Microbiological stains – Simple, Gram’s and differential staining -Microbiology

Microbiological Stains (acidic, basic and special) and principles of staining, Simple, Gram’s and differential staining

Microbiological staining is a technique employed in microscopy to improve the visibility and contrast of microscopic images.

Stain

A stain is a substance that adheres to a cell or specimen or section, providing color and contrast for enhanced visibility under a microscope.

• Stains and dyes are commonly utilized in biological tissues for observation, often with the assistance of various microscopes.

• These stains can be used to examine and define bulk tissues, such as muscle fibers or connective tissue, classify different cell populations like blood cells, and even reveal organelles within individual cells.

• For effective microscopic observation, proper preparation of specimens is crucial, involving fixation and staining to increase visibility, to notice specific morphological features, and preserve them for future reference.

Important terms related to staining:

1. Chromophore

The part of the stain molecule responsible for its color.

2. Auxochrome

A group in the stain molecule that enhances the ability of the dye to bind to tissues or cells (e.g., amino, carboxyl groups).

3. Fixative

A chemical substance used to preserve biological tissues or cells before staining (e.g., formaldehyde, alcohol).

4. Mordant

A substance that enhances the binding of the stain to the tissue, fixing the dye in place (e.g., iodine in Gram staining).

5. Primary Stain

The first stain applied during a multi-step staining process (e.g., crystal violet in Gram staining).

6. Counterstain

A lighter stain applied after the primary stain to provide contrast, helping visualize cells or structures that didn’t retain the primary stain (e.g., safranin in Gram staining).

7. Decolorizer

A chemical (often alcohol or acetone) used to remove the primary stain from certain cells or components, allowing differential staining.

8. Simple Stain

A staining technique using a single dye to color cells uniformly.

9. Differential Stain

A staining technique that uses more than one dye to distinguish between different types of cells or cell structures (e.g., Gram stain, acid-fast stain).

10. Positive Staining

The technique where the stain colors the cells or tissues themselves, making them visible.

11. Negative Staining

The technique where the background is stained, leaving the cells or tissues clear or transparent, often used for observing cell morphology or capsules.

12. Acidic Stain

A negatively charged dye that binds to positively charged cell components (e.g., eosin).

13. Basic Stain

A positively charged dye that binds to negatively charged cell components (e.g., methylene blue).

14. Neutral Stain

A stain made of both acidic and basic dyes, which colors different parts of the cell different colors.

15. Histological Staining

Staining used specifically for studying the microscopic structure of tissues (e.g., H&E stain – Hematoxylin and Eosin).

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Microbiological stains (acidic, basic andspecial)

Stains are colored organic compounds (salts) used for staining cells, tissues, microorganisms, etc.

Stains contain ions which impart color, these ions are called chromophores.  

If the chromophore is a positive ion, then the dye/stain is called a basic stain.

If Chromophore is negative, then the dye/stain is known as acidic stain.

According to pH, stains can be classified into:

Acidic Stains:

These stains have a negative charge, hence they bind to positively charged cellular structures like some proteins.

Acidic dyes are not very often used in a microbiology lab, except to provide background staining like in negative staining.

Examples include nigrosine, picric acid, eosin, carbol fuchsin, etc.

Application: Used for negative staining to observe bacterial shapes and sizes, particularly useful for visualizing capsules.

Basic Stains: 

These stains have a positive charge, hence they bind to negatively charged molecules such nucleic acids and acids in bacterial cell walls (teichoic acids in gram-positive cells and phospholipids in gram-negative cells).

Since the surface of bacterial cells are negatively charged, basic dyes are most commonly used in bacteriology.

Examples include: crystal violet, methylene blue, safranin, basic fuchsin, etc.

Application: Used in simple staining and differential staining techniques to visualize bacteria and other cells.

Neutral Stains:

These stains are usually formed from precipitation, when aqueous acidic and basic stains are combined. Neutral dyes stain nucleic acids and cytoplasm.

Examples include: eosinate of methylene blue, Giemsa stain, etc.

Different stains exhibit selective affinities for distinct organisms or their components, enabling the differentiation of various organisms or the visualization of specific parts.

