APPENDIX III: Reagent composition
1. 1% Soft agar
1 g of agar was weighed and added to 100 mL of dH2O and sterilised using autoclave at 121˚C
for 15 min.
2. 0.85% saline
0.85 g of sodium chloride was weighed and dissolved completely in 80 mL of distilled water
and made up to 100 mL with dH2O and sterilised using autoclave at 121˚C for 15 min.
BIOCHEMICAL CHARACTERIZATION REAGENTS
3. Gram’s Crystal Violet
20 g of crystal violet was dissolved in small volume of dH2O, to this 8 g of ammonium oxalate
was added and made up to 800 mL. Further 200 mL of ethanol was added and stored in amber
colour bottle and allowed for maturation for period of 48 h at RT. Filter through paper prior to
use.
4. Gram’s Iodine
3.3 g of iodine and 6.6 g of potassium iodide was dissolved completely in small volume of
dH2O and then made up the volume to 1000 mL and stored in amber colour bottle and allowed
for maturation for period of 48 h at RT.
5. Counter staining Safranin
2.5 g of Safranin was dissolved in small volume of dH2O and made up the volume to 900 mL.
To this 100 mL of ethanol was added and stored in amber colour bottle and allowed for
maturation for period of 48 h at RT.
6. 2 mol L-1 sodium hydroxide
8 g of sodium hydroxide was weighed and dissolved completely in 80 mL of distilled water
and made up to 100 mL with same. The solution was sterilised by steam under pressure.
7. 2 mol L-1 hydrochloric acid
1.8 mL of concentrated hydrochloric acid was added 15 mL of distilled water and mixed well.
It was then made up to 100 mL with distilled water. The solution was steam sterilised.
8. 20 mmol L-1 citrate buffer pH 3.0
0.36 g of citric acid was dissolved in 30 mL of distilled water. 0.09 g of tri sodium citrate was
dissolved in another 30 mL of distilled water. Both were mixed together and made up to
100 mL with distilled water.
9. Acid protease preparation
6 mg of acid protease was dissolved in 2 mL of citrate buffer and filter sterilised.
10. 40% potassium hydroxide (KOH)
4 g of potassium hydroxide was dissolved completely in 5 mL of distilled water and made up
to 10 mL with same.
11. Barritt’s Reagent
0.5 g of  – Naphthol was dissolved completely in 4 mL of absolute ethanol and made up to
10 mL with the same.
12. 3% hydrogen peroxide solution
1 mL of 30% hydrogen peroxide solution was diluted to 10 mL with distilled water and kept in
brown bottle. This was prepared fresh before use.
13. 6N perchloric acid
5.1 mL of 70% perchloric acid was pipetted and made up to 10 mL with distilled water.
14. 6N sodium hydroxide
12 g of sodium hydroxide was dissolved completely in 20 mL of distilled water and made up
to 50 mL with same.
15. Hydrazine - glycine buffer
3 mL of hydrazine hydrate was pipetted out and dissolved in 20 mL of water. 3.7 gm of
glycine was dissolved completely in 30 mL of distilled water. Both the solution were mixed
together and the pH was adjusted to 9 and finally made to 100 ml with distilled water.
16. NAD solution
10 mg of NAD was weighed and dissolved completely in 5 mL of hydrazine- glycine buffer.
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GENOMIC DNA REAGENTS
17. 1 mol L-1 Tris pH 8 (1 mol L-1)
Add 12.11 g of Tris in 80 mL of distilled water adjust the pH to 8 with concentrated HCl and
make the final volume to 100 mL with distilled water. Sterilise the solution by autoclaving at
121˚C for 15 min.
18. EDTA pH 8.0 (0.5 mol L-1)
Add 18.61 g of disodium EDTA to 80 mL of distilled water. Stir vigorously on a magnetic
stirrer. Adjust the pH to 8.0 with 6 mol L-1 NaOH and make the final volume to 100 mL with
distilled water and sterilise by autoclaving. The disodium salt of EDTA will not go into
solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.
19. Wash Buffer (25 mmol L-1 Tris-10 mmol L-1M EDTA, pH 8.0)
Mix 1.25 mL of 1 mol L-1 Tris and 1 mL of 0.5 mol L-1 and make to 50 mL of with distilled
water.
