Procedure for Curation of Routine Microbiological Sample

Integrated Ocean Drilling Program (IODP)
Center for Deep Earth Exploration (CDEX)
Kochi Core Center (KCC)

Version 1.9. 2012

TABLE OF CONTENTS
1. Introduction (What is RMS?) ……………………………………………..……….…….…. 2
2. Onboard processing …………………………………………………………...……….…… 3
2-1. Sampling of whole round core (WRC) as RMS ……………………....…….……. 4
2-1-1. Materials ………………………………………………………………...… 4
2-1-2. Method …………………………………….……………………...….……. 5
2-2. RMS sampling from WRC by using a syringe ………...………….……….…..…. 8
2-2-1. Materials ………..…………………………………………….…….….…..82-2-2. Method …….……………………………………..……………………..….. 9
3. Shipping …………………………..……………………………….……………..….….…. 12
3-1. Materials ………………………………………………………………..…..……. 12
3-2. Method …………………………………………………………….……..….…… 13
4. Onshore curation ………………………………………………….……………............….. 14
4-1. Materials …………………………………………………………………...….…. 14
4-2. Method ……............................................................................................................ 15

Optional: Chemical fixation procedure ………………………………………….…...….…… 17
Materials …………………………………………………….……………...….…. 17
Method ……............................................................................................................. 18
References …………………………………………………………………….….…….…..… 19
Appendix ……………………………………………………………………..…….......…….. 20

1

Procedure for Curation of Routine Microbiological Sample
Following the Scientific Technology Panel (STP) recommendation and preliminary
outcomes of the pilot study being conducted at the Kochi Core Center (KCC) for curation of
Routine Microbiological Sample (RMS), a procedure for handling of the RMS has been drafted.
This document provides an overview of the RMS curation and concisely describes various
steps involved in onboard sampling, QA/QC, shipment, and onshore storage and sub-samplingof the RMS.

1. Introduction (What is RMS?)
RMS is the cored material (or portions thereof) from the subseafloor which is adequately
preserved soon after recovery in frozen condition for future microbiological analyses. Figure 1
provides an overview of activities related to RMS curation. During the long history ofscientific drilling, most of the cores have been stored at around 4°C in walk-in storage rooms.
However, contaminating microbes from air and/or from bare hands of scientists easily grew on
the cores under these storage conditions (Masui et al.), and thus caused serious damage to corequality. Moreover, dramatic change in ambient conditions easily kills subseafloor microbes.
After cell death, fragile bio-molecules such as RNA, enzymes, sugar chains, and intact polar
lipids are rapidly degraded via abiotic hydrolysis and enzymatic reactions. Thus, storage ofcore samples under frozen condition for preventing such degradation is of great importance toprovide opportunities for future molecular analyses arising from rapid biotechnologicaldevelopments (D’Hondt et al.).

2

Fig. 1. Overview of RMS curation activities from onboard sampling to onshore storage

2. Onboard processing
Figure 2 shows a decision making flow chart for RMS sampling, which has been preparedin accordance with the STP recommendation 0908-09 (Appendix I). Final decision on RMS
sampling and sampling method for the RMS will be made by onboard Sample Allocation
Committee (SAC). Precise operational definitions of various terms relevant to handling of
RMS are provided in Table T1 (Appendix II).

Will interstitial water (IW) sample be collected?

NO

YES

There is no need to collect an RMS.

Will the RMS sampling interfere withthe objectives of the cruise?

YES

NO

There is no need to collect an RMS.

An RMS whole round core (WRC) is
sampled next to the IW-WRC.

Is there sufficient number of staff ?

NO

YESIs the RMS-WRC lithified?

YES

The RMS-WRC is preserved as RMS.

NO
RMS is collected from the WRC with syringes.

Fig. 2. Decision making flow chart for RMS sampling onboard ship3

2-1. Sampling of whole round core (WRC) as RMS
2-1-1. Materials(1) Spray bottle with 70% ethanol(2) Clean disposable gloves
Clean disposable gloves must be used during all handling procedure (human skin is a
primary contamination source).
(3) Sterilized Spatula
Rinse spatula with distilled water, wrap its head with aluminum foil, and autoclave at
121ºC for 20 min. (Fig. 3a). After autoclave, put spatula into oven (60˚C) to dry it for
about 12 hours. After drying up, let spatula cool to room temperature prior to use. If
autoclaving is not feasible because of high frequency of its use, spray the head of spatulawith 70% ethanol, and wipe it with clean tissue paper. Perform this cleaning of spatula justbefore cutting the RMS-WRC.
(4) Clean end-caps
End-caps currently used cannot be sterilized by autoclaving, so these are cleaned with
70% ethanol. Rinse inside of the end-caps with distilled water to remove dust, and put onclean bench so that inside of the cap faces upward (Fig. 3b). Spray inside of the cap with
70% ethanol, and let it dry up for 1 hour by exposing to UV. After dry up, put clean caps
into a clean bag, and keep it in the laboratory ready for use. Clean bag is prepared bywiping inner side of it with 70% ethanol. If above preparation is not feasible due to limited
onboard staff and time, spray inside of an end-cap with 70% ethanol, and wipe it withclean tissue paper. Perform this cleaning of cap just before capping the RMS-WRC.
(5) Aluminum bag (similar to retort bag) (Fig. 3c)
Optimum bag dimensions are [210 mm(H) x 150 mm(W)].
(6) Transparent Teflon tape (e.g., NITOFLON® Adhesive Tape)
For keeping labels pasted on samples under ultra-low temperature condition