Special Stains:

Principle: Special stains are used for identifying specific structures or features of microorganisms, such as endospores, flagella, and capsules.

Examples:

Endospore Stain:

Malachite Green (stains spores) and Safranin (stains vegetative cells).

Capsule Stain: 

India Ink or Nigrosin for negative staining, and basic dyes for the cells.

Flagella Stain:

Tannic acid combined with crystal violet.

Application: To study specific microbial features (endospores, flagella, and capsules).

Some Steps Associated With Staining

1. Preparation of Smear 

Smear is a sample of cells or tissue or other material taken from a biological context, spread thinly on a microscope slide for examination.

It is prepared as follows:

• Place a small drop of the sample (i.e., blood, microbial culture, etc.) one side of a clean slide, about 1-2 cm from one end.
• Place another slide (spreader) at an angle of 45° from the slide and move it back to make contact with the drop. The drop should spread out quickly along the line of contact of the spreader with the slide.
• Spread the film by rapid smooth forward movement of the spreader. The film should be 3-4 cm in length.

2. Fixation of Smear

Fixation is a process that kills the bacteria, firmly attaches the smear to the microscope slide, and allows the sample to more readily take up the stain.

Before fixation, the smear is air-dried so that it fixes to the glass to avoid it being washed when treated with liquid stain.

Fixation denatures the bacterial enzymes and prevents autolysis (enzymatic digestion of cells) and ensures bacterial adherence to a microscopic slide. Soon after fixing, the slide can be stained.

Fixation can be done in one of two ways:

Heat Fixation: 

The smear is passed through the top part of the Bunsen burner flame 2-4 times taking care the glass slide is quite hot but bearable to one’s hand skin.

Chemical Fixation:

The smear can be chemically fixed by covering the smear with 95% methanol for 1-5 minutes.

Some the materials used in microbial staining techniques as follows:

• Stains & Reagents
• Staining Rack
• Glass Slides & Cover Slips
• Inoculation Loop
• Dropper
• Wash Bottle
• Bunsen Burner
• Compound Microscope

Principles of Staining

• The principle of staining involves using dyes to selectively bind to cell components, enhancing visibility under a microscope.

• Acidic stains bind to positively charged structures, while basic stains bind to negatively charged ones.

• Fixation preserves the cells, and mordants may be used to fix the dye more firmly.

• Simple staining uses one dye, while differential staining uses multiple to distinguish cell types.

• Decolorization removes the primary stain from some cells, followed by counterstaining for contrast.

• Factors like time, dye concentration, and pH affect staining intensity and specificity.

Simple staining

Simple staining is a technique used in microbiology to make bacteria more visible under a microscope by using a single dye to color cells. This helps to observe the shape, size, and arrangement of microorganisms. Here’s the step-by-step process:

Materials Needed

1. Bacterial smear
2. Staining dye (commonly used stains: Methylene blue, Crystal violet, or Safranin)
3. Microscope slide
4. Inoculating loop
5. Bunsen burner or heat source
6. Distilled water
7. Microscope
8. Bibulous paper or blotting paper

Procedure

1. Prepare the Bacterial Smear:

   – Take a clean glass microscope slide.

   – Use a sterilized inoculating loop to transfer a small sample of bacterial culture onto the slide.

   – Add a drop of distilled water to the slide if the sample is from solid media (skip this step if from liquid media).

   – Spread the bacteria evenly into a thin layer on the slide.

2. Air Dry the Smear:

   – Allow the smear to air dry completely at room temperature. This prevents the bacteria from being washed away during the staining process.

3. Heat Fixation:

   – Once the smear is dry, pass the slide through the flame of a Bunsen burner 2-3 times. This kills the bacteria and makes them stick to the slide.

4. Staining:

   – Cover the heat-fixed smear with a few drops of staining dye (Methylene blue, Crystal violet, or Safranin).

   – Let the dye sit on the smear for about 30 to 60 seconds, depending on the stain used.

5. Rinse the Slide:

   – Hold the slide at an angle and gently rinse it with distilled water to remove excess dye. Be careful not to over-rinse.

6. Dry the Slide:

   – Gently blot the slide dry with bibulous paper or allow it to air dry.

