Bacterial Staining Methods
Bacterial Staining Methods
Sprirochete Staining
1. Fontana's method
Solutions required:
1. Fontana's
fixative (see fixatives)
2. Absolute
ethyl alcohol
3. Fontana's
mordant
4. Phenol
(melted) Tannic acid Distilled water to
5. Dissolve
tannic acid in water then add melted phenol.
6. Fontana's
stain.
Add
10 per cent ammonia solution to 0.5 per cent aqueous silver nitrate solution
drop wise, until the precipitate formed just redissolve. Slowly add more silver
nitrate solution until the light precipitate reformed and remain stable.
Procedure:
1.
Prepare
the smear and air dry.
2.
Treat
the smear with solution a for three times, each time for 30 seconds.
3.
Wash
the smear with solution b and treat with the same solution for 3 min.
4.
Remove
solution b and dry the slide in air.
5.
Treat
the smear with solution c and heat the slide from below i till steam rises for
30 seconds. Cool.
6.
Wash
the slide with distilled water and air dry.
7.
Treat
the smear with solution d and heat the slide until steam rises and smear
becomes brown in colour.
8.
Wash,
dry and examine under oil immersion lens.
Result: Spirochetes appear brownish black against yellow background.
2. Becker's method (modified)
Solution required:
a. Fontana's
fixative (see fontana's method)
b. Mordant
(as in fontana's method)
c. staining solution.
|
Basic fuchsin (Saturated alcoholic solution) Shunk's mordant B (95 or 100% ethanol |
45.0 ml |
|
aniline oil 4 ml) |
18.0 ml |
|
Distilled water to |
100.0 ml |
Mix the Shunk's mordant and the alcoholic fuchsin and then add the distilled water. (the glassware should be dry). Filter before use.
Procedure:
a. Prepare the smear and dry in air.
b. Treat the smear with solution a for 1 to
3. minutes.
c. Wash in water.
d. Treat the smear with solution b for 3-5
minutes, wash in water.
e. Treat the smear with solution c for 3 to 5
minutes.
f. Wash in water and dry. Observe under oil
immersion lens.
3.Rue's method
Solutions required:
a. l%Formalin
b. 5% NaHCO,
c. Basic fuchsin
Basic fuchsin
95% ethyl alcohol 25.0 ml Dilute the stain 1:9 before use.
Procedure:
1. Prepare
a smear and air dry.
2. Treat
the smear with solution a for 1 minute and dry.
3. Place
a drop of solution b on the smear and then add 10 drops of solution c. Mix well
and treat the smear with this mixture for 3-5
minutes.
4. Wash with water, dry and examine under oil immersion lens.
3. Levaditi's method
(Staining of spirochetes in tissues)
pyridine
modification
Solutions required:
a. a.10% formalin
b. %-98% alcohol
c. Silver nitrate pyridine solution
i. Pure pyridine 10.0 ml
ii. Silver nitrate (1 %
solution) 90.0 ml
d. d.10% pyriding solution
e. Reducing fluid
|
Acetone (pure) |
10.0 ml |
|
Pyridine (pure) |
15.0 ml |
|
Formalin (4%) |
100.0 ml |
Procedure:
1. Keep
the small piece of tissue (1 mm thick) in solution a for 24 hrs.
2. Wash
the tissue for 1 hr in water and then place it in solution b for 24 hrs.
3. Treat
the tissue by solution c at room temperature for 2 hrs and then at about 50°C
for 4 to 6 hrs.
4. Wash
the tissue section with solution d.
5. Treat
the tissue immediately by solution e for 48 hrs in the dark.
6. 'Wash with distilled water, dehydrate with increasing strength of alcohol.
