Bacterial Staining

All the bacterias are colorless and have some refractive index to the aqueous salt present in the surrounding.So it is difficult to observe structural details until the microscope is optimally adjusted.To overcome this problem the bacterias are colored with stains.

Fixation:Fixation is the process by which the internal and external structures of cells and microorganisms are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation.

Heat fixation is routinely used to observe procaryotes. Typically, a film of cells (a smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology but not structures within cells.
Chemical fixation is used to protect fine cellular substructure and the morphology of larger, more delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, to render them inactive, insoluble, and immobile. Common fixative mixtures contain such components as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Bacterial stains are organic compounds called dye, consisting of Chromophore and Auxochrome groups attached with a benzene ring.

The chromophore is an atom or group whose presence is responsible for the color of a compound. Chromophore is a Greek word that means “Colour bearer”.Molecules absorb some wavelengths of the visible spectrum and reflect other wavelengths that are visible to us.

Auxochrome is a group of atoms attached to the chromophore of a particular molecule. However, it modifies the ability of the chromophore to absorb light. Auxochrome is a Greek word that means “to increase the color”. Auxochromes themselves fail to produce the color, but their presence along with chromophores in an organic compound intensifies the color of the chromogen.Furthermore, some examples of auxochromes include the hydroxyl group (−OH), the amino group (−NH2), the aldehyde group (−CHO), and the methyl mercaptan group (−SCH3). Therefore, auxochromes are functional groups with one or more lone pairs of electrons.

For instance, benzene does not display color as it does not have a chromophore, but nitrobenzene is a pale yellow color because of the presence of a nitro group (−NO2), which acts as a chromophore. Similarly, p-hydroxynitrobenzene exhibits a deep yellow color, in which the −OH group acts as an auxochrome. In this, the auxochrome (−OH) is conjugated with the chromophore −NO2. Also, similar behavior is seen in azobenzene, which has a red color, but p-hydroxyazobenzene is dark red in color.

On the basis of the Auxochrome group,dyes can be Acidic,Basic or Neutral.

Acidic: Anionic(-ve)ly charged , Used to detect Cell cytoplasm

E.g.: Eosin

Basic: Cationic(+ve)ly charged, Stain Cellular constituent

E.g.: Methylene blue,Crystal Violet,Carbol fuchsin, Methyl violet

Neutral: Complex of salts of dye acid & dye base

E.g.: Eosinate of Methylene Blue

Bacterial cell surfaces are negatively charged in neutral pH so it attracts basic stains.Negatively charged molecules in the cell are DNA & RNA.

Types of Staining-

Simple Staining
Differential Staining
Acid fast staining
Negative staining
Endospore staining
Flagellar staining

Simple Staining

Simple staining involves directly staining the bacterial cell with a basic(+ve)ly charged dye in order to see bacterial detail.Some stains commonly used for simple staining include crystal violet, safranin, and methylene blue.Simple stains can be used to determine a bacterial species’ morphology (cell shape),size and arrangement (single, chains, clusters, etc.), but they do not give any additional information.

Gram’s staining

This is the most useful and frequently employed staining technique in bacteriological laboratories. It is a differential staining.It gets its name from the Danish bacteriologist Hans Christian Gram who first introduced it in 1882.Bacteria generally categorized as either Gram positive or Gram negative.

Principle: Gram-positive bacteria have a thick peptidoglycan layer and less lipid.With the use of solvent(alcohol), the lipid layer is dissolved. When alcohol is used for destaining, only narrow pores are formed in the cell wall of gram positive bacteria.As a result they retain the primary stain (Crystal violet) after decolorization. Gram-negative bacteria on the other hand possess very thin peptidoglycan layer but thicker lipid layer.When alcohol is used for destaining, lipid dissolves in alcohol making wide- pores in the cell wall through which primary stain leaks out. As a result they become totally decolorized after alcohol washing and finally retain the counter stain (safranin/dilute carbol fuchsin).Some laboratories use safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely than safranin. Similarly, Haemophilus spp., Legionella app, and some anaerobic bacteria stain poorly with safranin.

The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria.

Reagents (Composition):
a. Hucker’s ammonium oxalate crystal violet
Solution 1
Crystal violet 2 g
Ethanol (95%) 20 ml
Solution 2
Ammonium oxalate 0.8 g
Distilled water 80 ml
Mix solution 1 and solution 2 and filter.

