Reagents
Reagents
Chrome-acetic acid fixative
10% aqueous chromic
acid 2.5 ml
10 % aqueous acetic acid 5.0 ml
Distilled water to 100 ml
Use: For
fixation of chromosomes, nucleoli and centrioles of fungi.
Flemming fluid
Osmic acid 0.1 g
Chromic acid 0.2 g
Glacial acetic
acid 01 ml
Distilled water to 100 ml
The
osmic and chromic acids when mixed will keep for only3-4 weeks, The acetic acid
should be added immediately before use.
Use:
Used as a fixative for tissue
section.
Fontan's fixative (Ruge's solution)
Glacial acetic
acid 1.0 ml
Formalin (37-40%
formaldehyde) 2.0 ml
Distilled water to 100 ml
Use: Used
in spirochete staining.
Fomalin-acetic acid alcohol (FAA) mixture
60% Alcohol 90.0 ml
Glacial acetic acid 5.0 ml
Formalin 5.0 ml
Use: For fixation of plant material before staining.
Mercuric chloride-formalin fixative
Saturated aqueous 9.0 g
Solution of mercuric
chloride 280.0
ml
Commercial formalin 20.0 ml
Use: The minimum amount of destoration and
fairly good cytological details are obtained, particularly in staining of various
inclusion bodies if above fixative is used.
Moller's fixation
Lead acetate 9.0 g
Distilled water to 2800 ml
Formalin 20.0
ml
Precaution: Store
in tightly stoppered bottle.
Use: Used
in capsule (method of Moller) staining.
Newcomer's fixative.
Dioxane 10.0 ml
Acetone 10.0 ml
Petroleum ether 10.0 ml
Propionic acid 30.0 ml
Isopropyl alcohol 60.0 ml
Use: Most acetic and alcohol mixtures result in to loss of nuclei staining property and dissolution of mitochondria. This fixative overcomes this difficulty during fixation of smear.
Schaudinn's fixative
Absolute ethyl alcohol 33.0 ml
Glacial acetic acid 1.0 ml
Saturated solution of HgC12
(6.9g/100 ml at
20°C) in water 66.0 ml
Use:
Used to fur the smear
in staining of protozoa.
Used in nuclear staining (method of Robinow)
Susa's Fixative
Mercuric chloride 45.0 g
Distilled water to 800 ml
Sodium chloride 5.0 g
Trichloroacetic acid 20.0 g
Acetic acid (Glacial) 40.0 ml
Formalin (40% formaldehyde) 200 ml
Use: Fixative for normal and pathological specimens before staining.
This fixative give better result.
Zenker's fluid
Mercuric chloride 5.0 g
Potassium bicarbonate 2.5 g
Sodium sulphate 1.0 g
Distilled water to 100 ml
Immediately before use, add 5 ml of glacial acetic acid per 100 ml of above mixture.
Use: It is a fixative for animal tissue staining.
Zenker-formal fixative
This is similar to Zenker's fluid except that the 5 ml of formalin is added instead of acetic acid.
Salines
Azide saline
Sodium azide 0.8 g
Physiological saline OR buffered saline 100 ml
Use: Azide
prevents microbial decomposition. It is used as a diluting fluid.
Borate-calcium saline
NaCl 8.0 g
CaCl2 1.0 g
H3BO3 1.2 g
Na2B4O7.10H2O 0.052 g
Distilled water to 1000 ml
Use: It is
used as a cell suspension and diluting fluid tor haemagglutination experiments,
where calcium is required and phosphate should be absent.
Buffered saline
NaCl 8.0 g
K2HPO4 1
: 21 g
KH2PO4 0.34 g
Distilled water to 1000 ml
Use: This solution gives a pH of about 7.3 and also
provides potassium and phosphate ions. It is general diluent and suspending
fluid. NaCl can be diluted in different buffer solution giving desired pH.
Formal saline
Sodium chloride (0.9%) solution 90.0
ml
40% formaldehyde 10.0 ml
The pH is adjusted at 7.0 by addition of CaCO3 granules.
