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.