Download Metabolic Interactions of aPurple Sulfur Bacterium and a Facultative

Metabolic Interactions of a Purple Sulfur Bacterium
and a Facultative Anaerobe
Laura K. 1
Baumgartner and Adam C. 2
Martiny
Microbial Diversity 2001
Marine Biological Laboratory
Woods Hole, MA
.
1
U
niversity of Connecticut Department of Marine
Sciences, 1084 Shennecossett
Rd, Groton CT 06340, LKB00002Uconn.edu
2
University of Denmark, BioCentrum-DTU, Buil
ding 301, 2800 Lyngby,
Denmark, [email protected]
.
Introduction
Purple Sulfur Bacteria: Overview and An
Unusual Co-Culture
Purple sulfur bacteria (PSB) are generally
anaerobic photolithoautotrophs that
conduct anoxygenic photosynthesis with sulf
ide as the electron donor (Pfenning and
Truper 2001). However, some strains hav
e been shown to grow heterotrophically
as
well as lithotrophically (Bauld et al. 1987),
and some strains can utilize acetate, mal
ate,
pyruvate, and succinate (Krassilnikova
and Kondratyeva, 1984). PSB are commo
n in
marine environments such as microbial mat
s (van Gemerden 1993).
We examined an unusual colony formatio
n that arose in PSB enrichments
•- 1) from Sippewisset Salt Marsh, Cr”? Cod
, Massachusetts. Under the
.
Figure 1: Satellite colonies in the prim
ary enrichment. The white bacteri
um is surrounded by a
halo of purple bacterium displaying
enhanced growth.
dissecting microscope, the initial colonies
consisted of a white disc-shaped colony
surrounded by several different color
morphs of red to purple colonies that
are
presumed to be PSB. These satellite
colonies developed in all but the highes
t dilution
of the shake tubes, and their developmen
t appeared to occur in stages: first the
(darker) PSB appeared as small colonies
throughout the tube, then the white colo
(strain ACMOI) appeared, and finally
nies
the PSB colonies nearest the ACMOI
gre
w
relative to the more distant PSB colonies
and more colonies appeared near AC
MOI.
This phenomenon was interesting for
two reasons. First, because the media was
highly limiting (see methods and Append
ix A), the metabolic strategy of AC
MOI was
unclear. Second, the enhanced growth
and spatial distribution of the PSB imp
lied some
interaction between the two strains.
2
•
We were interested in several questions. First, what is the iden
tity of ACMOI?
Second, is ACMOI growing on some product of the PSB, or is
it growing from a
component of the media (including agar)? Third, what is the inter
action between
ACMOI and the PSB that resulted in the satellite colony form
ation? These questions
were examined with both traditional microbiological and mole
cular analyses.
Methods
Sample source and Enrichment
The bacteria were enriched and isolated from a sediment samp
le from
Sippewisset Salt Marsh, Cape Cod, MA. The sediment was
dark pink and is known to
be enriched for PSB. A small inoculum (—1 g) was initially
enriched in CR medium (see
Appendix A). After nine days of growth, 0.5 mL of the enric
hment was diluted in shake
tubes with CR medium in 1% agar (deep agar dilution series,
see Appendix A). Further
liquid cultures of the PSB also utilized CR medium as a base
, and all enrichments
grown in the light were given broad-spectrum light from stand
ard incandescent bulbs.
Specific Shake Enrichments
Further shake enrichments were carried out to examine the
metabolism of PSBI
and ACMOI. Mixed cultures (predominantly ACMOI) were
grown in the dark to assess
the phototrophic requirements of ACMOI and PSBI. For PSB
I, enrichments included
the addition of galactose, glucose, or 0.2 im filter-sterilize
d supernatant from an ACMOI
culture. ACMOI was also grown in shake tubes with 0.1%
bromothymol blue to assess
pH change (pH-indicator, blue at 7.6 and yellow at 6.0).
