2901 South Loop Drive Suite 3300 Ames, IA 50010
Real-time enumeration and viability of organisms used as expression vectors using the RBD2100.
(515)296-6600 phone
A.Oppedahl*, A.M. Steger, K.R. Harkins, Advanced Analytical Technologies, Inc., Ames, IA.
(515)296-6789 fax www.aati-us.com
MATERIALS MATERIALS
There are many industrial applications using bacteria and yeast as
Bacterial Cell Lines: Escherichia coli ATCC #25922, Bacillus
expression vectors to produce desired proteins for use as vitamin
subtilis var. niger ATCC #9372, and Saccharomyces cerevisiae
supplements, food additives and vaccine productions to name a
ATCC #2601 (ATCC, Manassas, VA). Tryptic Soy Broth (TSB),
few. Cell concentration and biological status are important to
Tryptic Soy Agar (TSA), Yeast and Malt Extract (YM) broth and
monitor during a fermentation process as protein production is
agar (Difco, Sparks, MD).
dependent upon cell conditions. Three organisms commonly used
Cytometry/Tagging: Syto 62, Syto 59 and ToPro3 are nucleic
as
acid
expression
vectors
(Bacillus
subtilis,
E.
coli,
and
RESULTS and DISCUSSION RESULTS and DISCUSSION Syto 62 is a nucleic acid stain that labels live and dead cells in a
(Molecular
provides information on cell viability.
BRAG3 is a proprietary
solution that helps decrease non-specific debris fluorescence in a sample and has the added advantage of assessing viability in Bacillus
Probes,
Eugene,
OR);
Saccharomyces cerevisiae), were enumerated using the RBD2100.
cetyltrimethylammonium bromide (CTAB) (Fisher Scientific,
For microbial cells counts, samples were diluted and labeled with
Pittsburgh, PA). BRAG3 is a proprietary solution developed by
100nM of Syto 62 dye (total cell label) for 5 minutes prior to
Advanced Analytical Technologies, Inc (AATI). The RBD2100
counting on the RBD2100. Yeast cells were diluted and labeled
flow cytometer was designed and built by AATI (Ames, IA).
An RBD2100 viable cell count for E. coli can be
resulting in excellent count correlations (Figure 1).
Staining
3
2
RBD #406 (R = 0.999) RBD #426 (R= 0.993) 1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
Plate Counts (Log10 CFU/ml)
Bacillus subtilis with Syto 62, adding BRAG3 and incubating an additional 10 minutes provided RBD viable cell counts with excellent correlation to traditional methods (Figure 2). Figure 3 displays RBD based counts from two different instruments of Saccharomyces cerevisiae tagged with Syto 59 (another nucleic
METHODS METHODS
4
1
determined by staining with the total cell tag and then staining
prior to analyzing. Total cell tag minus dead cell tag gave viable
5
determined by subtracting the ToPro3 count from the Syto 62 count
with 250nM Syto 59 dye for 10 minutes. E. coli viability was
another aliquot with 20nM ToPro3 (dead cell tag) for 5 minutes
6
sample, whereas ToPro3 labels membrane compromised cells and
subtilis. stains
Figure 3. Correlation of RBD2100 vs. Standard Plate Counts of Saccharomyces cerevisiae
RBD2100 Counts (Log10 counts/ml)
ABSTRACT ABSTRACT
Figure 4. RBD2100 bitmaps of organisms tested A. Saccharomyces cerevisiae Syto 62
C. E. coli stained with
B. Bacillus subtilis ToPro3
D. E. coli stained with
acid stain) resulting in excellent correlation with plate counts.
organism counts. Unstained aliquots were plated for count comparisons. Counts correlated well between the RBD2100 and
Staining/Cytometry:
the traditional method.
diluted in phosphate buffer (PB) followed by nucleic acid labeling
a rapid and cost effective means of determining the condition of microorganisms during a fermentation run.
Figure 1. E. coli Correlation of RBD2100 Viable Cell Counts vs. Standard Plate Counts.
methods.
A
B
C
6.5
6.0
Bacillus subtilis viability: Samples were stained with 100nM Syto 62 for 5 minutes followed by the addition of 22µM BRAG3 and incubated an additional 10 minutes at room temperature (RT). E. coli viability: Samples were stained with either 5µM Ctab and
5.5 RBD2100 Viable Counts (Log 10 counts/mL)
enumerates and assesses viability of organisms tested and provides
Three organisms (18 hour culture) were
CONCLUSIONS CONCLUSIONS
5.0
4.5
4.0
3.5
1. The RBD2100 accurately enumerates viable cells counts in 15 minutes with two commonly used bacterial expression vectors.
3.0 RBD#432 (R = 0.999)
100nM Syto 62 or 20nM ToPRo3 and incubated for 5 minutes at
2.5
2.0
room temperature and then analyzed on the RBD2100. Saccharomyces cerevisiae total counts: Samples were stained with a final concentration of 250nM Syto 59, 5µM CTAB and incubated
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Plate Counts (Log10 CFU/mL)
2. A large dynamic range (100-150000 CFU/ml) of Saccharomyces cells were accurately counted by the RBD2100.
Figure 2. Correlation of RBD2100 Viable Cell Counts vs. Standard Plate Counts of Bacillus subtilis var niger.
at room temperature for 10 minutes prior to analysis on the
3. Methods used to assess viability are applicable to other gram positive and gram negative bacterial species.
6.0
instrument.
Serial Dilution Study: A serial dilution was performed on each of the organisms (102-106) in PB. Samples were labeled and analyzed as above including a negative control of PB and staining solution (no organism). Unlabeled dilutions were plated for standard enumeration comparisons.
5.5
R B D2 1 0 0 V iab le C ell C o u n ts (L o10g C o u n ts/m L )
The RBD2100 rapidly (
