methylene violet is a reagent that penetrates into
yeast cells, dead cells are stained violet while viable cells contain enzymes that reduce the violet colour to colourless.
MuseTM system is evaluated here as a rapid method that uses small volume of sample and that is based on flow cytometry. Differentiation
between viable and non-viable cells is dependent on their permeability to a DNA-binding stains present in the reagent. One reagent stains
cells that have lost their membrane integrity (the nucleous of dead and dying cells are stained). Second reagent is a membrane-permeant DNA
stain that stains all cells with nucleous (is useful for differentiate cells from debris or non-nucleaded cells). In the instrument, a couple of plots
are obtained with this information (figure 1). Furthermore, the instrument is compact and easy to use for trained people. The result is
obtained in few minutes, with high reliability.
Equivalence between haemocytometer and MUSETM was evaluated for cell count and viability for both lager and ale strains.Figure 1. Example of results from MUSETM
MATERIALS AND METHODYeast strains: lager and ale strains from Mahou-San Miguel company.
Methods:
- Reference method: EBC Methods 3.1.1.1 Yeast Cell count-Haemocytometer and 3.2.1.1
Yeast Viability-Methylene Blue/Violet Stain
- Alternative method: MUSETM protocol: Prepare samples with a concentration range of
1x104cells/ml to 5x105 cells/mL using MUSETM Count & viability reagent
Experimental design:
RESULTS part 1: Prepared laboratory samplesIn figure 2 the correlation between MUSETM system and haemocytometer+MV for both
lager and yeast cells in different cell count concentrations and in different levels of
viability is shown . Each point in the plot shows the mean of triplicates and their standard
deviation.
Figure 1. Example of results from MUSETM
system (from supplier)
R² = 0,95021,2E+07
1,6E+07
2,0E+07
cells
/m
l in
Mu
se
Cell count in Lager yeast
R² = 0,98611,2E+07
1,6E+07
cells
/m
l in
Mu
se
Cell count in Ale yeast
Experimental design:
Part 1: Prepared lab samples. To obtain a range of yeast cell counts, different dilutions
were prepared from both lager and ale yeast slants. Furthermore, an aggressive
treatment with ethanol was done to kill all the cells (addition of ethanol 96% for 15min).
After this treatment, a range of dilutions from different viability were prepared.
Each prepared sample was evaluated in triplicate with both haemocytometer +
methylene violet (VM) and MUSETM system
Part 2: Real samples. Samples from fresh yeast slants, propagation, fermentation and
yeast slurry were used from both lager and ale yeasts. Each sample was analysed once
with both methods. Moreover, some samples were analysed in quintuplicate in order to
calculate the coefficient of variation (CV) for each method. 37 samples were analysed for
lager yeast and 10 samples for ale yeast.
0,0E+00
4,0E+06
8,0E+06
1,2E+07
0,0E+00 4,0E+06 8,0E+06 1,2E+07 1,6E+07
cells
/m
l in
Mu
se
cells/ml in Haemocytometer + MV
0,0E+00
4,0E+06
8,0E+06
0,0E+00 4,0E+06 8,0E+06 1,2E+07 1,6E+07
cells
/m
l in
Mu
se
cells/ml in Haemocytometer + MV
R² = 0,9939
0
20
40
60
80
100
0 20 40 60 80 100
% v
iab
le c
ells
in
Mu
se
% viable in Haemocytometer + MV
Viability in Ale yeast
R² = 0,9918
0
20
40
60
80
100
0 20 40 60 80 100
% v
iab
le c
ells
in
Mu
se
% viable in Haemocytometer + MV
Viability in Lager yeast
lager yeast and 10 samples for ale yeast.
RESULTS part 2: Real samplesIn figure 3 the correlation between both methods using real samples from the brewery is shown. Cell count in samples from lager yeast shows a correlation of R2=0.73 between both
methods. In viability the correlation in lager yeast samples is R2=0.82. For ale yeast the correlation in cell count is higher (R2=0.98) than in lager yeast. However, correlation for viability is
lower R2=0.69 .
