Staining
Staining is a technique used in microbiology to enhance the contrast of cells and microorganisms under a microscope, making them easier to see and identify. The type of stain used depends on the sample and the cells or organisms being observed
Types of Staining Techniques
- Gram staining
- Ziehl Neelsen Staining
- Giemsa staining
1. Gram staining
Principle
The bacterial cell wall and cytoplasmic membrane’s capacity to permeate the dye-iodine combination is necessary for the reaction to occur. The iodine complex and crystal violet dye combine to generate a big molecule that precipitates inside Gram-positive bacteria.
Materials
- Clean grease free glass slide
- Bacterial growth
- Normal saline
- Inoculum loop
- Bunsen burner
- Pipette / dropper
Gram Staining Reagents
- Crystal violet (Primary stain)
- Iodine (Mordant)
- Decolorizer alcohol (Decolorizer)
- Safranin (counterstain)
Procedure
- Using the inoculum loop, distribute the colony on the glass slide and create a smear after adding a drop of normal
- After applying two to three drops of crystal violet for a minute, the glass slide was rinsed with tap water.
- Next, for one minute, apply two to three drops of iodine to the glass slide, and then rinse with tap water.
- After 30 seconds, apply two to three drops of decolorizer alcohol to the glass slide, and then rinse with tap water
- Finally, paint the glass slide with two to three drops of counterstain safranin for one minute, and then rinse with tap water.
- Observed with a 100X oil immersion microscope.
Result
- Gram negative rods have a pink appearance.
- Gram-positive rods have a purple appearance.
- Ziehl Neelsen Staining
Principle
A bacteriological stain called Ziehl Neelsen (ZN) staining is used to detect Acid-Fast organisms, primarily Mycobacterium Tuberculosis (TB). Mycobacterium’s cell wall contains high lipid content, including alcohol, lipid, fatty acid, and mycolic acid. Due to this, the cell wall becomes rigid and doesn’t easily capture primary stains. That’s why specialized staining techniques are required for it.
Materials
- Clean grease free glass slide
- Bacterial growth
- Normal saline
- Inoculum loop
- Bunsen burner
- Pipette / dropper
Ziehl Neelsen Staining Reagents
- Carbol fuchsin (Primary stain)
- 20% sulphuric acid for tuberculosis/tubercle or 5% sulphuric acid for Lepra bacilli acid alcohol (Decolorizer)
- Methylene blue or malachite green (counterstain)
Procedure
- Using a drop of normal saline on a glass slide, distributed the colony using the inoculum loop, create a smear, and heat-fix it.
- Cover the smear with carbol fuschin, flood it, and then slowly heat it till odors are produced.
- After letting it stand for five minutes, rinse it off with slowly running tap water. Then, add the acid alcohol and let it sit for one to two minutes. Continue doing this until the smear takes on a pink hue.
- Used water to wash the acid off
- Apply a thick layer of methylene blue dye to the smear, let it sit for two to three minutes, and then rinse it with water.
- Allow the stain to air dry before examining it with an oil immersion lens.
Result
- Non-acid fast bacteria absorb the methylene blue dye and become blue
- Acid-fast bacteria stain pink and retain the Primary stain, carbol-fuschin.
- Giemsa staining
Principle
A differential staining method called Giemsa staining is used to see and distinguish distinct parts of cells, especially in tissue slices, bone marrow, and blood smears. Giemsa staining allows for selective staining by taking use of variations in the chemical makeup of cellular constituents. The method includes:
- Acidic structures (cytoplasm, erythrocytes) are stained pink by an acidic dye (eosin).
- Basic structures (nuclei, basophils) are stained by basic dyes (methylene blue and azur). Purple-blue.
Giemsa stain uses
- Giemsa stain uses peripheral blood smear
- Bone marrow specimens
- Erythrocytes stain pink
- Platelets show a light pale pink
- Lymphocytes cytoplasm stains sky blue
- Monocyte cytoplasm stains pale blue
- Diagnosis of malaria
Materials
- Performed peripheral blood test with Giemsa stain
- Clean dry glass slide
- Spreader for slide preparation
- Patient sample in EDTA vial
- Adjustable pipette
- Tap water for dilution
Giemsa staining Reagents
- Giemsa stain (Commercially prepared)
- Methanol 70% for fixation
Procedure
- Put one drop of Blood on slide.
- Use spreader to spread the blood on slide makes a thin smear.
- Wait for air dry.
- Dip the smear (2-3 dips) into 70% methanol for fixation of the smear.
- Leave to air dry for 30 sec.
- Making the dilution ( 1 part stain + 10 part Tap water)
- Using the filter paper to filtrate the stain.
- Flood the slide with Giemsa stain solution for 20-30 minutes.
- Flush with tap water and leave to dry.
- Observe under the microscope first at 40x then using oil immersion lens.
Result
- Neutrophils stained with Wright stain
- The cytoplasm and granules of blood cells appear red in color while nucleus appears blue-purple in color
- The erythrocytes will appear pink in color