Appendix II

(1) Determination of ferrous iron

Standard ferrous solution

Weigh out accurately about 14.Og of ferrous sulphate ciystals; dissolve in
450mL of 5% (w/v) sulphuric acid in a 500 mL volumetric flask and make
up to the mark with distilled water. Shake well titrate 25.0 ml with the
standard 0.1N potassium dichromate using diphenylamine as internal
indicator.

Standard 0.1N Potassium dichromate

Weigh accurately 4.9g of potassium dichromate and transfer the salt
quantitatively to a 1 L measuring flask. Dissolve salt in the flask in waste
and make up to the mark, shake well. Calculate exact normality by dividing
the actual weight of the potassium dichromate employed by the theoretical
weight for 1 L of normal solution (49.035). An exactly 0.1N solution may be
prepared by weighing out 4.904g of the salt and dissolving in water and
diluting to 1L in a volumetric flask.

Diphenylamine indicator
Weigh l.Og diphenylamine indicator and dissolve in lOOmL of concentrated
sulphuric acid.

Procedure
Use 3 drops of indicator, add 200ml of 25% (v/v) sulphuric acid followed by
5 ml of 85% orthophosporic acid and titrate slowly, whilst stirring constantly
with standard dichromate until the solution assumes a bluish green or
greenish blue tint near the end point. Continue the titration, adding the
dichromate solution drop wise and maintaining an interval of a few seconds
between each drop, until the addition of one drop causes the formation of an
intense purple or violet blue coloration, which remains permanent after
shaking an is unaffected on the further addition of the dichromate. Carry
out two or at the most three titrations, this should agree with in 0.1 mL.

311
Appendix II

Use of phosphoric acid lowers the oxidation potential of the ferric-ferrous

system by forming complex Fe (HPO4 ") with the ferric ions so that the
equivalence potentials coincides more nearly with that of the indicator.
The action of diphenylamine as an indicator depends upon its oxidation first
into colorless diphenylbenzidine which is the real indicator and is reversibly
further oxidized to diphenylbenzidine violet.
Diphenylbenzidine violet undergoes further oxidation if allowed to stand
with excess of dichromate solution the excess of oxidation is irreversible and
red or yellow products of unknown composition are produced.

lml 0. IN K2Cr20 7 -------------------- ► 5.585 mg Fe+2

(2) Determination of total iron by 1.10- Phenanthroline

spectrophotometric method

Reagents
Stock iron solution
Ferrous ammonium sulphate
Slowly add 20ml of concentrated H2SC>4 to 50mL distilled water and dissolve
1.404g of ferrous ammonium sulphate (Fe(NH4 )2 (S0 4 )2 6 H2 0 ).
Add 0.1 N potassium permanganate drop wise until a faint pink color
persist. Dilute to lOOmL and mix.
lml = 200 pg Fe

Standard Iron solution

Pipette 50mL stock solution in to lOOOmL volumetric flask and dilute to the
mark with distilled water.
lmL = 10jug Fe

Hydroxylamine solution
Dissolve lOg NH2OH.HCl in 100ml distilled water

312
Appendix II

Ammonium acetate buffer

Dissolve 250g NH4 C2 H3 O2 in 150 mL distilled water. Add 700mL
concentrated (glacial) acetic acid. Final volume will be slightly more than
lOOOmL.

1,10-phenanthroline solution
Dissolve lOOmg of 1,10-phenanthroline monohydrate C 12H8N2 .H2 O in
lOOmL distilled water by stirring and heating to 80°C. Do not boil. Discard
the solution if it darkness. Heating is unnecessary if 2 drops of concentrated
HC1 added.

Procedure
Dilute samples that contain iron in range of 20-200|iig.
Add distilled water to make volume 33mL in 50mL volumetric flask.
Add 2mL concentrated HC1 and 1ml NH2 OH.HCI solution.
Add glass beads heat it up to 15 minutes.
Cool to the room temperature and add lOmL NH3 C3 H2 O2 buffer solution and
4mL phenanthroline solution and dilute to mark with distilled water.
Mix thoroughly and allow it at least 10-15 minutes for maximum color
development.
Measure optical density v /s total iron (jig).

131 Acidity

Reagents
Standard sodium hydroxide 0.02N
Disslove 0.8g NaOH and dilute to lOOOmL using CO2 free distilled water
store in air-tight, rubber Stoppard pyrex/coring glass bottle to protect from
atmospheric CO2 . Standardize against 0.02N potassium biphthalate.

