STRUCTURE

OF
THE BACTERIAL CELL

BACTERIAL ENDOSPORES

ACID-FAST ZIEHL NEELSEN STAIN

REACTION 

Department of Microbiology
 BACTERIAL SPORES
• Spores are the most resistant life-forms
known
• They are remarkably resistant to heat (they
survive boiling), drying, freezing,
desiccation, ultraviolet light, action of toxic
chemicals and radiation and poor nutrient
supply
• But they can be killed by autoclaving (that is
subjecting the spores to temperatures above
121°C at elevated pressure)
 BACTERIAL SPORE

• Also called as endospore

• Allows the bacteria to remain dormant for
extended amount of time
• Once in the soil, the endospores remain in
inert state many years
• Helps the bacteria to survive in
impermissible conditions
• It is significant in the spread of the diseases
and indicator of sterility of materials
 BACTERIAL ENDOSPORES

• When environmental conditions again

become favorable, the endospore
germinates, resulting in a new vegetative
cell
 BACTERIAL ENDOSPORES
• Endospore formation is a distinguishing feature of
the family Bacillaceae, which includes members of
the aerobic genus, Bacillus and the anaerobic
genus, Clostridium
• Some saprophytic species living in the soil, water
and bodies of animals are also capable of
sporulation
THE BACTERIAL ENDOSPORE

• It is a highly retractile body formed within the

vegetative bacterial cell at a certain stage of
growth

• It contains a complete copy of the

chromosome, the bare minimum concentration
of essential proteins and ribosomes, and a high
concentration of calcium bond to dipicolinic
acid
 STRUCTURE OF THE BACTERIAL
ENDOSPORE
• Core- it’s the spore protoplast. Contains
nucleus, protein synthesizing apparatus,
energy generating system. Contains large
amounts of calcium dipicolinate responsible
for resistance
• Spore wall- innermost layer surrounding
the inner spore membrane. Made of
peptidoglycan and forms cell wall
• Cortex- thickest layer made of
peptidoglycan. Role in spore germination
 STRUCTURE OF THE
BACTERIAL ENDOSPORE
• Coat- keratin like protein containing many
intermolecular disulphide bonds.
Impermeable and provides resistance to
antibacterial agents

• Exosporium- composed of lipids, proteins

and carbohydrates
Structure of the bacterial spore
Structure of the bacterial spore
Structure of the bacterial spore
 BACTERIAL ENDOSPORES
• The size, shape, and position of the spore are
relatively constant characteristics of a given
species and are therefore, of some value in
distinguishing the kind of bacillus from
another
• The position of the spore may be central, sub
terminal or terminal
• It may be the same diameter as the cell,
smaller, or larger causing a swelling of the
cell
 BACTERIAL ENDOSPORES
Arrangements of
spores:
1.No bulging of cell 2.Bulging of cell wall
wall • Oval sub terminal
• Oval central • Oval terminal
• Oval sub terminal • Spherical terminal
• Spherical central • Free spore
 POSITION OF THE SPORE
• In some species of sporulating microorganisms,
the spore diameter is greater than the width of the
bacterial cell. If the spore is located subterminally,
the microbes take on the form of a spindle
• In tetanus clostridia the spore diameter is also
greater than the width of the vegetative cell, but
the spore is located terminally, and hence the
drum-stick appearance
 POSITION OF THE SPORE

•Centrally (1,4)- in B.anthracis (causative agent of

anthrax)
•Terminally (5)- at the end of the cell- in C.tetani
(causative agent of tetanus);
•Subterminally (3)- towards the end- in
C.botulinum (causative agents of botulism)
POSITION OF THE SPORE
POSITION OF THE SPORE
 SPORULATION
• Any bacteria form only one spore

• The ability to form endospores, a

process called sporulation
 SPORULATION

• Process by which spores are formed

• Involves production of many new
structures, enzymes and metabolites along
with disappearance of many vegetative cell
components- differentiation
• Sporulation process takes about 8-10 hrs
under laboratory conditions
 SPORULATION
• During maturation, the spore dehydrates and
the spore becomes refractile and resistant to
heat, radiation, pressure, desiccation, and
chemicals; these properties correlate with the
cortical peptidoglycan and the presence of
large amounts of calcium and dipicolinic acid

• In the dormant, inert endospore state, bacteria

do not metabolize or reproduce, but exist in a
type of suspended animation, much like the
seeds of plants do
SPORE FORMATION
 STAGES OF GERMINATION