Figure 2. Correlation between haemocytometer+VM and MUSETM system for cell count and viability in prepared samples
R² = 0,731
1,0E+07
2,0E+07
3,0E+07
4,0E+07
cell
/m
l in
MU
SE
Cell count in Lager yeast
R² = 0,8174
70
80
90
100
110
% v
iab
le c
ell
s in
MU
SE
Viability in Lager yeast
R² = 0,6975
96
97
97
98
98
99
99
% v
iab
ilit
y i
n M
US
E
Viability in Ale yeast
R² = 0,9868
4,0E+08
6,0E+08
8,0E+08
1,0E+09
1,2E+09
cell
s/m
l in
MU
SE
Cell count in Ale yeast
Figure 3. Correlation between
<
CV cell count in
traditional
CV cell count in
MUSE TM
CV viability in
traditional
CV viability in
MUSETM
Yeast slant 6,68 4,17 1,66 2,09
Fermentation 7,58 4,04 1,36 0,70
Yeast slurry 7,74 7,04 1,11 1,67
End propagation n.a 7,06 n.a 0,40
Coefficient of variability for each method in samples from different parts of the brewing process
for lager yeast is shown in table 1. Results for ale yeast are not shown.
In general, viability in MUSETM system is lower than in haemocytometer method
60
65
70
75
80
85
90
95
100
105
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
% v
iab
ility
Figure 4. Comparison of %viability
0,0E+00
1,0E+07
0,00E+00 1,00E+07 2,00E+07 3,00E+07
cells/ml in Haemocytometer + MV
50
60
70
50 60 70 80 90 100 110
% v
iab
le c
ell
s in
MU
SE
% viable cells in Haemocytometer + MV
95
96
96
95 96 96 97 97 98 98 99 99 100 100
% v
iab
ilit
y i
n M
US
E
% viability in Haemocytometer + MV
0,0E+00
2,0E+08
0,0E+00 5,0E+08 1,0E+09 1,5E+09
cell
s/m
l in
MU
SE
cells/ml in Haemocytometer + MV
Figure 3. Correlation betweenhaemocytometer+VM and MUSETM
system for cell count and viability in reallager and ale samples
CONCLUSIONS-When yeast cells are killed with ethanol, yeast cells are completely dead and high correlation in viability between both methods using lager and ale yeast is obtained. However, these
treatments are not from a realistic situation. High correlation between MUSETM and traditional methods is also found in cell count .
- Lower correlation is found in real samples than in prepared samples both for cell count and viability. The exception is cell count for ale yeasts probably due to low number of samples
analysed.
- Furthermore, methylene violet gives higher viabilities than MUSETM system. As it is known, methylene violet and methylene blue may not stain all dead cells, thus giving overestimated
number of live cells (O’connor-Cox et al., 1997).
-For both lager and ale yeast, the coefficient of variability for traditional cell count is in general higher than for MUSETM system for each type of samples. The coefficient of variability for
viability is very similar in both methods (table 1).
End propagation n.a 7,06 n.a 0,401 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
different type of samples
Viability Haemocytometer + MV (%) Viability in MUSE (%)
Figure 4. Comparison of %viability
between both methods Table 1. Coefficient of variability for both methods in cell count and viability for lager yeast.
viability is very similar in both methods (table 1).
- Correlation between MUSETM system and traditional method, both for cell count and viability, is considered acceptable by Mahou-San Miguel. Moreover, the MUSETM system gives lower
variability, it is easy to use, rapid and reduces the human error in cell counts and colour distinction.
-MUSE system is the recommended method and is already in use in Mahou San Miguel.
REFERENCESEuropean Brewery Convention (2005). Section 3.Yeast Analysis. 3.1.1.1 Yeast Cell count-Haemocytometer. EBC Analytica Microbiologica, pp 133-138, Fachverlag Hans Carl, Nürnberg (Germany)
European Brewery Convention (2005). Section 3.Yeast Analysis. 3.2.1.1 Yeast Viability-Methylene Blue/Violet Stain. EBC Analytica Microbiologica, pp 147-150, Fachverlag Hans Carl, Nürnberg (Germany)
O’Connor-Cox, E., Mochaba, F. M., Lodolo, E. J., Majara, M., Axcell, B. (1997) Methylene Blue Staining: Use at your own risk. Technical Quarterly Master Brewers Association of the Americas 34: 306:312
Pawlowsky, K. (2007). Evaluation of the Nucleocounter YC-100. BRI Review, Issue 9