Phenolphathalein indicator
Dissolve 0.5g in 500ml 95% ethyl alcohol. Add 500mL distilled water. Add
drop wise 0.02N NaOH till faint pink color appears.

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Appendix II

Methyl orange indicator

Dissolve 0.5g and dilute to lOOOmL with CO2 free distilled water.

Procedure
Measure suitable volume of sample (50 or 100ml) in 250ml conical flask or
beaker depending upon the method to be followed.
Add 2 drops of methyl orange and titrate with standard 0.02N NaOH till
colour changes to faint orange characteristic of pH 4.4 to 4.3.
Note down the volume of NaOH required mark the reading as A.
Add 2-3 drops phenolphthalein indicator and continue titrating with NaOH
till faint colour appears indicating pH-8.3.
Note down the volume of additional NaOH required mark the reading as B.

Calculations
Each mL of 0.02N NaOH = 1 mg CaC0 3 Therefore, acidity mineral or due to
CO2 .

mg/1 CaC0 3 = mL 0.02N NaOH required x 1000

mL sample

In case if normality of NaOH is other than 0.02N calculate as follows.

Acidity mineral or due to CO2 as mg/1 CaCOs = A/B x N x 50000
mL of sample

Where : A = mL NaOH required for sample to raise pH upto 4.4-4.3

B = mL NaOH required for sample to raise pH from 4.4 to 8.3
N = normality of NaOH used.

(4) Sulfate

Reagents
Conditioning reagents
Mix 50mL glycerol with a solution containing 30mL concentrated HC1,
300ml distilled water, lOOmL 95% ethyl alcohol and 75g NaCl.

314
Appendix I!

Barium Chloride
Crystals, 20-30 mesh.

Standard sulphate solution

Dissolve 147.9 mg anhydrous NaaSCUand dilute to lOOOmL.
lmL = 100pg SO4

Procedure
Take suitable volume of sample in 250mL conical flask and dilute to lOOmL.
Add 5mL conditioning reagent accurately, mix well.
Keep the flask constantly stirred with the help of stirrer. Add BaCl2 crystals
while stirring; continue stirring for 1minute, after addition of BaCk.
Measure the turbidity developed after every 30sec for/minute on colorimeter
at 420nm. After 2 minutes stirring reading will remain constant. Note this
reading for calculation purpose.
Prepare standard curve by carrying standard sulfate solution through entire
procedure.
Read mg SO4 present in the sample from the standard curve.

Calculation

Mg/L SO4 = mg SO4 x 1000

mL sample

(5) Determination of arsenic As tVl and As <1111

Material : 12 mL plastic tubes, micropipettes, 1 to 10 cm glass cuvette,

spectrophotometer.

Detection range : 0.005 pmoles As and 0.05 pmoles As and 0.05 pmolesto
0.5 pmoles As when a 10 cm path length cuvette and a 1 cm path length
cuvette are used, respectively. This corresponds to a lowest detection limit of
37.5 (ig/L (10 cm path length cuvette).

315
Appendix II

Chemicals and solutions

Oxidizing reagents:
(1) KIO3 unsaturated solution at room temperature. Dissolve 0.535 g KIO3
in 50 mL dd H2 O
(2) HC1 IN : Dilute 100 ml HCL fuming (37%) in 0.91 dd H20.

Reducing reagents
(1) Dissolve 4.2 g Na2 S2 0 5 , 5H20 in 30 mL dd H20 (0.74 M or 14%).
(2) Dissolve 0.42 g Na2 S20 3 in 30mL dd H20 (0.056 M or 1.4%).
(3) Add slowly (exothermic) 18mL H2SC>4 cone. (1.82 g/L) to 812 mL dd
H20 (=H2 S0 4 3.5 N). Allow cooling and store in a glass bottle.

Mixed Reducing reagent : Mix slowly 5 mL H2SC>4 3.5 N in 10 mL Na2S2Os

solution, avoiding excessive bubbling caused by the liberation of S02
(ventilation hood). Finally mix the 15 mL solution thus obtained with the
lOmL of the Na2S2C>3 solution. Prepare freshly. Prepare freshly before use.
This provides enough reagents for about 25 assays and in enough for the
determination of a set of 10 samples (only 1/3 of the assays has to be
reduced).

