SPORE STAINING METHODS

DR. KOMAL

LOHI

HISTORY

Endospores were first described by Cohn (1872) in Bacillus

subtilis and later by Koch(1876) in the pathogen, Bacillus

anthracis.

Cohn demonstrated the heat resistance of endospores in B.

subtilis, and Koch described the developmental cycle of

spore formation in B. anthracis.

HISTORY CONTD….

In 1922, Dorner published a method for staining endospores. It

employed a lengthy heating step but resulted in differential

staining of endospores and vegetative cells in the same sample.

Shaeffer and Fulton modified Dorner’s method in 1933 to

make the process faster.

ENDOSPORES…..

Endospores are so named because they are formed intracellularly,

although they are eventually released from this mother cell or

sporangium as free spores.

An endospore is a spherical/oval, thick-walled, highly resistant

structure formed in certain bacterial species that represent a

dormant or resting stage in the growth cycle of an organism. e.g.

Bacillus sp., Clostridium sp.

ENDOSPORES…..

Exhaustion of nutrients (Carbon and Nitrogen)

Sub-optimal temperatures

NOT a method of reproduction

The endospore consists of the bacterium's DNA and part of

its cytoplasm, surrounded by a very tough outer coating.

Triggering

factors

COVERINGS OF SPORE

Spore wall :

• delicate membrane from which cell wall of future vegetative bacterium develop

Spore cortex

Spore coat • tough, multi-layered

Exosporium

RESISTANT TO….

Ultraviolet radiation

Desiccation

High temperature

Extreme freezing

Chemical disinfectants

Starvation

DESTROYED BY….

Autoclaving

Tyndallisation

ENDOSPORES…..

Germination of spores-

Increase in metabolic

activity of spore

Endospore swells

Germ cell appears by

rupture or absorption of

spore coat

Elongates to vegetative

bacterium

Distend bacillary body –

Clostridium spp.

Do not distend bacillary body –

B. anthracis

Central – B. anthracis,

C. bifermentans

Terminal - C. tetani, C. tertium

Sub-terminal – C. perfringens

(club-shaped)

Oval - B. anthracis, C. tertium

(tennis racket)

Spherical - C. tetani. (drum-stick)

SHAPE, POSITION AND SIZE OF SPORE RELATIVE TO PARENT CELL

SHAPE, POSITION AND SIZE OF SPORE

Clostridium species in the pus sample

Bacillus species

Clostridium species

Thermobacillus

Thermoflavimicrobium

Sporolactobacillus

Geobacillus

Geosporobacter

Oxobacter

Acetonema – Gram negative bacilli

Sporosarcina – Gram positive cocci

SPORE FORMING BACTERIA

Gram

positive

bacilli

METHODS OF DEMONSTRATION OF SPORES

Endospores are best observed in unstained wet films

under the phase contrast microscope.

They appear as large, refractile, oval or spherical within

the bacterial mother cells or elsewhere free from the

bacteria.

PHASE CONTRAST MICROSCOPY

B. thuringiensis by Phase contrast microscopy

GRAM STAINING METHOD

Vegetative cell - violet

Spore – unstained clear area

Air dry and observe under oil immersion.

Safranine, 30 seconds, wash

Acetone, water

Gram’s iodine, 1 minute, wash

Crystal violet, 1 minute, wash

Heat fix the bacterial smear.

GRAM STAINING METHOD

MODIFIED ZIEHL-NEELSEN STAINING METHOD

Spores – red , Bacteria – blue

Counterstain - Loeffler’s methylene blue for 1-2 min

Decolourise using 0.25% to 0.5% sulphuric acid for 7-10 min

Wash with water

Heat the slide intermittently until steam rises for 5 min

Cover the slide with carbol fuchsin

Heat fix the smear

DORNER METHOD

Vegetative cells – colourless Endospores – red Background – black.

Observe under oil immersion lens

Dry a thin even film of nigrosin(10%) on the slide

Rinse with water and blot dry

Decolorize with acid-alcohol for 1 minute

Wash with water

Cover the smear with carbol fuchsin and intermittently heat the slide for 5 min. Do not

allow the stain on the smear to dry

Heat fix the slide

VARIATION IN DORNER METHOD

Vegetative cells – colourless Endospores -red Background – black.

Observe under oil immersion lens.

Mix a loopful of nigrosin on a glass slide with one loopful of the boiled carbol

fuchsin-organism suspension and air dry to a thin film.

Immerse the tube in a boiling water bath for 10 minutes.

Mix an aqueous suspension of bacteria with an equal volume of carbol fuchsin in a

test tube.

SCHAEFFER-FULTON METHOD / MODIFIED ASHBY METHOD

Vegetative cell – pink or red and Spore – green.

Counterstained with safranine 1 min, vegetative cells stained.

After cooling, outer layer makes the spore resistant to the action of decolorizing agent (water), but water can easily decolorize the vegetative

cells.

Smear taken from the steam bath and allowed to cool.