Liquid Cultures
Both strains were maintained in CR medium. Mixed cultu
res (predominantly
ACMO1) were grown in liquid CR medium in the dark to asses
s the phototrophic
requirements of ACMOI and PSB1. Metabolic preferenc
es of ACMOI and PSBI were
assessed by enrichment on various substrates. Concentr
ated (by centrifugation)
ACMOI or PSBI monoculture were added to 4 ml vials cont
aining CR medium and one
of the following substrates (2mM): acetate, butyrate, form
ate, fumarate, galactose,
glucose, lactate, malate, propionate, succinate, as well
as 5% supernatant, ‘10%
supernatant, 25% supernatant, 50% supernatant, and
CR medium (control) for the
PSBI (the supernatant percentages represent the perc
entage of filter-sterilized
supernatant from a galactose-enriched ACMOI culture
in the total media volume). After
three days of growth, the optical density of each enrichme
nt was measured
spectrometrically as absorbance at either 550nm (ACM
OI) or 660 nm (PSBI).
Plating
ACMOI was plated aerobically and anaerobically on seaw
ater and freshwater
1.5% agar plates, with and without malate, (all combina
tions, see Appendix A for
media). PSBI was plated on freshwater malate and
seawater POB plates aerobically
and anaerobically in continuous light and dark condition
s. However, no growth was
achieved on any PSBI plates, and the results will there
fore not be discussed.
DNA Extraction and Amplification
Satellite colonies were extracted from the 102 dilutio
n of the original shake series
by removing the agar from the tube and extracting individ
ual colony formations with
suction. DNA was extracted from these colonies with
Mo Bio Ultra Clean Soil DNA kits
and amplified with 8F and 1495 reverse primers at
55 °C annealing temperature.
3
•
•
.
Cloning, Restriction Fragment Length Pol
ymorphism, and Sequencing
The amplified 168 rDNA was cloned with the Invit
rogen TOPA TA Cloning kit.
Initial clones were grown on ampicillin-X-gal plate
s for 48 hours at 37°C. Fifty clones
were picked at random and transferred to another
ampicillin-X-Gal plate and allowed to
grow for 48 hours at 37 oC. PCR from 35 of thes
e clones was then amplified using
MI3F and MI3R primers. This PCR product was
then digested with Rsal and Hpall for
2 hours at 37°C and separated on a 2% agarose
gel for comparison of digested bands.
Products that appeared to have distinct band patte
rns were sent to Accugenix for
sequencing.
16S rDNA from ACMO1 was extracted from pure
culture with Promega Wizard
Genomic DNA kit and amplified via similar meth
ods, and the full 16S rRNA was
sequenced in 6 runs by Accugenix. Phylogeny
for all sequences was conducted with
ARB using Ribosomal Database Project databas
es.
Results and Discussion
Purple Sulfur Bacterium (PSBI)
Microscopic, Spectral Observations
At higher magnification (400-l000x), the purple
sulfur bacteria isolates are short
rods or long cocci with internal sulfur granules,
many of which are dividing and all of
which are motile (Figure 2), possibly Chromatium
(Jane Gibson, personal
communication). Cells have been observed
to reverse direction of rotation, and under
transmission electron microscopy, the cells have
a single polar flagellum. Spectral
analysis of PSBI revealed peaks at 590, 800,
and 850 nm, all of which are indicative of
bacteriochlorophyll a.
PSBI rapidly utilized sulfide, and sulfide depl
eted cultures (no internal granules)
regained internal granules within 10 hours with
the addition of 2mM S
2 (as Na
S,
2
pH7). Also, PSBI displayed chemotaxis with
sulfide gradients, moving away from high
concentrations of added sulfide on a microsco
pe slide to an intermediate concentration,
and then moving back towards the point of addi
tion as sulfide was consumed.
Figure 2: PSBI at 400x and I000x. The
left image is from a sulfide-starved cultu
re, and
the bacteria contain no internal granules.
The right image is from a sulfide-enrich
ed
culture, and internal granules are evid
ent as highly refractive spheres.