Reagents for the Molybdenum complex

(1) Dissolve 15g (NH4)e Mo7 0 2 4 - 4H20 (ammonium paramolybdate) analytical
quality in 500 mL dd H20. Store in a plastic bottle out of direct sunlight.
The .solution is stable in definitely. ---- -
(2) Add 140 mL of concentrated H2S04, analytical grade, d=1.82, to 900 mL
dd H20 (=H2S04 2.6 M). Allow to cool and store in a glass bottle.
(3) Dissolve 27g of good quality ascorbic acid in 500 mL dd H20 . Store the
solution frozen in portions of 50 mL in plastic tubes at -20°C.
(4) Dissolve 0.34g of good quality potassium antimonyltartarate in 250mL of
ddH20, warming if necessary. Store in a glass or plastic bottle. The
solution is stable for many months.

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Appendix II

Mixed molybdenum complex reagent

Mix together 15mL ammonium molybdate,, 37.5 mL sulfuric acid, 15 mL
ascorbic acid and 7.5 mL potassium antimonyl-tartarate solutions. Do not
store for more than about 6 h. This quantity is suitable for about 72
determinations comprising 12 standards and 60 essays (determination of 10
samples).

Procedure
(1) Prepare 10 mL aliquots containing 0.05 pmoles (0.375 pg) to 0.5
pmoles (37.5 pg) As in 12 ml plastic tubes.
(2) Into untreated aliquots add lmL dd H2O.
(3) Into aliquots to be reduced pipet 1 mL of the mixed reducing reagent
and mix well.
(4) Into aliquots to be oxidized add 72.5 pL dd H2O, 250 pL of HC1 1 N
and then 25 pL of 50% saturated KIO3 solution and mix well.
(5) After 15 mins add 1 mL of the mixed molybdenum complex reagent to
each aliquot and mix. It is important to mix the sampled thoroughly
otherwise variability will occur in the absorbance.
(6 ) Measure the absorbance at 865 nm in a 1 to 10 cm path length
cuvette (depending on concentration of the samples to be determined)
against air or water after 90min and within 3h after addition of the
molybdenum reagent.

Calibration curve Complete aliquots containing O7 TO, 20, 40, 60, and 80
pL of a 5 mM As (V+) (samples containing 0.05 pmoles to 0.5 pmoles) or of a
0.5mM As (V+) (samples containing 0.005pmoles to O.Spmoles As) solution
to 11 mL with dd H20 and add 1 mL of molybdenum reagent. Measure as
described under point 6 of the procedure described above.

Calculation There is a linear relationship, y = ax + b, between absorbance

(y) and quantity of As (V+) or phosphate (x) in the samples. Determine the
parameters a and b for the calibration curve (for example with Excel and
sigma plot) and calculate x for all absorbance values y measured for the
samples. Calculate the As (V+) quantity using the relation As (V+) = (As (V+)

317
Appendix II

+ phosphate ) - phosphate (untreated sample - reduced sample) and the As

(III +) quantity by subtracting the value of the untreated samples from the
value of the oxidized samples. Convert the quantity of As (V+) and As (III)
into concentrations taking into account the dilution factor of the samples.
Determine also the correlation factor r2 of the calibration curve, which will
indicate the precision of the measurements.

(6) Determination o f protein with the Folin-Ciocalteau reagent

The phenol reagent of Folin and Ciocalteau which is essentially a

phosphotugstic-phosphomoybdic acid solution is reduced by phenols to
molybdenum which may be reduce the phenol reagent which may therefore
be used for their determination.

Standard
Protein content from samples were detemined using the following protocol
with Bovine serum albumin as standard. A standard calibration curve in the
range of 10-200 pg/mL was used.

Reagents
Reagent I
(1) 5% Na2C03
(2) 0.5% CUSO4 .5 H2 O in 1% sodium potassium tartarate
To 50mL-of—(1) add 2mL of (2) prepare immediately-before use and do not
keep.
Dilute Folin-Ciocalteau reagent: Dilute it 1:1 to get IN solution

Procedure
(1) Measure 0.5mL of washed cell suspension (ca. 100 fag of wt. of cell)
(2) To this add 0.5mL of 1.0 N NaOH place in a boiling water for 5
minutes and cool in cold water.
(3) Add 0.5mL of Folin-Ciocalteau reagent.
(4) After standing 30 minutes to allow full colour development. Measure
O.D. at 750 nm.

