STAINING

METHODS

Significance of staining
Dyes give colour to microbes.
Iincrease visibility.
For recognition;
features.

accentuate

Preservation for future study.

specific

morphological

Dyes

ACIDIC DYES
Negative charged groups
Binds to positively charged cell
structures
Acid fushin, rose bengal and eosin

BASIC DYES
Positively charged
groups
Binds to negatively
charged cell
structures
Methylene blue, Basic
fuchsin,
Crystal voilet.

Staining methods for bacteria

Simple staining

Differential staining

highlight entire microorganism.

Grouping of bacteria.

One reagent

Multiple reagents

Methylene blue or basic fuchsin. Gram staining and Acid

fast staining

Structural/ special staining:

Multiple reagents are used .
Used to identify and study the structure of bacteria

Alberts staining
Volutin granule

EndoSpore staining
Flagellar staining
Capsular Staining(Negative

staining)

Staining for parasites

Iron-haematoxylin stain

Trichrome stain

Modified acid-fast stain

lishman
and giemsa staining

Staining of fungus
Calcoflour staining
For tissues PAS ( Periodic acid- schiff )
GMS ( Grocott-gomori methanamine-silver )

Staining for viruses-

Inclusion bodies
staining

Immunoperoxidase

GRAM STAINING
1884
HANS CHRISTIAN GRAM

(DANISH BACTERIOLOGIST

1938)

1853-

Types

Gram positive
negative

Gram

Exceptions

1. Chlamydia -exist exclusively within host

cells
2. Mycoplasma, Ureaplasm-Lack cell wall
3. Spirochaetes-Insufficient dimension to
be
resolved by light
microscopy.

When stained with basic dye and iodine

Decolourisation with acetone or alcohol

Resist- gram positive

gram

Do not resistnegative

Cell wall theory

Basic dye + Iodine
Dye iodine complex
Attach to bacterial cell wall.
1. Gram positive- thick and dense
peptidoglycan layer which is less
permeable to dye-iodine complex.
2. Iodine binds with peptidoglycan and
decreases its permeability.
3. Also Teichoic acid combines with basic dye.

Gram positive
1. Gram positive- thick and dense
peptidoglycan layer which is less
permeable to dye-iodine complex.
2. Iodine binds with peptidoglycan and
decreases its permeability.
3. Also Teichoic acid combines with
basic dye.
4. Protoplasmic theory- Gram positive
have acidic protoplasm which helps
in retaining basic dye.

Gram negative
Lipid theory- Gram negative have more lipids in
their cell wall which are easily dissolved by
decolouriser.thereby, increasing cell wall
permeability

Magnesium ribonucleate theory- Gram

negative has more magnesium ribonuclease which
helps in forming a dye-iodine complex. This dyeiodine magnesium ribonuclease complex is not
easily removable by decolouriser.

Ideal conditions
YOUNG CULTURE
THIN SMEAR
FRESH REAGENT
CONTROL CULTURE

Steps
Prepare a smear.
Fixing either physical and chemical.
Application of PRIMARY STAIN (Violet dye).
Application of MORDANT (Iodine solution).
Application of DECOLOURIZER.
Application of COUNTERSTAIN (Pink/Red dye).

GRAM
POSITIVE
BACTERIA
(VIOLET)

GRAM
NEGATIVE
BACTERIA
(RED)

Both
Gram
positive
+
Gram
negative

ORIGINAL METHOD OF
GRAM (1884)

REAGENT

PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZER

COUNTER
STAIN

ANILINE
GENTIAN
VIOLET

IODINE
+
POTASSIU
M IODIDE
+
WATER

ABSOLUTE
ALCOHOL

BISMARCK
BROWN

45 sec

1 min

Until colour
caeses to
come

45 sec

TIME

Kopeloff & Beermans Modification (1922)

REAGENT

TIME

PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZER

COUNTER
STAIN

METHYL
VIOLET
STAIN (1%)
+
SODIUM
BICARBONA
TE
(5%)

IODINE
+
SODIUM
HYDROXID
E
(4%)

ACETONE

BASIC
FUSCHIN

5 MINS

2 MINS

2 3 SECS

( 100 %)
(0.05%)

30 SECS

ADVANTAGE:
1) sodium bicarbonate strengthens
( gram-positive staining )
2) sod. Hydroxide - more alkaline sol. (stronger
gram positive staining)
3) Acetone - fastest and most specific.
4) Recommended for anaerobic organisms.

