Simplified Methods for Algal Characterization
Using Microcapillary Flow Cytometry
Katherine Gillis, Khadiza Chowdhury, Julie Clor, Kamala Tyagarajan
MilliporeSigma, Hayward, CA, United States
Abstract
Background
 The evaluation of algal concentration and lipid content is important in optimizing and
monitoring growth conditions in a variety of environments but very few dedicated solutions
exist for algae analysis today.
Mix and Red Algae Nile Red Kit Comparable to Predicate
A.
80
0
easyCyte
Methods
Figure 5. Relative lipid content in algae is an important part of culture and strain optimization. The utilization of Nile Red
for evaluation of the intracellular lipid has been widely published in the literature. The mix-and-read format of the Muse®
Nile Red Assay provides a rapid assessment of the relative lipid content. Evaluation using Nile Red was done on
Chlamydomonas Reinhardtii (CR)on the Muse (A), easyCyte (example plots not shown), or FACS (B) as well as Chlorella
Vulgaris (ChV) (Muse C or FACS D), and Nannochloropsis Oculata (NaO) (example plots not shown). Dot plots above
show results for either unstained or Nile Red stained samples. The graph (E) shows the MFI ratios for the three strains
and three instruments. While Chlamydomonas Reinhardtii showed a larger MFI ratio on the FACS compared to the other
instruments, clear separation between the stained and unstained was observed. Staining profiles and MFI ratios for the
remaining strains were similar on all three platforms.
Simplified Relative Lipid Content Evaluation with Multiple Algae Strains
A.
B.
E.
200
Incubate 5 minutes at room temp. D.
C.
NaO
ChV
80
CR
40
0
easyCyte
Figure 1. MilliporeSigma offers
multiple kits to assist in algal
research. All assays are mix
and read, using 5µL of dye
staining solution with 195µL of
diluted cell samples. Each kit
provides instructions for
staining samples varying from
5x104 to 2x107 cells/mL. The
mix and read aspect of the
assays with low staining time
allows for rapid, easy evaluation
of count and viability or relative
lipid content. Each assay can
be acquired on either the
Muse® or the Guava® systems.
Associated outputs (right panel)
will display assay specific plots
and statistics evaluating cell
size and cellular fluorescence.
Mix thoroughly and run on Muse® Cell Analyzer or the Guava® easyCyte Systems. Add 195µL diluted cell suspension to each tube or well. FACS
Figure 6. The utilization of Lipid Screen Green (Difluoro{2-[(3,5-dimethyl-2H-pyrrol-2-ylidene-N)methyl]-3,5-dimethyl1H-pyrrolato-N}boron) for evaluation of the intracellular lipid droplets has been widely published in the literature. The
mix-and-read format of the FlowCellect® Lipid Screen Green Kit for relative lipid content allows for quick, reliable results.
Staining using the Lipid Screen Green was done on Chlamydomonas Reinhardtii on the easyCyte (A) or FACS (B) as well
as Chlorella Vulgaris (example plots not shown), and Nannochloropsis Oculata (easyCyte C and FACS D). Dot plots above
show results for either unstained or Lipid Screen Green stained samples. The graph (E) shows the MFI ratios for the
three strains and both instruments. Similar staining profiles and MFI ratios were seen on all three platforms.
Precise Evaluation of Relative Lipid Content Using Multiple Markers
Lipid Screen Green
3.23%
Nile Red Muse® 2.86%
Nile Red Guava® 2.04%
Figure 7. Excellent precision was obtained for relative lipid content evaluations using either the Guava® or Muse®
Systems with the Algae Nile Red and Lipid Screen Green Assays. MFI ratios for samples stained with either Algae Nile Red
Kit or the Lipid Screen Green kit compared to corresponding unstained samples using Chlamydomonas Reinhardtii was
evaluated. Samples were acquired on either the Guava® or the Muse®. %CVs were mined from the MFI ratios of ten
individual samples. As the data shows, all %CVs were below 5% for both assays on both platforms.
