• Labtop for Microbiology LaboratoryTable of Contents
S. No.
1.

Page number
9

6.
7

Title
Rates in centrifugation
a. Settling rate
b. RCF
c. Volume and centrifugal force
d. Fixed versus swing out rotor
Glass ware cleansing solution
Common tissue preservatives
Trypan blue solution for intravenous use
Buffers used for suspending live cells, bacteria and other
live materials
Buffers/solution for plasmid/chromosomal DNA isolation
Solutions for plasmid isolation by alkaline lysis

8.

Buffers/solutions for SDS-PAGE

13-14

9.

Recipes for making discontinuous dissociating gels

14

10.

Recipes for making discontinuous non-dissociating gels

14

11.

Recipes for making continuous non-dissociating gels

14

12.
13.
14.

Gel processing solutions:
ELISA-reagents and buffers
Solutions for estimation of protein

15
15-17
17-18

2.

3.
4.
5.

10
10
10
10-11
11-12
12-13

A. Modified Lowry’s method
B. UV spectrophotometery
C. Biurate method
D. Bicinchoninic acid method
E. Dye binding method
F. Silver binding method
15.

pH of standard solutions of common chemicals used in 19

16.

laboratory
Physical characteristics of common acids and alkalies 19

17

used in laboratory
Buffers for specific purposes

a. Mcilvaine’s buffer 20
b. Phthalate buffer
c. PBS, 0.025 m, pH 6.0 and 6.8
d. PBS, 0.15 m (pH 7.0)
e. PBS, pH 7.2
f. PBS, pH 7.4
g. PBS, pH 7.3
h. Azide saline, pH 7.3
i. Borate calcium saline, pH 7.3

20-25

1


j. Veronal-NaCl diluent 5x
k. Sorensen’s citrate buffer
l. Citrate phosphate buffer for pH 2.6 to 7.0
m. Citrate buffer pH 3.0 to 6.2
n. Phosphate buffer for pH 5.7 to 8.0 (0.25m)
o. Phosphate buffer for pH 5.8 to 8.0 (0. 5 m)
p. Potassium phosphate buffer for pH 5.8 to 8.0 (0. 1 m)
q. PBS (bacteriological)
r. Tris buffer
s. Barbital buffer (0.05 m) pH 6.8 to 9.2
t. Borate buffer (0.0125 m) pH 8.1 to 9.0
u. Borate buffer (0.0125 m) pH 9.3 to 10.7
v. Glycine buffer (0.05 m) pH 8.6 to 10.6
w. Sodium carbonate bicarbonate buffer (0.05 m) pH 9.2
18.

19.
20.

21.

22.
23.
24.

25.

to 10.8
x. Carbonate buffer (0.025 m) pH 9.7 to 10.9
Antibiotic solutions
Sugar solutions
Dye solutions and indicators
A. Common dyes
B. Andrade’s indicator
C. Litmus solution
Common culture media used in microbiology
A. Tryptic soy broth
B. Semisolid phosphate buffered agar
C. Gelatin agar
D. Organic acid media
E. Decarboxylase test media
F. Luria bertani broth
G. 1% peptone water
H. RPMI-1640 growth medium
I. MEM (minimum essential media)
J. M-9 agar

K. Aro mix
Clinical sample transport media
Pre-enrichment media and resuscitation media
Storage of cultures for future use
A. On/in media
a. Dorset egg medium
b. Buffered semisolid nutrient agar
c. Glycerol broth
d. Stock culture agar
e. In sterilized powder of multani mitti (fullers earth)
B. Freezing the cultures
C. Freeze-drying
Some special media and reagents required in bacteriology
A. Media for blood cultures in cases of typhoid
B. Swarm-agar (Guard plate)

25-26
26
27

28-30

30
31
31-33

33
332
33-35


2


26.

C. Semi-solid agar in u tubes
D. Worfel Ferguson medium for capsule enhancement
E. Minimal salt medium
F. Glucose minimal salt agar medium
G. Minimal agar medium
H. Deca strength phage broth (DSPB)
Bacteriological
media
used
in
biochemical 36-43
characterization of bacteria
A. MR-VP test medium
B. Dextran and levan production medium
C. Sucrose broth
D. Starch agar
E. Aesculin broth
F. Aesculin blood agar
G. Capsulation medium for Bacillus anthracis (serumbicarbonate agar)
H. Casein agar
I. Christensen’s citrate medium
J. Castaneda medium:
K. Koser’s citrate medium
L. Simmons’ citrate
M. Organic acid medium

N. Decarboxylase medium
O. Hippurate hydrolysis medium
P. KCN broth
Q. Loeffler’s serum slants
R. LJ medium (lowenstein-jensen medium for mycobacteria)
S. Lecithinase agar/ lecithinovitellin agar
T. Malonate-phenylalanine medium
U. Litmus milk
V. Purple milk
W. Purple milk
X. Motility agar
Y. Sulphide indole motility agar
Z. MRS lactobacillus agar
Aa. Nitrate reduction test media
Ab. Nitrite medium
Ac. O/F test medium
Ad. ONPG broth
Ae. Sodium potassium magnesium (SPM) broth
Af. Phenolphthalein phosphate agar
Ag. Phenylalanine agar
Ah. Polyhydroxybutyrate agar
Ai. Pyruvate fermentation medium
Aj. Salt broth
Ak Selenite broth
Al. Soil extract agar
Am. Streptomyces extraction medium for preparing of
streptococcal group antigen
An. Todd-Hewitt broth
Ao. Media for antigen extraction from streptococci for
grouping


3


27.

28.

29.
30.
31.
32.

Ap. TSI (triple sugar iron) agar
Aq. Tyrosine hydrolysis agar
Ar. Urea medium
a. Christensen’s medium
b. Christensen’s broth medium
As. Xanthine and hypoxanthine agar
Some basic media used in microbiology
A. Nutrient broth (NB)
B. Nutrient agar
C. Semisolid NA
D. Peptone water
E. PW agar
F. Robertson’s cooked meat medium (RCM)
G. Brain heart infusion broth (BHI)
H. BHI agar
I. Mueller Hinton agar
J. Blood agar (BA)

K. Layered BA
L. Chocolate agar
M. Serum agar
N. Serum glucose agar
O. Fildes agar and broth
P. Glucose broth
Q. Glycerol broth
R. Mac-Conkey broth double strength
S. Laural tryptose broth, double strength
T. Brilliant green lactose bile broth
U. Tryptone water
V. Membrane lauryl sulphate broth
Media for detecting pigment production ability of
bacteria
A. Chromobacterium
B. Mycobacterium
C. Pseudomonas
D. Serratia marcescens
E. Micrococcus spp
F. Melaninogenicus oralis
G. Clostridium difficile
H. Potato slopes
I. Mannitol yeast extract agar
J. King’s medium for pyocyanin
K. King’s medium for fluorescine
Mc-Farland standard preparation
Some important parameters for experimental animals
used in microbiology
Sizes of hypodermic needles used in microbiology
Precipitation of protein antigens without denaturation

A. Salting out with ammonium sulphate
B. Precipitation with water miscible organic solvent as
ethanol and acetone
C. Precipitation with water miscible organic polymers as
polyethylene glycol

43-45

45-46

46
47
47
48

4


33.