(a)

(b)

(a)

(b)

(c)

4

Push

1
2

Fig. 3. Tools for RMS sampling (a) Spatula sterilized with autoclave, (b) Cleaning of end-caps in clean bench, (c)Aluminum bags

2-1-2. Method
After retrieval, the core is transferred to core cuttingarea for cutting into sections, taking void gas and/or sample
for safety monitoring, capping sections with end caps, andlabeling (Fig. 4). During these processes, sections arevisually inspected for their quality. If the core quality is
judged to be inferior and interstitial water (IW) sample will
not be collected, the RMS need not be collected. Usually, ittakes approximately 15-20 min. from core retrieval (“coreon deck” call) to transfer of core into core cutting area.

Fig.4. Core in core cutting area
onboard D/V Chikyu

Sequence of steps for taking a whole round core (WRC) as RMS is shown in Fig. 5 anddescribed as follows;
Core cutting area
(1) Select sampling point for the RMS based on discussion with onboard
geochemist in order to check core quality and avoid conflict with cruise objective.
(2) Wipe the core liner around selected points (3-5 cm in width) with a tissue paper
soaked with 70% ethanol.
(3) Mark the range of the RMS-IW sample with felt marker. Range for RMS isusually 5-10 cm.
(4) Cut the liner at bottom side of RMS-IW-WRC with a standard liner cutter.
(5) Cut the sediment at bottom side of the WRC with a usual spatula.
(6) Cap the bottom side of the WRC with a usual white end-cap.
(7) Cut the liner at topside of the WRC with the liner cutter.
(8) Cut the sediment at top side of the WRC with a sterilized spatula.
(9) Cap the topside of the WRC with a clean blue end-cap. Transfer the WRC tolaboratory for CT scanning.
Laboratory(10) After CT scanning, wipe the RMS-IW-WRC core liner around RMS-IWboundary (3-5 cm in width) with a tissue paper soaked with 70 % ethanol, and cut
the WRC with a liner cutter and sediment in liner with a sterilized spatula.
(11) Cap the bottom side of the RMS-WRC with a clean yellow end-cap, and seal thecaps and liner with vinyl tapes. Give IW-WRC to lab. Tech.
(12) Print two labels after registration of RMS-WRC in J-CORES, and paste one on
end-caps and one on aluminum bag (or similar product).
(13) Cover the labels with transparent Teflon tape.
(14) Put the RMS-WRC into the aluminum bag (or similar product).
(15) Store the RMS-WRC into deep freezer (-80°C).

5The RMS-WRC sampling takes approximately 30 min.

6

Fig. 5. Sequence of steps for taking a whole round core (WRC) as RMS at core cutting area

Fig. 5. (continued) Sequence of steps for taking a whole round core (WRC) as RMS at laboratory7

2-2. RMS sampling from WRC by using a syringeCore liner is not sterile and contamination of outer rim of the core cannot be avoided
during drilling, therefore, it is preferable to sub-sample middle part of a WRC with asyringe with the tip removed. However, when sub-sampling is not possible (e.g., because oflimited staff or indurated nature of the core), syringe sampling should be skipped.
2-2-1. Materials(1) Tip cut-off sterilized syringes (20 and 30 cc)
Cut off the luer lok end of syringe with a heated knife. Wrap the tip cut-off syringe inaluminum foil, and autoclave at 121ºC for 20 min. After autoclaving, put syringe into oven
(60˚C) to dry it up for about 12 hours. After drying up, let the syringe cool to room
temperature (Fig. 6a). Store the sterilized syringes in a clean bag. If above preparation is
not feasible due to limited onboard staff and time, sterilized syringes can be preparedonshore and shipped to the drilling platform.
(2) Sterile centrifuge tube (50 cc) (e.g., FALCON® tube)
For storing syringe-sampled RMS(3) Cooling gel packFor keeping the RMS-WRC under low temperature during sampling by tip cut-off
sterilized syringe
Gel packs are to be stored at -20˚C prior to use.
(4) Styrene foam box
For keeping the RMS-WRC and cooling gel packs in place during sampling of the(a)WRC by sterilized syringe (b)(c)
Inner size of box should be [250 mm (L) x 150 mm (W) x 150 mm (W)].
(5) Syringe adapter
For inserting the sterilized syringe into somewhat hard RMS-WRC (Fig. 6b)(6) All materials mentioned in “Sampling of whole round core (WRC) as RMS”

(a)

(b)

Push

(i)
8

(ii)

Fig. 6. Tools for RMS sampling (a) Tip cut-off sterilized syringe, (b) Syringe adapter

