Microbiology

Dyes or stains may be divided into two groups: basic and acidic.

Basic dye: If the color portion of the dye resides in the positive ion, as in the above case, it is called a basic dye(examples: methylene blue, crystal violet, safranin).

when using a basic dye, the positively charged color portion of the stain combines with the negatively charged bacterial cytoplasm (opposite charges attract) and the organism becomes directly stained (see Fig. 1).

Acidic dye: If the color portion is in the negatively charged ion, it is called an acidic dye (examples: nigrosin, congo red).

An acidic dye, due to its chemical nature, reacts differently. Since the color portion of the dye is on the negative ion, it will not readily combine with the negatively charged bacterial cytoplasm (like charges repel). Instead, it forms a deposit around the organism, leaving the organism itself colorless (see Fig. 1). Since the organism is seen indirectly, this type of staining is called indirect or negative, and is used to get a more accurate view of bacterial size, shapes, and arrangements.

Direct Stain using a Basic Dye

In direct staining, the positively charged color portion of the basic dye combines with the negatively charged bacterium, and the organism becomes directly stained.


Organisms
Your pure cultures of Staphylococcus epidermidis (coccus with staphylococcus arrangement) or Micrococcus luteus (coccus with a tetrad or a sarcina arrangement) and Escherichia coli (small bacillus) or Enterobacter aerogenes (small bacillus).

Procedure

1.  Heat-fix a smear of either Escherichia coli or Enterobacter aerogenes as follows:

a.  Using the dropper bottle of distilled water found in your staining rack, place a small drop of water on a clean slide by touching the dropper to the slide.

b.  Aseptically remove a small amount of the culture from the agar surface

c.   Burn the remaining bacteria off the loop. (If too much culture is added to the water, you will not see stained individual bacteria.)

d.  Using the loop, spread the suspension over the entire slide to form a thin film.

e.  Allow this thin suspension to completely air dry.

f.    Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat-fix.

2.  Place the slide on a staining tray and cover the entire film with safranin. Stain for 1 minute.

3.  Pick up the slide by one end and hold it at an angle over the staining tray. Using the wash bottle on the bench top, gently wash off the excess safranin from the slide. Also wash off any stain that got on the bottom of the slide.

4.  Use a book of blotting paper to blot the slide dry. Observe using oil immersion microscopy.

5.  Prepare a second direct, this time using either Staphylococcus epidermidis or Micrococcus luteus as the organism.

a.  Heat-fix a smear of the Micrococcus luteus or Staphylococcus epidermidis by following the directions under step 1.

b.  Stain with methylene blue for 1 minute.

c.   Wash off the excess methylene blue with water.

d.  Blot dry and observe using oil immersion microscopy.

6.  Prepare a third slide of the normal flora and cells of your mouth.

a.  Using a sterile cotton swab, vigorously scrape the inside of your mouth and gums.

b.  Rub the swab over the slide (do not use water), air dry, and heat-fix.

c.   Stain with crystal violet for 30 seconds.

d.  Wash off the excess crystal violet with water.

e.  Blot dry and observe. Find epithelial cells using your 10X objective, center them in the field, and witch to oil immersion to observe the normal flora bacteria on and around your epithelial cells.

Indirect Stain using an Acidic Dye

In negative staining, the negatively charged color portion of the acidic dye is repelled by the negatively charged bacterial cell. Therefore, the background will be stained and the cell will remain colorless.


Organism
Your pure culture of Staphylococcus epidermidis or Micrococcus luteus.


Procedure

1.  Place a small drop of nigrosin on a clean slide.

2.  Aseptically add a small amount of Staphylococcus epidermidis or Micrococcus luteus to the dye and mix gently with the loopUsing the edge of another slide, spread the mixture with varying pressure across the slide so that there are alternating light and dark areas. Make sure the dye is not too thick or you will not see the bacteria!

3.  Let the film of dyed bacteria air dry completely on the slide. Do not heatfix and do not wash off the dye.

4.  Observe using oil immersion microscopy. Find an area that has neither too much nor too little dye (an area that appears light purple where the light comes through the slide). If the dye is too thick, not enough light will pass through; if the dye is too thin, the background will be too light for sufficient contrast.

Procedure of Gram’s Staining Technique:

1.Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. (# Methanol fixation preserves the morphology of host cells, as well as bacteria, and is especially useful for examining bloody specimen material).

An easy way to remember the steps of the Gram stain

2.   Application of the primary stain (crystal violet). Crystal violet stains all cells blue/purple

3.   Application of mordant: The iodine solution (mordant) is added to form a crystal violet iodine (CV-I) complex; all cells continue to appear blue.

4.   Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells.
The organic solvent such as acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin walled gram negative bacteria to a greater degree than from the lipid poor, thick walled, gram-positive bacteria.  The gram negative bacteria appear colorless and gram positive bacteria remain blue.

5.   Application of counter stain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

Principle of Gram Staining

Image 2: Cell wall of Gram Positive and Gram Negative Bacteria

The differences in cell wall composition of Gram positive and Gram negative bacteria accounts for the Gram staining differences. Gram positive cell wall contain thick layer of peptidoglycan with numerous teichoic acid cross linking which resists the decolorization.

In aqueous solutions crystal violet dissociates into CV+ and Cl – ions that penetrate through the wall and membrane of both Gram-positive and Gram-negative cells. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.

When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet iodine (CV-I) complexes within the cytoplasm and outer layers of the cell.

The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram negative bacteria.

The outer membrane of the Gram-negative cell (lipopolysaccharide layer)  is lost from the cell, leaving the peptidoglycan layer exposed. Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of gram-positive cells. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.

The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. The multi-layered nature of the peptidoglycan along with the dehydration from the ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple in color, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Acid-Fast Stain- Principle, Procedure, Interpretation and Examples

It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen stainingtechniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.

The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.

This method is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.

Principle of Acid-Fast Stain

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Then after all cell appears red. Then the smear is decolorized with decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene blue. Only decolorized cells absorb the counter stain and take its color and appears blue while acid-fast cells retain the red color.

Procedure of Acid-Fast Stain

1.   Prepare bacterial smear on clean and grease free slide, using sterile technique.

2.   Allow smear to air dry and then heat fix.
Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite (bleach) treated sputum and will not be stained immediately. M. tuberculosis is killed by bleach and during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas alcohol-fixation is bactericidal.

3.   Cover the smear with carbol fuchsin stain.

4.   Heat the stain until vapour just begins to rise (i.e. about 60 [1]C). Do not overheat. Allow the heated stain to remain on the slide for 5 minutes.
Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is carried out over a tray or other container in which highly flammable chemicals have collected from previous staining. Only a small flame should be applied under the slides using an ignited swab previously dampened with a few drops of acid alcohol or 70% v/v ethanol or methanol. Do not use a large ethanol soaked swab because this is a fire risk.

5.   Wash off the stain with clean water.
Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.

6.   Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e. pale pink.
Caution: Acid alcohol is flammable, therefore use it with care well away from an open flame.

7.   Wash well with clean water.

8.   Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin.

9.   Wash off the stain with clean water.

10.   Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).

11.   Examine the smear microscopically, using the 100 X oil immersion objective.