4
Metabolic Observations
PSB and ACMOI were grown in mixed culture in shake tubes in the
dark with no
apparent PSB colonies forming. When the enrichments were place
d in constant light,
PSB colonies appeared, indicating that PSB may be an oblig
ate phototroph. However,
PSBI grown axenically did not form the large colonies that
were observed in mixed
culture. The addition of 0.2 jim filter-sterilized supernatant from
ACMO1 cultures
enhanced colony growth while media alone had no affect (micro
scopically, the
supernatant did not contain any ACMOI). This experiment will
be discussed in greater
detail below, but the result hinted that some metabolic product
of ACMOI was
stimulating PSBI growth. Therefore PSB were enriched with vario
us carbon sources
(sugars, fatty acids, and different concentrations of supernata
nt from ACMO1) and the
optical density of the enrichments was measured after two days
(Figure 3)
Enrichments with fatty acid additions showed enhanced growth
over the control,
as did enrichments with sugars and supernatant. However, the
greater degree of
enhancement displayed by the fatty acid enrichments appe
ars to indicate that PSBI
utilized these substances more easily or gained more from them.
Shake tubes of PSBI
with various carbon additions were also carried out, but these
shakes had no apparent
growth within the time of this study.
.
Optical Density after 2 Days
E
4.ICD
.cD
0.6
0.5
0.4
0.3
—o
0.2
.11.0 0.1
0.
0
Substrate
Figure 3: Optical density of PSBI cultures at 660 nm
after 2 days. Sugars and supematant from
ACMOI slightly enhanced growth over the control, while
enrichments with fatty acids showed
strongly enhanced growth.
Isolation and characterization of strain ACMOI
ACMO1 was isolated from the white central colony in the
shake-tube. This was
done by removing (sucking) a piece of the colony and trans
ferring it to liquid media and
shake tubes and incubate it in the dark. Light microscopy
showed a non-motile, black
5
(
rod 2-10 m long (depending on growth conditions),
which was consistent with earlier
observations of the mixed culture.
The pure culture was incubated under anaerobic cond
itions with various
carbon/energy sources and was shown to grow on agar
and agarose as the sole energy
source as well as hexoses (see Table 1).
Table 1. Conditions that initially promoted
cirowth of ACMOI
Media (incu. dark)
Media (incu. light)
Glucose
Galactose
Agar
Agarose
Glucuronic acid
+
+
+
+
ACMOI was streaked aerobically on seawaterand freshwater-base plates with
and without malate. The strain grew only on seaw
ater-based plates both in the
presence and absence of malate. The strain clear
ly degraded agar and produced a
small halo around the colony (see Figure 4).
.
Figure 4: An ACMOI colony on a agar plate.
Note the darkened
halo of degraded agar.
The combined data suggested that the strain
was able to utilize agar as a sole
carbon/energy source. Gas was produced durin
g degradation of agar and gas bubbles
were formed in the shake tubes (see Figure
5a). These bubbles could be CO
2 or H
.
2
6
.
Figure 5a (left) and b (right): a) Gas bubbles in a shake of
ACMOI. b) Shake dilutions of
ACMOI with bromothymol blue. Note the yellow color indicat
ing increased acidity in the
lower dilutions.
Bromothymol blue was added to measure a pH-change during
degradation of
agar (see Figure 5b). The results show a decrease in pH at high
densities of ACMOI,
which is contributed to the production of fatty acids. Attempted
identification of this acid
using GC and HPLC was unsuccessful.
Since the strain did not grow on freshwater-based plates, it poin
ted toward a
requirement of a certain salt concentration. This was tested by
incubating ACMOI in
galactose containing media with various salt concentrations
(see Figure 6).
Figure 6: ACMOI enrichments with varied salt concen
trations. A minimum salt
concentration of 0.5-1% is required for growth.
7
ACMO1 was grown on various substrates to further investiga
te and characterize
its metabolic properties under anaerobic conditions (see Figu
re 7). The results show
increased growth on galactose compared to various fatty acids
.