DISADVANTAGE:
1) Methyl Violet & Sodium Bicarbonate
precipitate within few days
cannot be kept.
2)Acetone - evaporate fastly
short period of exposure
difficult to control-many
slides

PRESTON & MORRELLS

MODIFICATION (1962)

REAGENT

PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZE
R

COUNTER
STAIN

CRYSTAL
VIOLET

IODINE
+
POTASSIUM
IODIDE
+
DISTILLED
WATER

ACETONE
+
LIQUOR IODI
FORTIS
(IODINE +
POT IODIDE
+
METHYLATED
SPIRIT +
DISTILLED
WATER)
(0.35%
iodine)

ZIEHL
NEELSENS
CARBOL
FUSCHIN
+
DISTILLED
WATER

30 SECONDS

30 SECONDS

+
METHYLATE
D SPIRIT
+
AMMONIUM
OXALATE 1
%

30 SECONDS 30

(5%)

Advantage:
1)Gram positive staining can be
strengthened by addition of ammonium
oxalate.
2)Addition of small conc. of iodine to
acetone slows its rate of decolourization.
Disadvantage:
1) Iodine-Acetone produces an irritant
aerosol when expelled.

MODIFIED PRESTON & MORRELLS

METHOD (1962)
PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZE
R

COUNTER
STAIN

REAGENT

CRYSTAL
VIOLET
+
ABSOLUTE
ALCOHOL
+
DISTILLED
WATER

IODINE
+
POTASSIUM
IODIDE
+
DISTILLED
WATER

WEAK
IODINE
ACETONE
SOLUTION
(0.035 %)

ZIEHLNEELSENS
CARBOL
FUSCHIN
+
DISTILLED
WATER

TIME

30 SECONDS 30 SECONDS 10 SECONDS

30
SECONDS

Advantage : Reduced conc. of Iodine in

Acetone to one-tenth (0.035%).

JENSENS MODIFICATION

REAGENT

PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZER

COUNTER
STAIN

METHYL
VIOLET
+
DISTILLED
WATER

IODINE
+
POTASSIU
M IODIDE
+
DISTILLED
WATER

ABSOLUTE
ALCOHOL
(100 %
ETHANOL)

NEUTRAL
RED
+
1 % ACETIC
ACID
+
DISTILLED
WATER

(0.5%)
(0.1%)
TIME

30 SECONDS 30
SECONDS

UNTIL COLOR
CEASES TO
COME

12
MINUTES

For examination of smears for Gonocococci

Meningococci.
Excellent results with freedom from deposit.
When organisms are scanty SANDIFORDS
COUNTERSTAIN used.

(Malachite green,Pyronine,DW)

WEIGERTS MODIFICATION
PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZER

REAGENT

SATURATED
ALCOHOLIC
SOLN. OF
GENTIAN
VIOLET
+
5%
PHENOL

IODINE
ANILINE
+
+
POTASSIUM XYLOL
IODIDE
+
DISTILLED
WATER

CARMINIC
ACID
+
POTASSIUM
ALUM
+
DISTILLED
WATER

TIME

23
MINUTES

1 MINUTE

10 MINUTES

UNTIL STAIN
CEASES TO
COME OUT

COUNTER
STAIN

Advantage:
Recommended for
staining sections of
tissue(aniline- Xylol as
decolourizer)

Disadvantage: Primary stain mixture-tends to

precipitate

QUICK GRAMS METHOD FOR SINGLE

SLIDES

REAGENT

PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZE
R

COUNTER
STAIN

CRYSTAL
OR
METHYL
VIOLET

IODINE
SOLUTION

ACETONE

BASIC
FUSCHIN

(1%)

