Saline-Saturated DMSO-EDTA as a Storage 

Medium for Microbial DNA Analysis from Coral 

Mucus Swab Samples

Lisa A. May, Julie L. Higgins, and 

Cheryl M. Woodley

February

2011

 

NOAA Technical Memorandum NOS NCCOS 127

NOAA Technical Memorandum CRCP 15
This report has been reviewed by the National Ocean Service of the National Oceanic and 

Atmospheric Administration (NOAA) and approved for publication. Mention of trade 

names or commercial products does not constitute endorsement or recommendation for 

their use by the United States government.

Citation for this Report

May, L. A., Higgins, J. L., and Woodley, C. M. 2011. Saline-Saturated DMSO-EDTA 

as a Storage Medium for Microbial DNA Analysis from Coral Mucus Swab Samples. 

NOAA Technical Memorandum NOS NCCOS 127 and CRCP 15. 14 pp.
Saline-Saturated DMSO-EDTA as a Storage Medium 

for Microbial DNA Analysis from Coral Mucus 

Swab Samples

Lisa A. May1

, Julie L. Higgins1, and Cheryl M. Woodley2

1

JHT, Inc., NOAA, National Ocean Service

National Centers for Coastal Ocean Science

Center for Coastal Environmental Health and Biomolecular Research 

Hollings Marine Laboratory

331 Fort Johnson Road

Charleston, South Carolina 29412

2

NOAA, National Ocean Service

National Centers for Coastal Ocean Science

Center for Coastal Environmental Health and Biomolecular Research 

Hollings Marine Laboratory

331 Fort Johnson Road

Charleston, South Carolina 29412

NOAA Technical Memorandum NOS NCCOS 127

NOAA Technical Memorandum Coral Reef Conservation Program 15

February 2011

 

 

 United States Department of National Oceanic and National Ocean Service

 Commerce Atmospheric Administration

 Gary F. Locke Jane Lubchenco David Kennedy

 Under Secretary of Commerce

 for Oceans and Atmosphere, 

 Secretary NOAA Administrator Assistant Administrator
i

TABLE OF CONTENTS

LIST OF FIGURES...................................................................................... ii

LIST OF TABLES....................................................................................... ii

INTRODUCTION....................................................................................... 1

EXPERIMENT 1: Test of DNA Recovery and PCR integrity of

Acropora palmata Mucus Swab Samples.................................................... 2

EXPERIMENT 2: Long-term SSDE Coral Mucus Swab Storage............................. 5

EXPERIMENT 3: Temporal Study of SSDE as a Storage Medium 

for Coral Mucus Swab Samples.............................................................. 7

DISCUSSION............................................................................................ 11

CONSIDERATIONS AND RECOMMENDATIONS.......................................... 12

REFERENCES.......................................................................................... 13

ACKNOWLEDGEMENTS........................................................................... 14
ii

LIST OF FIGURES

Figure 1. Poly A RNA detection following FastDNA® Spin Kit for Soil extraction............ 4 

Figure 2. DGGE results of the DNA extraction test for SSDE-stored coral mucus swabs...... 5 

Figure 3. DGGE results of long-term storage experiment........................................... 7

Figure 4. Swab method of Montastraea annularis from the NOAA Coral Culture 

and Collaborative Research Facility............................................................ 8

Figure 5. Amplification results of DGGE PCR....................................................... 10

Figure 6. Results of temporal storage study using denaturing gradient gel electrophoresis... 11 

LIST OF TABLES

Table 1. Acropora palmata coral colony mucus swab sample detail, Hawksnest Bay,

St. John, US Virgin Islands...................................................................... 2

Table 2. DNA recovery of SSDE samples from coral mucus swabs stored for 3 weeks 

at room temperature............................................................................... 3

Table 3. DNA recovery from SSDE samples stored 4.5 months at room temperature.......... 6

Table 4. DNA recovery from coral swab SSDE samples in the temporal study................. 9
1

Introduction:

The mucus surface layer of corals plays a number of integral roles in their overall health and 

fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier 

against physical invasions and trauma, and serves as a medium to host a community of 

microorganisms distinct from the surrounding seawater. In healthy corals the associated 

microbial communities are known to provide antibiotics that contribute to the coral’s innate 

immunity and function metabolic activities such as biogeochemical cycling. 

Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent 

methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; 

Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change 

with changes in the environment and health condition of the coral. These changes may suggest

that changes in the microbial associates not only reflect health status but also may assist corals in 

acclimating to changing environmental conditions. With the increasing availability of molecular 

biology tools, culture-independent methods are being used more frequently for evaluating the 

health of the animal host. Although culture-independent methods are able to provide more indepth insights into the constituents of the coral surface mucus layer’s microbial community, their 

reliability and reproducibility rely on the initial sample collection maintaining sample integrity. 

In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab 

method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe 

sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen

in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a 

cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry 

shipper. The samples are then sent to a laboratory for analysis. After the initial collection and 

preservation of the sample, the duration of the sample voyage to a recipient laboratory is often 

another critical part of the sampling process, as unanticipated delays may exceed the length of 

time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust 

prematurely. In remote areas, service by international shipping companies may be non-existent, 

which requires the use of an alternative preservation medium. Other methods for preserving 

environmental samples for microbial DNA analysis include drying on various matrices (DNA 

cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl 

alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, 

however, they add expense and permitting requirements for hazardous liquid components, and 

the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et 

al., 2010). 

A method to preserve coral mucus samples without cold storage or use of hazardous solvents, 

while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, 

especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic 

acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a 

means of storing tissue of marine invertebrates at ambient temperatures without significant loss 

of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology 

would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the 

coral microbiota DNA would be adversely affected by this storage medium either by degradation 
2

of the DNA, or a bias in the DNA recovered during the extraction process created by variations 

in extraction efficiencies among the various community members. Tests to determine the 

efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are 

presented here. 

Experiment 1. Test of DNA Recovery and PCR integrity of Acropora palmata Mucus Swab 

Samples

Coral mucus was collected from five Acropora palmata colonies exhibiting both healthy and 

disease states using DNA collection swabs (Catch-All Sample Collection Swabs, Epicentre 

Biotechnologies, Madison, WI) at Hawksnest Bay, St. John, US Virgin Islands on 9 June 2008 (a 

total of 10 collected samples). Two swabs were collected per coral, one from the disease lesion 

and one from the healthy-appearing tissue distant from the lesion. Individual swabs were placed 

in 2.0 ml cryovials containing 1.0 ml SSDE solution (sufficient to cover the applicator end of the 

swab). The swab handle was snapped off prior to capping the vial and all samples were shipped 

to the Hollings Marine Laboratory in Charleston, SC for analysis (Table 1). 

Table 1. Acropora palmata coral colony mucus swab 

sample detail, Hawksnest Bay, St. John, US Virgin 

Islands. “Healthy” refers to normal-appearing tissue on 

a part of the colony away from the disease lesion.

DNA isolations were performed on samples after 3 weeks at room temperature. To determine 

the best DNA isolation protocol for optimal nucleic acid recovery, three methods were 

employed: 1) a bacterial pellet recovery and extraction, 2) swab extraction, and 3) swab 

extraction with supplemental poly A and final elution in 10 mM Tris-1mM EDTA (TE, pH 8.0). 

Sample vials 1-4 (healthy and diseased samples from colonies 1 and 2) were vortexed for 3 min 

to release bacterial cells from the swab into the suspension and then centrifuged at 14,000 rcf for 

15 min at 4oC. The swab was carefully removed; the end of the swab then was cut off with 

sterile scissors and placed into a separate sterile 1.5 ml Eppendorf tube for the direct DNA 

isolation from any adherent cells. From the initial centrifugation, most of the supernatant was 

aspirated from the cryovial, leaving approximately 200 μl over the bacterial pellet. This

Vial # Colony # Healthy(H)/Diseased (D)

1 1 H

2 1 D

3 2 H

4 2 D

5 3 H

6 3 D

7 4 H

8 4 D

9 5 H

10 5 D
3

remaining volume was used to resuspend the bacterial pellet (not visible) for DNA extraction. 