Use: In staining of virus inclusion bodies.
Phenolized saline
Sodium chloride 8.5 g
Phenol 5.0 g
Distilled water to 1000 ml
Physiological saline
Sodium chloride 8.5 g
Distilled water to 1000 ml
Use: Used
for preparation of bacterial suspensions. It prevents osmolysis of bacteria.
VDRL, buffered saline solution (pH 6.0)
Formaldehyde (neutral,
c.p.) 0.5 ml
Na2HPO4.12H2O 0.093 g
KH2PO4 0.170 g
NaCl 10.0
g
Distilled water to 1000 ml
Dissolve
all the constituents in distilled water. Check the pH of the solution. Store
the reagent in screw capped bottles.
Use: Used
for preparation of VDRL antigen.
Vernol-NaCI diluents
NaCl 8.5 g
Barbitone (diethyl-barbituric acid) 0.575 g
Sodium barbitone 0.2 g
MgCl2.6H2O 0.168 g
CaCl2 0.028 g
Distilled water to 1000 ml
Preparation: stock solution concentrated x 5 is made by dissolving 5.75 g barbitone
in 500 ml hot distilled water. Add 85 gm, NaCl and make up the volume to about
1400 ml. Dissolve 2.0 gm sodium barbitone in 500 ml distilled water and add it
to the NaCl barbitone solution. Make up to 2000 ml. Add 1.68 g MgC12.6H20 and
0.28 g CaCl2. For use dilute 1 in 5 with distilled water.
Use: This
saline may be used for complement fixation tests and gives more reproducible
results.
IMViC Test Reagents
Kovac's reagent
n-amyl alcohol 75.0 ml
Hydrochloride acid (conc.) 25.0 ml
P-dimethylamino-benzaldehyde 5.0 g
Add
aldehyde to a flask containing alcohol and dissolve by gently warming to 5S°C
in a water bath. Cool and add HCI. Store the reagent in a dark glass bottle in
a refrigerator.
Use: Kovac's
reagent develop red colour in presence of indole.
Methyle red solution
Methyl red 0.04 g
Ethyl alcohol
(absolute) 40.0 ml
Distilled water to 60.0 ml
Dissolve methyl
red in ethyl alcohol and add water.
Use: Used in methyl red test. Add about 5 drops of the
methyl red reagent in to inoculated and incubated liquid medium. Mix. Positive
tests are bright red and negative are yellow.
Barritt's reagent (Voges Proskauer test)
Solution A: 5g alpha-naphthol in 100 ml of 95% ethyl alcohol.
Dissolve the alpha naphthol in the ethyl alcohol with constant stirring.
Solution B: 40g
potassium hydroxide in 100 ml water.
Use: Used in Voges Proskauer test. Add 1 ml of Potassium hydroxide and 3 ml of alpha naphthol in the inoculated and
incubated liquid medium. A positive reaction is detected by the development of
an eosin-pink colour usually in 2-5 minutes.
Caution: Avoid all contact with human tissues, alpha-naphthol is considered to be carcinogenic.
Other Reagents
Andrade's indicator
Preparation: Prepare
0.5% solution of acid fuchsin. Add 1 N NaOH to it, slowly, till colour turns
slight yellow.
Use: Used
as an indicator in sugar fermentation tube. It becomes dark red in colour at or
below pH 5.5.
Gelatin Hydrolysis
Mercuric chloride solution (Frazier's solution)
Mercuric chloride
15.0 g
Hydrochloric acid (conc.) 20.0 ml
Distilled water to 100.0 ml
Use: Used for detection of gelatin breakdown. After flooding the plate with mercuric chloride solution gelatin liquifying microorganisms show clear zone around colony against opaque background.
Tannic acid solution
Tannic acid 1.0 g
Distilled water to 100.0
ml
Use: To test gelatin hydrolysis. Tannic acid causes relative opacity around gelatin liquifying colonies, quick to develop but fading as the medium also becomes opaque.
Trichloroacetic acid
Trichloroacetic acid (C.P.)