7. Examine Under Microscope:

   – Place the slide under the microscope and start viewing with a lower magnification, then move to higher magnifications for better visualization of bacterial shape and arrangement.

Result:

The bacteria will appear colored against a clear or lightly stained background, making it easier to observe their morphology.

Gram Staining Technique

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938).

Aim

To study the basic classification of bacterial suspension by gram staining method.

Principle

The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet.

Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with.

Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process.

Gram-Positive Staining

Gram-positive bacteria have a thick peptidoglycan layer in their cell walls, which allows them to retain the crystal violet-iodine complex during the Gram staining process, resulting in a purple color under the microscope.

Steps and Process for Gram-Positive Staining:

Reagents used

1. Crystal violet (primary stain)
2. Iodine solution/Gram’s Iodine (mordant that fixes crystal violet to cell wall)
3. Decolorizer (Ethanol)
4. Safranin (secondary stain)

Requirement

1. Bacterial suspension (Sample),
2. Slides,
3. Microscope,
4. Distilled water.
5. Blotting paper

Procedure/ Steps

1. Place a drop of bacterial suspension on one edge of clean glass slide.
2. And make a thin smear of bacterial culture on glass slide. The cells are then fixed to a slide by passing slightly above the Bunsen burner.
3. Add a drop of Crystal violet stain on the heat fixed bacterial smear, allow it for 30-40second.
4. Then slide is rinsed with the water to remove the excess stain of Crystal violet. In this stage all cell appear purple under the microscope.
5. The gram Iodine solution is added on a slide and retains it for about 1min. The Iodine combines with crystal violet to form di-iodine complex there by decrease solubility within the cell.
6. The drop wise ethanol is added to cell for decolorize, but the cells retain crystal violet stain due to thick peptidoglycan layer.
7. Excess ethanol is then washed with the water.
8. The slide is dried with filter paper. And observed under microscope.

Observation

Presence of Cocci (round) and Bacilli few shaped bacteria with purple color observed under microscope.

Result

Positively strained Purple colored Bacilli and Cocci shaped bacteria were obtained.

Conclusion

Gram positive bacteria were observed under microscope.

Gram Negative Staining Bacteria

The Gram staining technique for Gram-negative bacteria is the same process used for Gram-positive bacteria. The difference in results comes from the structural differences in their cell walls.

Aim

To observe the morphology of bacterial cell by negative staining technique.

Principle

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes. Lipopolysaccharide (LPS) is the most abundant antigen on the cell surface of most Gram-negative bacteria. The stain that stains the background and does not stain bacteria is called negative staining. Adding Alcohol, Gram negative bacteria loses color of crystal violet as crystal violet bound lipid containing outer membrane gets dissolved in alcohol.

Requirement

1. Crystal violet,
2. Bacterial suspension,
3. Inoculation loop,
4. Bunsen burner,
5. Slides,
6. Microscope,
7. Iodine solution,
8. Ethanol,
9. Safranin.

Procedure

1. Place a drop of bacterial suspension on one edge of the clean glass slide.
2. And make a thin smear of bacterial culture on a glass slide. The cells are then fixed to a slide by passing slightly above the Bunsen burner.
3. Add a drop of Crystal violet stain on the heat fixed bacterial smear, allow it for 30-40 seconds.
4. Then the slide is rinsed with the water to remove the excess stain of Crystal violet. In this stage all cells appear purple under the microscope.
5. The solution of gram Iodine solution is added on a slide and retains it for about 1 min. The Iodine combines with crystal violet to form a di-iodine complex thereby decreasing solubility within the cell.
6. Few drops of ethanol is added on the slide for decolorize the cells and lose the crystal violet stain.
7. Excess ethanol is then washed with the water.
8. Cells are covered by counterstain Safranin for 20-30 seconds. This stain stains gram negative bacteria pink.
9. Then excess of stain is rinse with water and slide is dried with filter paper
10. And observed under a microscope.

Observation

Presence of Gram negative bacteriaResult: Gram negative stained bacteria were obtained.

Conclusion

Gram negative bacteria were observed under microscope.

The difference between Gram-negative and Gram-positive bacteria in this staining method is due to the thickness of their peptidoglycan layers and the presence of an outer membrane in Gram-negative bacteria.