34.

35.

36.

Precipitation of protein antigens by denaturation
A. High temperature
B. Extreme pH
C. Organic solvents

Reagents required for different bacteriological tests
A. Phenylalanine test reagent
B. Gelatinase and caseinase test reagents
C. MR reagent
D. Nitrate test reagent
E. VP test reagents
F. Ehrlich’s reagent for indole test
G. Benedict’s reagent for presence of reducing sugars
H. Acid ferric chloride for phenylpyruvic acid
I. Acid mercuric chloride for proteinases
J. Nessler’s reagent
Bacteriological test strips impregnated with reagents
a. For detection of H2S
b. For indole test
c. PPA test
d. For H2O2
e. For oxidase test
f. O/129 discs for Vibrionaceae
g. Optochin discs
h. Liquoid discs for inhibition of Peptococcus
anaerobius & Streptobacillus moniliformis
i. X-factor discs
j. V-factor discs
Common rapid tests for identification of bacteria
a. Acetyl-methyl carbinol production test (VP test)
b. Aesculin bile test for aesculin hydrolysis by
streptococci
c. Bile solubility test
d. Pseudocatalase
e. Coagulase:

f. Rapid coccal transformation
g. Decarboxylase test
h. DNase test
i. Gelatinase test
j. Hippurate hydrolysis test for enterobacteriaceae
k. Indole test
l. Gluconate oxidation test
m. Gluconate utilization broth
n. Malonate utilization
o. Niacin (nicotinic acid) test
p. ONPG (o-nitrophenyl-β-d-galactopyranoside) test
q. Oxidase test (cytochrome oxidase)
r. Catalase test
s. Phenylalanine test
t. Levon/ dextran production test
u. Porphyrin test (for determining requirement of factor
X)
v. Phosphatase test

48-49

49-50

50-51

52-60

5



w.
x.
y.
z.

37.

38.

Tween hydrolysis
Urease test
X & V factor requirement test
Carbon dioxide requirement for bacterial growth
(candle jar)
aa. Nitrate reduction test
Ab. Temperature tolerance and growth temperature
Ac. Co-aggregation test
Ad. Carboxymethyl cellulose hydrolysis test
Ae. Pyrase test
Af. Benzidine test
Stains and reagents used in microbiology
A. Acetone-iodine solution
B. Strong iodine solution
C. Acid alcohol
D. Albert’s stain
E. Ammonical silver nitrate
F. Ammonium oxalate crystal violet stain
G. Different common dyes.
H. Aqueous iodine (BP)
I. Liquor iodi fortis (BP)

J. Carbol fuchsin
K. Giemsa stain stock
L. Kirkpatrick’s fixative
M. Loeffler’s methylene blue (polychrome methylene blue)
N. Lugol’s iodine
O. Albert’s iodine
P. Iodine acetone decolourizer
Q. Weak-iodine acetone decolourizer
R. Muir’s mordant
S. Plimmer’s mordant
T. Ryu’s mordant
U. Rhodes’ mordant
V. Neisser’s staining solution
W. Ryu’s flagella stain
X. Sudan black
Y. Buffers for dilution of Leishman’s stain and washing of
slides
Staining methods in microbiology
A. Simple staining
B. Gram’s staining
C. Acid-fast staining (Ziehl Neelsen’s method)
D. Auramine-phenol stain for fluorescence method
E. Spore staining
i. Moeller’s method
ii. Schaeffer and Fulton’s method
iii. A modified Ziehl-Neelsen staining procedure
F. Capsule staining
i. Muir’s method
ii. Giemsa method
iii. India-ink method (negative staining)

G. Staining of lipid granules

60-63

63-67

6


39.
40.

41.
42.
43.

44.
45.

46.
47.
48.
49.
50.
51.
52

i. Holbrook and Anderson method
ii. Burdon’s method
H. Metachromatic granule (volutin granules) staining

i. Albert’s method
ii. Neisser’s method
I. Flagella staining
i. Ryu’s method
ii. Cesares-Gill’s method
iii. Rhodes’ method
J. Fungal staining (for cell polysaccharides)
i. Direct microscopy
ii. Parker-blue staining
iii. PAS (per-iodic acid Schiff) staining
K. Staining for spirochetes
i. Fontana’s method for films
ii. Levaditi’s method for staining spirochetes in tissues
DNA staining
Fixatives used in microbiology
a. Formalin
b. Susa’s fixative
c. Bouin’s fluid
d. Schaudinn’s fluid
e. Flemming’s fluid
General protocol for embedding the fixed tissues
Inhibitors for swarming
Sterilization
A. Heat
B. Chemical
C. Radiation
D. Filtration
Media used for sterility testing
Indicator organisms
a. Coliforms

b. Faecal coliforms
c. Faecal E. coli
d. Faecal streptococci
e. Sulphite reducing clostridia
f. Pseudomonas
g. Bacteriophages
Sampling plan for polluted waters
Membrane filtration test for indicator microbes
Air sampling
A. Settle plate method
B. Slit sampler
MPN table for indicator bacteria
Some important conversion factors
Equivalents
Index

67
67-68

68
68-69
59-70

70
70-71

71
71-72
72-73
73-77

78
78
79-83

7


Acknowledgements
Help rendered in terms of facilities to work by the Indian Council of Agricultural Research, India is
thankfully acknowledged. I am thankful for moral support from my wife (Geeta), daughters
(Sumedha and Richa) and Dr. BP Bhatt (Joint ICAR Research Complex for NEH Region, Nagaland
Centre, Jharnapani).

It is dedicated to my father, who put so many efforts for my education but
could not see myself as I am today in his lifetime.

• Labtop Book for Microbiology-

8


1. RATES IN CENTRIFUGATION:
a. Settling rate (cm/second)=2a2g(dp-dm)/9η
Where, dp=density of the particle, g=981 cms-2, dm=density of medium, η =viscosity (in cgs
unit) of medium
b. Relative centrifugal force (RCF, in g)= 1.118×10-5×R×N2
g=9.81ms-2, R=radius of the centrifuge rotor in cm, it is distance between center of centrifuge
shaft and tip of the tube, N=revolution per min.
500g means 500 times the force of gravity i.e., the particle will settle 500 times faster than at
the bench top.