TIME

5 SECONDS

(0.5%)

5 SECONDS

2 SECONDS

5 SECONDS

GRAM METHOD FOR MULTIPLE

SLIDES
PRIMARY
STAIN

IODINE
SOLUTION

DECOLORIZER

COUNTER
STAIN

REAGENT

METHYL
VIOLET

LUGOLS
IODINE 1
%

IODINE
ACETONE(slo
w acting
decolourizer)

BASIC
FUSCHIN

TIME

30 SECONDS 30
SECONDS

30 SECONDS

30 SECONDS

STAINING OF ACID FAST

BACILLI
EHRLICHS

METHOD.

ZIEHL-NEELSEN

METHOD.

EHRLICHS METHOD
Original method(1882).
Used Aniline-Gentian Violet
stain.
Strong Nitric Acid as
decolourizer.

DISADVANTAGE:
Ordinary aniline dye sol. do not readily
penetrate
tubercle bacilli.
rest structures and cells
counterstain

ZIEHL-NEELSENS METHOD
Given in (1885).
Powerful staining solution used containing
Phenol.
Application of heat(Do not boil).
Any strong acid as Decolourizer.
Counter stain.

principal

ACID FAST
BACILLI

PRIMARY
STAIN

RED

TYPES
ZN Method A (single smears).

ZN Method B (multiple
smears).

METHOD
A

REAGENT

TIME

PRIMARY STAIN

DECOLOURIZER

BASIC FUCHSIN
( 1%)
+
PHENOL
+
ALCOHOL
+
DW
+
Heat(do not boil)

CONCENTRATED LOEFFLERS
SULPHURIC
METHYLENE
ACID
BLUE
(20%)
OR
+
MALACHITE
DW
GREEN

5 min

10 min

COUNTERSTAIN

15-20 sec

METHOD
B

PRIMARY STAIN

DECOLOURIZER

COUNTERSTAIN

ZN Carbol
fuchsin
+
Heat(Do not
boil)

Acid-Alcohol

Dilute
Malachite
Green

(MULTIPLE
SLIDES)

REAGENT

20% sulphuric
acid
Again
20%H2SO4
3 min

TIME

6 min

5 min
5 min

20-30 sec

IMPORTANT POINTS
Alcohol as secondary decolourization:
1)Decolourization is completed more
quickly.
2)Identification of Tubercle bacilli(some
acid-fast bacilli are decolourized by alcohol).
3)Kept for 2 min.
Acid-Alcohol as decolourizer:
1) 3%HCL in 95% ALCOHOL.
2)Expensive,less corrosive,convienient to make.

MODIFICATIONS OF ZN
METHOD
A. (Conc. Of H2S04 Varies )
BACTERIA

PRIMARY
STAIN

DECOLOURIZE
R

COUNTERSTAI
N

LEPROSY
BACILLI

SAME

5% H2SO4

SAME

NOCARDIA

SAME

1%H2SO4

SAME

0.25%0.5%H2SO4

SAME

SAME
SPORES

B. Cold Method (Kinyouns Method)

Heating is not required.
Phenol concentration in carbol fuchsin is
increased.
Duration of carbol fuchsin staining is more.
C. Malachite green can be used as
counterstain.
D. Acid-alcohol can be used as decolouriser.

Alberts Staining
To demonstrate the metachromatic granules of
Corynebacterium diphtheriae
Stain:
Alberts stain {Toludine blue,Malachite
green, Glacial acetic
acid,
Alcohol }
Alberts iodine.

Steps

Make film, dry, fix.

Cover slide with Alberts stain for 3-5min.
Wash in water.
Cover slide with Alberts iodine for 1min.
Wash and blot dry.
RESULTS:
Granules---------BLUISHBLACK
Organisms-------LIGHT GREEN

principle

ALBERTS
STAINING

thanks