BSc Microbiology – Advanced
Practical Notes (Part 2)
Differential Staining Techniques
Acid-Fast Staining (Ziehl-Neelsen Method)
Acid-fast staining is an important differential staining technique used to identify bacteria
that have mycolic acid in their cell walls. These bacteria are called acid-fast bacteria
because they retain the primary stain even after treatment with acid-alcohol decolorizer.
Principle:
Mycobacteria have a waxy cell wall containing mycolic acid which makes them
impermeable to ordinary stains. Carbol fuchsin (the primary stain) is a lipid-soluble dye
that can penetrate this waxy layer when heat is applied. Once stained, acid-fast bacteria
resist decolorization by acid-alcohol due to high lipid content in their cell walls. Non-acid-
fast bacteria lose the primary stain during decolorization and take up the counterstain.
Materials Required:
Clean glass slides
Carbol fuchsin (primary stain)
Acid-alcohol (3% HCl in 95% ethanol - decolorizer)
Methylene blue (counterstain)
Bunsen burner or steam bath
Blotting paper
Microscope with oil immersion lens
Bacterial culture (Mycobacterium tuberculosis or M. smegmatis)
Procedure:
Step 1: Smear Preparation
Take a clean grease-free glass slide. Add a drop of distilled water on the slide. Transfer a
small amount of bacterial culture using an inoculating loop and spread it evenly to make a
thin smear. Allow the smear to air dry completely. Pass the slide through a flame 2-3 times
to heat-fix the bacteria to the glass surface.
Step 2: Primary Staining
Place a piece of blotting paper or filter paper over the smear. Flood the slide with carbol
fuchsin stain covering the entire smear. Heat the slide gently over a Bunsen burner or place
on a steam bath. Do not allow the stain to boil. The stain should steam for about 5-7
minutes. Add more stain if it begins to dry out. Remove the blotting paper after heating.
,Step 3: Washing
Allow the slide to cool to room temperature. Wash the slide gently with tap water or distilled
water to remove excess stain. Tilt the slide to drain water completely.
Step 4: Decolorization
Flood the slide with acid-alcohol decolorizer. Allow it to act for 2-3 minutes or until the pink
color stops flowing from the smear. Wash immediately with water to stop the decolorization
process. This step is critical - over-decolorization can remove stain from acid-fast bacteria
while under-decolorization leaves non-acid-fast bacteria pink.
Step 5: Counterstaining
Apply methylene blue counterstain to the smear. Allow it to act for 1-2 minutes. Wash gently
with water to remove excess counterstain. Blot the slide with blotting paper or allow to air
dry completely.
Step 6: Microscopic Examination
Place a drop of immersion oil on the dried smear. Observe under oil immersion objective
(100X) of the microscope. Acid-fast bacteria appear bright red or pink against a blue
background. Non-acid-fast bacteria appear blue.
Results and Interpretation:
Acid-fast bacteria retain the red color of carbol fuchsin = RED/PINK cells
Non-acid-fast bacteria take up methylene blue = BLUE cells
Clinical Significance:
This staining technique is extremely important for diagnosing tuberculosis (TB) caused by
Mycobacterium tuberculosis. It is also used to identify Mycobacterium leprae which causes
leprosy. Other acid-fast bacteria include Nocardia species. The presence of acid-fast bacilli
(AFB) in sputum samples is a presumptive diagnosis of tuberculosis.
Important Points to Remember:
Heat application is essential for carbol fuchsin penetration
Mycolic acid in cell wall makes bacteria acid-fast
Over-decolorization can give false-negative results
Under-decolorization can give false-positive results
Always use fresh reagents for accurate results
Sputum samples should be concentrated before staining
At least 100 oil immersion fields should be examined before reporting negative
Endospore Staining (Schaeffer-Fulton Method)
Endospore staining is a differential staining technique used to visualize bacterial
endospores. Endospores are highly resistant dormant structures formed by certain bacteria
under unfavorable environmental conditions. They can survive extreme heat, desiccation,
radiation, and chemical disinfectants.