Growth Studies in Combination with Lipid and Counting Assays Provide Culture
Insights
55.30%
Algae Viability
Algae Viability
44.70%
Algae Viability
Figure 2. Muse® Algae Count & Viability Assay utilizes a proprietary mix DNA intercalating dyes in a single reagent. One
of the dyes is membrane permeant and will stain all cells with a nucleus. The second dye only stains cells whose
membranes have been compromised and are dying or dead. This combination allows for the discrimination of nucleated
cells with either low or high levels of chlorophyll from those without a nucleus or debris, and live cells from dead or dying
resulting in both accurate cell concentration and viability results. Figures show high or low viability for Chlamydomonas
Reinhardtii(A&B) or Chlorella Vulgaris (C&D) when treated with H202 and mixed at a known concentration with live cells.
Clear separation is seen of live and dead cells for both strains shown.
Accurate Viabilities and Cell Concentrations from Muse® Algae Count & Viability
80
60
40
20
0
50
100
250
500
25
75% Viable
50
100
250
50% Viable
500
Algae Count & Viability
Concentration (cells/mL)
B.
100
6.E+06
5.E+06
Muse: y = 1.0623x - 55464
R² = 0.9936
4.E+06 Guava: y = 0.981x - 29735
R² = 0.9956
3.E+06
2.E+06
1.E+06
0.E+00
0.00E+00
Sample
2.00E+06
4.00E+06
6.00E+06
Hemocytometer Concentration
Figure 3. The Muse® Cell Analyzer provides accurate cell concentration measurements, comparable to results from
other analysis methods, across a variety of cellular concentrations and viabilities. Samples from Chlorella Vulgaris at 5
concentrations and 2 viabilities, were prepared in triplicate and analyzed by either Manual Hemocytometer or, Count &
Viability Assay using the Muse® Cell Analyzer or the Guava® easyCyte. Viabilities (A) and Cell concentrations (B) and
viabilities from the Muse® system were compared to those obtained by the manual count. Excellent correlation between
the different methods was seen with percent differences less than +/-10%.
Precise Results Obtained with Muse® Algae Count & Viability Kit
%CV
5
3
Cell Concentration
% Viability
0
Chlorella Vulgaris
Chlamydomonas
Thalassiosira
Reinhardtii
Psuedonana
Algae Strain
Nannochloropsis
Oculata
Figure 4. The Muse® Cell Analyzer provides excellent precision for cell concentration and viability measurements. Data
show both %CV for either cell concentration (blue) or percent viability (purple) across 4 strains of algae. Data are based
on ten samplings at two cell concentrations and viabilities for each strain acquired on the Muse® Cell Analyzer. As the
data shows, the %CV for the concentrations for all strains was below 5% and the %CV for the viabilities was below 3%.
The Muse® Algae Count & Viability shows excellent precision across multiple strains.
180
2.5E+06
120
120
60
0.0E+00
D.
MFI
Ratio
84.00%
C.
180
MFI
Ratio
16.00%
B.
5.0E+06
60
0
Day 0
Day 1
Day 2
Day 3
5.0E+07
2.5E+07
E.
0
Day 1
Day 2
Day 3
100
75
75
50
50
25
0.0E+00
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
Day 1
Day 2
Day 3
25
0
Day 0
F.
100
MFI
Ratio
23.56%
Cell Concentration (cells/mL)
Algae Viability
76.43%
Nucleated Cells
Nucleated Cells
70.20%
D.
C.
B.
29.80%
Nucleated Cells
A.
Cell Concentration (cells/mL)
A.
Muse® Algae Count & Viability Assay is Applicable to Multiple Algae Strains
MFI
Ratio
Results
Nucleated Cells
FACS
120
• Determination of relative lipid content using Lipid Screen Green
• For use on the Guava® easyCyte Systems
% Viable
Muse
1
• For use on the Muse® Cell Analyzer or Guava® easyCyte Systems
FlowCellect™ Lipid
Screen Green
25
CR
20
160
Muse® Algae Nile Red
Kit
A.
ChV
40
Product Description
• Determination of cell concentration and viability
• Determination of relative lipid content using Nile Red
• For use on the Muse® Cell Analyzer or Guava® easyCyte Systems
Add 5µL of dye staining solution to each tube or well. NaO
60
D.
C.