Optical Density
4-’
(U
0.21
U)
0
C
(U
.0
0.18
I
0.15
0
(I)
0.12
.11(0
0.09
C
U)
0.06
to
(U
0
4-’
0.
0.03
0.00
0
?
-C’
.
C,
Substrate
Figure 7: Optical density of ACMOI at 560 nm indicating
different cell densities on different
substrates.
Biolog plates were also used for a more complete investigation
of the strains
metabolic properties under aerobic and anaerobic condition
s. Unfortunately, no color
development was observed after 4 days of incubation indicat
ing either no growth or no
production of reducing equivalents. Therefore, it is not clear
whether the strain can
perform aerobic respiration or is simply aerotolerant.
Molecular Analysis
.
The restriction fragment length polymorphism yielded 17
distinct band patterns
that were sent for sequencing (Figure 8). A phylogen
etic analysis of the sequences
using the neighbor-joining algorithm from these RFLP-gr
oups fell into 6 groups:
cytophagas, chromatiaceae, rhodobacters, vibrios, spiro
chaetes, and
psuedoalteromonads (Figures 9-11, products noted as
PS#, with numbers
corresponding to Figure 8). The sequence from ACMOI
also fell into the cytophaga,
near Cytophaga fermentans. The Chromatium sequence
s may be representative of
PSBI. Vibrios and rhodobacters are common in mat envi
ronments, as are
spirochaetes, which are also known to utilize agar degradati
on products. While Figure 9
shows all of the products together, each sequence was careful
ly examined in smaller
trees to ensure accurate positioning.
8
.
Figure 8: RFLP pattern from clone library DNA. Numbered lanes
were judged to have distinct band patterns and PCR product
corresponding to these lanes was sequenced. Numbers correspon
d
to the numbers in the phylogenetic trees. “L” denotes a ladder.
Phylogenetic tree
Cytophage
Purple Non-sulfur Becteria
Chromatie,
Purple Sulfur acteria
Psuedoalteromonads
Vibrios
Spirochaetes
0.1
Figure 9: Phylogenetic tree from clone library (“PS”)
and ACMOI PCR products. Note that
sequences similar to ACMOI appeared in the clone
library, as did purple sulfur- arid purple non
sulfur bacteria, a vibrio, a spirochaete, and a psue
doalteromonad.
9
4
Phyogenetic tree
BzetIine3
Cytophaga fermentans
1
P
512
JLP524
P519
PS29
I
.hRCI1e11198313489
I
SUCCIflIC8flS
odoratum
odoratus
.
• 8zeti i rier
Anaeroflexus maritimus
Figure 10: Phylogenetic positioning of the Cytophaga-related
sequences, including the full
sequence from ACMOI.
10
Chromatium purpuratum
Unknown 1144, phototrophic
90Th I nus
—
PS2
PSi 5
L_PS31
Phototrophic Bacteria
Ppc8acte
—
1
P
pcBaet5
I PpcBack3
99LRosea
PpcBact2
PpcUact4
578Purpu
Unkno4
Gamma
—Gamma. 1
•
SuuEndos
Rbmtlar In
—
—
Unknoli43
S78Roseu
35TV 101 a
tepidum
Chromatium vinosum
cBact6
nromatium okenii
Thiocystis qelatinosa
.
0. 1
Figure 11: Phylogenetic positioning of the Chromati
um-related sequences. PSBI is believed
to be a Chromatium.
Interaction
Both the strain from the central white colony and the
purple sulfur bacterium were
isolated. The two strains did therefore not have an oblig
ate syntrophic relationship. It
was more likely that one strain provided a substrate that
promoted growth of the other.