5.0 g
Distilled water to 100.0
ml
Dissolve the acid in the water with constant stirring.
Use: Used to test gelatin hydrolysis by microorganisms on the gelatin agar.
Pectin Hydrolysis
Hexadecyltrimethyl ammonium 1.0 g bromde
Distilled water to 100.0 ml
Use: To detect pectin hydrolysis. It
precipitates intact pectin.
Starch Hydrolysis
Lugol's Iodine (Gram's
Iodine)
Use: After flooding the plate of starch agar with iodine solution amylolytic microorganisms show
clear zone around the colony against
purple background.
Oxidase test reagent
Dimethyl-p-phenylene
diamine hydrochloride 1.0 g
Distilled water to 100.0 ml
The
reagent should be made fresh daily. It should not be stored longer than one
week in the refrigerator.
If
the preparation becomes darkened, discard. Tetramethyl-p-phelylenediamine
dihydrochloride (1%) is even more sensitive but it is more expensive and
difficult to obtain.
Use: To test presence of certain oxidises in bacteria, the dye is reduced to deep purple colour if the test is positive.
Methylene blue (1:25000) solution
Methylene blue dye 0.04 g
Distilled water to
1000 ml
Preparation: Dissolve the methylene blue in the distilled water and dispense into regular staining bottles.
Use: Used in methylene blue reduction test (for determining the quality of milk.) Dilute 10 times before use.
Motility studies reagent
Carboxy-methyl-cellulose 2.0 g
Sucrose (0.2
M) 98.0 ml
Distilled water to 1000 ml
Preparation: Prepare
0.2 M sucrose solution by adding 68.4 g. of sucrose in 1000 ml of distilled
water.
Use: If a bacterial culture growing on a
solid medium is to be examined for motility, a loopful of culture should be
mixed with a drop of 2% CMC at the centre of coverslip.
Methyl cellulose reagent
Carboxy methyl
cellulose 10.0 g
Distilled water to
90.0 ml
Reparation: Dissolve
the methyl cellulose in warm distilled water.
Use: For
microscopic observation of protozoa.
Significance: Methyl cellulose slows down the movement of protozoa.
Resazurin (1:20,000) solution
Resazurin
Distilled water to 1000 ml.
Preparation: Dissolve the resazurin in distilled water.
Use: Used
in resazurin reduction test (for determining the quality of milk.)
Cleaning solution for glassware
(Sulphuric acid dichromate solution)
Sodium dichromate 25.0 g
Sulphuric acid (conc.) 1000 ml
Distilled water to 50
ml
Preparation: Dissolve
the dichromate crystals in 50 ml of warm water. Cool to, room temperature and
add acid slowly to the preparation. Use: This preparation is used for the
removal of residual organic matter from laboratory glassware. Glassware should
be soaked in this solution for a number of days. Then rinsed in running tap
water at least 10 times, then rinsed twice in single distilled water and
finally rinsed once in double glass distilled water.
Ferric chloride reagent
FeCl36H2O 12.0 g
2% Aqueous
HCl 100.0 ml
Made
up the 2% aq. HCl by adding 5.4 ml of concentrated HCl (37%) to 94.6 ml H2O.
Use: Used in phehyyalanine deaminase test. Development of green colour indicate positive phenylalanine deaminase test.
Diphenylamine reagent (nitrate test)
Diphenylamine 0.7 g
Sulphuric acid (conc.) 60 ml
Distilled water
28.8 ml
Preparation: Dissolve diphenylamine in a mixture of sulphuric acid
and water. Cool and add slowly 11.3 ml of concentrated hydrochloric acid. After
the solution has stood for 12 hours some of the base separates, showing that
the reagent is saturated.
Use: Used in detection of nitrate in the medium. This
reagent produces a blue black colour in the presence of either nitrites or
nitrates. It is necessary to make sure that no nitrites are present by
Trommsdorf -regent when it is used as a test for nitrates.