2-2-2. Methods
Sequence of steps for taking RMS from a WRC by using sterilized syringes is shown in
Fig. 7.
Core cutting area
(1) Select sampling point for the RMS based on discussion with onboard
geochemist in order to check core quality and to avoid conflict with cruise
objective.
(2) Wipe the core liner around selected points (3-5 cm in width) with a tissue paper
soaked with 70% ethanol.
(3) Mark the range of the RMS-IW sample with felt marker. Range for RMS isusually 5-10 cm.
(4) Cut the liner of bottom side of RMS-IW-WRC with a standard liner cutter.
(5) Cut the bottom side of RMS-IW sediment from the core with sterilized spatula.
(6) Cap the bottom side of the RMS-IW-WRC with a usual white end-cap.
(7) Cut the liner of topside of RMS-IW-WRC with the liner cutter.
(8) Cut the RMS-IW sediment from the topside core with sterilized spatula.
(9) Cap the topside of the RMS-IW-WRC with a clean blue end-cap. Transfer the
WRC to laboratory for CT scanning.
Laboratory(10) After CT scanning, cut RMS-IW-WRC with a liner cutter and sediment in linerwith a sterilized spatula.
(11) Cap the bottom side of the RMS-WRC with a clean yellow end-cap, and store at
4°C.
(12) Print labels after registration of RMS-WRC in J-CORES, and paste them on blue
end-cap of RMS-WRC, sterile centrifuge tubes and aluminum bag (or similar
product).
(13) Cover the sample labels with transparent Teflon tape.
(14) Put the WRC into a styrene foam box with frozen cooling gel packs in clean
bench. Remove the blue end-cap of the WRC.
(15) Insert three tip cut-off sterilized syringes in middle part of the WRC. Push eachsyringe into sediment so that it samples the entire length of the WRC (e.g., 10 cm
in the case of 10 cm WRC; volume of syringe is not important.). Use syringe
adapter when sediment in WRC is somewhat hard for inserting a syringe into it(see Fig. 6b). Take out the sediment-filled syringe from the WRC by gently
rotating and pulling action.
(16) Transfer the sediment from the syringe into the sterile centrifuge tube, and returnthe syringe without piston back into the hole by gently rotating and pushing action
to prevent the hole from collapsing.
9(17) After taking RMS by all 3 syringes, cap the residual WRC with the blue end-cap,and seal the caps and liner with vinyl tapes.
(18) Put the centrifuge tubes with RMS and residual WRC into separate aluminumbags (or similar product), and seal them.
(19) Store the RMS and residual WRC into deep freezer (-80°C).
The syringe sampling takes 40 min to 1 hour.
Steps (1)-(10) of Fig. 5 precede this sequence.

(11)

(12)

(13)
Teflon tape

J-CORESlabel(14) Cooling gel
packs

RMS
R

RMS-WRC

Aluminum bag

Centrifuge tube

RMS

RMS

(15)

Styrene foam box(16)

Tip cut-off
syringe

R

R

R

R

(17)

(18)

(19)
-80˚C

RMS

Deep freezer

Vinyl tape
RMS

RMS

Residual WRC
Fig. 7. Sequence of steps for taking RMS from a WRC by using sterilized syringes

10Notes• To minimize changes in quality of RMS, all handling should occur as quickly as
possible.
• Safety goggles and lab coat must be put on for personal protection.
• Although steps (1) to (7) can be changed depending upon objectives of expedition, theRMS is finally sampled next to the IW-WRC.
• Depending on sequence of RMS and IW in a WRC (RMS-IW or IW-RMS), use clean
end-cap of appropriate color only for the RMS-WRC (blue or yellow cap toward top andyellow or white cap toward bottom).
• Depending on shipboard freezer capacity, the frequency of RMS sampling may need tobe reduced if whole-rounds or half-rounds are often collected. Final decision on sampling
frequency for the RMS will be made by onboard SAC.
• In the case of larger diameter core (e.g., diameter is 10 cm), 30 cc tip cut-off sterilized
syringe can be used for taking RMS from a WRC.
• In the case of sampling by syringe, if it deprives opportunities to obtain samples for
onboard scientists, the residual WRC should not be frozen, and instead should be given
to the curatorial staff for discrete sampling. Final decision on sampling quantity such as
three 20 cc syringes or one 30 cc syringe for the RMS will be made by onboard SAC.
• Before returning the syringe without piston into the hole in a WRC, adjust the length ofsyringe by cutting luer lok end with a clean knife so that length of the syringe becomes
nearly same as that of WRC. This will prevent sediment from collapsing inside thecapped WRC. This adjustment in syringe length is not required, if WRC is 10 cm long,
because syringe is originally ca. 10 cm long.

113. Shipping
RMS are shipped with dry ice in order to maintain their frozen condition. To maintainfrozen condition surely, volume of dry ice should be more than twice * that of the RMS.
Temperature logger may be included in each microbiological or biogeochemical shipping
container to get the thermal history of the samples during shipment. Alternatively, dry ice
remaining in the container at the destination can be used as an indicator of frozen condition ofsamples during the shipment.
* During the training cruise CK09-03, RMS-WRC were shipped with dry ice in order tomaintain frozen condition (approximately -80˚C) of the samples from Shizuoka (unloadingport) to KCC. Approximately 10 kg of chipped dry ice and twelve of 10 cm long
RMS-WRC (approximately 5 kg) were put into a styrene foam box [dimension: 55×35×70(cm) (Fig. 8a)] so that the WRC were surrounded by the dry ice. Next day, the samples
were delivered to KCC with limited loss of dry ice (Fig. 8b).

