This document provides information on various staining techniques used to visualize bacteria and fungi under a microscope. It discusses simple staining techniques that use a single dye to highlight structures, as well as differential staining techniques like Gram staining, acid-fast staining, and Albert staining that distinguish between bacterial groups. Special staining methods are described to visualize specific structures like endospores, flagella, and capsules. Fungal staining techniques including lactophenol cotton blue, KOH, calcofluor white, and various histopathological stains used to identify fungi are also outlined. The document aims to enhance contrast and visibility of microscopic specimens through selective staining of tissues and cellular components.

1. Name : Anusaya Nayak
Branch : MSc Applied Microbiology( 1st Sem)
Topic : MICROBIAL STAINING TECHNIQUES FOR
BACTERIA AND FUNGI

2. CONTENTS
1. SIMPLE STAINING Positive staining And Negative staining
2. DIFFERENTIAL
STAINING
Gram stain , Zn- Stain( Acid- fast stain) , And Albert Stain
3. SPECIAL STAIN
Capsular stain
Flaggelar stain
Endospore stain
4. FUNGAL STAIN LPCB
KOH
Calcofluor white stain
Histopathological stain

3. .1
A technique used to enhance and
contrast biological specimen at
microscopic level.
Staining is used to highlight
important features of the tissue as
well as to enhance the tissue
contrast.
Numerous staining techniques are available for
visualisation,differentiation and separation of
bacteria in terms of morphological characteristics
and cellular structures
INTRODUCTION
TO
STAINING
1.
2.
3.
1.
2.
3.

4. SIMPLE STAINING
( For examination of shape, size
& arrangement of bacterial cells )
DIFFERENIAL STAINING
( For differentiating
bacterial groups )
SPECIAL STAINING
( For visualising the bacterial
external and internal structure )
BACTERIAL STAINING METHODS

5. 01
SIMPLE STAINING
• A single is used to emphasize particular structure.
• Basic dye such as methylene blue , crystal violet and acidic
dye such are eosin,Indian ink,nigrosin are used in simple
stain against a specimen.

6. DIFFERENTIAL STAINING
• Here 2 stains are used which imparts different colours
to different bacteria or bacterial structures ,which helps
in differentiating bacteria.
• The most commonly differential stains used are Gram
stain, Acid-fast stain and Albert’s stain
02

7. A. Gram Stain
Gram stain was devised by Christian Gram, in
1884.It is an essential procedure that is used in
identification of bacteria.The stain differentiate
bacteria into 2 broad groups : Gram positive and
Gram negative bacteria .
The Gram staining method essentially consists
of 4 steps :
(I)Primary staining with basic dyes, such as
crystal violet
(ii)Mordant is used in the form of dilute
solution of Iodine
(iii)Decolourization with ethanol, acetone
(iv)Counterstaining with acidic dyes such as
Carbol fuchsin,Safranin or neutral Red.

9. • 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 staining techniques.
• 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.
• The main aim of this staining is to differentiate bacteria into acid fast group and non-acid
fast groups.
• Zn Stain : Carbol fuchsin (basic)
• Decolourizer : Sulphuric acid, acid alcohol
• Counter stain : methylene blue, malachite green
B. ACID – FAST Staining ( Zn- Staining)

10. Fig : Microscopic view of acid fast stain
Fig : Procedure of Acid- fast staining

11. C . ALBERT STAINING
• Albert stain is a kind of differential stain used for staining and identifying metachromatic
granules.
• Albert stain is no different. Albert stain distinctly identifies metachromatic granules that
are found in Corynebacterium diphtheriae.
• It is named metachromatic because of its property of changing color i.e. when stained with
blue stain they appear red in color. When grown in Loffler’s slopes, C. diphtheriae
produces a large number of granules.
• Albert stain is made up of two staining solutions; designated as Albert Solution 1 and
Albert Solution 2, their compositions being;
• Albert Solution 1: toluidine blue, malachite green, glacial acetic acid, and alcohol
• Albert solution 2 : Iodine and Potassium iodide in water .

12. Fig : Microscopic view of Corynebacterium
diptheriae

13. SPECIAL STAINING
A . ENDOSPORE STAINING
• In 1922, Dorner published a method for special staining of
bacterial endospore.
• The main purpose of endospore staining is to differentiate
bacterial spores from other vegetative cells and to
differentiate spore formers from non-spore formers.
• Reagents used for Endospore Staining :
• Primary Stain: Malachite green
• Decolorizing agent : Tap water or Distilled Water
• Counter Stain: Safranin , Stock solution (2.5% (wt/vol)
alcoholic solution)
03

14. Fig : Microscopic view of endospore staining
Fig : Procedure of spore staining

15. B. FLAGELLAR STAINING C. CAPSULAR STAINING
This technique is used to visualize the presence
and arrangement of flagella for the presumptive
identification of motile bacterial species.
Result: Bacterial cells appear
pink which are surrounded by
deeply stained flagella; the
flagella may be monotrichous,
amphitrichous, peritrichous, or
lophotrichous.
• 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 cell and that surrounds and adheres
to the cell wall.
• Reagents: 1% crystal violet and 20% copper sulfate
(CuSO4.5H2O)
Capsule: Clear halos zone against dark
background No Capsule: No Clear halos zone

16. FUNGAL STAIN
FUNGAL STAINING METHODS
LACTOPHENOL
COTTON BLUE
KOH MOUNT CALCOFLUOR WHITE
STAIN
HISTOPATHOLOGICAL
STAIN
PERIODIC ACID SCHIFF (PAS)
STAIN
It is the recommended
stain for detecting fungi. PAS positive
fungi appear deep pink, whereas die
nuclei stain blue .
GOMORI METHENAMINE SILVER
(GMS) STAIN
It is used as an alternative to PAS for
detecting fungi. lt stains both live and
dead fungi, as compared to PAS which
stains only the live fungi. GMS stains
the polysaccharide component of the
cell wall. Fungi appear black whereas
the background tissue takes
pale green color.
HEMATOXYLIN AND EOSIN STAIN
(H&E) STAIN
H&E is the combination of two histological
stains: hematoxylin and eosin. The
hematoxylin stains cell nuclei a purplish
blue, and eosin stains the extracellular
matrix and cytoplasm pink, with other
structures taking on different shades, hues,
and combinations of these colors

17. LPCB STAINING

18. THANK YOU !