318
Appendix II

(7) Total arsenic was analysed by using atomic absorption

spectrophotometer model ELICO-194 by standard procedure

Standard solution preparation (1000 ppm).

Dissolve 1.3203g of dried AS2O3 in the minimum volume of 20% NaOH
solution practicable and neutralize with nitric acid dilute to 1 L.
Atomic absorption : Lamp Current - 8.0 mA
Flame type - Nitrous oxide-Acetylene
(Stoictimetric)
Flame emission : Arsenic is not normally determined by emission

(8) Bacterial genomic DNA isolation using CTAB

(1) Grow the cells overnight in LB broth.

(2) Take 1.5mL of cell is microfuge or centrifuge at 10,000 rpm for 5
minutes.
(3) Resuspend cells in TE buffer adjust OD600 approximately 1.0.
(4) Add 20|iiL of lysozyme (cone. lOOmg/mL). Mix well.
(5) Incubate for 5 minutes, at room temperature.
(6) Add 40juL of 10% SDS. Mix well.
(7) Add 8pL of proteinase K (lOmg/mL)
(8) Incubate for 1 h at 37°C.
(9) Add 100 pL of 5N NaCl. Mix well. -----
(10) Add lOOpL CTAB/NaCl (heated to 65°C) for 10 min.
(11) Incubate at 65°C for 10 minutes
(12) Add 0.5mL of chloroform : isoamylalcohol (24:1). Mix well.
(13) Spin at max speed for 10 min at room temperature.
(14) Transfer aqueous phase to clean eppendrof (should not be viscous).
(15) Add 0.5mL of phenol : chloroform : isoamylalcochol (25:24:1). Mix
well.
(16) Spin at maximum speed for 10 min at room temperature.
(17) Transfer aqueous phase and add 0.6 vol isopropanol (~20°C).
(18) Incubate at room temperature for 30 min.

319
Appendix II

(19) Spin at maximum speed for 15 minutes.

(20) Wash pellet with 70% ethanol, spin at max speed for 5 min.
(21) Discard the supernatant and let pellet dry for 5 - 10 min at room
temperature.
(22) Resuspend in 20pL TE plus RNAse (99pL TE + lpL RNAse (10
mg/mL)).
(23) Transfer to sterile microcentrifuge tubes.
(24) Incubate at 37°C for 20 min.
(25) Run 1 pL in a 1% agarose gel with concentration standards.

(9) Method for plasmid isolation (mini-prep) using alkali lysis method
(saunders, S.E. and J. F. Burke)

Reagents
Solutions should be made with high quality water, such as MiliQ water and
all solution should be autoclaved as indicated before use to inactivate any
DNAses if present.
Solution I :
5 mL of 1M glucose stock
2.5 mL of a 1M Tris-HCl, pH 8 stock at pH 8
2 mL of 0.5M EDTA stock to 91.5 mL autoclaved dHaO
Solution H :
0.9 g NaOH to 90 mL dHaO autoclave and then add 10 mL of 10% SDS
solution ------ ------
Solution III:
Dissolve 29.45 g potassium acetate in50mL dHaO
add 11.438 mL glacial acetic acid
make up to lOOmL with dHaO and autoclave

Procedure
(1) Resuspend the pellet from 1.5 mL active culture grown overnight on
LB in 100 pL solution I. If using autotrophic microbes, increase the
culture volume 50 to 100 fold.

320
Appendix II

(2) Add 200 pL of solution II and gently mix and swirl to ensure complete
lysis. Do not mix too vigorously otherwise chromosomal DNA will
contaminate the preparation.
(3) Add 150 pL of solution III and immediately mix contents completely.
Do not mix too vigorously otherwise chromosomal DNA will
contaminate the preparation.
(4) Centrifuge for 5 min in a microfuge.
(5) Transfer 380 pL of the supernatant to a clean microfuge tube and add
720 pL isopropanol.
(6) Centrifuge for 15 min in a microfuge.
(7) Carefully remove the supernatant leaving the pellet at the bottom of
the tube.
(8) Wash the pellet with lmL of ice-cold 70% ethanol. Centrifuge for 5
min at 12,000 g and remove the supernatant. Be very carful here as it
is easy to lose the pellet.
(9) Diy the pellet and then resuspend it in 49 pL of dH20 and add 1 pL of
RNase 1 pL of this should be plenty for agarose gel analysis and for
restriction enzyme analysis.

321