MFI Ratio
Muse® Algae Count &
Viability Kit
E.
100
 In this study we present data from new dedicated reagent kits for algae on MilliporeSigma’s
Muse® and Guava® platforms based on microcapillary cytometry that require small sample
sizes, generate less waste and provide precise results.
Product
B.
MFI
Ratio
The initial screening and assessment of algae quality for parameters such as count, viability and
lipid content can be critical in the overall proliferation of algae cultures. Traditionally, such
testing has been limited due to the cumbersome and time consuming protocols. In this study,
we present results from a simplified method for the characterization of multiple strains of algae
on either the Muse™ Cell Analyzer or the Guava® easyCyte lines. The methods utilize simple
mix and read assays, provides quick assessment of count and viability measurements and the
overall lipid content on algae strains Chlorella Vulgaris and Chlamydomonas Reinhardtii. Our
results indicate that good accuracy and linearity data on count and viability can be obtained for
the algae strains in a wide concentration range using sample sizes as low as 20 µL. Precise
results were obtained for all assays tested as shown by the low coefficient of variation
(CV<10%). The simplified assay format allows for low operator to operator variability with interoperator CV’s of <10%. Data on impacts on proliferation caused by changes in temperature,
light exposure, and culture concentration will be presented. The combination of rapid methods
with a simplified platform will empower researchers to easily select optimal culture conditions
for downstream experiments.
0
Day 1
Day 2
Day 3
Figure 8. Growth studies are often important in understanding optimal culture conditions and best times to perform
critical studies. A growth curve study was performed on Chlamydomonas Reinhardtii (A-C) and Chlorella Vulgaris (D-F).
Cells were split on day 0 at 2x105 cells/mL. Cells were then cultured for 3 days with either no light exposure or 12 on 12
off light exposure. On days 1, 2, and 3 cell concentrations were evaluated (A&D) as well as Nile Red Staining (B&E) or the
Lipid Screen Green Staining (C&F). The data shows that while both lines were split initially at similar concentrations, by
day 1, Chlorella Vulgaris showed an almost 10 fold higher concentration when compared to Chlamydomonas Reinhardtii.
Additionally, as expected, the lack of exposure to light, greatly decreased the cell concentrations for both lines. Chlorella
Vulgaris (B) showed little to no growth day to day without the light exposure, while the Chlamydomonas Reinhardtii (A)
showed a slight increase in cell concentration day to day without light exposure, but overall cell proliferation was
dramatically decreased. Relative lipid content was also dramatically decreased on both lines with both lipid markers,
although due to the two dyes staining slight different cellular components, slightly different results were seen on both
lines. Day 1 on Chlamydomonas Reinhardtii with Nile Red (B) there was a large difference in the relative lipid content
with and without light exposure, where the Lipid Screen Green (C) showed little difference. Interestingly, the relative
lipid content with both markers increased day to day for the Chlamydomonas Reinhardtii (B&C), but decreased day to day
for the Chlorella Vulgaris (E&F). Combining cell concentration determinations with relative lipid content can greatly
increase the understanding of appropriate culture conditions.
Summary
 Algae research requires dedicated and simple solutions that can quickly provide
cell concentration, viability and lipid content related information.
 Our data demonstrates that new dedicated assays from Millipore Sigma present
easy to use solutions for the algae researcher. The Algae Count and Viability and
Lipid Content Related Assays were shown to be applicable to multiple strains, and
provide accurate, and precise data.
 These assays were designed in combination with the easy to use Muse® or Guava®
systems to enhance algae research flexibility in either the single tube or plate based
option.
 Availability of dedicated assays and software modules for algae researchers can facilitate the
easy screening and evaluation of algae growth conditions.
MilliporeSigma, Muse and Guava are registered trademarks of Merck KGaA, Darmstadt, Germany. All trademarks belonging to third parties are the
property of their respective owners. © 2016 EMD Millipore Corporation, Billerica, MA USA. All rights reserved.
The life science business of Merck KGaA, Darmstadt, Germany operates as MilliporeSigma in the U.S. and Canada
Corresponding Authors: kamala.tyagarajan @emdmillipore.com
www.emdmillipore.com