7. Proceed for section cutting according to standard procedure.
Flagella Staining
Special care should be taken in cleaning
slide and preparing bacterial smear in flagella staining.
a. Cleaning
the slide: Wash the slide with dichromic acid cleaning solution. Wash with
distilled water. Pass the slide through Bunsen flame until flame shows yellow colour. I
b. Preparation
01 smear: Take agar slant having actively growing young culture. (18- 20 hrs
old). Add slowly about 2-4 ml of
sterile saline through the side of tube keeping slant in upward position.
Rotate the tube slowly between palms or 5 minutes. Incubate the tube at optimum
temperature for 40 minutes. Take a small drop of saline suspension from the
tube by capillary pipette and put at the one end of slide. Tilt the slide so
that the drop will slowly run over the slide and form a smear. Allow the slide
to air dry. Do not heat fix the smear. Stain the slide the suitable staining method.
Method of Bailey (modified) Solutions required
Bailey's staining solution a.
1.
6% aqueous FeC13.6H2O --- 6.0 ml
2. 10% solution of tannic acid Mix. Filter before use 18.0 ml
Bailey's
staining solution b
|
Bailey's
solution a 0.5% basic fuchsin |
3.5 ml |
|
in ethyl alcohol |
0.5 ml |
|
Concentrated HCl |
0.5 ml |
|
Formalin |
2.0 ml |
3. Mix. Filter before use. Use freshly.
Carbol fuchsin (see acid fast staining)
Procedure:
Prepare the smear as given above.
Treat the smear by solution a for about
3-5 minutes.
Remove the solution a and treat the
smear by solution b for 7 minutes.
Wash with distilled water. Do not dry.
Treat the smear with solution c
immediately and heat it from below till steam rises for 1 min.
Wash the slide with water, air dry ad examine.
Result:
Flagella and cell wall appear red in
colour.
Method
of Patel, Kulkarni and Gaikwad
Solutions required:
a. Tannic
acid solution
|
Tannic acid |
0.2 gm |
|
Distilled water to |
100 ml |
b. Iodine
solution
|
Iodine crystal |
2.0 g |
|
1 N NaOH |
10.0
ml |
c. Basic
fuchsin solution
Basic
fuchsin Absolute alcohol Distilled water.
Procedure
1. Prepare
a smear as mentioned earlier.
2. Treat
the smear with 4-6 drops of solution a immediately followed by equal drops of
solution b.
3. Heat
the smear from below gently for 1-2 minutes.
4. Add
2-3 drops of solution c to the smear, and again heat for
5. Minutes.
(do not allow to dry the steam).
6. Wash
the slide with water, air dry and examine tinder oil immersion lens.
Result:
Bacteria appek red, flagella appear pink in colour.
Method of Gray
Solutions required:
Gray's staining solution
Solution I
|
Saturated solution of
aluminium potassium sulphate |
5.0 ml |
|
20 per cent tannic acid
saturated solution of |
2.0 ml |
|
Mercuric chloride |
2.0 ml |
Solution II
|
Basic
fuchsin |
6.0
g |
|
Ethanol |
100.0 ml |
Complete Solution
|
Solution I |
- |
|
Solution II |
9.0
ml |
|
Prepare fresh |
0.4
ml |
Procedure:
1. Prepare
smear as given earlier.
2. Treat
the smear by solution a for 10 minutes.
3. Wash
with water air dry and examine under oil immersion lens.
Result: Flagella appear light red and cells appear dark red in colour.
Method of Gray-modified
Solutions required:
a.
Similar as Gray's
staining solution except the concentration of basic fuchsin is doubled.
Procedure:
a)
Prepare a
bacterial suspension as given earlier.
b)
Place a loopful
of suspension on a clean slide and place a cover slip over it.
c)
After ten minutes
place two drops of solution a at one edge of coverslip. Capillary
d)
action will such
the solution below the coverslip.
e)
Observe the slide
after a minute.
Result: Similar as Gray's method.
Note: In this method it is possible to observe the m o w and presence of flagella.