Trommsdorf's reagent
Zinc, chloride solution (20%) 100.0 ml
Starch 4.0 g
Potassium iodide 2.0 g
Distilled water 100.0 ml
Preparation: Prepare 100 ml of 20% aqueous ZnC12 solution and add
slowly with constant stirring to a mixture of 4.0 g of starch in water.
Dissolve completely by heating. Dilute with water and add the potassium iodide.
Made the volume to 1 liter by additions of more water, filter and store in
brown-stoppered bottle.
Use: For
detection of nitrites. This reagent produce intense blue-black colour in
present of nitrite.
Nitrite test reagents
Solution A:
Sulfanilic acid 8.0 g
5 N acetic acid (1 part glacial acetic acid to
2.5 parts water) 1000 ml
Solution B:
Dimethyl-alpha-napthylamine 5.0 g
Immediately before use, mix equal volumes of solution
A and solution B.
Use: Add 0.1 nil of test reagent to 5 ml of broth. A development of red colour indicate presence of nitrite.
Nessler's reagent (Ammonia test)
|
Potassium iodide |
50.0 g |
|
Ammonia free distilled water (cold) |
35.0 ml |
|
Mercuric chloride
solution (saturated) |
35.0 ml |
|
Potassium hydroxide (50% aqueous solution) |
400 ml |
|
Distilled water |
530 ml |
Dissolve potassium iodide in ammonia free distilled water. Add a saturated solution of mercuric chloride to it. (slight precipitate persists.) Add solution of potassium hydroxide. Dilute to 1 litre, allow to settle for one week and decant the supernatant for use. Store in brown bottle.
Buffers
Acetic buffer
Solution required
A: 0.2 M solution of acetic acid (11.55
m1 in 1000 ml).
B: 0.2 M solution of
sodium acetate (16.4 g of C2H3O2Na or 27.2 g
of C2H3O2Na.3H2O in 1000 ml). X ml of A + Y ml of B, diluted to a total of 400 ml
|
X |
Y |
pH |
|
185.2 |
14.8 |
3.6 |
|
176.0 |
24.0 |
3.8 |
|
164.0 |
36.0 |
4.0 |
|
147.2 |
52.8 |
4.2 |
|
122.0 |
78.0 |
4.4 |
|
102.0 |
98.0 |
4.6 |
|
80.0 |
120.0 |
4.8 |
|
59.2 |
140.8 |
5.0 |
|
42.0 |
158.0 |
5.2 |
|
35.2 |
164.8 |
5.4 |
|
19.2 |
180.8 |
5.6 |
Barbitone (veronal) buffer
Solutions required:
A: 0.2M solution of sodium barbitone (sodium diethyl barbiturate) (7.37 g in 1000 ml). B: 0.2M HCl 100 ml of A+ X of B, diluted to a total of 400 ml
|
X |
pH |
|
3.0 |
9.2 |
|
5.0 |
9.0 |
|
8.0 |
8.8 |
|
12.0 |
8.6 |
|
18.0 |
8.4 |
|
25.4 |
8.2 |
|
35.0 |
8.0 |
|
45.0 |
7.8 |
|
55.0 |
7.6 |
|
65.0 |
7.4 |
|
78.0 |
7.2 |
|
86.0 |
7.0 |
|
90.0 |
6.8 |
Bicarbobnate-CO2 buffer
Concentration of CO2
in gaseous phase
|
5% |
10% |
20% |
|
7.4 |
7.1 |
6.8 |
|
7.8 |
7.5 |
7.2 |
|
Concentration 0.02 M |
|
Of NaHCO3 0.05 M |
Above solution give desired pH only at 370C.
Borax-NaOX buffer
A: 0.05 M solution of borax (19.05 g in 1000 ml, 0.02 M in terms of sodium borate) B: 0.2 M NaOH 100 ml of A + X ml B, diluted to a total of 400 ml
|
X |
pH |
|
0.0 |
9.28 |
|
14.0 |
9.35 |
|
22.0 |
9.4 |
|
35.2 |
9.5 |
|
46.0 |
9.6 |
|
58.0 |
9.7 |
|
68.0 |
9.8 |
|
77.2 |
9.9 |
|
86.0 |
10.0 |
|
92.0 |
10.1 |
Boric acid-Borax buffer
Solution required:
A: 0.2 M solution of boric acid (12.4 g
in 1000 ml).