RPM versus RCF Conversion Table
Speed
RPM
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9500
10000
10500
11000
11500
12000
13000
13500
14000


4
45
101
179
280
402
548
716
906
1118
1353
1610
1889
2191
2516
2862
3231
3622
4036
4472
4930
5411
5914
6440
7558
8150
8765

5
56

126
224
349
503
685
894
1132
1398
1691
2012
2362
2739
3144
3578
4039
4528
5045
5590
6163
6764
7393
8050
9447
10188
10956

RCF with Rotor Radius (from center of rotor to sample) in centimeters
6
7
8

9
10
11
12
13
67
78
89
101
112
123
134
145
151
176
201
226
252
277
302
327
268
313
358
402
447
492
537
581
419

489
559
629
699
769
839
908
604
704
805
906
1006
1107
1207
1308
822
959
1096
1233
1370
1507
1643
1780
1073
1252
1431
1610
1789
1968
2147

2325
1358
1585
1811
2038
2264
2490
2717
2943
1677
1957
2236
2516
2795
3075
3354
3634
2029
2367
2706
3044
3382
3720
4058
4397
2415
2817
3220
3622
4025

4427
4830
5232
2834
3306
3779
4251
4724
5196
5668
6141
3287
3835
4383
4930
5478
6026
6574
7122
3773
4402
5031
5660
6289
6918
7547
8175
4293
5009
5724

6440
7155
7871
8586
9302
4847
5654
6462
7270
8078
8885
9693
10501
5433
6339
7245
8150
9056
9961
10867 11773
6054
7063
8072
9081
10090 11099 12108 13117
6708
7826
8944
10062 11180 12298 13416 14534
7396

8628
9861
11093 12326 13559 14791 16024
8117
9469
10822 12175 13528 14881 16233 17586
8871
10350 11828 13307 14786 16264 17743 19221
9660
11269 12879 14489 16099 17709 19319 20929
11337 13226 15115 17005 18894 20784 22673 24562
12225 14263 16300 18338 20376 22413 24451 26488
13148 15339 17530 19722 21913 24104 26295 28487

14
157
352
626
978
1409
1917
2504
3170
3913
4735
5635
6613
7669
8804
10017

11309
12678
14126
15652
17256
18939
20700
22539
26452
28526
30678

15
168
377
671
1048
1509
2054
2683
3396
4193
5073
6037
7085
8217
9433
10733
12116
13584

15135
16770
18489
20292
22178
24149
28341
30563
32869

c. Volume is important factor to achieve results of centrifugation, it determines success of
centrifugation. If using low volume yeast, bacteria and viruses settle at 2,000g, 5,000g and
150,000g, respectively while on using large volumes it needs 5,000g, 20,000g and 200,000g,
respectively.
d. Vertical rotor needs to be run at low speed and for shorter duration than horizontal/ angled or
swing out rotors to achieve the same effect.

2. GLASS WARE CLEANSING:

9


Dichromate-Sulfuric acid solution: Dissolve 63 g of sodium or potassium dichromate by heating
in 35 ml water. Cool and add concentrate H2SO4 to make volume 1 lt. It is very corrosive and
must be handled with utmost care and if splashes come in contact of cloths or skin wash with
plenty of water and neutralize with sodium bicarbonate solution. The dirty glass items can be
directly dipped into it for 1 day or more and then cleaned with water.

3. COMMON TISSUE PRESERVING SOLUTION:
Formal saline solution (10.0% v/v): Used for preservation of pathological specimen and also to

kill bacteria. For killing bacteria 1% formalin is sufficient to preserve the surface antigens of
most bacteria.
Formaldehyde solution (35%)

100 .0 ml

Normal saline solution (0.85%)

900.0 ml

Both solutions were mixed and stored in a capped jar.

4. TRYPAN BLUE SOLUTION FOR INTRADERMAL ASSAY: Solution is
injected intravenously to determine hyper-permeability of blood vessels.
Trypan blue solution (0.1%)
Trypan blue

100 mg

Distilled water100 ml

5. BUFFERS USED FOR SUSPENDING LIVE CELLS, BACTERIA AND
OTHER LIVE MATERIALS
Aqueous solution of

Proportion of solutions to prepare buffer
Ringer
Locke
Krebs-Ringer
Plain

Bicarbonate
NaCl, 9 g/lt
100
100
100
100
KCl, 11.5 g/lt
4
4
4
4
CaCl2, 12.2 g/lt
3
3
3
3
KH2PO4, 21.1 g/lt
1
1
MgSO4.7H2O, 38.2 g/lt
1
1
NaHCO3, 13.0 g/lt
3
21
Phosphate buffer, 0.1M, ---

Phosphate
100
4

3
1
20

pH 7.4
Quarter Strength Ringer’s solution
NaCl, 2.25 g; KCl, 0.105 g; CaCl2, 0.12 g; NaHCO3 0.05 g dissolve in 1lt water and sterilize by
autoclaving (121oC for 15 min) in screw capped vials.
Alsever’s Solution
Glucose

1.866 g

10


Sodium chloride

0.418 g

Sodium citrate

0.8

Citric acid

0.055 g

Distilled water to


100

Sodium chloride

8.5 g

g
ml

NSS
Distilled water to make 1 lt.

6. BUFFERS/SOLUTION FOR PLASMID/CHROMOSOMAL DNA
ISOLATION
1 M Tris (pH 8.0)
Tris base

12.10 g

Distilled water

80.0

ml

Adjusted pH 8.0 with concentrated HCl, make final volume to 100 ml with distilled water.
0.5 M EDTA
Disodium ethylene diamine tetraacetate, 2H2O

18.61 g


Distilled water

80.0

ml

Adjust the pH 8.0 by adding Sodium hydroxide pellets (about 2 gm), EDTA dissolve
only when pH reaches 8, make final volume to 100 ml with distilled water. Sterilize by
autoclaving.
10% SDS
Sodium dodecyle sulphate

10.0

g

Distilled water to

100.0 ml

5M NaOH
Sodium hydroxide

20.0

g

Distilled water to


100.0 ml

5M Potassium Acetate
Potassium acetate

49.075 g

Distilled water to

100.0 ml

3M Sodium Acetate (pH 5.2)
Sodium acetate, 3H2O

40.81 g

Distilled water

80.0

ml

Adjust pH 5.2 with glacial acetic acid, make final volume in 100 ml.
5M NaCl
Sodium chloride

29.20 g

11



Distilled water to

100.0 ml

10% Cetyl Trimethyl Ammonium Bromide (C-TAB)
CTAB

10

g

Distilled water to

100.0 ml

Proteinase K
Stock solution of 20 mg/ml in Tris (pH 8, 50 mM) calcium acetate (1.5 mM) solution or in
distilled water is prepared aliquoted and kept at –20ºC.
RNase A (free from DNase)
Stock solution of 10 mg/ml in Tris chloride buffer (10mM Tris – HCl, pH 7.5 and 15mM
NaCl) or 2 mg in 2 ml of TE (100 mM Tris Cl, pH 7.6 and 10 mM EDTA), stored at –20ºC.