Both the spore and vegetative cells appear green.

Smear is heated over a steam bath for 5 min.

The primary stain, Malachite Green, is added over the heat fixed bacterial smear

SCHAEFFER-FULTON METHOD

SCHAEFFER-FULTON METHOD

MOELLER STAIN

Spores red; Bacteria-blue.

The slide is rinsed with acidified ethanol, and counter-stained with methylene blue

for 30 seconds.

The slide is then heated over a bunsenburner, or suspended over a hot water bath, covered with a paper towel, and

steamed for 3 minutes.

Carbol fuchsin is applied to a heat-fixed slide.

MODIFIED MOELLER STAINING METHOD

Rinse thoroughly in running water

Differentiate with 2 % sulphuric acid for 5-10 sec.

Rinse thoroughly in running water.

Flood with un-steamed Kinyoun’s carbol-fuchsin solution containing Tergitol7, and stain for 3 min.

Rinse thoroughly in running water.

Immerse in 5% chromic acid solution for 3 min.

Fix in absolute methyl alcohol for 1-3 min.

Spread a small drop of the specimen on a slide, and allow it to air-dry at room temperature.

MODIFIED MOELLER STAINING METHOD CONTD…

Microscopic examination using oil objective lens

Rinse with running water and allow it to air-dry.

Counterstain with 0.1 % Loeffler’smethylene blue for 1-2 min.

Rinse for 10 sec in running water.

Decolorize with 80 % ethanol until removal of excess stain dye from the

slides.

ABBOTT'S METHOD

Spores blue, bodies of the bacteria red.

Wash in water, dry and mount.

Stain for 8-10 seconds in aniline-fuchsin solution.

Wash in water.

Wash in 95% alcohol containing 0.2 to 0.3% HCl.

Wash in water.

Stain the slide deeply with methylene-blue, heating until the solution boils.

HOLBROOK AND ANDERSON LIPID/SPORE STAINING METHOD(1980)

Wash in water, dry and examine. Spores-green, vegetative bacilli-red, lipid granules-unstained.

Counterstain with 0.5% safranine for 20 sec.

Wash film with xylene for 5 sec and blot dry.

Stain with 0.3% sudan black B in 70% ethanol for 15 min

Stain the film with 5% malachite green for 2 min while the slide is held little above the surface of boiling water in a beaker. Then wash

with water and blot dry.

Air dry the film and fix with minimal flaming.

Many treatments are known which destroy the permeability barrier,

such as

Severe heat fixation (Bartholomew and 'Mittwer, 1950),

Acid hydrolysis (Robinow, 1951),

Ultraviolet light (Bartholomew and Mittwer, 1952),

Mechanical rupture (Fitz-James, 1953; Rode and Foster, 1960).

OTHER MODIFICATIONS OF DORNER’S AND SCHAEFFER-

FULTON METHODS….

BARTHOLOMEW AND MITTWER METHOD

SUMMARYMethod Primary stain Decoloriser Counterstain Interpretation

Grams stain Crystal violet Acetone Saffranin Spore-colourless

Bacteria-violet

Modified Ziehl-

Neelsen Stain

Carbol fuchsin 0.25%-0.5%

H2SO4

Loeffler’s

methylene blue

Spore-red

Bacteria-blue

Dorner stain Carbol fuchsin Acid alcohol Nigrosin Spore-red

Bacteria-colorless

Variation in

dorner stain

Carbol fuchsin Nigrosin Spore-red

Bacteria-colorless

Schaeffer-Fulton

stain

Malachite green Water Safranine Spore-green

Bacteria-red

Bartholomew and

mittwer method

Malachite green Water Safranine Spore-green

Bacteria-red

Abbott's Method Methylene blue Acid alcohol Aniline-fuchsin Spore-blue

Bacteria-red

Moeller stain Carbol fuchsin Acidified

ethanol

Methylene blue Spore-red

Bacteria-blue

Modified moeller

stain

Kinyoun’s

carbol-fuchsin

2%H2SO4

80% ethanol

Loeffler’s

methylene blue

Spore-red

Bacteria-blue

REFERENCES

Colour Atlas & Textbook of Diagnostic Microbiology by Koneman

Practical Medical Microbiology by Mackie & McCartney

Monica Cheesbrough

Textbook of Microbiology by Ananthanarayan

http://www.microbelibrary.org/component/resource/laboratory-test/3112-

endospore-stain-protocol

http://chestofbooks.com/health/disease/Pathology/Methods-For-Staining-

Spores.html#

http://www.generalmicroscience.com/microbial-laboratory-

techniques/endospore-staining-by-bartholomew-and-mittwers-method/

M. Hayama, K. Oana, T. Kozakai, S. Umeda, J. Fujimoto, H. Ota, Y.

Kawakami Proposal of a simplified technique for staining bacterial spores

without applying heat-successful modification of Moeller’s method. Eur J

Med Res (2007) 12: 356-359.