Background
Clay Clarkson, Rachel Weisend, Dr. Brandi Kiel Reese
Department of Life Sciences, Texas A&M University-Corpus Christi
Culturing the Unculturable:
Using Fluorescence Activated Cell Sorting to Obtain Viable Isolates
Figure 1. BD FacsJazz Cell Sorter.
• A homogenized sample was prepared:
1 mL of a deep subsurface sediment
slurry was diluted with 9mL 1X
phosphate buffered saline (1X PBS).
• Sample is gently vortexed to separate
cells from sediment matrix. Sample is
then centrifuged at 900 RPM for
30 seconds.
• Aliquots of sample supernatant were
dyed with TO and PI or left undyed.
• Control samples of non-viable cells
were lysed via sonication (50oC – 5
min).
• Samples run through BD FACSJazz
• Creation of population gates
Samples to determine gates
1. Undyed sample noise
2. PI sonicated non-viable cells
3. TO sonicated non-viable cells
4. Sample – PI only non-viable cells
5. Sample – TOPI viable AND non-viable cells
Gates were created following the processing of each sample.
Fluorescent dyes allow for distinct populations to be revealed and
can later be sorted based using the created gates.
Figure 3. Gated populations
from the recorded events
obtained from the BD FacsJazz
Cell Sorter.
Majority of microbial species are underrepresented in cultured isolates
and have yet to be characterized in laboratory settings. The involved
culturing issues stem from traditional methods that favor fast-growing
populations. This leaves many novel species deemed as ‘unculturable’.
Despite being termed unculturable, a select few have been cultured
using nutrient specific media, however; this method does not eliminate
competition between species.
To overcome obstacles associated with traditional culturing techniques,
we developed a new high throughput culturing (HTC) technique that
utilizes fluorescence-activated cell sorting (FACS) to sort cells into
targeted media. Membrane permeable and impermeable fluorogenic
dyes are incorporated into this method. Complexation of the dyes with
compromised and uncompromised cell membranes generate different
emission signals ensuring fluorescent distinction between viable and
non-viable cells. The viable cells are then separated and sorted into a
96-well plate. As a result, obtained cultured isolates will be more
representative of native samples.
Viable cell isolates can be further cultivated and characterized by their
metabolisms and relation with geobiological processes. In principle,
this HTC FACS method opens opportunities for developing unique
culturing methods for novel archaeal and bacterium species that were
once termed unculturable.
Methodology Gated Populations
Future DirectionsObjective
• Distinguish between viable and non viable cells using fluorescent dyes
• Sort viable cells for high throughput culturing trials
• Search for populations active in carbon cycling and methanogenesis
Approach
Sample U197A 2F1 116/136:
• Collected from Celestial Summit 2.3
meters below sea floor.
• Possible populations active in
methanogenesis, sulfate reduction, and
other geochemical processes.
Acknowledgments
I’d like acknowledge my McNair mentor, Dr. Brandi Reese, and her doctoral student, Rachel Weisend, for this
opportunity to be guided and be involved in with their research. I’d also like to give thanks to the McNair staff
members and work efforts for keeping the McNair program alive.References
1.Stewart, E.J., Growing Unculturable Bacteria. Journal of Bacteriology, 2012. 194(16): p. 4151-4160.
2.Vartoukian, S.R., et al., Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiology Letters, 2010. 309(1): p. 1-7.
3.Staley, J.T. and A. Konopka, Measurement of in Situ Activities of Nonphotosynthetic Microorganisms in Aquatic and Terrestrial Habitats.
Annual Review of Microbiology, 1985. 39(1): p. 321-346.
4.Shi, L., et al., Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry Part A, 2007. 71A(8): p. 592-598.
5.Berney, M., et al., Assessment and Interpretation of Bacterial Viability by Using the LIVE/DEAD BacLight Kit in Combination with Flow
Cytometry. Applied and Environmental Microbiology, 2007. 73(10): p. 3283-3290.
• Establish a standardized gate for live-dead assays
• Sort viable isolates using FACS for HTC
• Identify cultured isolates using the 16S rRNA gene
The BD FACSJazz sorts cells that are stained with the flourogenic
dyes. Based on the known function of the dyes, the resulting
emissions distinguish between viable and non-viable populations.
Figure 2. Basic depiction
of fluorescent activated
cell sorting (FACS)
live/dead assay.
• An undyed sample is
represented by the green dots
• Control gates are established
to represent signals of lysed
cells.
• A ‘NON VIABLE’ gate was
established from a sample
stained only with PI to select
for cells with compromised
membranes.
• A ‘VIABLE CELL’ gate
was determined from cells
showing signals outside the
gates and towards higher TO
emission intensity (ordinate).
Figure 4. Positive cell growth
on a 96 well plate from cell
isolates obtained by previous
flow cytometry methods.
The aim of this method is to sort viable cells into designed
media that matches in situ conditions.
Dyes:
• The fluorescent probes presented for this
live/dead assay are propidium iodide (PI) and
thiazole orange (TO).
• PI is a membrane impermeable fluorogenic dye
These gates can then be used as a guideline for future sorts
and therefore, future high throughput culturing trials.
• TO is a membrane permeable fluorogenic dye that provides signals for
cells with intact membranes (viable cells)
• By using TO/PI simultaneously, we can distinguish distinct viable
populations from a sample.
targeting cells with compromised membranes (non-viable cells) [4, 5].
Isolates can eventually be sequenced and matched to
known sequences in online databases. This can provide
insight into structure and function in geochemical
processes.