7. SOLUTIONS FOR PLASMID ISOLATION BY ALKALINE LYSIS
Solution A (5 ml) - Can be stored at 4ºC for 48 hr.
Sterile distilled water

4.525 ml

40% dextrose


0.25

1M Tris (pH 8)

0.125 ml

0.5M EDTA

0.1

ml

Lysozyme

25

mg

Sterile distilled water

4.3

ml

10% SDS

0.5

ml


5M NaOH

0.2

ml

Sterile distilled water

2.85

ml

5M Potassium acetate

6.0

ml

Glacial acetic acid

1.15

ml

ml

Solution B (5 ml) – Make Fresh

Solution C (10 ml) - Can be stored at room temperature


TBE Buffer (pH 8.3) 5X
54 gm Tris base, 27.5 gm boric acid, 20 ml of 0.5 M EDTA (pH 8.0)
TAE Buffer (pH 7.6)

10X

50 X

1X

Tris base

48.4 gm

242 gm

40 mM Tris acetate

Glacial acetic acid

11.4 ml

57.1 ml

1 mM EDTA

0.5M EDTA (pH 8)

20 ml


100 ml

Distilled water to

1000 ml

1000ml

TPE Buffer (1X= 90 mM Tris Phosphate with 2mM EDTA)

12


For 10 × solution
180 gm Tris base, 15.5 ml phosphoric acid (85%, 1.679 gm/ml) and 40 ml of 0.5 M EDTA
(pH 8), in 1 lt DW
TE Buffer (pH 8)
1M Tris HCl (pH 8)

1.0

ml

0.5M EDTA (pH 8)

0.2

ml


Distilled water to

98.8

ml

Tris Glycine buffer (1X= 25 mM TRis –HCl, 250 mM glycine, 0.1 % SDS)
5 × Solution is made by dissolving
15.1 gm Tris base, 9.4 gm Glycine and 50 ml of 10 % SDS in in 1 lt DW.
50× Ethidium Bromide
10 mg of ethidium bromide is dissolved in 1 ml of distilled water and stored at dark at 4ºC.
6× Gel Loading Buffer
Bromophenol blue

0.25%

Xylene cyanol 0.25%
Glycerol (v/v) 30%
Store at 4ºC.

8. BUFFERS/SOLUTIONS FOR SDS-PAGE
Acrylamide-bisacrylamide stock Solution (30% w/v)
Acrylamide

29

g

N,N’ methylenebisacrylamide
Distilled water to


100

1.0

g

ml

3.0 M Tris HCl (pH 8.8) resolving buffer stock
Tris base

36.3

Distilled water80

g
ml

Adjust pH 8.8 with 1 M HCl, (48 ml of 1 M HCl) make final volume to 100 ml.
0.5 M Tris HCl (pH 6.8) Stacking gel buffer stock
Tris base

6.0

Distilled water40

g
ml


Adjust pH 6.8 with 1 M HCl, (48 ml of 1 M HCl) make final volume to 100 ml.
0.25 M Tris (pH 8.3) Reservoir buffer stock (10X)
Tris

30.3 gm

Glycine
SDS

144 gm
10 gm

13


Dist water to make it 1 lt
1.50% APS
Ammonium per sulfate

0.15

Distilled water to

ml

10

g

Make fresh after 2-3 weeks if stored at 4oC.

2% SDS Loading Buffer
Tris HCl

100

mM

SDS

4

%

Glycerol

20

%

2 Mercaptoethanol

8

%

Bromophenol blue

0.2

%


9. RECIPES FOR MAKING DISCONTINUOUS DISSOCIATING GELS
Stock solution

Acrylamide-bisacrylamide
Stacking gel buffer stock
Resolving Gel buffer stock
10 % SDS
1.5 % APS
Water
TEMED

Stacking

Final acrylamide concentration in resolving gel

gel

(%)
20.0
20.0
---3.75
0.3
1.5

17.5
17.5
--3.75
0.3
1.5


15.0
15.0
---3.75
0.3
1.5

12.5
12.5
----3.75
0.3
1.5

10.0
10.0
----3.75
0.3
1.5

7.5
7.5
---3.75
0.3
1.5

5.0
5.0
---3.75
0.3
1.5


4.45
0.015

6.95
0.015

9.45
0.015

11.95
0.015

14.45
0.015

16.95
0.015

19.95
0.015

2.5
5.0
-0.2
1.0
11.3
0.015

10. RECIPES FOR MAKING DISCONTINUOUS NON-DISSOCIATING

GELS
SDS is not added at any of the stage of preparation of buffers or gels, and Volume is met by
DW.

11. RECIPES FOR MAKING CONTINUOUS NON-DISSOCIATING GELS :
Same as in number 10 but stacking gel is not used.

12. GEL PROCESSING SOLUTIONS:
Fixing Solution
Methanol
Glacial acetic acid
Distilled water

45
10
45

%
%
%

14


Staining Solution: It is 0.1% Coomassie brilliant blue R-250 in fixing solution. (0.2 µg to 0.5 µg of

any protein in form of sharp band cab be determined with this stain), Silver stain is 100 times
more sensitive than it detecting 0.38 ng of protein / mm2.
De-staining Solution
Methanol

Glacial acetic acid
Distilled water
Preservative Solution

30
10
60

%
%
%

Glacial acetic acid
Distilled water

7
93

%
%

13. ELISA REAGENTS AND BUFFERS
A. Coating Buffer
Tris buffer 0.1 M, pH 7.0 to 9.0
Or
0.05 M carbonate buffer, pH 9.6 with 0.02 % NaN3 (Sodium azide)
or
0.01 M PBS pH 7.4 with 0.02 % NaN3 (Sodium azide)
For better coating of antibodies and protein antigens: Dilute serum with equal volume of 0.1 M
glycine-HCl buffer, pH 2.5 with 0.1 M NaCl, incubate for 10 min, and neutralize with 0.1 M

Tris pH 9.0
Or
Make antigen in 0.05 M glycine-HCl buffer, pH 2.5 with 0.1 M NaCl, incubate for 10 min, and
neutralize with 0.05 M Tris pH 9.0
For lipid/ surface antigens, make suspension of antigen in sodium deoxycholate (1 mg/ml)
solution before coating.
b. Washing buffer
Phosphate buffered saline (pH 7.2)

1000 ml

Tween-20 (Polysorbate)