Principle:
Bacterial endospores have a tough outer covering composed of keratin-like protein and
, dipicolinic acid which makes them impermeable to ordinary stains. Malachite green is a
water-soluble dye that can penetrate the endospore wall only when heat is applied. Once
inside, the dye is trapped and resists decolorization with water. Vegetative cells lose the
green color during washing and take up the pink safranin counterstain.
Materials Required:
Clean glass slides
Malachite green (5% aqueous solution - primary stain)
Safranin (0.5% aqueous solution - counterstain)
Bunsen burner or steam bath
Blotting paper
Distilled water
Microscope
Bacterial culture (Bacillus subtilis, Bacillus cereus, or Clostridium species)
Procedure:
Step 1: Smear Preparation
Prepare a thin bacterial smear on a clean glass slide using standard aseptic technique.
Allow the smear to air dry completely at room temperature. Heat-fix the smear by passing
through the flame 2-3 times. Cooling is important before applying stain.
Step 2: Primary Staining with Malachite Green
Place a piece of blotting paper over the fixed smear. The blotting paper should cover the
entire smear area. Saturate the blotting paper with malachite green stain. Heat the slide
gently over a steam bath or Bunsen burner flame. Keep the stain steaming (not boiling) for
5-10 minutes. Add more malachite green stain periodically to prevent drying. The steam
helps drive the stain into the resistant endospore coat.
Step 3: Cooling and Washing
Remove the slide from heat and allow it to cool for about 2 minutes. Remove and discard the
blotting paper. Wash the slide thoroughly with tap water or distilled water for about 30
seconds. This step removes the malachite green from vegetative cells but not from
endospores. Tilt the slide to drain excess water.
Step 4: Counterstaining with Safranin
Apply safranin counterstain to the smear. Allow it to act for 30-60 seconds without heating.
Safranin stains the vegetative cells and any other cellular material that lost the green color
during washing. Wash gently with water to remove excess safranin.
Step 5: Drying and Observation
Blot the slide dry with blotting paper or allow to air dry. Examine under oil immersion
objective (100X) after adding immersion oil. Endospores appear green while vegetative
cells and other cellular material appear red or pink.
Results and Interpretation:
Endospores = GREEN (retained malachite green)
Vegetative cells = RED/PINK (stained with safranin)
Practical Notes (Part 2)
Differential Staining Techniques
Acid-Fast Staining (Ziehl-Neelsen Method)
Acid-fast staining is an important differential staining technique used to identify bacteria
that have mycolic acid in their cell walls. These bacteria are called acid-fast bacteria
because they retain the primary stain even after treatment with acid-alcohol decolorizer.
Principle:
Mycobacteria have a waxy cell wall containing mycolic acid which makes them
impermeable to ordinary stains. Carbol fuchsin (the primary stain) is a lipid-soluble dye
that can penetrate this waxy layer when heat is applied. Once stained, acid-fast bacteria
resist decolorization by acid-alcohol due to high lipid content in their cell walls. Non-acid-
fast bacteria lose the primary stain during decolorization and take up the counterstain.
Materials Required:
Clean glass slides
Carbol fuchsin (primary stain)
Acid-alcohol (3% HCl in 95% ethanol - decolorizer)
Methylene blue (counterstain)
Bunsen burner or steam bath
Blotting paper
Microscope with oil immersion lens
Bacterial culture (Mycobacterium tuberculosis or M. smegmatis)
Procedure:
Step 1: Smear Preparation
Take a clean grease-free glass slide. Add a drop of distilled water on the slide. Transfer a
small amount of bacterial culture using an inoculating loop and spread it evenly to make a
thin smear. Allow the smear to air dry completely. Pass the slide through a flame 2-3 times
to heat-fix the bacteria to the glass surface.
Step 2: Primary Staining
Place a piece of blotting paper or filter paper over the smear. Flood the slide with carbol
fuchsin stain covering the entire smear. Heat the slide gently over a Bunsen burner or place
on a steam bath. Do not allow the stain to boil. The stain should steam for about 5-7
minutes. Add more stain if it begins to dry out. Remove the blotting paper after heating.
,Step 3: Washing
Allow the slide to cool to room temperature. Wash the slide gently with tap water or distilled
water to remove excess stain. Tilt the slide to drain water completely.