(a)

(b)

Fig. 8. Shipping of RMS with dry ice (a) Styrene foam boxes with RMS and dry ice, (b) Delivered RMS withlimited loss of dry ice

3-1. Materials(1) Container (Plastic cool box or styrene foam box)
The container should have ventilation so that carbon dioxide gas from the dry ice does
not cause explosion of the package. Lid of the styrene foam box must be punctured (onehole of 1-2 mm diameter) to allow diffusion of the gas. Inner height of container should be
more than three times that of the RMS-WRC in order to put dry ice in reasonable amount
together with the RMS-WRC.
(2) Dry ice (chipped or block type)Dry ice is lost quickly with time, so it must be made available as close to the shipping
time as possible. It is preferable to use chipped dry ice for reducing the weight but
increasing the volume of dry ice.
(3) Airway bill or similar document12

3-2. Method(1) Put dry ice (chipped or block type) and RMS into container so that the samples are
surrounded by the dry ice (Fig. 9).
(2) Put temperature logger into the container (optional).
(3) Seal the container with the shipping tape, and paste airway bill for logisticcompany.
(4) Ship the container as quickly as possible.
(5) After receiving the container in repository, transfer RMS into deep freezer (-80˚C)quickly.
(6) Confirm the thermal history of container during shipment with temperature logger(optional) or by presence of remaining dry ice in the container.

SampleDry ice (Block)Dry ice (Chip)

Fig. 9. Examples of good and bad packing for RMS shipping

Note• Dry ice must be handled in a well-ventilated location because of the carbon dioxide
fumes. All handling should be progressed as quickly as possible.
• Safety cold gloves must be used during handling of dry ice because it will burn humanskin (cold burn).

13

4. Onshore curation
Storage of RMS under deep frozen condition is very important for preventing
degradation of fragile bio-molecules such as DNA, RNA, enzymes, carbohydrates, and intact
polar lipids caused by abiotic hydrolysis, enzymatic reaction, and possible contamination.
Moreover, aseptic sub-sampling and distribution of the RMS without thawing are highly
desired for quality assurance of the samples. KCC stores RMS in deep freezer (-80˚C) and/or
liquid nitrogen (LN) cooled tanks (-160˚C) (Fig. 10 a, b). For sub-sampling and distribution toscience community, RMS is cut with electric band saw system under HEPA-filter units (Masui
et al.) (Fig. 10 c-f). It is possible to slice the RMS as thin as 0.5 cm slabs without thawing
under clean condition, and it permits sharing the exact same horizon among multiple analyses.
RMS curation data, relevant for microbiological research will be made available to thescientific community via the KCC website.

(a)

(b)

(d)

(c)

(e)

(f)

Fig. 10. Apparatuses for long-term storage and ‘clean’ processing of RMS in KCC (a) Liquid nitrogen cooled
RMS storage tanks, (b) Deep freezers, (c) Overview of the band saw system, (d) Band saw machine in clean booth,(e) Open lid condition of the band saw machine, (f) RMS-WRC cut into half vertically

4-1. Materials(1) Perfluoroalcoxyalkane (PFA) jar (e.g., Savillex or Sanplatec)
The PFA jar is used for storing RMS in LN cooled tank (-160˚C) (Fig. 11a). The PFA
jars remain stable over a wide range of temperature from -196 to +200ºC, thus they can be
autoclaved and stored in the LN cooled tanks. Roughen the outer surface of PFA jar with asandpaper for securely pasting a sample label and preventing its detachement underultra-low temperature condition. Autoclave the jars at 121ºC for 20 min. (the jar must not
be tightly closed, otherwise it will be deformed by the vacuum effect.). After autoclaving,
put the jars into oven (60˚C) for drying up over night. After drying, allow the jars to cool
to room temperature.
14(2) Polypropylene 8 row reservoir (e.g., Porvair sciences)
This reservoir is used for storing RMS bars in the deep freezer (-80˚C). It can be
autoclaved and used for storing the bars in good order (Fig. 11b). Wrap a reservoir withaluminum foil, and autoclave it at 121ºC for 20 min. After autoclave, put the reservoir intooven (60˚C) to dry it up over night. After dry up, let the reservoir cool to room
temperature.

(a)

(b)

Fig. 11. Materials for storing RMS at very low temperature (a) Perfluoroalcoxyalkane (PFA) jars, (b)Polypropylene 8 row reservoir with bar shape RMS

4-2. Method(1) Turn on two HEPA-filter units on the clean booth one hour before operating bandsaw system.
(2) Spray and wipe saw blade, three wheels and sample cutting stage with 70%
ethanol.
(3) Fix the frozen RMS-WRC on the stage with fittings.
(4) Cut the sample into half vertically (Fig. 12a, b).
(5) Cut the halves into 10 mm thick slabs. Remove the contaminated outer edge (5 to
10 mm thick layer) from the slabs (Fig. 12c).
(6) Cut each slab into bars (5 mm x 10 mm in cross section) (Fig. 12d).
(7) Put all bars from one half into pre-labeled 8 row reservoirs in good order (Fig.
12e), and store in deep freezer (-80˚C). The bars should be put in the case in the same
direction such as make the left side as top of core.
(8) Put all bars from the other half in a pre-labeled PFA jar, and store LN cooled tank(-160˚C). The bars should be put in PFA jar in the same direction such as make the
top of the jar as the top of core.