0.5 ml

Sodium azide

0.2 gm

Stored at 4°C.
C. Blocking solution
Gelatin

1.0 g

PBS

100 ml

Or


15


2 % Bovine serum albumen in water with 0.02 % NaN3 (Sodium azide)
or
5% Skim Milk in water with 0.02 % NaN3 (Sodium azide)
D. Substrates for Horse radish peroxidase (HRPO) conjugate:
1. Ortho-phenylene-diamine (0.5 mg/ml) in Citrate-phosphate buffer, 0.1 M, pH 4.6 - 5.0),
made fresh and add H2O2 (of 30 %, 5 µl per 30 ml) just before use, avoid exposure to direct
light. Read at 492 nm.
Buffer Stock Solution A
Citric acid (C6H8O7.H2O) 2.10 g in Distilled water

100 ml

Buffer Stock Solution B
Sodium citrate (Na3C6H5O7.2H2O)

2.94 g in Distilled water

100 ml

Working Solution: 25.5 ml of solution A and 24.5 ml of solution B were mixed just before use.
Enzyme activity arrester (1 M, H2SO4)
Concentrated sulphuric acid

27.77 ml

Distilled water


72.33 ml

B. Tetramethyl benzidine (TMB): Dissolve 10 mg TMB in 1 ml DMSO and then dilute with 0.1
M sodium acetate citric acid buffer pH 6.0, add 30 µl H2O2 (30 %), read at 450 nm, bright
yellow colour after adding sulfuric acid.
Enzyme activity arrester (2 M, H2SO4)
3. 5-aminosalycilic acid
Add 100 mg in 100 ml of 0.01 M Sod. Phosphate buffer, pH 6.0 having 1mM EDTA, add 20 µl
H2O2 (30 %), read at 450 nm, brown colour after adding NAOH.
Enzyme activity arrester (1 M, NaOH)
e. Substrate for Alkaline phosphatase conjugate
1. p-Nitrophenylene phosphate: Substrate solution (1 mg/ml) is made in 0.05 M carbonate buffer
pH 9.8 with 0.001 M MgCl2. Read at 400 nm.
Enzyme activity arrester (1 M, NaOH)
Or
p-Nitrophenylene phosphate solution (1 mg/ml) can be made in 0.05 M diethanolamine buffer
pH 9.8. Read yellow colour at 405 nm.
Diethanolamine buffer: Diethanolamine, 9.7 ml; Water,

80 ml; Sodium azide 0.02 gm. Adjust

pH with 1M HCl to 9.8. Final volume is made with distilled water to 100 ml.
Enzyme activity arrester (3 M, NaOH)

14. SOLUTIONS FOR ESTIMATION OF PROTEIN

16



A. Modified Lowry’s Method (for 0.1 to 1 mg / ml protein)
Solution A:1% CuSo4.5H2O

1 g/ 100 ml distilled water

Solution B: 2% sodium potassium tartarate

1 g/ 50 ml distilled water

Solution C: 0.2 M, NaOH

0.8 g/100 ml

Solution D: 4% Na2CO3

4 g/100 ml

Reagent Mixtures
Solution E: 49 ml each of solution C and solution D and 1 ml each of solution A and solution B
were mixed prior to use.
Standard solutions
1% bovine albumin

0.1 g in 10 ml distilled water.

Folin Reagent: To 10 ml Folin Ciocalteau, add 10 ml Distilled water and use it as reagent F.
Procedure: To 0.5 ml sample containing up to 0.5 mg protein, mix 2.5 ml of reagent E and keep
for 10 min then add 0.25 ml of reagent F and incubate for 30 min at RT. Measure absorbance at
750 nm
B. UV Spectrophotometery (0.05 to 2 mg protein ml-1) :

Estimation must be made in quartz tubes. Protein in mg ml-1= 1.55 A280- 0.76 A260
Or
For increased sensitivity use far UV method (0.01 to 0.05 mg/ml)
Protein in mg ml-1= 27 + 120 (A280/ A205
C. Biurate method: (1 to 6 mg/ ml of protein)
Biurate reagent: Dissolve 1.5 gm copper sulphate CuSo4.5H2O and 6 gm of sodium potassium
tartarate in 500 ml water. Add 300 ml of 105 sodium hydroxide and make up to 1 lt. 1 gm
iodine can be added for long term storage of reagent.
Procedure: 0.5 ml sample is mixed with 2.5 ml of reagent and allow to stand for 30 min and
then read at 540 nm.

D. Bicinchoninic acid method: (0.5 to 10 µg/ ml of protein)
Solution A: 1.5 % sodium tartarate, 1.6 % NaOH, 8 % Na2CO3 solution is made and pH is
adjusted to 11.25 with solid NaHCO3
Solution B: 4 % (w/v) Sodium Bicinchoninic acid
Solution C: 4% CuSo4.5H2O

17


Solution D: 4 volumes of C are mixed with 100 volumes of B (made fresh)
Solution E: One volume of D 1 volume of A
Procedure: To one volume of sample add 1 volume of E, mix and allowed to stand for 60 min at
60 oC. Cool to Rt and read at 562 nm.
E. Dye binding Method: (0.2 to 1.4 mg/ ml of protein or 5-100 µg/ ml in micro assay)
Reagent: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of ethanol by vigorous
homogenization. Mix it with 100 ml of 85% (W/V) phosphoric acid and then dilute it to 1 lt
with DW. Stable at RT for 2 weeks.
Procedure: Add 5 ml of the reagent in to 0.1 ml protein sample containing 20-140 µg of protein,
mix and allow to stand for 5- 30 min. Measure absorbance at 595 nm.

Or
For micro assay add 0.8 ml of dye reagent to 0.2 ml of sample containing 1-20 µg of protein,
mix and allowed to stand for 5- 30 min. Measure absorbance at 595 nm.
F. Silver binding Method: For highly purified samples and tween-20 should be added to sample
for inhibiting adsorption of complexes to glass or plastic surfaces.
Reagents: A-7.5 % Tween-20 in 100 mM Tris, 100 mM sodium carbonate.
B- 2.5 % Glutaraldehyde (made fresh from stock 25 % solution stored at 4oC).
C- Add 1.4 ml of 20 % w/v sodium hydroxide and 0.2 ml of ammonium hydroxide (29%,
concentrated) to 18.2 ml of DW, then add drop wise 0.2 ml of 20 % (w/v) silver nitrate
D- 3 % sodium thiosulphate (made fresh).
Procedure: Add 11 µl of reagent A to 100 µl of sample containing protein from 15 ng to 2 µg
protein. Mix it and centrifuge at 450 g (1000 rpm) for 5 min through a 2 ml Bio-Gel P-2 preequilibrated in 1:10 diluted A and then drain of void volume. Add 0.9 ml of DW to make
volume to 1 ml. Then add 20 µl of reagent B and vortex for 2-4 seconds. Then add 200 µl of
Reagent C and vortex for 2-4 seconds. Allow to stand for 10 min at RT. Add 40 µl of reagent D
and read the absorbance at 420 nm against blank made similarly with 100 µl sample buffer.