Healthy and diseased samples from colonies 3-5 (healthy and diseased, vials 5-10) were 

subjected to a swab extraction with supplemental poly A RNA in the extraction mixture and 

elution with TE. From this point all samples were processed in a similar manner. The DNA was 

extracted from each sample set (pellet or swab) using the Fast DNA Kit for Soil (MP 

Biomedicals, LLC, Solon, OH), which incorporates mechanical disruption of the bacterial cells 

(bead beating). Tubes were designated 1p, 2p, 3p, 4p for the pellet extractions, and 1s, 2s, 3s, 4s 

for the swab extractions. A fluorescence-based DNA quantification technique (Quant-iT DNA 

Assay kit, High Sensitivity, Invitrogen/Molecular Probes, Eugene, OR) using a Qubit 

fluorometer revealed that almost no DNA was recovered from samples targeting only the 

bacterial pellets; however a sufficient amount of DNA was recovered when extractions were 

conducted using the intact swab, indicating that the mucus associated bacteria were tightly bound 

to the DNA swab (Table 2). Modifications of the DNA purification protocol incorporated 

supplemental poly A RNA (200 mg) in each sample to facilitate DNA recovery (Shaw, et al., 

2009), and substitution of 10 mM Tris-1mM EDTA (TE, pH 8.0) for the kit-supplied “DES

solution” to inhibit potential DNA degradation (Rozman and Komel 1994). All samples were 

used in a PCR assay (polymerase chain reaction) for denaturing gradient gel electrophoresis 

(DGGE) analysis. It should be noted that varying the amount of poly A added to the extraction 

tubes can significantly alter DNA recovery amounts, producing spurious results (Figure 1). 

With a typical recovery of microbial DNA from mucus in the range of 1 μg, the contribution of 

the poly A may be as much as 4% of the total quantity measured. However if the amount of 

supplemental poly A RNA is decreased to 20 μg, the contribution of the RNA to the total DNA 

increases to 8%, which may have negative effects on downstream applications (PCR).

Table 2. DNA recovery of SSDE samples from coral mucus swabs stored for 3 

weeks at room temperature. Samples 1p-4p were from vortexing and centrifuging 

the swab to dislodge and pellet sample bacteria prior to DNA purification. 

Samples 1s-4s were swab DNA extractions without prior bacterial centrifugation, 

but with added poly A RNA to facilitate DNA precipitation.

Sample Disease state

(H/D)

DNA Concentration 

(ng/mL)

1p H < 10

1s H 20

2p D < 10

2s D < 10

3p H < 10

3s H 35

4p D < 10

4s D 12

5 H 264

6 D 100

7 H 404

8 D 330

9 H 5600

10 D 316
4

Figure 1. Poly A RNA detection following FastDNA® Spin Kit for 

Soil extraction. To determine the potential contribution of poly A 

RNA to DNA quantification, varying amounts (0, 10, 20, 50, 100 

and 200 μg) of poly A RNA were added to 200 μl sterile water. 

These samples were purified using the MP Biomedicals kit 

(standard coral mucus extraction protocol). Following extraction,

the amount of poly A detected using the Qubit fluorometer was 

determined. At 200 μg initial concentration (our standard addition 

of poly A), only 0.036 μg was detected. 

The PCR amplification mixture contained 1 μl sample DNA, 5 μl 10X ExTaq buffer, 20 μM 

each primer, 1 μl dNTP mixture, 0.25 μl ExTaq polymerase, and sterile water to a final volume 

of 50 μl. As a positive control, a defined mixture of bacterial DNA from four putative coral 

pathogens (Serratia marcescens, Vibrio shiloi, Vibrio coralliilyticus, and Aurantimonas 

coralicida) was used. The negative control incorporated sterile water in the PCR mixture in 

place of template DNA. Bacterial specific primers for the 16S rRNA gene were 911F 5’-TCA 

AAT GAA TTG ACG GGG GC-3’ (Maidak, 1996) and 1406R 5’-CGC CCG CCG CGC CCC 

GCG CCC GGC CCG CCG CCC CCG CCC CAC GGG CGG TGT GTA C-3’ (Lane, 1988) 

with GC clamp (underlined). Touchdown PCR was performed as follows: 95 oC for 5 min; 20 

cycles of 94 oC for 1 min, 65 oC (-0.5 oC /cycle) for 1 min, 72 oC for 1 min; 20 cycles of 94 oC 

for 1 min, 55 oC for 1 min, 72 oC for 1 min; terminal extension at 72 oC for 10 min. DGGE gel