• Activation- spore coat gets damaged

• Initiation- autolysin is secreted that
degrades the cortex peptidoglycan. Water is
taken up releasing calcium dipicolinate and
degrades various spore components by
hydrolytic enzymes
• Outgrowth- degradation of cortex and outer
layers results in emergency of new
vegetative cell
Life cycle of spore forming bacteria
 THE BACTERIAL ENDOSPORES

• Bacterial spore is smooth walled and oval or

spherical in shape
• It doesn’t take up ordinary stains
• It looks like areas of high refractivity under light
microscope

Spores are detected by:

• Simple staining methods
• Special staining methods
Endospore formed bacteria imaged
with phase-contrast microscopy
 BACTERIAL ENDOSPORE
STAINING
• Endospores strongly resist application of simple
dyes, but once stained are quite resistant to
decolorization. This character suggests one way to
make the structure visible

• If simple stains are used, the body of the bacillus

is deeply colored, whereas the spore is unstained
and appears as a clear area in the organism

• By vigorous staining procedures the dye can be

introduced into the substance of the spore. When
thus stained, the spore tends to retain the dye after
treatment with decolorizing agents
 BACTERIAL ENDOSPORE
STAINING
• To make the distinction clear between the
spore and the vegetative portion of the cell, a
contrasting counter stain is usually applied
in the ordinary fashion and the resulting
picture shows the initial stain taken up by
the spore and the second stain appear in the
cytoplasm
• Thus, it makes for a very simple method of
distinguishing the endospore from the
vegetative cell
 BACTERIAL ENDOSPORE
STAINING

• Gram Stain- spore appears as clear unstained area

within the cell
• In order to stain these tough structures,
the endospore stain is used (Peshkov's method) 
 GRAM STAINING

• Make a smear of Bacillus cereus

• Do the steps in Gram staining
• Observe under the microscope
• You will see:
Gram positive rods
Spores are located centrally and they are
colorless
SPORE STAINING
 PESHKOV'S METHOD
The procedure for differentially staining endospores and
vegetative cells is as follows:

1) Place Methylene blue stain over the whole slide with a

smear
2) Heat the smear over the flame until boiling. Heating the
glass with burning swab with alcohol
3) Leave the slide for cooling
4) Rinse slide until water runs clear
5) Stain with Neutral red for 30 sec
6) Gently blot the slide dry
7) Observe under the microscope with oil immersion
 PESHKOV'S METHOD
Тhe endospores will appear blue, having retained the
primary stain, methylen blue
Vegetative cells (bacteria are in the active,
metabolizing state) will appear red, having retained
the counterstain, neutral red
Endospores are small, typically oval and you should
see numerous uniform examples on your slide
Vegetative, rod-shaped cells appear red and oval
endospores blue-green
 ACID-FAST ZIEHL-NEELSEN STAIN
REACTION 
• Many bacteria of the
genera Mycobacterium and Nocardia are medically
significant, causing infectious diseases such as
tuberculosis (Mycobacterium tuberculosis),
leprosy(Mycobacterium leprae) and other lung and skin
infections.  

• They have mycolic acids in their cell wall and they can
not be stained by the Gram method. Ziehl-Neelsen stain
method differentiates acid-fast bacteria than any other
that are non acid-fast. Is used in the diagnosis of
tuberculosis and other diseases associated with these
organisms
 STEPS IN ZIEHL-NEELSEN STAIN

1. Prepare a smear
2. Place a slip of filter paper over the the fixed slide
3. Place Сarbol fuchsin dye over the whole slide with
bacteria
4. Heat the slide over the flame of burning swab until steam
appears and leave it for cooling. Repeat heating three
times.
5. Leave the slide for cooling
6. Discard blotting paper
7. Rinse slide until water runs clear
8. Decolorize with 5% sulphuric acid for 10–15 seconds
and rinse with water
9. Decolorize with alcohol for 1 minute and rinse with water
10. Stain with Methylene blue for 3-5 minutes
11. Gently blot the slide dry
• It is now ready to be viewed under oil immersion
Acid-fast Bacteria are red colored while other bacteria
and tissue elements, leucocytes and lymphocytes are
blue.

Acid-fast Stain Reaction Explained:

Carbol fuchsin stains all bacteria red. Decolorizers do

not remove the Carbol fuchsin stain trapped within the
acid-fast cell wall, but remove the stain from bacterial
cells that do not have mycolic acid in their cell wall.
• (B)Acid fast: Bright red to intensive red, straight or slightly
curved rods, occurring single or in small groups, may appear
beaded

• (A)Non-acid fast: Blue color 

 ACID-FAST ZIEHL NEELSEN STAIN
REACTION 
• Acid-fast Bacteria (Mycobacterium tuberculosis)
are red colored while other bacteria and tissue
elements, leucocytes and lymphocytes are blue