MLS Bacteriology

Staining

Group 3 (MT 3rd year)

Arreola, Luces, Oke, Patanao, Salazar, Ulanday
 Content

1. Staining Techniques
2. Ionizable dyes used in staining
3. Gram staining
4. Acid fast staining
5. Modified acid fast staining
method
6. Special staining method
 Staining Techniques

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 staining technique: use of methylene blue

 Steps on
simple stain
 Simple Staining

Microscopic view of Bacillus (rod) shaped bacteria simple stained with crystal violet.
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Differential Staining

Hans Christian Gram in 1882

Gram stain procedure has been
widely used by microbiologists
everywhere
 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 staining technique are Gram staining & acid-fast

bacilli (AFB) staining.
Differential Staining Steps
 Differential Staining

Microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus

ATCC 25923, purple) and Gram-negative bacilli (Escherichia coli ATCC 11775, red)
 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 staining technique is the use of India ink or Nigrosin dye

Negative staining
Negative staining
Ionizable dyes used in
staining
DYES EXAMPLE

Basic Fuchsin
Methylene Blue
BASIC Crystal Violet
Malachite Green
Safranin

Rose Bengal
ACIDIC Eosin
Acid Fuschin
 BASIC DYE: 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
Rose bengal is used for microscopic analysis and
suppression of bacterial growth in several
microbiological media.
 Eosin
is most often use 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

 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
PRINCIPLE 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 GRAM
POSITIVE 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
PROCEDURE
 Precautions Rule
1. If the Crystal violet dye is rinsed too All cocci are Gram-positive except for
vigorously prior to application of the idoine it Neisseria, Veilonella and Branhamella
will not be retained and will leave the Gram-
(Moraxella)
Negative bacteria unstained.
2. If the decolorization is prolonged, the Gram- All bacilli are Gram-negative except for
Positive complex will be removed and the Actinomadura, Arcanobacterium, Bacillus,
Gram-positive becteria will not be stained Clostridium, Corynebacterium,
3. If the decolorization is insufficient, the Erysipelothrix, Gordona, Kuthria, Listeria,
organism may falsely apper Gram-positive
Mycobacterium, Nocardia, Rhodococcus,
cells
Streptomyces, Tropheryma whipplei and
4. If the safranin dye is left applied for more
than a minute, the Gram-positive complex Tsukamurella
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
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.
Modified Acid
fast staining
Acid Fast Stain
 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

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 Stain Methods

 Carbolfuchsin Staining
The carbolfuchsin staining comprises of the Ziehl-
Neelsen method and the Kinyoun method. In the Ziehl-
Neelsen 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:
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.
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.
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. 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. 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.
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. 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 dependant on the quality of methylene blue.
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:
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. Methylene blue or potassium
permanganate is used as a counterstain to provide background color. Potassium
permanganate is preferred as it provides a darker background giving it a better
contrast and sensitivity as compared to methylene blue. The last step is to gently
wash the slide with slow running water and letting it dry. Blotting the slide is
avoided as it may damage the stained smear.
Differential Characteristics of
Acid-Fast & Non-Acid Fast
Bacterias
 Non Acid Fast
Acid Fast Bacteria
Bacteria readily
Acid Fast bacteria resist the
resist the decolorizing decolorizing by acid
by acid after staining. after staining.
Non Acid Fast bacteria
Acid Fast bacteria
appear in blue color.
appear in pink or red
color. It retains the color of
counterstain,
It retains the color
methylene blue.
of primary stain,
carbol fuchsin. Mycolic acid is absent
in cell wall.
Mycolic acid is
present in cell wall. Non Acid Fast
Bacteria
 Special Staining Techniques

Special staining is performed to visualize selected tissue elements,

entities and microorganisms.
Provide valuable information in identification of microorganisms
and their cellular structure such as:
Cell wall
Capsule
Flagella

Staining Technique Stain Cellular Structure

Dyar Congo red Cell wall

Anthony's, Hiss' and

Gin's
Crystal Violet Capsule

Nigrosin Nigrosin dye Capsule

Methylene blue and

Neisser Metachromatic Granules
Crystal violet

malachite green and

Albert Metachromatic Granules
toluidine blue
Staining Technique Stain Cellular Structure

carbol fuchsin & nigrosin

Dorner dye
Endospore

malachite green &

Schaeffer-Fulton
safranin red
Endospore

carbol fuchsin and

Gray tannic acid (Mordant)
Flagella

Carbol Fuchsin, tannic

Leifson acid and methylene Flagella
blue
Staining Technique Stain Cellular Structure

Feulgen carbol fuchsin DNA

Levaditi's Silver nitrate Spirochetes

Fontana- Ammoniacal silver

nitrate and tannic acid
Spirochetes
Tribondeau
Indiana Ink Nigrosin HISS'
CAPSULE STAINING
METACHROMATIC GRANULES STAINING

Albert stain Neisser

ENDOSPORE STAINING
ENDOSPORE STAINING
DNA & SPIROCHETE STAINING
References:
Aryal, S. (2022, August 10). Negative Staining- Principle, Reagents, Procedure and Result. Microbiology Info.com. Retrieved
October 4, 2022, from https://microbiologyinfo.com/negative-staining-principle-reagents-procedure-and-result/

General Data Protection Regulation(GDPR) Guidelines BYJU’S. (2021, March 22). BYJUS. Retrieved October 4, 2022, from
https://byjus.com/biology/staining/

Libretexts. (2021a, May 26). 4.1: Introduction to Staining. Biology LibreTexts. Retrieved October 4, 2022, from
https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3
A_Staining_Techniques/4.01%3A_Introduction_to_Staining

Libretexts. (2021b, May 26). 4.1: Introduction to Staining. Biology LibreTexts. Retrieved October 4, 2022, from
https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3A_
Staining_Techniques/4.01%3A_Introduction_to_Staining

Libretexts. (2021c, May 26). 4.1: Introduction to Staining. Biology LibreTexts. Retrieved October 4, 2022, from
https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3A_
Staining_Techniques/4.01%3A_Introduction_to_Staining

Rodriguez, M. T. T. (2018). Review Handbook in Diagnostic Bacteriology (2nd edition). C&E Publishing, Inc.