15. pH OF STANDARD SOLUTIONS OF COMMON CHEMICALS USED IN
LABORATORY:
S.No. Solution
1
2
3

Acetic acid
Hydrochloric acid
Sulfuric acid

1N
2.4
0.1

0.3

pH values at
0.1 N
0.01 N
2.9
3.4
1.07
2.02
1.2
2.1

0.001 N
3.9
3.09
--

18


4
5
6
7
8

Citric acid
Ammonium hydroxide
Sodium hydroxide
Sodium bicarbonate

Sodium carbonate

-11.8
14
---

2.1
11.3
13.7
8.4
11.5

2.6
10.8
12.12
-11

-10.3
11.13
---

16. PHYSICAL CHARACTERISTICS OF COMMON ACIDS AND
ALKALIES USED IN LABORATORY
S.
No.

Substance

Specific
gravity


Percent
by
weight

Norm
ality

Ml/lt
to
make
1M

Eq.
Wt.

Mol.
Wt.

Mol
es/lt

Gms/lt

1

Ammonium
hydroxide
(stronger
Sodium hydroxde (saturated)


0.898

28.0

14.8

67.6

35.0

35.0

14.8

251

1.53
1.11
1.84

50.0
10.0
96.0

19.1
2.75
35.9

52.4

363.6
55.6

40.0

40.0

98.1

49.0

19.1
2.75
18.0

763
111
1766

1.42
1.40
1.37
1.18
1.05

71.0
67.0
61.0
36.0
10


15.99
14.0
13.3
11.6
2.9

62.5
67.1
75.2
86.2
344.8

63.02

63.02

15.99
14.0
13.3

36.46

36.46

1008
938
837
424
105


(syrupy)

1.71

85.0

45.0

55.2

80.0

18.1

1445

(glacial)

1.05
1.045

99.5
36

17.4
6.27

57.5
159.5


60.5

60.5

17.4
6.27

1045
376

1.67
1.54

70.0
60.0

9.2

85.8
108.7

100.5

100.5

11.65
9.2

1172

923

1.52
1.09

50.0
10.0

13.5
1.94

74.1
515.5

126.05
56.10

63.03
56.10

13.5
1.94

757
109

106.00

53.00


381.44

190.72
23.4

1080

2
3
4

Sulfuric acid H2SO4
Nitric acid (concentrated)
HNO3

5

Hydrochloric
(concentrated)
Dilute
Phosphoric acid
H3PO4
Acetic
acid
CH3COOH

6
7

acid


8

Perchloric acid HClO3

9
10

Oxalic acid
Potassium hydroxide

11

Sodium
carbonate
(anhydrous)
Borax (Na2B4O7.10H2O)
Formic acid HCOOH

12
13

1.20

90.0

23.4

42.7


46.02

17. BUFFERS FOR SPECIFIC PURPOSES
a. McIlvaine’s buffer:
Solution A: 0.1 M-citric acid ((2.1015 g of citric acid C6H8O7.H2O, in 100 ml water)
Solution B: 0.2 M-disodium phosphate (Na2HPO4, 2.8394 g in 100 ml water)
To prepare pH 4.0 solution, add 61.45 ml of solution A and 38.55 ml of solution B.
To prepare pH 6.0 solution, add 38.65 ml of solution A and 63.15 ml of solution B.
b. Phthalate buffer (0.0125 M, pH 5.0):
Solution A: 0.05 m-Potassium hydrogen phthalate (C6H4(COOH)COOK, 1.02115 g in 100 ml
water)

19


Solution B: 0.05 M sodium hydroxide (NaOH, 200 mg in 100 ml water)
To prepare pH 5.0 buffer, add 50 ml of solution A and 23.85 ml of solution B, dilute with
water to make 200 ml then add 5 ml of 1% ethanolic bromocresol purple.
c. PBS, 0.025 M, pH 6.0 and 6.8
SolutionA: 0.025 M Na2HPO4 (Na2HPO4.12H2O, MW 358.16)
Solution B: 0.025 M-KH2PO4 (MW 136.09)
To prepare pH 6.0 buffer add 12 ml of A and 88 ml of solution B
To prepare pH 6.8 buffer, mix 50 ml of slolution A and 50 ml of solution B.
(For use in urease or malonate test add 5 ml of 0.1% ethanolic phenol red to 100 ml buffer.)
d. PBS, 0.15 M, pH 7.0
Sodium chloride
Potassium chloride
Di-sodium hydrogen phosphate
Potassium dihydrogen phosphate
Distilled water to

e. PBS, pH 7.2

8.0
0.2
1.15
0.20
1000

Sodium chloride (NaCl)

8.0 g

Potassium dihydrogen phosphate (KH2PO4)

0.20 g

Disodium hydrogen phosphate (Na2HPO4)

1.16 g

Potassium chloride

0.20 g

Distilled water

1000 ml

g
g

g
g
ml

The solution was autoclaved at 121°C, 15 lbs pressure for 30 min and stored at 40° for
further use.
f. PBS, pH 7.4
Sodium chloride

8.0 g

Potassium chloride

0.20 g

Potassium dihydrogen orthophosphate

0.20 g

Disodium hydrogen phosphate

1.16 g

Distilled water

1000 ml

g. PBS, pH 7.3, Oxoid tabs)
Sodium chloride


8.0 g

Dipotassium hydrogen phosphate

1.21 g

Potassium dihydrogen orthophosphate

0.34 g

Distilled water

1000 ml

h. Azide saline (pH 7.3, Oxoid tabs). Azide act as preservative preventing microbial degradation.

20


Sodium chloride

8.0 g

Dipotassium hydrogen phosphate

1.21 g

Potassium dihydrogen orthophosphate

0.34 g


Sodium azide

0.8 g

Distilled water

1000 ml

i. Borate Calcium saline (pH 7.3). It is used for haemagglutination experiments.
NaCl 8g, CaCl2 1 g, H3BO3 1.2 g, sodium borate Deca hydrate (Na 2B4O7.10H2O) 0.052 g in
1 lt-distilled water.
j. Veronal-NaCl diluent 5X (used in CFT): Dissolve 5.75 g barbitone in 500 ml hot distilled
water, add 85 g of NaCl and make up to 1400 ml. Dissolve 2 g sodium barbitone in 500 ml
distilled water and add NaCl-barbitone solution to make upto 2 lt Add 1.68 g MgCl 2.6H2O
and 0.28 g CaCl2. For use dilute in 1 in 5 with distilled water.
k. Sorensen’s citrate buffer
Solution A: 0.1 M-citric acid (2.1015 gm of citric acid C6H8O7.H2O, in 100 ml water)
Solution B: 0.1 M-trisodium citrate (Na3C6H5O7.2H2O 2.9411 gm in 100 ml water)
To prepare buffer of pH 5.6 mix 13.7 ml of solution A and 36.3 ml of Solution B and adjust
the volume to 500 ml with water.
l. Citrate Phosphate buffer for pH 2.6 to 7.0 (0.25 M)
Stock Solution A: 1M solution of citric acid (192.1 gm/lt)
Stock solution B: 1 M Disodium hydrohen phosphate (Na 2HPO4. 7H2O 536.5 gm/ lt or
Na2HPO4. 10H2O 717.1 gm/ lt)
A in ml + B in ml to make it 50 ml (i.e. 50 ml-A in ml), dilute to make 100 ml
pH
2.6
2.8
3.0

