Staining techniques

● Stains are chromogenic solutions that are essential in the microscopic identification & differentiation of
bacterial cells.

● Staining, in microbiology, can be defined as a technique which is used to enhance and contrast a biological
specimen at the microscopic level. Stains and dyes are used to highlight the specimen at the microscopic level to
study it at higher magnification for histopathological studies and diagnostic purposes.

Importance why we need to stain

Most types of cells do not have much natural pigment and are therefore difficult to see under the light microscope unless
they are stained. Several types of stains are used to make bacterial cells more visible. In addition, specific staining
techniques can be used to determine the cells’ biochemical or structural properties, such as cell wall type and presence or
absence of endospores. This type of information can help scientists identify and classify microorganisms, and can be used
by health care providers to diagnose the cause of a bacterial infection.

Staining techniques
1. Simple staining
- In this procedure, a single stain is used
- It is directed towards coloring the forms & shapes of the
cells
- The inoculum size is 10^5 CFU/mL
- An example of this staining technique is the use of
methylene blue

● in which only one stain is used, and all types of bacteria appear as the
color of that stain when viewed under the microscope.
● 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 and
arrangement, but they do not give any additional information.
● Living bacteria are almost colorless, and do not present sufficient contrast with the
water in which they are suspended to be clearly visible. The purpose of staining is
to increase the contrast between the organisms and the background so that they are more readily seen in
the light microscope.
● In a simple stain, a bacterial smear is stained with a solution of a single dye that stains all cells the same color
without differentiation of cell types or structures. The single dye used in lab is methylene blue, a basic stain. Basic
stains, having a positive charge, bind strongly to negatively charged cell components such as bacterial nucleic
acids and cell walls.

2. Differential staining
- It divides bacteria into separate group
- It is directed towards coloring the components of
the elements present
- The inoculum size is 10^5 CFU/mL
- Some examples of this staining technique are
Gram staining & acid-fast bacilli (AFB) staining.
The steps in differential staining are as follows:
- Application of the primary stain
 - Application of the mordant (performed to increase the binding of primary stain to the cell wall)
- Application of the decolorizing agent
- Application of the secondary stain/counterstain
● This very commonly used staining procedure was first developed by the Danish
bacteriologist Hans Christian Gram in 1882 (published in 1884) while working with
tissue samples from the lungs of patients who had died from pneumonia. Since then,
the Gram stain procedure has been widely used by microbiologists everywhere to
obtain important information about the bacterial species they are working with.
Knowing the Gram reaction of a clinical isolate can help the health care
professional make a diagnosis and choose the appropriate antibiotic for
treatment.
● differential stain, as this allows them to gather additional information about the bacteria they are working with.
● Differential stains use more than one stain, and cells will have a different appearance based on their chemical
or structural properties. Some examples of differential stains are the Gram stain, acid-fast stain, and endospore
stain.

3. Negative staining

- It is utilized to demonstrate the presence of diffuse capsule

surrounding some bacteria
- It is an excellent technique for studying bacterial gas vacuoles & viral
morphology
- It results in the bacteria appearing as light-colored bodies against a
dark background since the cell surface repels the acidic stain as a
result of bacterial cells being negatively charged.
- An example of this staining technique is the use of India ink or Nigrosin
dye

● The main purpose of Negative staining is to study

the morphological shape, size and arrangement of
the bacteria cells that is difficult to stain. eg:
Spirilla. It can also be used to stain cells that are
too delicate to be heat-fixed.

● It is also used to prepare biological samples for

electron microscopy. It is used to view viruses,
bacteria, bacterial flagella, biological membrane
structures and proteins or protein aggregates, which
all have a low electron-scattering power. It is also
used for the study and identification of aqueous lipid aggregates like lamellar liposomes (le), inverted spherical
micelles (M) and inverted hexagonal HII cylindrical (H) phases by Negative staining transmission electron
microscopy.

● India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the
chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively
charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The
bacteria will show up as clear spots against a dark background.

PROCEDURE :
1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix.
 3. Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the
edge of the applied slide.
4. Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry.
Note: Do not heat fix the slide.
5. Examine the slides under oil immersion.