20. Protoplast buffer (25 mmol L-1 Tris, 10 mM EDTA, 50 mmol L-1 Glucose, 10 mg mL-1
lysozyme, 100 µg mL-1 RNaseA pH 8.0)
To 4 mL of wash buffer, add 0.465 g of dextrose, dissolve completely, add 50 µL of RNase
from 10 mg mL-1 stock and make to 5 mL with wash buffer. To this add 50 mg of lysozyme
and dissolve completely.
21. SDS 10% (w/v)
Dissolve 10 g SDS in 100 ml of distilled water with stirring. Avoid frothing of the preparation.
22. Tris saturated phenol
Crystalline phenol was melted at 68°C using water bath. To the melted phenol, equal volume
of 0.5 mol L-1 Tris-Cl (pH 8.0) buffer was added at RT and the mixture was stirred on a
magnetic stirrer for 15 to 30 min for phase separation. When the two phases are separated, the
upper aqueous phase was carefully removed. To this phenol again an equal volume of
0.1 mol L-1 Tris-Cl (pH 8.0) was added and the mixture was stirred on a magnetic stirrer for
phase separation. The upper aqueous phase again was removed as described above. The
extractions were repeated until the pH of the bottom phenolic phase was >7.5 (measured with
pH paper). The saturated phenol with the pH of 7.5 – 8.0 was preserved in amber bottle and
stored at 4˚C. Wearing of gloves is essential when carrying out this procedure.
23. Chloroform : isoamyl alcohol (24:1)
24 mL of chloroform is mixed with 1 mL of isoamyl alcohol. This reagent is freshly prepared
before use. It can also be stored at 4˚C protected away from light.
24. TE buffer
0.1 mL of 1 mol L-1 Tris of pH 8.0 and 0.02 mL of 0.5 mol L-1 of EDTA was added and made
up to 10 mL with distilled water.
25. TBE buffer 1X (v/v)
5.4 g Tris base, 2.75 g boric acid, 20 mL of 0.5 mol L-1 EDTA adjusted to pH 8.0 and made up
the volume to 1L and stored at 4˚C.
26. Ethidium Bromide Stock Solution (10mg/ml)
10 mg ethidium bromide was dissolved in 1ml distilled water and the solution was stored in
amber bottle at RT or at 4˚C. Wear gloves to avoid contact with ethidium bromide as it is
toxic.
27. 1% agarose
1 g of agarose was added to 100 mL of 1X TBE buffer. The solution was heated to boiling in
the microwave oven until agarose completely dissolves. Cool the solution to bearable heat and
add 2 µl of ethidium bromide, mixed well and poured in agarose gel plate to solidify.
28. 2% agarose
2 g of agarose was added to 1X TBE buffer. The solution was heated to boiling in the
microwave oven until agarose completely dissolves. Cool the solution to bearable heat and add
2 µL of ethidium bromide, mixed well and poured in agarose gel plate to solidify.
29. 0.1% Congo red
0.1 g of Congo red dissolved completely in 100 mL of distilled water
30. 1 mol L-1 NaCl
5.84 g of NaCl was dissolved completely in 100 ml of distilled water
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31. 25 mmol L-1 acetate buffer pH 5
0.17 g of anhydrous sodium acetate was dissolved in 40 mL of distilled water and to this
0.02 mL of glacial acetic acid dissolved in 20 mL of distilled water was mixed and made up to
100 mL with distilled water. The solution was sterilised by autoclave.
32. Dinitrosalicylic acid reagent
1 g of 3, 5 dinitrosalicylic acid was dissolved completely with solution containing 0.05 g
sodium sulphate, 18.2 g sodium potassium tartarate and 1 g sodium hydroxide and made up
the volume to 100 mL. It was stored at 4˚C in amber bottle.
33. 40% Rochelle salt
40 g of sodium potassium tartrate (Rochelle salt) was dissolved in 50 ml of distilled water and
finally volume made up to 100 mL.
34. 10 mmol L-1 of ethylenediaminetetraacetic acid
0.37 g of EDTA was dissolved completely in 70 mL of water and made up to 100 mL.
35. 0.2 mol L-1 phosphate buffer
Stock Solution A: 3.56 g disodium hydrogen phosphate was dissolved in small volume of
distilled water and make up the volume to 100 mL
Stock Solution B: 2.76 g of sodium dihydrogen phosphate dissolved in small volume of
distilled water and made the volume to 100 mL. The stock solutions can be stored at RT.
Working solution: 40.5ml of solution A is mixed with 9.5 ml of solution B and pH was
adjusted to 7.4 and finally made the volume to 5oml. The working solution must be prepared
fresh before use.