Gram’s iodine solution
Iodine crystal 1 g
Potassium iodide 2 g
Distilled water 200 ml

Dissolve the ingredients. Filter and store in an amber color bottle. It acts as ‘mordant’.
c. Decolourizer
It may be absolute alcohol or a mixture of acetone and alcohol (1:1 v/v)
d. Safranin (counter stain)
Safranin (2.5% solution in 95% ethanol) 10 ml
Distilled water 100 ml
Dissolve the stain and filter.

Materials:
1. Bacterial culture.
2. Reagent a, b, c, d

Procedure:
1. Prepare a bacterial smear upon a grease free slide and fix over flame following standard technique.
2. Flood the smear with ammonium oxalate crystal violet solution and allow it to act for 2-3 minutes.
3. Wash under gently running tap water.
4. Fixing of dye-Flood the smear with Gram’s iodine and keep it for 30 sec.Crystal violet- iodine complex is formed.
5. Wash with gently running tap water.
6. Decolourize with available decolourizer by gently agitating the slide till no color comes out from the smear. Wash with gently running tap water.
7. Flood the smear with safranin/dilute carbol fuchsin and keep it for 30 sec.
8. Wash under gently running tap water, air dry and examine under oil immersion lens.

Observations:
Gram positive bacteria will be deep violet and Gram negative bacteria will be red/pink in color.

Acid Fast Staining:

This is a special type of staining method for a few selective groups of bacteria which are having mycolic acid on their cell wall.This group is called CMN group.(Corynebacterium, Mycobacterium and Nocardia)

Certain bacteria like Mycobacterium sp. do not stain readily by simple staining or even Gram’s Method of differential staining.Stains are relatively impermeable to these cells because of the high lipid (mycolic acid) content of their cell wall.Their staining is facilitated by heat as it will help to penetrate the dye like concentrated Carbolfuchsin.Here heat act as Mordant.Once stained, they retain the color of the primary stain ( carbol fuchsin) even when treated with strong decolourizer(Acid-alcohol). These organisms are designated as acid fast. Counter stain MB or Malachite green is applied to create the contrasting background.

Paul Ehrlich first demonstrated the stain in 1882.Later it was improved by Ziehl-Neelsen,so it is called ZN staining.

Reagents (composition):
a. Concentrated carbol fuchsin
b. Decolourizer (Ethanol (95%) 97 ml and Concentrated HCL 3 ml)
c. Loeffler’s alkaline methylene blue
Materials:
1. A portion of formalin preserved tuberculous lung.
2. Staining solutions – a, b, c.

Procedure:
1. Cut a piece of tuberculous lung and make an impression smear upon a microscopic slide. Dry the smear and fix over the flame.
2. Flood the smear with concentrated carbol fuchsin. Apply heat from bottom of the slide but do not allow boiling. Stain should not dry on the slide. Keep it for 3-5 minutes.
3. Wash with running tap water.
4. Decolourize the smear with decolourizer till no stain comes out.
5. Wash with running tap water.
6. Counterstain with Loeffler’s alkaline methylene blue for about 15-20 seconds and then wash again with running tap water.
7. Dry it and examine it under oil immersion lens (100X).

Observations:
Acid-fast bacteria will show intense red color against blue coloured tissue debris.

Negative Staining

It is the method of staining the background instead of the microbe.The bacteria will appear stainless or transparent against a colorful background.

Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface the dye will not penetrate the cell.Indian ink or Nigrosin is used here.Exact cell size,morphology of bacteria remain same as NO HEAT is applied.

Use: The staining is used to detect Spirochete.

Endospore staining

Bacterial endospores are generally heat & chemical resistant due to presence of Spore coat or Exosporium.

In 1922, Dorner published a method for staining endospores. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain which selectively stains bacterial endospores. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from non-spore formers.

Principle: In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolorized with water. Safranin is then applied to counterstain any cells which have been decolorized. At the end of the staining process, vegetative cells will be pink, and endospores will be dark green.

Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical.

Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)
0.5 gm of malachite green
100 ml of distilled water

Decolorizing agent: Tap water or Distilled Water

Counter Stain: Safranin
Stock solution (2.5% (wt/vol) alcoholic solution)
2.5 gm of safranin O
100 ml of 95% ethanol

Result:

Endospores: Endospores are bright green.
Vegetative Cells: Vegetative cells are brownish red to pink.

Endospore forming bacterias:

Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus

Flagellar staining

Bacterial flagella are normally too delicate & thin to be seen under such conditions. The flagella stains employ a mordant to coat the flagella with stain until they are thick enough to be seen.

There are 2 methods, Wet method & Dry method

Wet method:

It is a more successful and easy method to practice for routine use. The wet mount method is also called the “Ryu method” because it uses Ryu flagella stains to examine the arrangement and number of flagella.