(30-60% denaturant, 7% polyacrylamide) was run for 16.5 hours at 60V (60oC) using the DCode 

Universal Mutation Detection System (Bio-Rad Laboratories; Hercules, CA USA). The gel was

stained with ethidium bromide (1 μg/mL) and destained with TAE buffer (40 mM Tris base, 20 

mM glacial acetic acid, 1 mM EDTA) prior to visualization. A digital image was captured using 

the FluorChemTM 8900 fluorescent imager (Alpha Innotech, San Leandro, CA), equipped with a 

UV transilluminator (365nm).

The resulting denaturing gradient gel (Figure 2) indicates that DNA recovery was sufficient only 

for healthy samples (1 and 3) using the pellet or swab purification methodologies, while no 

results could be obtained from the diseased samples in this group (even numbers). However it 

must be noted that more consistent results were obtained when the swabs were extracted with the 

addition of 200μg poly A RNA (samples 5-10). This method also yielded more bands per 

0 50 100 150 200

0.00

0.02

0.04

0.06

0.08

0.10

 Initial Poly A (μg)

Final Poly A Concentration (

μg)
5

sample, indicating increased diversity of mucus microbes revealed using this protocol. The lane 

9 sample (healthy swab from colony 5) had the highest DNA recovery, yet poor amplification 

yield as shown on the denaturing gradient gel. This illustrates that there is an optimum DNA 

template concentration range for the PCR, and that too much template may produce effects 

similar to insufficient template. The experimental results show that supplemental poly A in the 

DNA extraction mixture greatly improves microbial DNA recovery from the samples, and that 

microbial DNA can be recovered after sample storage in SSDE at room temperature after about 3 

weeks. However it is not clear how this compares to storing the samples in liquid nitrogen. 

Figure 2. DGGE results of the DNA extraction test for SSDEstored coral mucus swabs. Lanes 1p-4p: DNA from bacterial 

pellet from coral colonies 1 and 2 (healthy and diseased), Lanes 

1s-4s: DNA from swab extractions from coral colonies 1 and 2 

(healthy and diseased), Lanes 5-10: DNA from coral colonies 3-5 

(healthy and diseased) swabs using added poly A RNA, Lane (-): 

PCR negative control, Lane (+): PCR positive control using 

defined mixture of bacterial DNA. 

Experiment 2. Long-term SSDE Coral Mucus Swab Storage 

Six coral mucus samples collected with DNA swabs were taken from a single colony of 

Montastraea annularis located in the NOAA Coral Culture and Collaborative Research Facility, 

Charleston, SC. Samples were placed immediately into one of 6 screw-capped cryovials. Three

cryovials contained 1.0 ml SSDE (20% DMSO-0.25M EDTA, pH 8.0, saturated with NaCl) 
6

storage solution and were stored at room temperature (22oC) (D=SSDE samples). The three 

remaining tubes without SSDE solution were frozen in liquid N2, then transferred to a -80 ̊C 

freezer for long term storage (L=liqN2 samples). After 4.5 months, the six coral swab samples 

were subjected to a DNA isolation protocol using the methodology determined to maximize 

yield in the previous study (including the addition of poly A RNA to the extraction mixture). 

DNA recovery was determined using the Quant-iT DNA Assay kit, High Sensitivity

(Invitrogen/Molecular Probes, Eugene, OR) on a Qubit fluorometer. Sufficient DNA recovery 

occurred with all samples (Table 3).

Table 3. DNA recovery from SSDE samples stored 4.5 

months at room temperature. Samples L1-L3 were stored 

in liquid nitrogen for 4.5 months and samples D1-D3 were 

stored in saline saturated DMSO-EDTA (SSDE) solution 

for 4.5 months at room temperature (22oC). DNA recovery 

was sufficient for use in a PCR for all samples.