B:
0.2 M solution of borax (19.05 g in 1000 ml : 0.2 M in terms of sodium borate).
100
mi of A + X ml of B, diluted to a total of 400 ml.
|
X |
pH |
|
4.0 |
7.6 |
|
6.2 |
7.8 |
|
9.8 |
8.0 |
|
14.6 |
8.2 |
|
23.0 |
8.4 |
|
35.0 |
8.6 |
|
60.0 |
8.8 |
|
118.0 |
9.0 |
|
230 |
9.2 |
Cacodylate buffer
Solutions required:
A: 0.2 M solution of sodium cacodylate
[42.8 g of Na (CH3)2 AsO2.3H2O
in 1000 ml]. B: 0.2 M NaOH 100 ml of
A + X ml of B diluted to a total of
400 ml.
|
X |
pH |
|
5.4 |
7.4 |
|
8.4 |
7.2 |
|
12.6 |
7.0 |
|
18.6 |
6.8 |
|
26.6 |
6.6 |
|
36.6 |
6.4 |
|
47.6 |
6.2 |
|
59.2 |
6.0 |
|
69.6 |
5.8 |
|
78.4 |
5.6 |
|
86.0 |
5.4 |
|
90.0 |
5.2 |
|
94.0 |
5.0 |
Carbonate-Bicarbonate buffer
Solutions required:
A: 0.2 M solution of anhydrous sodium carbonate. (2.12
g in 100 ml). B: 0.2 M solution of sodium bicarbonate. (1-68 g in 100 ml).
X ml of A + Y ml of B, diluted to a total of 400 ml.
|
X |
Y |
pH |
|
8.0 |
92.0 |
9.2 |
|
19.0 |
81.0 |
9.4 |
|
32.0 |
68.0 |
9.6 |
|
44.0 |
56.0 |
9.8 |
|
55.0 |
45.0 |
10.0 |
|
66.0 |
34.0 |
10.2 |
|
77.0 |
23.0 |
10.4 |
|
85.0 |
15.0 |
10.6 |
Citrate buffer
Solutions
required:
A: 0.1 M solution of citric acid (19.21
g in 1000 ml).
B: 0.1 M solution of sodium citrate
(29.41 g C6H5O7Na3.2H2O
in 1000 ml).
X ml of A + Y ml of B,
diluted to a total of 400 ml.
|
X |
Y |
pH |
|
186.0 |
14.0 |
3.0 |
|
174.8 |
25.2 |
3.2 |
|
160.0 |
40.0 |
3.4 |
|
148.0 |
52.0 |
3.6 |
|
140.0 |
60.0 |
3.8 |
|
132.0 |
68.0 |
4.0 |
|
126.0 |
74.0 |
4.2 |
|
112.0 |
88.0 |
4.4 |
|
102.0 |
98.0 |
4.6 |
|
92.0 |
108.0 |
5.0 |
|
82.0 |
118.0 |
5.0 |
|
72.0 |
128.0 |
5.2 |
|
64.0 |
136.0 |
5.4 |
|
54.8 |
145.2 |
5.6 |
|
47.2 |
152.8 |
5.8 |
|
38.0 |
162.0 |
6.0 |
|
28.8 |
171.2 |
6.2 |
Citrate-Phosphate buffer
Solutions required:
A: 0.1 M solution of citric acid (19.21
g in 1000 ml).