Step 4: Decolorization
Flood the slide with acid-alcohol decolorizer. Allow it to act for 2-3 minutes or until the pink
color stops flowing from the smear. Wash immediately with water to stop the decolorization
process. This step is critical - over-decolorization can remove stain from acid-fast bacteria
while under-decolorization leaves non-acid-fast bacteria pink.
Step 5: Counterstaining
Apply methylene blue counterstain to the smear. Allow it to act for 1-2 minutes. Wash gently
with water to remove excess counterstain. Blot the slide with blotting paper or allow to air
dry completely.
Step 6: Microscopic Examination
Place a drop of immersion oil on the dried smear. Observe under oil immersion objective
(100X) of the microscope. Acid-fast bacteria appear bright red or pink against a blue
background. Non-acid-fast bacteria appear blue.
Results and Interpretation:
Acid-fast bacteria retain the red color of carbol fuchsin = RED/PINK cells
Non-acid-fast bacteria take up methylene blue = BLUE cells
Clinical Significance:
This staining technique is extremely important for diagnosing tuberculosis (TB) caused by
Mycobacterium tuberculosis. It is also used to identify Mycobacterium leprae which causes
leprosy. Other acid-fast bacteria include Nocardia species. The presence of acid-fast bacilli
(AFB) in sputum samples is a presumptive diagnosis of tuberculosis.
Important Points to Remember:
Heat application is essential for carbol fuchsin penetration
Mycolic acid in cell wall makes bacteria acid-fast
Over-decolorization can give false-negative results
Under-decolorization can give false-positive results
Always use fresh reagents for accurate results
Sputum samples should be concentrated before staining
At least 100 oil immersion fields should be examined before reporting negative
Endospore Staining (Schaeffer-Fulton Method)
Endospore staining is a differential staining technique used to visualize bacterial
endospores. Endospores are highly resistant dormant structures formed by certain bacteria
under unfavorable environmental conditions. They can survive extreme heat, desiccation,
radiation, and chemical disinfectants.
Principle:
Bacterial endospores have a tough outer covering composed of keratin-like protein and
, dipicolinic acid which makes them impermeable to ordinary stains. Malachite green is a
water-soluble dye that can penetrate the endospore wall only when heat is applied. Once
inside, the dye is trapped and resists decolorization with water. Vegetative cells lose the
green color during washing and take up the pink safranin counterstain.
Materials Required:
Clean glass slides
Malachite green (5% aqueous solution - primary stain)
Safranin (0.5% aqueous solution - counterstain)
Bunsen burner or steam bath
Blotting paper
Distilled water
Microscope
Bacterial culture (Bacillus subtilis, Bacillus cereus, or Clostridium species)
Procedure:
Step 1: Smear Preparation
Prepare a thin bacterial smear on a clean glass slide using standard aseptic technique.
Allow the smear to air dry completely at room temperature. Heat-fix the smear by passing
through the flame 2-3 times. Cooling is important before applying stain.
Step 2: Primary Staining with Malachite Green
Place a piece of blotting paper over the fixed smear. The blotting paper should cover the
entire smear area. Saturate the blotting paper with malachite green stain. Heat the slide
gently over a steam bath or Bunsen burner flame. Keep the stain steaming (not boiling) for
5-10 minutes. Add more malachite green stain periodically to prevent drying. The steam
helps drive the stain into the resistant endospore coat.
Step 3: Cooling and Washing
Remove the slide from heat and allow it to cool for about 2 minutes. Remove and discard the
blotting paper. Wash the slide thoroughly with tap water or distilled water for about 30
seconds. This step removes the malachite green from vegetative cells but not from
endospores. Tilt the slide to drain excess water.
Step 4: Counterstaining with Safranin
Apply safranin counterstain to the smear. Allow it to act for 30-60 seconds without heating.
Safranin stains the vegetative cells and any other cellular material that lost the green color
during washing. Wash gently with water to remove excess safranin.
Step 5: Drying and Observation
Blot the slide dry with blotting paper or allow to air dry. Examine under oil immersion
objective (100X) after adding immersion oil. Endospores appear green while vegetative
cells and other cellular material appear red or pink.
Results and Interpretation:
Endospores = GREEN (retained malachite green)
Vegetative cells = RED/PINK (stained with safranin)