15

Fig.12. An example of RMS-WRC sub-sampling(9) Clean the band saw parts, such as the saw blade, wheels and stage using asteam-heat cleaner (Fig. 13).
(10) Dry up saw’s parts by wiping.
(11) Spray and wipe saw blade, three wheels and cutting stage with 70% ethanol priorto next use.
(12) Prepare curation data sheet for RMS. Format of RMS curation data sheet isshown in Table 1.

(a)

(b)

Fig. 13. Cleaning of wheels (a) and saw blade (b) in the band saw machine

Table 1. An example of curation data sheet for RMS
Expedition

Site

Hole Core TypeSec.

Interval (cm)VolumeDepthContamination test

Top

Bottom(cc)(mbsf)

PFT(µg/gsediment)

FB(Beads/gsediment)

Storagetemperature

Cell density

Remarks(total cell/gsediment)

322

C0011

B

9

R

2

31.0

41.0

300

414

NC

NC

-80˚C

NC

WRC (already sampled by two 5 cc syringes in center)

322

C0011

B

14

R

4

36.5

46.5

300

464

NC

NC

-80˚C

NC

WRC (already sampled by two 5 cc syringes in center)

3XX

C000X

A

1

H

2

30.0

40.0

350

2

NC

NC

-80˚C

2.0 x108

One 30 cc subcores, residual WRC

3XX

C000X

A

1

H

4

30.0

40.0

350

5

NC

NC

-80˚C

1.8 x108

One 30 cc subcores, residual WRC

3XX

C000X

A

2

H

3

40.0

50.0

350

10

NC

NC

-80˚C

1.5 x108

One 30 cc subcores, residual WRC

3XX

C000X

A

3

H

3

60.0

70.0

350

20

NC

NC

-80˚C

8.0 x107

One 30 cc subcores, residual WRC

3XX

C000X

A

5

H

3

85.0

95.0

350

40

NC

NC

-80˚C

6.0 x107

One 30 cc subcores, residual WRC

3XX

C000X

A

7

H

3

10.0

20.0

350

60

NC

NC

-80˚C

3.0 x107

One 30 cc subcores, residual WRC

3XX

C000X

A

9

H

3

65.0

75.0

350

80

NC

NC

-80˚C

1.0x107

One 30 cc subcores, residual WRC

3XX

C000X

A

11

H

4

45.0

55.0

350

100

NC

NC

-80˚C

9.0x106

One 30 cc subcores

3XX

C000X

A

18

H

4

90.0

100.0

350

200

NC

NC

-80˚C

7.0x105

One 30 cc subcores

3XX

C000X

B

10

R

2

30.0

40.0

300

500

BD

0

-80˚C/-160˚C

NC

WRC (already removed scrappings from the outside)

3XX

C000X

B

20

R

2

20.0

30.0

300

600

0.05

100

-80˚C/-160˚C

NC

Ten 1 cc bars (After removing scrappings from the outside, WRC cut into bars)

3XX

C000X

B

30

R

1

80.0

90.0

300

700

0.08

200

-80˚C/-160˚C

NC

Ten 1 cc bars (After removing scrappings from the outside, WRC cut into bars)

3XX

C000X

B

40

R

2

10.0

20.0

300

800

BD

0

-80˚C/-160˚C

NC

WRC (already removed scrappings from the outside)

3XX

C000X

B

50

R

2

20.0

30.0

300

900

BD

0

-80˚C/-160˚C

NC

WRC (already removed scrappings from the outside)

3XX

C000X

B

60

R

1

120.0

125.0

150

1000

0.06

100

-80˚C/-160˚C

NC

Ten 1 cc bars (After removing scrappings from the outside, WRC cut into bars)

Note: PFT = perfluorocarbon tracer. FB = fluorescent beads. NC = not conducted. BD = below detection.
Cells in red frame show the RMS curation data relevant for microbiological analyses.

Notes• In advance, J-CORES labels must be pasted on PFA jars and 8 row reservoir. The labels
must be covered with transparent Teflon tape.
• Clean disposable gloves must be used during all handling procedure.
• Safety goggles and earplug must be put on for personal protection during cutting andcleaning operation.
• Cutting of RMS should be progressed as quickly as possible in order to avoid thawing.
• The WRC-half and slabs are stored temporarily in -50˚C freezer in the clean booth to
avoid unintentional thawing. PFA jars and 8 row reservoirs should be pre-cooled in thisfreezer so that no unintentional warming of the bars takes place.

16• In the case of syringe RMS, cut it into halves, and then cut each half into nearly equalvolume bars. Store half of the bars in deep freezer (-80˚C) and the remaining bars in LN
cooled tanks (-160˚C).