IONIZABLE DYES

BASIC FUCHSIN - a dye used for detection of acid-fast bacilli. It can also track proteins in acidic pH systems.
Methylene Blue - basic stain used for determining bacterial morphology. recommended for gram-negative
bacteria found in spinal fluid.
Crystal Violet - Primary stain used in Gram staining to differentiate between Gram-positive and Gram-negative
bacteria.
Malachite Green - effective against fungi and gram-positive bacteria.
Safranin - In Gram staining, safranin directly stains the bacteria that has been decolorized. with this stain, it is
easy to determine gram-negative bacteria from gram-positive bacteria.

ACIDIC DYE
● Rose bengal is used for microscopic analysis and suppression of bacterial growth in several
microbiological media.
● Eosin - is most often used as a counterstain to H&E(Hematoxylin & Eosin) staining. It is an acidic dye
that binds to basic components of a cell, mainly proteins located in the cytoplasm.
● Acid Fuchsin - is a cationic triphenylmethane used to detect acid-fast bacilli and is commonly used in
Ziehl Neelsen staining technique.

GRAM STAINING

● It is the most commonly used differential stain in the Clinical

Microbiology Laboratory
● It utilizes crystal violet as the primary stain while safranin is
the secondary or counterstain
● In this staining procedure, iodine acts as the mordant while acetone-alcohol mixture acts as decolorizing agent
● Principle: A bacteria with thick cell wall containg teichoic acid retain the crystal violet-iodine complex dye after
decolorization and appear purple, which means that they are GRAM-POSITIVE
● Other bacteria with thinner cell walls that contain lipopolysaccharides do not retain the dye complex and appear
deep pink or red, which means that they are GRAM-NEGATIVE
 GRAM POSITIVE GRAM NEGATIVE

● Actinomyces ● Escherichia coli (E. coli)

● Bacillus ● Salmonella
● Clostridium ● Shigella
● Corynebacterium ● Other Enterobacteriaceae
● Enterococcus ● Pseudomonas
● Gardnerella ● Moraxella
● Lactobacillus ● Helicobacter
● Listeria ● Stenotrophomonas
● Mycoplasma ● Bdellovibrio
● Nocardia ● Acetic acid bacteria
● Staphylococcus ● Legionella
● Streptococcus ●
● Streptomyces

Precautions:
1. If the Crystal violet dye is rinsed too vigorously prior to application of the idoine it will not be retained and
will leave the Gram-Negative bacteria unstained.
2. If the decolorization is prolonged, the Gram-Positive complex will be removed and the Gram-positive
becteria will not be stained
3. If the decolorization is insufficient, the organism may falsely apper Gram-positive cells
4. If the safranin dye is left applied for more than a minute, the Gram-positive complex will be eliminated
from the previously stained cells
5. Failure to leave the safranin dye for a sufficient time will result in unstained Gram-negative bacteria and
background materials

Rule:
● All cocci are Gram-positive except for Neisseria, Veilonella and Branhamella (Moraxella)
● All bacilli are Gram-negative except for Actinomadura, Arcanobacterium, Bacillus, Clostridium,
Corynebacterium, Erysipelothrix, Gordona, Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus,
Streptomyces, Tropheryma whipplei and Tsukamurella
MODIFIED ACID-FAST STAINING
● developed for rapid detection of Mycobacterium tuberculosis and its L forms, wherein carbol fuchsin and
dioxogen were mixed into the sputum smear.
PROCEDURE MODIFIED ACID FAST STAINING
1. Flood slides with Kinyoun carbol fuchsin for 5 minutes.
2. Rinse gently with water until the water flows off clear.
3. Flood slides with acid-alcohol (3% HCL in ethanol) for 3~5 seconds.
4. Rinse gently with water until the water flows off clear.
5. Flood slides with methylene blue for 3 minutes.
6. Rinse gently with water until the water flows off clear.
7. Allow slides to air dry before viewing.
ACID FAST STAINING
● The acid-fast stain is a laboratory test that determines if a sample of tissue, blood, or other body substance
is infected with the bacteria that causes tuberculosis (TB) and other illnesses. It is used in the demonstration
of acid-fast bacteria belonging to the genus 'mycobacterium', which include the causative agent for
tuberculosis.
PRINCIPLE IN ACID FAST STAINING
● 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.

