Witwatersrand Microbiology Project Field Manual
by
Duane
P. Moser
pACIFIC
nORTHWEST nATIONAL lABORATORY
S.M.
PfiffneR
uNIVERSITY
OF tENNESSEE
A.Welty
Northern
Arizona university
j.
wARD
UNIV.
OF TORONTO
T.C. ONSTOTTand J. HALL
pRINCETON
uNIVERSITY
Table of contents
Benches
and Workspaces
..
1
PReparation
of equipment
1
FIELD KITS CHECK
LIST.........................................
.
...
5
BOREHOLE WATER SAMPLING...........................................
.5
SAMPLE PROCESSING: PRESSURE
FILTRATION........................................
.10
PRESERVATION OF
WATER SAMPLES
..12
ONLINE FIELD
NOTEBOOK
..13
INOCULATION OF
ANAEROBIC MEDIA
13
FIGURES
..14
APPENDIX 1: GAS
SAMPLING PROCEDURE
...17
APPENDIX 2: D.I.
WATER (Labcon Waterstill System)
.20
PROPER USE OF A
SUB-ZERO FREEZER
....20
Benches and Workspaces
The
field lab is open to the outside air.
In winter a considerable load of dust accumulates on surfaces. In the summer, mold spores, pollen, etc. are
a threat to general lab cleanliness. As
a matter of routine, it is strongly recommended that before any lab work that
you spray down bench surfaces with Mr. Muscle or equivalent cleaner and wipe
down with paper towel.
PReparation of
SAMPLING equipment
All equipment that comes in contact with samples must be cleaned
thoroughly between applications. The
risk of cross contamination is to some extent reduced by autoclaving, but good
lab hygiene is still strongly encouraged. Equipment that is autoclaved and
stored wrapped will remain sterile for a long time, provided the equipment is
dry. Even autoclaved equipment stored
wet is prone to moldy growth (an obvious source of contamination). As a general rule of thumb, equipment should
be cleaned and stored in such a manner, as you would be comfortable EATING from
it.
MATERIALS:
1.
Vacuum pump
2.
Ar or N2
high pressure gas cylinders with sterile filter and tubing
3.
Autoclave
4.
Bunsen burner
5.
Sterile gloves
6.
Al foil
7.
Cornelius
canisters
8.
Sampling
manifold (pre-methanol rinsed and autoclaved)
9.
Sampling tubing
10.
Packers
11.
Serum vials
12.
Needles
13.
50 ml Falcon
tubes for FISH samples
14.
Filter forceps
15.
Flame and
ethanol for flaming forceps and canister quick connects
16.
15 ml falcon
tube for anions
17.
15 ml falcon
tube with 1.5 ml of concentrated nitric acid for cations.
HOSES/QUICK DISCONNECTS: There
are four hoses used in sampling. Two have
quick connects at both ends. One quick
connect attaches it to the Cornelius canister and the other to the sampling
manifold. The other two hoses have
quick connects for the sampling manifold at one end and syringes at the other
end. Hoses are easy to overlook as
sources of contamination between samples. The insides of hoses are out of sight
(and mind) and also difficult to clean.
Worse, the inside surfaces of hoses come into intimate contact with
samples both in the field and lab.
Contaminated hoses are very efficient means to transfer contamination
into an otherwise pristine sample, thus hoses deserve special care.
After every use, hoses must be thoroughly rinsed out first with soapy
water (hold ends and slosh around).
Soap can be delivered from a squirt bottle. This is followed by rinsing
with clean tap water, then DI and finally a small amount of HPLC grade methanol
to assure that samples are of PLFA quality.
Hoses attached to the female end of quick disconnects (e.g. Cornelius)
may in some circumstances be left attached for cleaning provided that the
plunger (Figure 1) is depressed to permit the inside of the disconnect to be
washed along with the hose. If you
really want to be sure, remove the disconnect every time and clean it
separately. It is very easy to leave
wash (or sample) water in dead spaces within valves and fittings where unwanted
microbes can grow to considerable density and lead to the unwitting
contamination of samples.
Once clean, wind them up, autoclave them and keep them in their
autoclave bag until at the sampling site underground.
FILTER/PACKER MANIFOLDS: These
must be thoroughly cleaned between samples.