Optional: Chemical fixation procedure
Cell counts on fixed samples is an IODP standard measurement for microbiological
QA/QC of samples. Therefore, it is preferable to collect a sub-sample from RMS-WRC forchemical fixation. Final decision on sampling for chemical fixation will be made by
Implementing Organization (e.g., CDEX).
Materials(1) Phosphate Buffer Saline (PBS buffer)(pH 7.2) (e.g., Ambion or Invitrogen)
Dilute commercial 10 x PBS pH 7.2 with distilled water, and autoclave or filter tosterilize.
(2) 4% performaldehyde solution (50 ml)
Preparation(i) Heat to 60˚C 30 ml of distilled water in Duran glass bottle.
(ii) Add 2 g of paraformaldehyde with a stirring bar to the water.
(iii) Transfer the solution to fume hood, maintain at 60˚C on a hot plate and keep
stirring (Be careful not to overheat the solution!).
(iv) Add 1.5 µl of 10 N NaOH to the solution.
(v) Add 15 ml of distilled water to the solution.
(vi) Remove from hot plate and add 5 ml of 10 x PBS (Confirm the pH with pH test
paper).
(vii) Filter the mixture with a 0.2 µm filter.
(viii) Cool the filtrate to room temperature or to 4˚C.
(3) Tip cut-off of sterilized syringe (1 cc) (Fig.14)
Cut off the luer lok end of syringes with a heated knife.
Wrap the tip cut-off syringe with aluminum foil, andautoclave at 121˚C for 20 min. After autoclave, put syringe
into oven (60˚C) to dry it up for about 12 hours. Afterdrying, let syringe cool to room temperature prior to use. Fig. 14. One cc tip cut-off
syringe (below) and 20 cc
This preparation can be done onshore, too.
syringe (above)(4) 13 cc round-bottom centrifuge tube with screw cap (e.g., Sarstedt)(5) 100 % ethanol(6) Centrifuge machine (Rotor for 15 cc tube, 3000 g)
17(7) Micro pipet and sterilized tip (5 cc)
For adding reagents such as PBS, 4% performaldehyde solution and 100 % ethanol
into centrifuge tube

Method(1) Print a label after registration of RMS-WRC in J-CORES, and paste it on a 13 cc
round-bottom centrifuge tube with screw cap.
(2) Cover the label with transparent Teflon tape.
(3) Add 4.5 ml of Phosphate Buffer Saline (PBS buffer) to the tube.
(4) Sub-sample sediment from middle part of RMS-WRC with a 1 cc tip cut-off
sterilized syringe so that it samples entire length of the WRC.
(5) Add 1 cc of sample to the buffer tube. Mix vigorously.
(6) Add 4.5 ml of 4% performaldehyde solution in draft chamber. Mix immediately.
(7) Incubate at 4 ˚C for 12 hours.
(8) Centrifuge the tubes at 3,000 g for 10 minutes at 4°C.
(9) Store supernatant in waste container and add 9 ml of PBS buffer to the tube in draft
chamber. Mix vigorously.
(10) Repeat steps (8) and (9) twice to remove performaldehyde.
(11) Centrifuge tubes at 3,000 g for 10 minutes at 4°C.
(12) Remove supernatant by decantation and add 4.5 ml of PBS buffer to the tube. Mix
vigorously.
(13) Adjust to 10 ml with 100 % ethanol. Mix vigorously.
(14) Store these buffered samples at –20°C.
Time requirement: Steps (1) to (6) 20 min., steps (8) to (14) 40 min.
The buffered frozen samples can be brought to an onshore laboratory for further
processing for cell counting by following the procedure described by Morono et al.

Notes• As performaldehyde may cause sensitization of skin and eye, and may be harmful if
inhaled and ingested, clean disposable gloves, safety goggle and mask are to be usedduring all handling procedure.
• As performaldehyde solution does not last long, prepare it at least once per week.
• Liquid wastes containing performaldehyde are separately stored in a Polypropylene
bottle. They should not be flushed into chemical drain.

18

References
D’Hondt, S., Inagaki, F., Ferdelman, T., Jørgensen, B. B., Kato, K., Kemp, P., Sobecky, P.,
Sogin, M., and Takai, K. (2007) Exploring subseafloor life with the Integrated Ocean
Drilling Program. Sci. Drilling, 5, 58-69.
Masui, N., Morono, Y., and Inagaki, F. (2009) Bio-archive core storage and subsampling
procedure for subseafloor molecular biological research. Sci. Drilling, 8, 35-37.
Morono, Y., Terada, T., Masui, N. and Inagaki, F. (2009) Discriminative detection andenumeration of microbial life in marine subsurface sediments. The ISME Journal, 3,503-511.