ACID FAST STAINING METHODS

CARBOLFUCHSIN STAINING
● The carbol fuchsin staining comprises of the ZiehlNeelsen method and the Kinyoun method. In the
ZiehlNeelsen method, smeared slides are first stained with carbolfuchsin. The Ziehl-Neelsen method of
staining is also called the hot method as it involves heating the carbolfuchsin stain. In contrast, the historic
method of staining called the Kinyoun method does not involve heating and is hence known as the cold
method. Currently, the cold method is already obsolete.

PROCEDURE CARBOL FUCHSIN STAINING

1. This is done by submerging the smear in a drop of carbolfuchsin and subsequently heating it using an
alcohol lamp until steam can be seen rising.
2. This facilitates the penetration of the stain inside each bacterium. Care must be taken that boiling does not
occur as that may alter the results of the test.
3. The stain and smear should remain in contact for approximately 10 minutes and be allowed to cool down
thereafter, thus, trapping the stain within the bacterial cell wall.
4. These steps make the entire smear, including acid-fast bacilli, red. After stain fixation, the second step
focuses on washing the excess stain off from the smear.
5. This is done by gently washing the smear in a stream of water and then covering it with acid alcohol for 2 to
3 minutes.
6. Acid alcohol has the ability to completely decolorize all non-acid-fast organisms, thus only leaving behind
red-colored acid-fast organisms, like M. tuberculosis.
7. The slides are then stained a second time with methylene blue that serves as a counterstain. The
recommended time for stain to smear contact is 1 minute but is largely dependent on the quality of
methylene blue.
8. Counterstaining creates an effective visual contrast of red acid-fast bacilli during microscopy.
FLUOROCHROME PROCEDURE
● It utilizes one of two dyes, the auramine-O dye, or the auramine-rhodamine dye. Auramine-O is a
hydrochloride dye that causes stained AFB to emit fluorescence (green or yellow) when viewed under a
fluorescence microscope. Unlike the Ziehl-Neelsen method, heating is not required for the penetration of the
stain into the bacteria. The stain to smear contact time, however, must be a minimum of 20 minutes for the
acid-fast organisms to pick up the stain properly.
PROCEDURE OF FLUOROCHROME
1. After the auramine dye has fully stained the smear, a drop of acid alcohol is applied for one to two minutes
to decolorize the smear.
2. Methylene blue or potassium permanganate is used as a counterstain to provide background color.
3. Potassium permanganate is preferred as it provides a darker background giving it a better contrast and
sensitivity as compared to methylene blue.
4. The last step is to gently wash the slide with slow running water and letting it dry.
5. Blotting the slide is avoided as it may damage the stained smear

ACID FAST BACTERIA NON ACID FAST BACTERIA

● Acid Fast bacteria resist the decolorizing by
acid after staining.
● Acid Fast bacteria appear in pink or red color.
● It retains the color of primary stain, carbol
fuchsin.
● Mycolic acid is present in cell wall.
● Non Acid Fast Bacteria readily resist the
decolorizing by acid after staining.
● Non Acid Fast bacteria appear in blue color.
● It retains the color of counterstain, methylene
blue.
● Mycolic acid is absent in the cell wall.