Metachromatic Granules (volutin) Staining
Method of Gohar
Solution required:
Loeffler's methylene blue
(see monochrome staiing)
|
b. 0.1 % H2SO4 |
|
1% eosin Y. |
|
b. 0.1 % H2SO4 |
Procedure:
1.
Prepare the smear
and heat fm.
2.
Treat the smear
with solution a for 5 minutes.
3.
Wash with water.
Treat the smear with solution b for 15-30 seconds
4.
Wash with water.
Treat smear with solution c for 1 minute.
5.
Wash with water.
Treat the smear with solution d for 1 min.
6.
Wash with water,
dry and examine under oil immersion lens.
Result: Metachromatic granules appear black. Cytoplasm appears pink.
Method of Pugh I
Solutions required:
Staining solution
|
Toluidine blue (C1 No. 52040) |
1.0 g |
|
Ethyl alcohol, absolute |
20.0 ml |
|
Acetic acid, glacial |
50.0 ml |
|
Distilled water |
950.0 ml |
Combine the acetic acid with the
distilled water and dissolve the dye in the alcohol. Mix. Filter.
Procedure:
a) Prepare
a smear, air dry and heat fix.
b) Treat
the smear with solution a for 2-3 minutes.
c) Wash
with water, air dry and observe under immersion lens.
Result: Metachromatic granules appear reddish purple in colour. Remaining organism appears light blue.
Method with methylne blue
Solution required:
Loffler's methylene blue (see
monochrome staining)
Procedure:
1.
Prepare a smear
and heat fix.
2.
Treat the smear
with solution a for 5 min.
3.
Wash with water.
Air dry and examine under oil immersion lens.
Result: Metachromatic granules appear red against pale blue cytoplasm.
Method of Albert (Laybourn modification)
Solutions required:
a. Albert's
staining solution
|
Toiuidine blue-0 |
0.15 g |
|
Methyl green or Malachite green |
0.2 g |
|
95% ethyl alcohol |
2.0 g |
|
Glacial acetic acid |
1.0 ml |
|
Distilled water |
100.0 ml |
Dissolve toluidine blue and methyl green
in ethyl alcohol. Add this to distilled water containing glacial acetic acid.
Store the reagent for one day and then filter.
b. Lugol's
iodine (Grams iodine) see gram staining method.
Procedure:
1. Prepare
the smear and heat fix.
2. Treat
the smear with solution a for 5 minutes.
3. Remove
the solution a, do not wash-with water.
4. Treat
the wet smear with solution b for 1 minute.
5. wash
with water, air dry and examine under oil immersion lens.
Result: Metachromatic granules appear black. Cytoplasm appears light green.
Lipid Staining (Budon's method)
Solutions required:
a)
Sudan black B
staining solution Sudan black B
a.
70% ethyl alcohol
Shake before use.
b)
Xylene
c)
0.5% safranin.
Procedure:
1.
Prepare a smear
and heat fix.
2.
Treat the smear
with solution anfor about 10.15 minutes.
3.
Remove solution a
and dry it. Do not was with water.
4.
Wash the smear
with solution b.
5.
Treat the smear
with solution c for 5-10 seconds.
6.
Wash yith water,
air dry and examine under oil immersion lens.
Result: Lipid granules appear deep blue against light pink cytoplasm.
Cytoplasmic Membrane Staining
Solutions required:
a)
Potassium nitrate
b)
Distilled water
to
c)
Bouin's fixative
(see fixatives)
d)
Victoria blue solution
Victoria
blue 0.4 g
Distilled
water to 100.0 ml
Procedure:
1.
Prepare a smear
and heat fix..
2.
Immerse the slide
in solution a for 15 minutes.
3.
Treat the slide
with solution b for 15 minutes.
4.
Wash the slide
with water. Treat the slide with solution c for 30 seconds.
5.
Wash the slide
with water. Dry and examine under oil immersion lens.
Result: The cytoplasmic membrane appears as a deep blue outer layer covering a contracted, irregular shaped body, the cytoplasm.
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