Remove all filter housings and open all valves. Clean out with a bottlebrush and use soapy
water for an initial cleaning. Rinse
well with DI water and final with HPLC grade MeOH. Allow to air dry and autoclave with the valves open. Packer manifold male quick connect ends need
not be removed every time, but valve should be propped open and the various
prescribed rinses performed. Keep
wrapped in autoclave bag with tape until underground at borehole sampling site.
BOREHOLE PACKERS: After a great
deal of experimentation, three major types of borehole packers have become
standard equipment. Types 1 and 2 (See figure 2) are adaptations of locally
available steel telescopic packers produced by CEM Engineering of South Africa.
Both types seal the hole using exactly the same approach. A threaded wrench is used to apply end
pressure on a series of rubber disks, which expand and seal the hole. Water and
gas are forced to travel through the interior of the packer, either inside the
bore of the packer itself or through a plastic liner.
Type 1 uses the standard steel packer (various sizes 1 inch up to 80
mm) commonly used in the mines, but with an all plastic insert designed by
George Rose of Princeton University to remove the influence of metal in the
sampling system. Type 1 (2 sizes)
packers are the most useful in lower flow situations and where high confidence
gas samples are required.
Type 2, employs either the standard steel telescopic packer design or a
stainless steel version constructed by CEM Engineering on special order. The latter version is generally used for
high flow situations and fits the largest of the sampling manifolds in our
collection.
Type 3 is the expansion drain plug version. This employs a commercially available plastic drain plug (United
States Plastics, Inc.), which is fitted into the smaller of the two sizes of
sampling manifolds (3/4 I.D.) via a plastic adaptor constructed for this
purpose in the Princeton University shop.
A custom-made Teflon washer (white) is useful to prevent leakage between
the commercial drain plug and the adaptor. All packer supplies are kept in the
corner cabinet behind the main lab door or in one of the small parts toolbox
labeled packers.
It is essential to assemble the packer you intend to use underground
prior to autoclaving and understand how the system works. Then wrap up packer and handle in autoclave
bags, autoclave and keep in autoclave bag until you are ready to use them
underground at sampling site.
CORNELIUS CANISTERS: In many
ways, these are your most critical piece of equipment. These must be cleaned well between samplings
not only to negate the potential for cross contamination, but also to assure
that corrosive samples do not damage these canisters.
1.
Empty canisters
are first dissembled to facilitate cleaning.
Both outlets are removed with the proper box-end wrench and the straw is
removed from the outlet and cleaned. Be sure to note that the straw is used
only on the outlet side. The canister is rinsed with normal tap water, and then
washed with lab soap and a long handled bottlebrush to remove all visible
particulates from the prior sampling. Next, the canister is rinsed with DI
water several times, as are the straw and other parts.
2.
The canister is
reassembled and about 100 ml of HPLC grade methanol or absolute ethanol is
added and the screw cap tightened down.
The fittings for the inlet and outlet are different. The inlet fittings have an indentation cut
into nut portion of the fitting. With
the alcohol inside, the canister is sloshed around with care to cover all
inside surfaces.
3.
It is essential
to clean the inside of the straw and outlet Cornelius fittings (black) with
alcohol as well. Adding a slight over
pressure to the canister using a bike pump or Ar or N2 gas cylinders
does this. An adaptor for the former is
stored in the lab tool drawer. The
adaptor has a Schrader valve on one end and a gray inlet Cornelius fitting on
the other. Add about 10 pumps with the
bicycle pump and then attach the black outlet fitting. Most of the alcohol will squirt out of the
canister rinsing the inside of the water passage as it does so. Finally, invert
the canister with the screw lid off and allow the inside to dry. Drying can be performed by attaching a
sterile, clean hose to the canister inlet valve from either the compressed Ar
or N2 cylinders and blowing out thru the outlet valve. Make sure that the hose has an autoclaved
filter in line to scrub the Ar or N2.
4.
Once dry and
fully assembled, the canisters are refitted with their screw lids and
autoclaved. It is critical to remove
the plastic handle on the one canister so equipped, as it will melt in the
autoclave. Black vinyl caps are
available in one of the small parts boxes which fit the inlet and outlet
couplings perfectly. These fittings
should be capped prior to autoclaving.
The heavy plastic cap should be loose during autoclaving. Autoclave on dry goods setting at 121oC.
5.
Once the
autoclave cycle is finished, remove than canister and tighten the canister lid
down. It is vented, so there is no
concern about not being able to remove it later.
6.