3.2
3.4

A
44.6
42.2
39.8
37.7
35.9

pH
3.6
3.8
4.0
4.2
4.4

A
33.9
32.3
30.7
29.4
27.8

pH
4.6
4.8
5.0
5.2
5.4


A
26.7
25.2
24.3
23.3
22.2

pH
5.6
5.8
6.0
6.2
6.4

A
21.0
19.7
17.9
16.9
15.4

pH
6.6
6.8
7.0

A
13.6
9.1

6.5

m. Citrate Buffer pH 3.0 to 6.2 (0.25 M)
Stock Solution A: 1M solution of citric acid (192.1 gm/lt)
Stock solution B: 1 M Sodium Citrate (C6H5O7Na3. 2H2O 294.1 gm/ lt)

21


A in ml + B in ml to make it 50 ml (i.e. 50 ml-A in ml), dilute to make 100 ml
pH
3.0
3.2
3.4

A
46.5
43.7
40.0

pH
3.6
3.8
4.0

A
37.0
53.0
33.0


PH
4.2
4.4
4.6

A
31.5
28.0
25.5

pH
4.8
5.0
5.2

A
23.0
20.5
18.0

pH
5.4
5.6
5.8

A
16.0
13.7
11.8


pH
6.0
6.2

A
9.5
7.2

n. Phosphate buffer for pH 5.7 to 8.0 (0.25 M)
Stock Solution A: 1 M Disodium hydrohen phosphate (Na 2HPO4. 7H2O 536.5 gm/ lt or
Na2HPO4. 10H2O 717.1 gm/ lt or Na2HPO4. 2H2O 178.05 gm/ lt )
Stock solution B: 1 M Sodium dihydrohen phosphate (NaH 2PO4. H2O 138.35 gm/ lt or
NaH2PO4. 2H2O 156.05 gm/ lt )
A in ml + B in ml to make it 100ml (i.e. 100 ml-A in ml), dilute to make 200 ml
pH
5.7
5.8
5.9
6.0

A
6.5
8.0
10.0
12.3

pH
6.1
6.2
6.3

6.4

A
15.0
18.5
22.5
26.5

pH
6.5
6.6
6.7
6.8

A
31.5
37.5
43.5
49.0

pH
6.9
7.0
7.1
7.2

A
55.0
61.0
67.0

72.0

pH
7.3
7.4
7.5
7.6

A
77.0
81.0
84.0
87.0

pH
7.7
7.8
7.9
8.0

A
90.5
91.5
93.0
94.7

o. Phosphate buffer for pH 5.8 to 8.0 (0. 5 M)
Stock Solution A: 1 M Sodium hydroxide (NaOH 40.0 gm/ lt)
Stock solution B: 1 M Potassium dihydrohen phosphate (KH2PO4. 136.00 gm/ lt)


A (NaOH) in ml + in 50 ml of B, dilute to make 100 ml
pH
-5.8
5.9
6.0

A
3.6
4.6
5.6

pH
6.1
6.2
6.3
6.4

A
6.8
8.1
9.7
11.6

pH
6.5
6.6
6.7
6.8

A

13.9
16.4
19.3
22.4

pH
6.9
7.0
7.1
7.2

A
25.9
29.1
32.1
34.7

pH
7.3
7.4
7.5
7.6

A
37.0
39.1
40.9
42.4

pH

7.7
7.8
7.9
8.0

A
43.5
44.5
45.3
46.1

p. Potassium Phosphate buffer for pH 5.8 to 8.0 (0. 1 M)
A: Volume of 1 M K2HPO4 (ml) to make 100 ml with 1 M KH2PO4 and then finally dilute with
distilled water to make 1 lt.
pH
5.8
6.0

A
8.5
13.2

pH
6.2
6.4

A
19.2
27.8


pH
6.6
6.8

A
38.1
49.7

pH
7.0
7.2

A
61.5
71.7

pH
7.4
7.6

A
80.2
86.6

pH
7.8
8.0

A
90.8

94

q. PBS (Bacteriological) for 1 lt.
8 gm (137 mM) NaCl, 0.2 gm (2.7 mM) KCl, 1.44 gm (10 mM) Na2HPO4,

0.2 gm (2

mM) KH2PO4

22


Dissolve in 800 ml Dist. Water, adjust pH 7.4 with HCl and make it to 1 lt.
If required it may be supplemented with CaCl2, 1 mM and MgCl2 0.5 mM
r. Tris Buffer
To make 0.05 M Tris buffere ad 0.1N HCl to desired level in 0.1 M Tris base 50 ml and
finally dilute to make 100 ml with Distilled water. (More the concentration more the pH,
10mM and 100mM buffers differ by 0.1 pH unit)
pH
HCl
pH
HCl
pH
HCl
pH
HCl
pH
HCl pH
HCl
7.1

45.7
7.4
42.0
7.7
36.6
8.0
29.2
8.3
19.9 8.6
12.4
7.2
44.7
7.5
40.3
7.8
34.5
8.1
26.2
8.4
17.2 8.7
10.3
7.3
43.4
7.6
38.5
7.9
32.0
8.2
22.9
8.5

14.7 8.8
8.5
To make 1 M Tris buffer: 121.1 gm Tris base, dissolve in 800 ml distilled water add required
quantity of concentrated HCl and make it a lt with DW.
pH
7.4

HCl
70 ml

pH
7.6

HCl
60 ml

pH
8.0

HCl
42 ml

s. Barbital Buffer (0.05 M) pH 6.8 to 9.2
Stock Solution A: 0.2 M solution of sodium barbital (41.2 gm/lt)
Stock solution B: 0.2 M HCl
In 50 ml of A add desired volume of B and finally dilute to make 200 ml
pH
6.8
7.0
7.2


B
45.0
43.0
39.0

pH
7.4
7.6
7.8

B
32.5
27.5
22.5

pH
8.0
8.2
8.4

B
17.5
12.7
9.0

pH
8.6
8.8
9.0


B
6.0
4.0
2.5

pH
9.2

B
1.5

t. Borate Buffer (0.0125 M) pH 8.1 to 9.0
Stock Solution A: 0.025 M solution of sodium borate Na2B4O7. 10H2O (9.525 gm/lt)
Stock solution B: 0.1 M HCl
In 50 ml of A add desired volume of B and finally dilute to make 100 ml
pH
8.1
8.2