Samples were PCR-amplified for DGGE using approximately 10 ng DNA according to the 

protocol detailed in Experiment 1. Following the PCR, 100 ng of amplified product was loaded 

onto a denaturing gradient gel (7% polyacrylamide, and 35-60% denaturant) and electrophoresed 

at 60 ̊C and 60V for 16.5h. The denaturing gradient was altered slightly (30-60% to 35-60%) to 

improve band resolution for the samples. The gel was stained for 20 min in 1.0 μg/mL ethidium 

bromide in 1 X TAE buffer for 20 min and destained in buffer alone for 20 min prior to imaging 

on a UV transilluminator using the FluorChemTM 8900. Amplified banding patterns were more 

prominent in the samples stored in liquid nitrogen (Figure 3). It is possible that the SSDE 

solution may not be entirely eliminated in the DNA extraction process and may inhibit PCR to a 

small extent, or prevent accurate DNA quantification. Adjusting the template concentration for 

the SSDE sample PCR could eliminate these problems.

Sample DNA concentration

(ng/mL)

L1 9000

L2 12000

L3 9300

D1 7500

D2 7000

D3 700
7

Figure 3. DGGE results of long-term storage experiment. 

Samples stored in liquid nitrogen (L1-L3) had stronger 

banding patterns than samples stored in SSDE (D1-D3). 

Lane M contains a defined mixture of known bacteria 

from Serratia marcescens, Vibrio shiloi, Vibrio 

coralliilyticus, and Aurantimonas coralicida as a positive 

control. The negative control (sterile water) is designated 

(-). 

Experiment 3. Temporal Study of SSDE as a Storage Medium for Coral 

Mucus Swab Samples.

Nine swab samples from different colony areas (approximately 2 cm2) were taken from the 

cultured Montastraea annularis used in Experiment 2 (Figure 4). Swab samples for standard 

long-term storage (n=4) were immediately placed in liquid nitrogen (liqN2), and transferred to a 

-80°C freezer. SSDE swab samples (n=4) were immediately placed in 1.0 ml solution, and 

stored at room temperature (22oC). The final sample taken was placed on ice, and used for a 

DNA purification control within 20 min. 
8

Liquid N2-1 Liquid N2-2 Liquid N2-3

Liquid N2-4 SSDE-1 SSDE-2

SSDE-3 SSDE-4 20 minute ice

Figure 4. Swab method of Montastraea annularis from the NOAA Coral Culture and Collaborative 

Research Facility. Mucus swabs were taken at nine different sites on the colony, and used in a comparative 

time course study of liquid nitrogen and saline saturated DMSO-EDTA solution storage methods. Photos 

courtesy of Thomas Bartlett.

At 1 week, 2 weeks, 4 weeks, and 8 weeks post-sampling, DNA purifications were prepared 

from one liqN2 and one SSDE sample using the FastDNA® SPIN for Soils (MP Biomedicals, 

LLC, Solon, OH) according to the protocol developed above for maximum recovery (i.e.,

extraction from the swab alone and addition of poly A RNA). DNA concentration was 

quantified with the Quant-iT DNA Assay kit, High Sensitivity (Invitrogen/Molecular Probes, 

Eugene, OR) on a Qubit fluorometer (Table 4). The sample used immediately in a DNA 

extraction resulted in the highest amount of nucleic acid recovered from any sample. Low 

recovery of DNA occurred from the 1-week SSDE sample, most likely as an artifact resulting 

from a DNA pellet lost during the drying process. Nucleic acid samples were stored at -20°C 

until all had been processed.
9

Table 4. DNA recovery from coral swab SSDE samples in the 

temporal study. *Low recovery of DNA from this sample is likely 

an artifact resulting from a DNA pellet lost during the drying 

process.

Sample DNA was amplified using primers specific for the 16S rRNA gene with the following 

reaction parameters: 5 μl 10 X buffer, 5 μl 25 mM MgCl2, 4 μl dNTP solution, 1 μl 911F (20 

pmol), 1 μl 1406R-GC (20 pmol), and 0.25 μl ExTaq polymerase. The remaining reaction 

volume contained 1, 2, or 5 μl template DNA (2, 5, or 10 μl for 1-week SSDE sample) and 

sterile water to 50 μl. The amplification parameters were: 94°C for 5 min; 20 cycles of: 94°C for 

1 min, 65°C for 1 min (-0.5°C/cycle), 72°C for 1 min; and 20 cycles of: 94°C for 1 min, 55°C for 

1 min, 72°C for 1 min; and a final extension of 72°C for 10 min. Five microliters of each 

reaction (one-tenth volume) was checked on a 1%-TAE agarose gel (75 ml volume) with 0.5

μg/mL ethidium bromide at 100V for 35 min. DNA size markers (Promega 100 bp DNA ladder)

were used for size and concentration estimation. Gel was imaged on a UV transilluminator using 

the FluorChemTM 8900 Digital Imaging System. Agarose gel electrophoresis of the 16S rDNA 

PCR products indicated that all coral mucus samples amplified (Figure 5). Variability in product 

yield among triplicate samples is due to the different amounts of template used in the reaction 

mixture. 