Through the
OUTLET fitting (fits gray adaptor) attach the clean and filtered Ar line. Set the second stage of the regulator for 50
kPa. Pull up on the spring-loaded cap vent and lodge it sideways (open
position). Start the Ar flowing and
flush the canister for 2 5 minutes depending on the flow and level of
anaerobicity required. Note that Ar is
heavier than air and filling from the bottom (through the outlet) aids in the
displacement of air. Once the canister
is done flushing, close the vent valve by pulling up, turning 90o,
and letting the spring pull the valve closed.
Since the Ar line is still running, the canister will develop a pressure
of 50 kPa, and can be stored this way until ready to sample. The seals on these units are of high quality
and the pressure will be maintained indefinitely (although a good policy is to
check by pulling on the vent valve to see if there is still a positive gas
pressure in the canister).
7.
It is a good
idea to label the canister as being sterile (autoclave tape), clean and Ar
filled at this point. You cant tell by looking at it after all. Leave the
black vinyl caps on the quick connect fittings. These are the only part of the canister that needs to be kept
scrupulously clean. The rest of the
outer surfaces can (and will) get dirty due to handling in the mine
environment.
MICROBIAL SAMPLING SERUM VIALS:
Prepare at least 4 to 6 120 ml serum vials for anaerobic microbial
sampling.
HANNA PROBES: Calibrate probes
prior to each sampling trip. AFTER
SAMPLING TRIP MAKE SURE TO RINSE PROBES IN D.I. WATER AND STORE PH PROBE TIP IN
STORAGE SOLUTION (THIS IS LOCATED IN REFRIGERATOR). YOU MUST DO THIS. IF
RESIDUAL SULFIDIC SALINE WATER DRYS ON PROBE IT WILL CORRODE THEM AND THEN
THEYLL NOT FUNCTION PROPERLY.
FIELD KITS CHECK LIST
(Check Table 1)
ICE CHEST: if planning an overnight stay after sampling or a long
distance travel
AUTOCLAVED GEAR:
1.
TWO Cornelius
canisters with black caps and filled with Ar or N2 gas.
2.
Sampling
manifolds with connectors and can also connect tubing to reduce contamination
if borehole is certainty. All wrapped
in autoclave bags.
3.
Long
telescoping packers w/ handles and small compression packers, all wrapped in
autoclave bags.
4.
TWO x tubes w/
Quick connects and w/ canister connectors
5.
TWO x tubes
w/Quick connects and syringe for bio samples
6.
Biosampling
Kit: 4 to 6 x 120 ml serum vials w/N2
gas and drop of H2O + 10 x 20.5 gauge needles (big yellow) + 10 x
22.5 gauge needles (smaller)
7.
bag of random
tubes, fittings and hose clamps for uncertain borehole situations
8.
ONE 1 liter
bottle for service water sample-this can be carried in a portable ice chest
MORE BIOSAMPLING GEAR:
1.
TWO x 50 ml
falcon tubes (Fish)
2.
Biofilm kit-6 x
15 ml falcon tubes + pH strips + plastic bulb syringe + 10 x Cryovials +
spatula + surgical gloves
3.
Syringe kit
(for no borehole access, just crack)-1 x 60 ml syringe+ 1 x 10 ml syringe+ 10 x
syringe needles large and small + stainless steel canola + 2 syringe filters
4.
Sterilization
kit-ethanol tube+ chemwipes + latex gloves + tube of bleach
5.
Take mattress
pump, Tygon tubing, one or two filter holders, MeOH pre-rinsed forceps,
Whirlpack bags, latex gloves, sharpie, two muffled Al foil with glass fiber
filters (2 filters per pack). The glass
fiber filters in Al foil pockets are used for ONLY for air sampling and extra
muffled Al foil pockets are used to store air samples. These samples are collected using mattress
pump in drawer. Use Teflon tubing to
connect to filter apparatus. Put
holders on the intake to the pump so that air is being pulled through the
filter first then into Tygon tubing and into pump. This avoids contaminating the filters with the pump. Do two air filters, one for PLFA and one for
DNA and a third optional for enrichments and fourth for FISH.
FIELD MEASUREMENT GEAR:
1.
Portable Black
Plastic Probe kit w/ Hanna pH, temperature, conductivity and Eh probes, Allen
key set for packers, pH 4, 7 and 10 solution to check calibration, pH strips.
2.
Thermometer
(for back up)
3.