19

Appendix I
STP Recommendation 0908-09: STP Recommendations for Routine Microbiological Sampling on
IODP Expeditions. The STP recommends the following approach to assist routine microbiologicalsampling on IODP expeditions (including those for which microbiology is the primary scientificobjective) so that samples are adequately and consistently preserved for future microbiologicalanalysis. The panel further recommends that a microbiologist sail as part of the science party with each
expedition in order to oversee the proper sampling, preservation, and integration of these materials into
specific expedition objectives.
To assist the IOs in implementing the collection of routine microbiological samples (RMS) on IODP
expeditions, the STP recommends the following:• Recommendation 1. On IODP expeditions, a minimum of one RMS should be collected per IW
sample acquired from each site that is sampled1*. The RMS should be collected in close proximity(within a few centimeters) to the IW sample. For reference, see STP Consensus Statement 0807-12.
The RMS sampling interval should be included in the scientific prospectus for the expedition.
Consideration of the scientific objectives of each cruise is encouraged in order to determine how
microbiological analysis of RMS can help to accomplish IODP objectives.
The candidate sampling procedure, based upon the Subseafloor Life Task Force (SLTF)
recommendation (STP consensus statement 0807-12), is as follows:
1) Bulk sediment should be collected in triplicate (e.g., with 30 cc sterile cut-off syringes; thereafter
frozen) and also scrapings from the outside of the core to be placed into a centrifuge tube (for use
as an indigenous tracer) where possible, or2) When #1 is not possible (e.g., because of limited staff or indurated nature of the core) thensuccessive whole- or half-round cores should be preserved as deep frozen samples. Depending on
shipboard freezer capacity, the frequency of RMS sampling may need to be reduced if wholerounds or half-rounds are often collected.
3) If the core quality is judged to be inferior and an IW sample will not be collected, or if the RMS
sample will interfere with the objectives of the cruise (e.g., if the sample would be taken near acritical interval), then there is no need to collect an RMS.
For preservation of RMS, the contents of the sterile syringes should be extruded into separate
centrifuge tubes followed by storage at -80 C. The whole- or half-rounds should be packaged inalumibags (or similar product) and frozen at -80 C. Freezing of the RMS should occur as soon as
possible after subcoring or collection (D’Hondt et al. 2007).
• Recommendation 2. An on-board microbiologist or a technician trained in aseptic samplingtechniques should implement these recommendations and the RMS sampling itself. The technician
will be responsible for maintaining the equipment required for routine sampling, maintaining aclean microbiology working space, and for maintaining an adequate inventory of needed sampling
supplies on board.
•1 Recommendation 3. Regular (e.g., annual) review of the methods adopted for RMS should be*performed
Based on threesites
each AsfromODP
202 & IODP
Expedition
tying
this RMS
schemetotothe
the collection of IW
by theSLTF.
partof Legthe review,if new
methods307,
come
to light
in relation
samples
will entail
taking ~20 to 40
19-59, bemean= 35) for for
eachadoption.
category at the average site focused oncollection
and preservationof samples
samples,(range
these=should
consideredor sedimentary
processes.
will entail removal
sampleplugsrecommendations
from the center of,shouldor whole rounds•paleoceanography
Recommendation
4. Questions
relatedThis
to implementation
ofofanyof these
totaling,
~3.5
m
of
core
at
each
site.
Assuming
three
full-length
holes
per
site
(range
=
3-5
for
202
and
307)
and verybe directed to the SLTF for guidance.
conservative 70% coring recovery, sediment will be removed from 3.5 of 504 m of core (~0.7%) per site. Much of the• Recommendation 5. Clarification of the final archival disposition of the RMS and the shipping
sediment in these 3.5 m will remain after sampling. (These residual sediment samples may not be of optimal use.) A 24 ft3 procedure at the conclusion of the expedition needs to be addressed by the IODP Curators.
80C freezer will hold ~430 packages of triplicate 30cc samples. This is almost four times the capacity required for IODPLeg 307 and about 1.33 times the capacity required for ODP Leg 202.

Voting Record: 13 For, 1 Against (Thomas), 1 Abstention (Young), 1 Absent (Paul Johnson)
Priority: HighSTP suggests this be forwarded to IODP-MI, SPC, and the IOs
Background to STP Recommendation 0908-09: This consensus statement follows the report from
IODP-MI that considers the issues related to implementing routine sampling for microbiologicalsamples aboard all IODP expeditions. Many of the20
concerns have been addressed and such sampling isimminent. This consensus statement references relevant recommendations generated by the 2003 IODP
Microbiology Working Group Report, the IODP Deep Biosphere Workshop held in Vancouver, BC(October 2006) and the manuscript resulting from that workshop (D’Hondt et al. Scientific Drilling.

centrifuge tubes followed by storage at -80 C. The whole- or half-rounds should be packaged inalumibags (or similar product) and frozen at -80 C. Freezing of the RMS should occur as soon as
possible after subcoring or collection (D’Hondt et al. 2007).
• Recommendation 2. An on-board microbiologist or a technician trained in aseptic samplingtechniques should implement these recommendations and the RMS sampling itself. The technician
will be responsible for maintaining the equipment required for routine sampling, maintaining a

1* Based on three sites each from ODP Leg 202 & IODP Expedition 307, tying this RMS scheme to the collection of IW
samples will entail taking ~20 to 40 samples (range = 19-59, mean = 35) for each category at the average site focused on
paleoceanography or sedimentary processes. This will entail removal of sample plugs from the center of, or whole rounds
totaling, ~3.5 m of core at each site. Assuming three full-length holes per site (range = 3-5 for 202 and 307) and very
conservative 70% coring recovery, sediment will be removed from 3.5 of 504 m of core (~0.7%) per site. Much of thesediment in these 3.5 m will remain after sampling. (These residual sediment samples may not be of optimal use.) A 24 ft3 80C freezer will hold ~430 packages of triplicate 30cc samples. This is almost four times the capacity required for IODPLeg 307 and about 1.33 times the capacity required for ODP Leg 202.

Appendix
IICIENTIFIC PARTYSHIPBOARD S

CHAPTER 5, EXPLANATORY NOTES

81

Table T1. Definitions of terms used in microbiology sampling.
Term

Definition

Aseptic

A clinical definition referring to freedom from pathogens. Clinical conditions are not applicable to ODP operations.

SterileThe most stringent category used during Leg 201. Sterile indicates the absence of contaminating prokaryotic cells that are capable ofmetabolism and growth and their nucleic acids.