Special staining - Based on classical dye staining methods

● Cell wall
○ Dyar method
○ Congo red is an acidic stain which is a negatively charged stain, the cell wall by contacting the positive
ions on the surface of bacterial cells red in color. Methylene blue stains the cytoplasm blue in color.
○ Result: Under the oil immersion lens, we can easily observe the red coloured cell wall and blue coloured
cytoplasm.
● Capsule stain
○ diagnostically useful since it is a virulent factor
○ are non-ionic, so neither acidic nor basic stains will adhere to their surfaces.
○ best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, congo red) and
to stain the cell itself using a basic stain (e.g.,crystal violet, safranin, basic fuchsin, and methylene blue).
Which is known as the negative staining
○ Negative stain
○ requires the use of acid stains such as the Indian ink or nigrosin.
 ○acid stain with its negatively charged chromogen will not penetrate the cells because of the negative
charge on the surface of the bacteria.
■ India ink
● Dyes: Crystal violet & india ink
● Results: capsule (if present) will appear clear against the dark background (capsule does
not take any stain).
● used for demonstrating Cryptococcus.
■ Nigrosin
● Nigrosin consists of a suspension of fine particles of carbon. These form a dark
background.
● Result: An organism with a capsule will show a halo around the cell.
● Used for observation of capsules in Cryptococcus neoformans, Klebsiella pneumonie,
Streptococcus pneumoniae, etc
○ Positive Stain
■ Anthony’s
● Dye: crystal violet as primary stain
● Mordant: 20% copper sulfate
● It in this procedure do not HEAT FIX to avoid destroying capsule or causing shrinkage and
DO not blot as it will remove the fixated bacteria from slide
● Results: Violet cells and faint blue capsule
■ Hiss’s Copper Sulfate Method:
● Dye: Crystal violet
● 20% Copper sulphate solution, which serves dual role of both the decolorizing agent and
counterstain
● Do not heat fix
● Results: Capsule appears as a faint blue halo around a purple cell
● METACHROMATIC GRANULES (VOLUTIN)
○ Albert stain
■ Mordant: Iodine and Potassium iodide in water
■ Results: stain bluish black while the rest of the microbial cell stains green
■ majorly used to identify the metachromatic granules found in disease-causing microorganisms like
Corynebacterium diphtheriae.
○ Neisser
■ Primary stain - methylene blue and crystal violet
■ Counter stain: Chrysoidine solution
■ Results: Polar bodies dark brown to black; Bacterium body brownish

● ENDOSPORE - cannot be stained by ordinary methods because the dyes do not penetrate the wall of the
endospore
○ DORNER
■ Primary stain: carbol fuchsin
■ decolorizer : acid alcohol
■ Counterstain: nigrosin
■ Carbol fuchsin, when applied to a heat-fixed slide and heated, softens the structure of the bacterial
spores and the basic fuchsin gets into the spores. When decolorized with acid alcohol, color
washes off the vegetative cells, making them colorless.
■ Results: Vegetative cells are colorless, endospores are red, and the background is black.
○ Schaeffer-fulton
■ Primary stain: malachite green - forced to permeate the spore wall by heating (mordant)
 ■ Secondary stain: safranin
■ Washing with water removes stain from vegetative cells, but not from spore wall.
■ vegetative cells (normal living cells) lose the primary stain and take the red color of the secondary
stain (safranin).
■ endospores thus retain the primary dye green
■ Result: Endospores are bright green. Vegetative Cells are brownish red to pink
● FLAGELLA
○ CARBOL FUCHSIN to build up the diameters of the flagella until they become visible under the light
microscope
○ TANNIC ACID SALTS cause a heavy precipitate to form on the cell walls and flagella
○ Gray’s method
■ Uses carbon Fuchsin and tannic acid as mordant
○ Leifson’s stain
■ Principle: When bacterial flagella absorbs this tannic acid and a dye(methylene blue), it forms a
colloidal precipitate as a result the flagella is colorized and increases in diameter.
■ Result: Bacteria and flagella will appear golden brown.
● DNA
○ Feulgen stain
○ dye/stain: carbol fuchsin
○ Principle: to dissociate the two strands of DNA through hydrolysis by a solution of molar HCl which
destroys the purine bases.
○ Result: magenta-stained nuclei

● SPIROCHETES
○ are spiral or corkscrew-shaped bacteria. These organisms are Spirochaeta, Treponema, Borrelia, and
Leptospira.
○ Silver stains are typically used to identify spirochetes, but Giemsa can also be used as a quick screen for
microorganisms.
○ Warthin-Starry silver stain: Demonstrates spirochetes by staining them black.
■ Levaditi’s
● for tissue sections
● processed in 1% silver nitrate for 2 hour at room temp and 4-6 hours at 50degrees celsius
and is washed with pyridine solution. It is transferred to reducing fluids for two days: 4%
formalin acetone and pyridine
● dehydrate tissue and embed it with paraffin.
■ Fontana-Tribondeau
● contains ammoniacal silver nitrate and when this stain is heated it forms silver oxide.
● silver oxide precipitates on the organism and increases the diameter of the cell and stains
the cell.
● Results: stain brownish black in color.
Aryal, S. (2022, August 10). Negative Staining- Principle, Reagents, Procedure and Result. Microbiology Info.com.

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https://microbiologyinfo.com/negative-staining-principle-reagents-procedure-and-result/

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Libretexts. (2021a, May 26). 4.1: Introduction to Staining. Biology LibreTexts. Retrieved October 4, 2022, from

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