B: 0.2 M solution of dibasic
sodium phosphate (28.39 g of Na2HPO4 or 71.7 g of Na2HPO4.12H2O
in 1000 ml). X ml of A + Y Ml of B, diluted to a total of 100 ml.
|
X |
Y |
pH |
|
44.6 |
5.4 |
2.6 |
|
42.2 |
7.8 |
2.8 |
|
39.8 |
10.2 |
3.0 |
|
37.7 |
12.3 |
3.2 |
|
35.9 |
14.1 |
3.4 |
|
33.9 |
16.1 |
3.6 |
|
32.3 |
17.7 |
3.8 |
|
30.7 |
19.3 |
4.0 |
|
29.4 |
20.6 |
4.2 |
|
27.8 |
22.2 |
4.4 |
|
26.7 |
23.3 |
4.6 |
|
25.2 |
24.8 |
4.8 |
|
24.3 |
26.7 |
5.2 |
|
23.3 |
26.7 |
5.2 |
|
22.2 |
27.8 |
5.4 |
|
21.0 |
29.0 |
5.6 |
|
19.7 |
30.3 |
5.8 |
Glycine-NaOH buffer
Solutions required:
A: 0.2 M solutions of &cine (15.01 g
in 100 ml).
|
X |
pH |
|
8.0 |
8.6 |
|
12.0 |
8.8 |
|
17.6 |
9.0 |
|
24.0 |
9.2 |
|
33.6 |
9.4 |
|
44.8 |
9.6 |
|
54.4 |
9.8 |
|
64.0 |
10.0 |
|
77.2 |
10.4 |
|
91.0 |
10.6 |
Maleate buffer
Solutions required:
A:
0.2M solutions of acid sodium maleate (8 g of NaOH+23.2 g of maleic acid of
19.6 g of maleic anhydride in 1000 ml).
B: 0.2MNaOH 100 ml of A + X pf B, diluted to a total
of 400 ml.
|
X |
pH |
|
14.4 |
5.2 |
|
21.0 |
5.4 |
|
30.6 |
5.6 |
|
41.6 |
5.8 |
|
53.8 |
6.0 |
|
66.0 |
6.2 |
|
76.0 |
6.4 |
|
83.2 |
6.6 |
|
88.8 |
6.8 |
Phosphate buffer
Solutions required:
A: 0.2 M solution of monobasic
sodium phosphate. (31.2 g NaH2PO4.2H2O in 1000
ml). B: 0.2 M solution of dibasic sodium phosphate. (28.39 g of Na2HPO4
or 7 1.7 g of N3HP0,. 12H20 in 1000 ml). X ml of A + Y of B, diluted to a total
of 200 ml.
|
X |
Y |
pH |
|
92.0 |
8.0 |
5.8 |
|
87.7 |
12.3 |
6.0 |
|
81.5 |
18.5 |
6.2 |
|
73.5 |
26.5 |
6.4 |
|
62.5 |
37.5 |
6.6 |
|
51.0 |
49.0 |
6.8 |
|
39.0 |
61.0 |
7.0 |
|
28.0 |
72.0 |
7.2 |
|
19.0 |
81.0 |
7.4 |
|
13.0 |
87.0 |
7.6 |
|
8.5 |
91.5 |
7.8 |
|
5.3 |
94.7 |
8.0 |
Succinate buffer
Solutions required:
A: 0.2 M solution of succinic acid (23.6 g in 1000 ml)
B: 0.2MNaOH 100 ml of A + X ml of B, diluted to a total of 400 ml.
|
X |
pH |
|
30.0 |
3.8 |
|
40.0 |
4.0 |
|
53.0 |
4.2 |
|
66.8 |
4.4 |
|
80.0 |
4.6 |
|
94.0 |
4.8 |
|
106.8 |
5.0 |
|
121.2 |
5.2 |
|
136.8 |
5.4 |
|
150.0 |
5.6 |
|
162.8 |
5.8 |
|
174.0 |
6.0 |
Tris (hydroxymethyl) aminomethane HCL (Tris HCL buffer)
Solution required:
|
X |
pH |
|
10.0 |
9.0 |
|
16.2 |
8.8 |
|
24.4 |
8.6 |
|
33.0 |
8.4 |
|
43.8 |
8.2 |
|
53.6 |
8.0 |
|
65.0 |
7.8 |
|
76.8 |
7.6 |
|
82.8 |
7.4 |
|
88.4 |
7.2 |
No comments:
Post a Comment