Preparation of Ryu stain: It involves the preparation of two solutions (Solution-I and II).

Solution-I includes the following components in a defined amount:

5% aqueous solution of phenol: 10 ml
Tannic acid: 2 g
An aqueous solution of aluminum potassium sulfate-12 hydrate: 10 ml

Solution-II contains a saturated ethanolic solution of crystal violet, in which 12 g of crystal violet is mixed in 100 ml of 95% ethanol.

The final stain is prepared by mixing solution-I and II in a ratio of 10:1. Then, separate the coarse precipitated particles from the stain via filtration using filter papers, and finally store the reagent at room temperature.

Procedure of Wet Mount Technique:

The organism needs to grow at room temperature in a blood agar medium for 16 to 24 hours.
Put a drop of saline onto the microscope slide.
Now take a sterilized inoculating loop and remove little inoculum from the culture plate by only touching the margin.
Then, add the inoculum into the drop of water placed on a glass slide.
After that, leave a glass slide undisturbed for about 15-20 minutes.
Afterwards, place the coverslip to the faintly turbid drop of water and immediately view it under the 40-50X of the objective lens.
If the motile cells are visible, the process is followed by staining the bacterial culture. Add a drop of Ryu flagella stain towards the one edge of the coverslip, which ultimately penetrates the bacterial suspension through capillary action.
Then, observe the glass slide after 10 minutes, under the light microscope upto the power of 100X.
Finally, note down the results by examining the presence, number and arrangement of the flagella.

Leifson’s Staining Method:

As flagella are too thin hair-like structures, so it’s not possible to visualize them under the microscope. It uses Leifson dye as a special reagent to color the thin flagella.

We can prepare Leifson’s reagent by simply mixing a mordant (tannic acid) and stain (basic fuchsin formed in an alcohol base).Once Leifson’s stain incorporates within the cell, the tannic acid available gers attach to flagella, and the available alcohol evaporates.

After evaporation of alcohol, the flagella thickness is increased due to the deposition of tannic acid. After the above treatment, we need to treat the cells with the methylene blue reagent, which eventually stains the cell.

Preparation of Leifson’s Stain: To prepare leifson stain, we require the following contents in the given amount:

Ammonium or potassium alum, saturated water solution: 20 ml
The tannic acid in 20% water solution: 10 ml
Distilled water: 10 ml
95% of ethyl alcohol: 15 ml
A saturated ethyl alcohol solution of basic fuchsin: 3 ml

Procedure of Leifson’s Staining Technique

Firstly, take flagellated cell culture slant and put two to three droplets of distilled water into the culture slant dropwise by using a sterile pipette without disturbing the cell growth.
Now incubate the slant for 20 minutes after adding water into it.
Afterwards, take one drop from the above-prepared suspension and put it on a clean slide. Then keep a slide in an inclined position.
The drop needs to flow from one end to another end of the slide to restrict the flagella folding on the cell.
Now allow the smear to air dry.
After the liquid completely evaporates, flood a glass slide with Leifson’s stain until you observe a shiny thin film.
Then wash the slide gently with water.
Afterwards, treat a glass slide with 1 % methylene blue for one minute.
Observe the glass slide by putting a drop of oil immersion after washing the slide with water and air-drying.

Result Interpretation

Wet mount staining method: It stains the flagella purple.
Leifson’s staining method: It stains the flagella red and the bacterial cells blue.

Capsular staining

The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. A capsule is a gelatinous outer layer secreted by bacterial cells that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. The capsule stain employs an acidic stain and a basic stain to detect capsule production.

Principle of Capsule Staining:

Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained.

Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. India ink is difﬁcult to obtain nowadays; however, nigrosin is easily acquired.

A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell walls take up the dye. Capsules appear colorless with stained cells against a dark background.

Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope.

Reagents used for Capsule Staining

Crystal Violet (1%)
Crystal Violet (85% dye content) = 1 gm
Distilled Water = 100 ml
Nigrosin
Nigrosine, water soluble = 10 gm
Distilled Water = 100 ml

Procedure of Capsule Staining

Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on the slide.
Congo Red is easier to see, but it does not work well with some strains. India Ink generally works, but it has tiny particles that display Brownian motion that must be differentiated from your bacteria. Nigrosin may need to be kept very thin or diluted.
Using sterile technique, add a loopful of bacterial culture to the slide, smearing it in the dye.
Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let it stand for 5-7 minutes.
Allow to air dry (do not heat fix).
Flood the smear with crystal violet stain (this will stain the cells but not the capsules) for about 1 minute. Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it air dries .
Examine the smear microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.