Clean

Defined as giving careful consideration and effort to avoid cross-contamination. May involve ethanol washing, flaming instruments,
etc., and may approach sterile conditions.

Anaerobic

A property of organisms indicating the ability to live without oxygen. Also a property of a method (“anaerobic cultivation”) or oflaboratory cultivation equipment (“anaerobic chambers”).

AnoxicProperty of an oxygen-free environment such as used in a glove bag or N2-flushed sampling bucket.

Shipboard Scientific Party
Chapter 5, explanatory notes, P 81.
Proceedings of the Ocean Drilling Program, Initial Reports Volume 201. Published in 2003.

21

Appendix III
Coring contamination tests by using fluorescent microspheres during CK09-03 Expedition 904
Kochi JAMSTEC Noriaki Masui
Onboard processing
Contamination tests were conducted for two coring systems (HPCS and EPCS) by attachingplastic bags containing 50 ml of fluorescent microspheres suspension (approximately 1 × 1010
microspheres/mL) inside a core catcher (Fig. 1). Fluoresbrite 0.5 μm microspheres(Polysciences Inc.) were used as particulate tracer of contamination.
In the HPCS coring, the test was done for Core C9010D-3H. Ruptured empty plastic bag was
found in the core liner of Core 3H (Fig. 2). In the EPCS coring, the experiment was done forCore C9010D-13X. Ruptured empty bag was found at Female Quick Release at the top of thelower inner barrel assembly. Ruptured bags prove that microspheres were successfully released
in core barrel during coring activities. Samples for contamination tests were taken as 10 cm
whole round core (WRC) per section from the 2 above mentioned cores. Each WRC wassealed in a Ziploc bag and stored in -20 C freezer.

Fig.1. Plastic bags containing suspension offluorescent microspheres, attached in core catcher

Fig.2. Ruptured plastic bag (in red circle) in arecovered coreSample collection and counting procedure
Sample collection procedure is shown in Fig. 3. A round slice sample (10 mm thick) was
sub-sampled from WRC by using a band-saw system (Fig. 4). Three portions of sliced sample(center, intermediate and outer edge) were sampled by using tip-cut syringe. Each sediment
sample was then mixed with an equal volume of saturated sodium chloride solution. The
solution was centrifuged (5 min, 2800 x g), and the supernatant was filtered onto black
polycarbonate filters (0.2-μm pore size). Fluorescent microspheres on the filter were counted
under ultraviolet (UV) light, and data were reported as numbers of microspheres per gram ofsediment.

22

Cutting
WRC

66 mm width
10 mm thick(30 mm width)(15 mm width)(3 mm width)

CenterIntermediate

Outer edge

SubsamplingMix and centrifuge

Filtration

Microsphere counting

Fig. 3. Procedure of sample collection and counting

Fig. 4. Cutting core sample by using a band-

Fig. 5. WRC C9010D-3H-4 covered with

saw system

sandy particles

Contamination assessment for samples obtained by HPCS and EPCS coring system
Results of contamination assessment are shown in Table 1. Microspheres were detected fromthe outer edge of all samples. In contrast, no microsphere was detected from center andintermediate zone of samples except for WRC C9010D-3H-4, which was mainly composed ofsilt and covered with sandy particles (Fig. 5). Most of the WRC, in which the microspheresfrom center and intermediate zones were not detected, were composed of mainly clay or silt.
Therefore, microspheres detected in center and intermediate zones of the C9010D-3H-4 seemto have diffused in from the sandy core surface. When band-saw system is used to subsample
cored material, contamination is likely to spread among subsamples. However, in the case of
WRC subsampled by using the band-saw, microsphere was not detected from center andintermediate zones. This suggests that band-saw system will not cause cross-contamination
during subsampling of a frozen sample. Sample processing by band-saw system in the Kochi
Core Center shows minimum risk of cross-contamination (Fig. 4).

23

There is no influence of difference of coring system (HPCS and EPCS) on contamination ofcore sample, especially in the intermediate and central zones. Number of microspheres fromouter edge of C9010D-13X-1 and 13X-2 (8.5 102 and 9.0 102 (microspheres/g)) are lower
than that of WRC C9010D-3H (2.5~7.5 103 (microspheres/g)). The lower number ofmicrospheres in outer edge of C9010D-13X can be attributed to washing and scraping of thecore during drilling and recovery. Application of chemical tracer such as perfluorocarbon may
demonstrate a different level of contamination of samples in contrast to that observed here byusing microspheres because chemical tracers are much smaller in size, and therefore are easy todiffuse into sediment, than the microspheres.
Table 1.
Core section
HPCS
C9010D-3H-1
C9010D-3H-2
C9010D-3H-4*
C9010D-3H-8
EPCS
C9010D-13X-1
C9010D-13X-2

Fluoresent microspheres in C9010D cores
Number of microspheres
Outer edge
Intermediate(microspheres/g)
Center

3.5
2.5
7.5
4.5

103
103
103
103

ND
ND
1.0 102
ND

ND
ND
1.0 102
ND

8.5
9.0

102
102

ND
ND

ND
ND
ND = None Detected

Delivery of microspheres was confirmed by detecting microspheres in scrapings and water samples
from inner wall of core liner.
*Surface of WRC C9010D-3H-4 was covered with sandy particles.

24