36. 2% glutaraldehyde
5 mL of 0.2 mol L-1 phosphate buffer containing 1.2 mL of 25% glutaraldehyde solution and
3.8 mL-1 of distilled water. This reagent must be prepared fresh before use.
37. 1% osmium tetroxide
0.1 mL of osmium tetroxide was made the volume to 1 mL with 0.2 mol L-1 phosphate buffer
pH 7.4. Osmium tetroxide is highly volatile so must be prepared fresh whenever for use in
minimal quantity and preserved in an air tight container. It is toxic and irritant so handled with
care.
PROBIOTIC PROPERTIES
38. Gastric juice
0.01 g of NaCl was dissolved in small volume to distilled water whose pH was adjusted to 2.5
with 1 mol L-1 of HCl and made up to 10 mL with distilled water. To this 30 mg of pepsin was
added and filter sterilised.
39. Intestinal Juice
0.01 g of NaCl was dissolved in small volume to distilled water whose pH was adjusted to 8
with 1 mol L-1 of NaOH and made up to 10 mL with distilled water. To this 10 mg of pepsin
and 0.03 g of bile were added and filter sterilised.
40. 33% KOH
16.5 g of KOH was dissolved in 20 mL of water and made up to 50 mL with distilled water
41. o- phthalaldehyde reagent
50 mg of o- phthalaldehyde was dissolved in 100 mL of glacial acetic acid. This reagent has to
be prepared just at the moment
42. Phosphate buffered saline (PBS) (1X)
8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g disodium hydrogen phosphate,
0.24 g potassium dihydrogen phosphate dissolved completely in small volume of water,
adjusted the pH to 7.4 using HCl and finally made up the volume to 1L.
43. Z - buffer
4.27 g of disodium hydrogen phosphate, 2.75 g sodium dihydrogen phosphate, 0.375 g of
potassium chloride and 0.125 g of magnesium sulphate was dissolved in small volume of
distilled water. The pH was adjusted to 7.0 and finally made the volume to 500 mL with
distilled water. This buffer was stored at 4˚C. Prior to use in the experiment freshly mix 50 mL
of Z buffer and 0.14 mL of β – mercaptoethanol.
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44. ONPG reagent (4 mg/mL)
80 mg of ONPG was dissolved in 20 mL of appropriate buffer. This reagent was prepared
fresh before use.
45. 1% sodium carbonate
0.5 g of sodium carbonate was dissolved completely in 50 ml of distilled water and stored at
4˚C until use.
46. Trypsin-EDTA solution
0.05 g of trypsin and 0.02 g of EDTA was dissolved in sterile PBS solution and made the
volume to 100 mL with the same buffer. The solution can be stored at 4˚C.
47. Trypan blue dye 0.04% (w/v)
0.04 g trypan blue was dissolved in 100 mL PBS without calcium chloride or magnesium
chloride salts and solution is stored at RT
48. Acridine orange staining solution
0.1 g of acridine orange was dissolved in distilled water and made up to 100 mL with the
same.
PURIFICATION OF ANTIMICROBIAL SUBSTANCES
49. 1% sodium deoxycholate
0.1 g sodium deoxycholate is dissolved in 10 mL of distilled water.
50. Biuret reagent
Solution A: 0.75 g of copper sulphate was dissolved in 100 mL of distilled water.
Solution B: 3 g of sodium potassium tartarate was dissolved in 100 mL of distilled water.
Solution C: 15 g of sodium hydroxide was dissolved in 200 mL of distilled water.
Solution A and Solution B were mixed together. This mixture was added to solution C and
made up to 500 mL.
51. Standard BSA 10% (w/v)
0.1 g BSA is dissolved in 10 mL of distilled water and stored at 4˚C.
52. 5 mmol L-1 acetate buffer pH 5.2
0.35 g of anhydrous sodium acetate was dissolved in distilled water followed with addition of
0.04 mL of glacial acetic acid dissolved in water and made up to 1L with distilled water.
53. Chromatographic matrix preparation
The Sephadex was weighed and slowly added to acetate buffer with slow stirring with glass
rod and heated in water bath at 60˚C. The matrix was allowed to swell for overnight and
packed in glass column.
54. Acrylamide solution (42.5%T; 6%C)
46.5 g of acrylamide is dissolved completely in 40 mL of double distilled water. Then 3 g of
Bis acrylamide added and dissolved completely. The final volume was made up to 100 mL
with distilled water. Filter with Whatmann filter paper No 1 if essential.