As isolated culture ACMOI formed large colonies in
shake tubes and grew up overnight
in liquid media containing galactose. In contrast, PSB
I formed tiny colonies compared
to the mixed culture. This suggested that ACMO1
provided a substrate from
degradation of agar that promoted growth of PSB
. To test this hypothesis, supernatant
from galactose- and glucose-containing media inoc
ulated overnight with ACMOI was
sterile-filtrated and added to a 7 day old shake-tub
e culture of PSB. As a control,
normal media was added to a higher density shak
e.
.
11
.
Figure 12: Shake tubes of PSB with supematant or media added
superficially. The supernatant of
galactose and glucose degraded by ACMOI enhanced the growth
of PSB. The top-liquid was
inspected microscopically to ensure that no ACMOI bacteri
a was transferred and proliferated.
This experiment was repeated in liquid culture (see Figure 3).
Here one can see
that an increase in the amount of supernatant enhances the growth
of PSB, and thereby
confirming the result. Additionally, Figure 3 shows that the growth
of PSB is generally
improved by addition of short-chain fatty acids. Combined with
the fact that acid is
produced during degradation of agar, these results could point
toward an excretion of a
short-chain fatty acid by ACMOI, which in turn is then utilized,
possibly assimilated, by
PSB during growth.
Another explanation could be that the gas formed during degr
adation of agar by
ACMOI is hydrogen, which provides PSB with reducing equivalen
ts that could be used
for reduction of CO
. Hydrogen has not been directly measured but one might
2
expect
that produced CO
2 would dissolve and not produce bubbles. On the othe
r
hand
, if
larger amounts CO
2 is generated it could form bubbles. Only a direct meas
urem
ent of
the gas could discriminate between the two possibilities.
To test if hydrogen could be a possible metabolic linkage betw
een ACMOI and
PSB, a liquid PSB culture was incubated with either a 2
/C0 or 2
N
/C0 headspace.
H
Additionally, acetate was added (Figure 13).
12
Figure 13: Optical density of PSBI grown with nitrogen or hydrog
en and with or without acetate.
Both acetate and hydrogen can be used growth by PSB and either
one or both of
the substrates are possibly transferred between ACMO1 and PSB.
Since the
supernatant significantly enhanced growth where one would expect
a low concentration
of H
, it is reasonable to say that at least a soluble compound is involv
2
ed, presumably a
short-chain fatty acid.
Finally, a constant number of PSBI was added to shake tubes with
a dilution of
ACMOI in an attempt to regenerate the satellite formation. PSBI
only formed satellite
colonies at low densities of ACMOI. This indicates that a chemic
al gradient of a
compound or relative cell densities may influence this phenomenon.
Conclusions
Phylogenetic analysis based on a full-length 168 rDNA sequence
placed ACMOI
in the genus Cytophaga. Cytophagas are known to be small
rods, that possess gliding
motility, are able to cleave 3-glycosidic bond, grow aerobic as
well anaerobic, and some
of which vary in length depending on growth conditions (pleom
orphic) (Reichenbach
2001). This is consistent with the observations of ACMOI, even
though gliding motility
was not tested. Additionally, members from this group have appe
ared in clone libraries
from a sulfide rich salt marsh in California. Agar degradati
ons is commonly observed in
this group and it has been reported that hydrogen as well short-c
hain fatty acids are
possible fermentations products. Based on molecular and phenot
ypic data, there is
strong evidence that ACMOI belongs to the Cytophaga.
Phylogenetic data generated from clone library and 16S sequ
encing showed an
organism belonging to the group Chromatium. Morphological
examination of PSBI
revealed a highly motile rod with internal sulfur granules and
bacteriochlorophyll-a that
is strictly phototrophic, all characteristic of Chromatium (Pfennig
and Truper 2001).
Combined, these results indicate that PSBI belong to the Chrom
atiaceae.
There is strong evidence for transfer of a metabolic product
generated by ACMO1
from degradation of agar to PSBI. The presence of the fermen
tation product enhances
13
growth of PSBI. It was not possible to directly identify the compound but eviden
ce
points towards either hydrogen or a short-chain fatty acid. Direct measurement
s with
either GC or HPLC are needed for a conclusive answer. The formation of PSB
colonies
surrounding a white colony of ACMOI is therefore generated by the gradien
t of one (or
both) of these compounds and is dependent of the density of both strains.