Small plastic
beaker
CHEMISTRY SAMPLING GEAR:
1.
TWO x tubes
w/Quick connect for sampling manifold and Leurlock fittings on other end.
2.
TWO x 1 liter
bottles (for 14C, 36Cl, T and d13C organic)
3.
ONE x 500 ml.
Nalgene bottle (for phosphorus, partially full, HCl washed)
4.
TWO x 20-40 ml
serum vials (for acetate/formate partially full, d18O full, not washed, no preservative)
5.
THREE x 60-120
ml serum vials (for dD-no preservatives, and
for d13C DIC and DIC, preservatives added later)
6.
TWO x 60-120
serum vials evacuated (for filtered d15N ammonia, unfiltered d15N ammonia)
7.
TWO x 40-60 ml
Quorpak (for S species w/ Zn acetate, TOC+DOC w/HCl or phosphoric acid)
8.
TWO x 60 ml
syringes with Leurlocks (for S isotopes on sulfate and sulfide). Preloaded with 10 ml of Zn acetate, HgCl2
and NaOH solution.
9.
TWO x 15 ml
Falcons filtered (cations w/HNO3, Fe w/HCl)
10.
ONE x Nalgene
filtered (anions, partially full)
11.
ONE x bag of
blue stoppers, Al caps, crimper, 0.2 ΅m syringe filters (to attach to tubes for
filtered samples) and small syringe needles.
GAS SAMPLING GEAR:
1.
TWO x Cu tubes
loaded in one rack
2.
ONE x green bag
of valve, fittings, and ratchet wrench
3.
ONE x big red
bucket
4.
ONE x small
plastic beaker with fitting for syringe needle
5.
ONE x big hose
6.
SIX x 120 ml
pre-evacuated serum vials
TOOL KIT:
Screw driver, spanner, Leatherman tool, tape, sharpies, and Bungie
cords.
Everything should fit into 3 large backpacks (Fig. 1b).
Table 1. Sample containers, labels and preservatives for geochemistry and microbiology
|
Labeling and
preservatives added as indicated below. |
|||
|
Type (Label) |
Container |
Preservative |
|
|
Geochemistry
filtered |
||
|
Anion |
0.22΅ filter into a 50ml Nalgene bottle or 15 ml Falcon tube |
3/4 fill and
freeze |
|
|
Cation |
0.22΅ filter into a 50ml Nalgene bottle or 15 ml Falcon tube already prepped
with HNO3 |
1 ml conc. HNO3/20
ml sample |
|
|
Diss. NH3 |
0.22΅ syringe filter into
a 20-60 ml serum vial |
50΅l conc. H2SO4/20
ml sample |
|
|
Diss. Fe |
0.22΅ filter into a 50ml Nalgene bottle or 15 ml Falcon tube prepped with HCl |
1 ml conc.
HCl/25 ml sample |
|
|
SPE |
ODS, silica, and 2 JW
cartridges |
On surface: Pass filtrate
from DNA/PLFA filters through SPE cartridges,
record sample and volume filtered and store in refrigerator or freezer. |
|
|
Geochemistry unfiltered |
|||
|
DIC |
50 ml amber septum bottle
or serum vial |
50΅L sat. HgCl2 |
|
|
d13C DIC |
50 ml amber septum bottle or serum vial |
50΅L sat. HgCl2 |
|
|
d15N NH3 |
20-60 ml serum vial |
50΅L conc. H2SO4/20
ml sample |
|
|
Formate/acetate |
45 ml Teflon septum
glass vials or serum vials |
partially fill and freeze |
|
|
DOC |
45 ml Teflon septum
glass or serum vials |
1 ml conc. HCl or
Phosphoric acid/25 ml sample |
|
|
TOC |
45 ml Teflon septum glass
or serum vials |
1 ml conc. HCl or
Phosphoric acid/25 ml sample |
|
|
d13C
DOC |
1 L amber glass bottles w/
Teflon caps |
1 ml sat. HgCl2 |
|
|
Sulfur species |
20 ml serum vial |
transfer anaerobically
into 50΅L 2N ZnAc loaded 5 ml serum vials, partially fill and freeze |
|
|
d34S |
2 x 60 ml syringes |
Preloaded with 10 ml D.I.,
500΅L 2N ZnAc, 50΅L sat. HgCl2 |
|
|
d18O |
1 x 15-40 ml serum vial |
none |
|
|
dD |
|||