B
19.7
18.8

pH
8.3
8.4

B
17.7

16.8

pH
8.5
8.6

B
15.2
13.5

pH
8.7
8.8

B
11.6
9.4

pH
8.9
9.0

B
7.1
4.6

u. Borate Buffer (0.0125 M) pH 9.3 to 10.7
Stock Solution A: 0.025 M solution of sodium borate Na2B4O7. 10H2O (9.525 gm/lt)
Stock solution B: 0.1 M NaOH
In 50 ml of A add desired volume of B and finally dilute to make 100 ml

pH

B

pH

B

pH

B

pH

B

pH

B

23


9.3
9.4
9.5

3.6
6.2
8.8


9.6
9.7
9.8

11.1
13.1
15.0

9.9
10.0
10.1

16.6
18.3
19.5

10.2
10.3
10.4

20.5
21.3
22.1

10.5
10.6
10.7

22.7

23.3
23.8

pH
10.4
10.6

B
19.6
22.75

v. Glycine Buffer (0.05 M) pH 8.6 to 10.6
Stock Solution A: 0.2 M solution glycine (15.1 gm/lt)
Stock solution B: 0.2 M NaOH
In 25 ml of A add desired volume of B and finally dilute to make 100 ml
pH
8.6
8.8

B
2.0
3.0

pH
9.0
9.2

B
4.4
6.0


pH
9.4
9.6

B
8.4
11.2

pH
9.8
10.0

B
13.6
16.0

w. Sodium Carbonate bicarbonate Buffer (0.05 M) pH 9.2 to 10.8
Stock Solution A: 0.1 M solution of sodium carbonate, Na2CO3. 10H2O (28.62 gm/lt)
Stock solution B: 0.1 M solution of sodium bicarbonate, NaHCO3 (8.40 gm/lt)
Add desired volume of B and make it to final volume of 100 ml with solution A
pH

at pH

20oC
9.2
9.4
9.5


pH at 20oC

pH

9.8
9.9
10.1

37oC
9.5
9.7
9.9

at B

37oC
8.8
9.1
9.4

90
80
70

at B
60
50
40

pH at 20oC


pH

10.3
10.5
10.8

37oC
10.1
10.3
10.6

at B
30
20
10

x. Carbonate Buffer (0.025 M) pH 9.7 to 10.9
Stock Solution A: 0.05 M solution of sodium bicarbonate, NaHCO3 (4.20 gm/lt)
Stock solution B: 0.1 M NaOH
Add desired volume of B and make it to final volume of 100 ml with solution A
pH
9.7
9.8
9.9

B
6.2
7.6
9.1


pH
10.0
10.1
10.2

B
10.7
12.2
13.8

pH
10.3
10.4
10.5

B
15.2
16.5
17.8

pH
10.6
10.7
10.8

B
19.1
20.2
21.2


pH
10.9

B
22.0

18. ANTIBIOTIC SOLUTIONS (must be stored at -20oc in aliquots)
Antibiotic
Amoxicillin
Amphotericin B
Ampicillin

Conc.
mg/m
l
50
5
50

Aztreonam
Carbenicillin
Cefotaxime

30
50
25

Dissolved in


Sterilized through

PBS, 0.1 M, pH 6
Dimethylformamide
Distilled water, PBS 0.1 M
pH 8
Aqueous NaHCO3
Distilled water
Dimethyl Sulfoxide

Filtration 0.22 µm filter
Filtration 0.22 µm filter
Filtration 0.22 µm filter
Filtration 0.22 µm filter
Filtration 0.22 µm filter
Filtration 0.22 µm filter

24


Ceftazidime
30
PBS, 0.1 M, pH7.0
Filtration 0.22 µm filter
Cephelothins &
30
PBS, 0.1 M, pH 6.0
Filtration 0.22 µm filter
Cephalosporins
Chloramphenicol

34
Ethanol/ methanol
Not needed
Cinoxacin
20
NaOH, 0.1 M
Filtration 0.22 µm filter
Clavulanic acid
10
Distilled Water
Filtration 0.22 µm filter
Enoxacin
10
NaOH, 0.1 M
Filtration 0.22 µm filter
Erythromycin
25
Distilled Water
Filtration 0.22 µm filter
Gentamicin
50
Distilled water
Filtration 0.22 µm filter
Kanamycin
10
Distilled water
Filtration 0.22 µm filter
Moxalactum
30/ 50 HCl, 0.04 – 0.08 M
Filtration 0.22 µm filter

Nalidixic acid
30/ 50 NaOH, 0.1 M
Filtration 0.22 µm filter
Norfloxacin
30
NaOH, 0.1 M
Filtration 0.22 µm filter
Ofloxacin
30
NaOH, 0.1 M
Filtration 0.22 µm filter
Oxolinic acid
10
NaOH, 0.1 M
Filtration 0.22 µm filter
Polymyxin B sulphate
10
PBS, 0.1 M, pH-7.0
Filtration 0.22 µm filter
Rifampin
30
Methanol/ DMSO
Not needed
Streptomycin
10
Distilled water
Filtration 0.22 µm filter
Sublactum-sodium salt
50
Distilled Water

Filtration 0.22 µm filter
Sulfonamides
200
NaOH, 0.1 M
Filtration 0.22 µm filter
Tetracycline
50
Ethanol
Not needed
Trimethoprim
30/ 50 HCl, 0.05 M
Filtration 0.22 µm filter
Mg++are antagonistic to tetracycline therefore, use media lacking Mg++ while using tetracycline for
selection.

19. SUGAR SOLUTIONS (concentration in % w/v) (can be stored at -20oc in
aliquots)
Class
Pentoses
Methyl
pentose
Hexoses

Disachharide

Trisaccharide

Sugar
Arabinose
Xylose

Rhamnose/
isodulcitol
Fructose
Galactose
Glucose/dextrose
Mannose
Sorbose
Cellobiose
Lactose
Maltose
Melibiose
Sucrose
Trehalose
Melezitose
Raffinose

Concentration
40
50
40
50
30
50
50
40
10
15
50
10
50

25
10
10

Class
Polysachharides

Glycosides

Alcohols

Non-carbohydrate

Sugar
Glycogen
Inulin
Starch

Concentration
5
20
Soluble in hot

Aesculin
Amygdalin
Arbutin
Salicin
Adonitol
Dulcitol
Erythritol

Glycerol
Mannitol
Sorbitol
Inositol

water only
0.1, 7.5 in hot
7.5
10
3
40
5, on heating
40
Any proportion
15
50
15

Sterilized either through filtration, tydallization or a few by autoclaving for 10 min at 110 oC.

25