Future Work
Continued examination of this phenomenon would focus mainly on what
compounds are created by ACMOI and exchanged with PSBI. The compo
unds could
potentially be identified with HPLC and GC as well as some wet chemistry
analysis.
Enrichments of ACMOI for gas production/identification analysis would identif
y whether
2 or CO
H
2 is being produced, and labeled galactose could be followed through both
cultures to examine the products and their utilization. Also, growth rate
experiments for
both cultures on various substrates would further clarify their metabolism, and
molecular
analysis of PSBI would definitively identify it as a purple sulfur- or purple non-su
lfur
bacterium.
Acknowledgements
We would like to acknowledge all of the faculty and students of Microbial
Diversity 2001 for their help and support, particularly Jane Gibson, Alfred
Spoorman,
Carrie Harwood, Bianca Brahamsha, John Waterbury, Dan Buckley, Jochen
Mueller,
and Louie Kerr.
.
14
Literature Cited
Bauld J, Favinger JL, Madigan MT, Gest H. 1987. Obligately haloph
ilic Chromatium
vinosum from Hamelin Pool, Shark Bay, Australia. Curr. Microbiol.
14(6): 335339.
Krassilnikova EN, Kondratyeva EN. 1984. Growth of different purple
bacterial species
belonging to the genus Ectothiorhodospira in the dark. Mikrobiolog
iya 53(3):
526-528.
Pfennig N and Truper HG. 2001. The Family Chromatiaceae. in:
The Prokaryotes
Online. Springer-Verlag. http:link-springer-ny.com:6635
Reichenbach H. 2001. The Order Cytophagales. In: The Prokaryotes
Online.
Springer-Verlag. http:Iink-springer-ny.com:6635
van Gemerden H. 1993. Microbial mats: a joint venture. Mar. Geol.
113: 3-25.
0
15
Appendix A:
PS Medium, shake instructions, fw and sw malate plates
.
16
__
-
Enrichment and isolation of anoxygenic phototrop
liic bacteria
Anoxygenic phototrophic bacteria are physiologically and
phylogenetically diverse.
In the following series of enrichments, different incubation cond
itions will be used in order to
obtain a wide variety of purple sulfur, purple nonsulfur, and gree
n phototrophic bacteria.
Parameters changed include the type of electron donor adde
d (organic/inorganic), the pH, and
light quality. After isolation in pure culture, strains will be
characterized based on their j,igment
composition, the formation and localization of elemental sulfu
r globules, cell morphology and
(partial) I 6S rRNA gene sequence.
1. Green and purple sulfur bacteria
Green and purple sulfur bacteria grow preferentially phot
olithoitotrophically by oxidizing
sulfide, elemental sulfur, polysulfides or thiosulfate as elect
ron-donating substrate in the light.
This growth mode will thus be used to selectively enrich these
bacteria from a variety of
environmental samples.
.
Green sulfur bacteria grow at pH values between 6.5 and 7.0,
at high sulfide concentrations of
up to 5 mM, absorb light of wavelengths below 760 nm, and
are able to exploit low light
intensities. In contrast, purple sulfur bacteria grow only at lowe
r sulfide concentrations, at pH
values above 7.1, absorb light in the infrared wavelength rang
e (up to 1030 nm), and require
higher light intensities. Consequently, different light sources
(fluorescent light, ER light), as
well as different pH values and sulfide concentrationsof the
growth medium are employed to
selectively enrich either for green sulfur bacteria or purple
sulfur bacteria.