Sample DNA Recovery 

(ng/mL)

Immediate prep 8800

1 wk liquid N2 1400

1 wk SSDE 70*

2 wk liquid N2 1300

2 wk SSDE 2900

4 wk liquid N2 2700

4 wk SSDE 1500

8 wk liquid N2 3100

8 wk SSDE 1300
10

 

Figure 5. Amplification results of DGGE PCR. Lanes 1, 16, 17 and 30: Promega 100 bp 

DNA ladder, Lanes 2: Immediate DNA prep, Lane 3: negative PCR control, Lanes 4-6: 1-

week LN2, Lanes 7-9: 1-week SSDE, Lanes 10-12: 2-week LN2, Lanes 13-15: 2-week SSDE, 

Lanes 18-20: 4-week LN2, Lanes 21-23: 4-week SSDE, Lanes 24-26: 8-week LN2, Lanes 27-

29: 8-week SSDE. Triplicate samples have increasing DNA template in the PCR. 16S DNA 

from all samples was successfully amplified.

Between 20-30 microliters of amplified product from each sample were run on a 7% 

polyacrylamide, 35-60% denaturing gel at 60V and 60°C for 16.5 h. The gel was stained in 

ethidium bromide (50 μg/mL in 1 X TAE) for 30 min and destained in 1 X TAE 5 min prior to 

image capture on the FluorChemTM 8900 Digital Imaging System. The one-week coral mucus 

samples (LN2 and SSDE) had dissimilar banding patterns on the DGGE gel (Figure 6). This is 

attributed to the partial pellet loss for the one-week SSDE sample in the DNA purification step. 

The SSDE-stored coral mucus samples were highly similar to LN2-stored samples for all other 

time points, both in terms of DNA recovery (Table 4) and banding patterns (Figure 6). While 

none of the archived samples exhibited the microbial diversity shown in the sample immediately 

processed, SSDE has been demonstrated to be a suitable medium for storage of coral mucus 

swab samples for use in microbial community analyses.
11

Figure 6. Results of temporal storage study using denaturing gradient gel 

electrophoresis. Lane 1: Immediate DNA prep (20 min ice), Lane 2: 1-week LN2, 

Lane 3: 1-week SSDE, Lane 4: 2-week LN2, Lane 5: 2-week SSDE, Lane 6: 4-week 

LN2, Lane 7: 4-week SSDE, Lane 8: 8-week LN2, Lane 9: 8-week SSDE, Lane 10: 

negative PCR control.

Discussion

DNA-based analysis of coral mucus microbial communities has become increasingly prevalent 

in recent years. This approach can give researchers a broad insight into the microbial ecosystem 

and the influences the community may have on coral health. However, isolation of microbial 

DNA from the coral surface mucopolysaccharide layer (SML) of sufficient quality for 

downstream applications (PCR) has been problematic. The SML is a nutrient-rich matrix 

composed of proteins, lipids and carbohydrates (Ducklow and Mitchell 1979). Various methods 

have been presented to minimize unwanted effects from coprecipitation of the DNA with 

polysaccharide byproducts, while improving DNA yield (Rohwer, et al., 2001; Harder et al., 

2002; Kellogg, 2004). Mechanical disruption ensures that microbes with cell walls (Gram 

positive bacteria and fungi) are represented in a community profile. Additionally, incorporation 

of supplemental poly A RNA to the extraction mixture can facilitate precipitation of the 
12

microbial DNA, increasing yield from a sample anticipated to have low numbers of 

microorganisms (Shaw, et al., 2009). Finally, selective alcohol precipitation and the use of a 

metal-chelating agent such as EDTA in the elution buffer, minimizes the presence of 

contaminating polysaccharides and the activity of degrading enzymes in solution. Once purified 

and in sufficient quantity, microbial DNA can be used for a community analysis. 