55. Acrylamide solution (42.5%T; 3%C)
48 g of acrylamide is dissolved completely in 40 mL of double distilled water. Then 1.5 g of
Bis acrylamide was added and dissolved completely. The final volume was made up to
100 mL with distilled water. Filter with Whatmann filter paper No 1 if essential.
56. Gel Buffer
Dissolve 33.33 gm of Tris in 50 mL of double distilled water and pH was adjusted to 8.42 with
conc. HCl. The final volume was made up to 100 mL and add 0.3 gm of SDS.
57. 10% ammonium persulphate
0.1 g of APS was dissolved in 1 mL of distilled water. This solution was prepared just before
use
58. 10X anode buffer
24.23 g of Tris was dissolved in 40 mL of double distilled water and pH is adjusted to 8.9 with
conc. HCl. Then the volume is made up to 100mL with double distilled water.
59. 10X cathode buffer
12.11 g Tris was dissolved in 30 mL of double distilled water. Similarly 17.92 g of Tricine
was dissolved in another 30 mL of double distilled water. The two solutions were mixed and
make up to 100 mL with double distilled water. Then add 1gm of SDS to this solution.
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60. 4X SDS sample buffer (150 mmol L-1 Tris pH 7, 30% glycerol, 6% mercaptoethanol, 4%
SDS (w/v), 0.05% Coomassie blue G)
0.45 g of Tris (150 mmol L-1) was dissolved in 10 mL of distilled water and pH was adjusted
to 7 with concentrated HCl. To this 7.5 mL (30%) of glycerol was added and dissolved
completely followed with addition of 1.5 mL of mercaptoethanol and made up to 25 mL with
distilled water. To this 1 g of SDS was added and dissolved completely after which 0.013 g of
CBB-G was added. One volume of this sample buffer was mixed with 3 volume of water or
appropriate concentration of sample buffer.
61. 5% glutaraldehyde
20 mL of gluteraldehyde (25% solution) was made up to 100 mL with distilled water. This
solution was prepared just before used.
62. Coomassie brilliant blue staining solution
0.025 g of Coomassie brilliant blue G was dissolved in 10 mL of glacial acetic acid (10%) and
made to 100 mL with distilled water. The solution was stored in amber bottle at RT.
63. Destaining solution
10 mL of glacial acetic acid (10%) made up the volume to 100 mL with distilled water
64. Isopropanol - Acetic acid mixture
20 mL of isopropanol (20%) and 10 mL of glacial acetic acid (10%) was made up the volume
to 100 mL with distilled water. This solution was prepared just before use.
65. Sep-Pak Activation
The Sep-Pak C18 cartridge was washed with 5 mL of 100% acetonitrile followed with 15 mL
of water wash.
66. Salkowski reagent
2.03 g of FeCl3 was dissolved in 500 mL of distilled water and 300 mL of concentrated
sulphuric acid was added. This solution is stable indefinitely.
EXOPOLYSACCHARIDE REAGENTS
67. 5% phenol
5 g of phenol was dissolved completely in distilled water and made to 100 mL with distilled
water. The reagent was stored at room temperature in amber bottle.
68. 50% trichloroacetic acid
25 g TCA was dissolved in distilled water and made up to 50 mL with the same.
69. Methanolic DPPH
0.008 g of DPPH was dissolved in methanol and made up to 100 mL with same. This reagent
was prepared fresh.
70. 6% hydrogen peroxide
4 mL of H2O2 (30% w/v) was made up to 20 mL with distilled water. This reagent was
prepared fresh in dark bottle.
71. 10 mmol L-1 ferrous ammonium sulphate-EDTA complex
0.078 g of FAS was dissolved in distilled water to which 0.074 g of disodium salt of EDTA
was added and made up to 20 mL
72. Safranine
5 mg safranine was dissolved in distilled water and made up to 10 mL with distilled water in
10 mL of distilled water.
73. 1% potassium ferricyanide
0.1 g of potassium ferricyanide was dissolved in distilled water and made up to 10 mL with
distilled water
74. 0.1% ferric chloride
0.05 gm of ferric chloride was dissolved in distilled water and made up to 50 mL with distilled
water
75. 100 µmol L-1 Congo red
0.7 g of Congo red was dissolved in distilled water and made up to 10 mL with same.
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