Subsamples from different sources (laminated mats from sand
flats, black mats, pink
“berries”, fluffy red/pink cell material from puddles, pink sand
from sand bars in tidal rivers)
are inoculated into mineral media CR or CL:
I
Media (per liter)
CR
NaC1
-
S
CL
20g
—
S
3
Ca
2
2
O
H
CI
PO
2
KH
4
KC1
NHCI
0.15 g
0.20 g
0.50g
0.25 g
After autoclaving and cooling under an 2
/C0 (90/10) atmosphere, add sterile solutions:
N
NaHCO
3
17 mM (final)
N
S
O
2
9H
a
1 mM (final)
2.5 mM (final)
12 vitamin sol.
I ml
1 ml
Trace element sol.
SLI2
SLIO
Final pH
7.2-7.3
6.7-6.8
Light source
>800nm
fluorescent light
>960nm
(selects for BChlb)
-L—
Y”/L.’
--
Composition of solutions used in construction of media during MBL 2000
lOOx FW-Base for Freshwater media (S-. C-. and N-free)
Water
NaCI
MgC
6
O
2
H
l
CaC
•
0
2
2H
l
PO
2
KH
4
KCI
Keep on shelf in clean bottle
I liter
bOg
40 g
10 g
20 g
50g
lx SW-Base for Marine media (S-. C-, and N-free)
Water
20 liter
NaCl
400 g
MgC
6
O
2
60 g
H
l
CaC
•
0
2
2H
3g
l
P0
2
KH
4
4g
KCI
log
Keep in Clean Na/gene plastic bottle
lOOx Ammonium Chloride solution for freshwater or marine media
Water
CI
4
NH
Store in clean bottle
I liter
25 grams
I 000x EDTA-Chelated Trace Elements Stock Solution
Water
EDTA
Adjust pH to 6.0 with NaOH
Add the following:
7H
4
FeSO
O
2
B0
H
3
MnC
•
0
2
4H
I
CoC
6
O
2
H
l
NIC
•
2
6H20
I
CuC
•
0
2
2H
I
ZnS
•
4
0
2
7H
O
N
M
•
4
0
2
2H
oO
a
Sodium Vanadate
N
S
5
3
O
2
eO
H
a
W
Na
•
4
0
2
2H
O
Filter sterilize
987 ml
5200 mg
2100 mg
30 mg
100 mg
190 mg
24 mg
2 mg
144 mg
36 mg
25 mg
6 mg
8 mg
12-Vitamin Solution
Q
Phosph
100 ml
l000x ate buffer, 10mM,
pH7.2
Riboflavin
10 mg
Thiamine•HCI
100 mg
L-Ascorbic acid
100 mg
D-Ca-pantOtheflate
100 mg
Folic acid
100 mg
Niacinamide
100 mg
Nicotinic acid
100 mg
4-Aminobenzoic acid
100 mg
Pyridoxine•HCI
100 mg
Lipoic acid
100 mg
NAD
100mg
Thiamine pyrophosphate
100 mg
Titrate with NaOH until dissolved; Filter sterilize and freeze in 10 ml aliquots
I 000x Vitamin B Solution
Water
100 ml
Cyanocobalamin
100 mg
Titrate with HCI until dissolved; filter sterilize and freeze in 10 ml aliquots
Sundry Solutions:
I M MOPS Buffer, pH 7.2, filtered
I M MES Buffer, pH 6.8, filtered
I M MES Buffer, pH 6.5, filtered
I M Sodium Sulfate, store in clean bottle on slelf
I M Sodium Bicarbonate, autoclaved under 100% 2
CO in serum bottles
I M Methanol, filtered
1 M Sodium Lactate, filtered
1 M Sodium Acetate, autoclaved
0.5 M 4-Hydroxybenzoic acid, titrated with NaOH until crystals dissolved, filtered
0.5 M Trimethoxybenzoic acid, titrated with NaOH until crystals dissolved, filtered
1 M Sodium Sulfide, stored under N
2 at 40 C (throw away when no longer clear)
I % (wlv) Resazurin Dye, stored on shelf in clean bottle
0.4 M Fe(lll) oxyhydroxide, prepared by neutralizing Fe(lll)Cl with NaOH, washed
3x with H
0
2