The results of our study indicate that SSDE is a viable preservative for coral mucus samples

taken by the DNA swab method after 4 months of room temperature storage. This preservation 

method also allows isolation of sufficiently pure DNA to support downstream PCR-based 

applications. It is important to note that most bacteria adhere to the sampling swab and are not 

easily removed, so any microbial DNA extraction protocol from DNA collection swabs, should 

incorporate the swab. Our results also indicate that high-yield PCR products from SSDE-stored 

coral mucus may require different amplification parameters from those of LN2-stored samples. 

Coral mucus extractions performed within 20 minutes of sampling produced the highest amount 

of microbial DNA yield from any sample. Supplemental poly A RNA (200μg/sample tube)

greatly improved yield of PCR-quality microbial DNA from coral mucus.

Considerations and Recommendations:

• Microbial DNA was successfully recovered from coral swab samples (A. palmata and M. 

annularis) up to 4.5 months after storage in SSDE. DNA preparations included bead 

beating the swab with 200 μl of SSDE storage solution using the MP Biologicals, LLC 

FastDNA® Kit for Soil. Additional poly A RNA (200 mg) improved microbial DNA 

recovery for PCR, especially for samples from diseased tissue. 

• Amplification of microbial DNA from SSDE-stored samples was successful for all 

storage times tested. The PCR results indicate that purified DNA from SSDE-stored 

samples may be of lower quality than DNA from LN2-stored samples. Variability of the 

microbial community at earlier time points of the SSDE-stored samples may be due to 1) 

differences in the coral microbial community sampled, 2) a result of some DNA 

degradation following purification (as the 1- and 2-week DNA samples were stored the 

longest prior to PCR analysis), 3) inherent PCR amplification bias, 4) differences in DNA 

recovery, or 5) dilution of bacteria in the SSDE solution. Liquid nitrogen stored samples 

were similarly variable over time.

• It is recommended that once coral mucus samples are extracted, DNA solutions not be 

stored at 4oC for longer than 24 hours prior to amplification, as it is possible that the 

DNA extraction process may not eliminate all compounds which could degrade the 

nucleic acid. If it is anticipated that the purified DNA will not be analyzed by PCR 

within this time period, the DNA should be precipitated with a high salt-ethanol 

(molecular biology-grade) solution, centrifuged to pellet the nucleic acid, and the pellet 

washed with 70% ethanol. The DNA can be archived (-20oC) as a lyophilized pellet (for 

detailed protocol, see Sambrook and Russell, 2001). Alternatively, amplification for 

temporal studies can be executed immediately and the PCR products stored at -20oC until 

all samples are ready for electrophoresis.
13

• Modifications to the PCR mixture which could improve yield for troublesome DNA 

template samples include: increasing primer concentration, the addition of supplementary 

MgCl2, increasing dNTP concentration, and varying template concentration.

• Based on the research detailed above, SSDE is a suitable medium for coral mucus swab 

samples, however results indicate that liquid nitrogen storage is preferable, resulting in 

increased quantities and higher amounts of PCR-quality DNA. If liquid nitrogen is 

unavailable, SSDE is an economical alternative storage medium, provided samples are 

processed in a timely manner once purified (i.e., sample DNA PCR-amplified within 24 h 

of DNA extraction). 

• For analysis of any given set of study samples, it is recommended that the same 

preservation method be used for all coral mucus swabs.

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Acknowledgements:

The authors wish to thank Dr. Christina Kellogg, St. Petersburg Coastal and Marine Science 

Center, USGS, for supplying the SSDE storage solution for the field samples and Dr. Caroline 

Rogers, Virgin Islands National Park, USGS, for performing the Acropora palmata mucus 

swabs. This project was funded in part by the NOAA Coral Reef Conservation Program. 
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United States Department of Commerce

Gary F. Locke

 Secretary

National Oceanic and Atmospheric Administration

Jane Lubchenco

Under Secretary of 

Commerce for Oceans and Atmosphere, 

NOAA Administrator

National Ocean Service

David Kennedy

Assistant Administrator