Spores are dormant, resistant structures formed by certain bacteria including Bacillus and Clostridium. Spores have complex multilayered structures that make them highly resistant to heat, radiation, chemicals and desiccation. Two important staining techniques are used to identify certain bacteria - Ziehl-Neelsen staining identifies acid-fast bacteria like Mycobacteria and Anjesky staining identifies bacterial spores. Both techniques use carbol fuchsin dye and specific heating and decolorization steps to differentiate bacteria based on their cell wall chemical composition and structures.

1. ACID-FAST BACTERIA. SPORES OF BACTERIA. COMPLETE METHODS OF STAINING. APPEARANCE
THE SPORES BY ANJESKY’S METHOD. THE METHOD OF STAINING ACID-FAST BACTERIA
ACCORDING TO ZIEHL-NEELSEN’S.
I. THEORETICAL QUESTIONS
1. Sporulation for bacteria:
a – to identify a difference between bacteria, bacilli and clostridia;
b – function of a sporulation process for bacteria
c – chemical composition of spores;
d – spores locating in pathogenic bacteria;
e – stages and condition of a sporulation;
f – influence of environmental factors on spores;
2. Acid-fast bacteria:
a – feature of chemical composition of the cell wall;
b – main acid-fast pathogens for human;
c – staining of acid-fast microorganisms.
3. Procedure and mechanism a staining of bacteria according to Ziehl-Neelsen’s method, its diagnostic value.
4. Procedure and mechanism a staining of bacteria according to Anjesky`s method, its diagnostic value.
1. Endospores. A number of Gram-positive bacteria can form a special resistant, dormant structure called
endospore. Endospores are formed within vegetative bacterial cells of several genera: Bacillus, Clostridium and other
(fig. 1). The process of forming endospore is called sporulation. Causative agents of anthrax,
tetanus, anaerobic infections, botulism are capable of sporulation. Spore formation only rarely
occurs in cocci {Sarcina lutea, Sarcina ureae) and in spiral forms (Desulfovibrio desulfuricans}.
Sporulation occurs in the environment (in soil and on nutrient media), and is not observed in
human or animal tissues. Sporulation is necessary for keeping the species intact in the
unfavourable enviroment.
Chemical structure and composition of the spores do their extraordinarily resistant to
environment stresses such as heat, ultraviolet radiation, chemical disinfectants and desiccation.
Some endospores have remained viable for over 500 years. Endospores often survive boiling for
an hour and more, therefore avtoclaves
must be used to sterilize many materials. Endospores survive in the solution of the disinfectants
for some hours also.
Endospores are small spherical or oval bodies formed within the cell. Spore position in the mother cell or
sporangium frequently differs among species, making it of considerable value of identification. Spores may be located:
a) centrally; b) close to one end (subterminal) or c) definitely terminal. Bacilli`s spores are small, but clostridia`s ones are
so large that they swell the sporangium.
The spore structure is complex. It consists of four layers and core. There are dehydrated cytoplasm with nucleoid
and ribosomes in the core. The layers of the spore are next:
a) Exosporium. It is thin delicate covering, surrounding the spore. (outer
layer)
b) Spore coat. It lies beneath the exosporium and may be fairly thick. It is
inpermeable and responsible for the spore`s resistant to stress factors. The
coat is composed of a keratinlike protein.
c) Cortex. Thick cover lying beneath the spore coat consists of changed
peptidoglycan.
d) Spore cell wall is inside the cortex and surrounds the core.
The chemical compositions of the spore such as dipicolinic acid complexed
with calcium ions are responsible for spore resistance to heat, desiccation and others.
Sporulation or sporogenesis is the spore formation.
The sporulation process begins when nutritional conditions become unfavorable and growth ceases due to lack of
nutrient or in case for anaerobs when the oxygen contacts with bacteria. It is complex process and may be divided into
seven stages:
a) Axial filament formation
b) Septum formation
c) Engulfment of forespore
d) Cortex formation
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2. e) Coat synthesis
f) Completion of coat synthesis, increase in refractility and heat resistance
g) Lysis of sporangium, spore liberation
Short descriptions this process: Morphologically, sporulation begins with the isolation of a terminal nucleus by
the inward growth of the cell membrane. The growth process involves an infolding of the membrane so as to produce a
double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope.
The growing points move progressively toward the pole of the cell so as to engulf the developing spore.
The two spore membranes now engage in the active synthesis of special layers that will form the cell envelope:
the spore wall and cortex, lying between the facing membranes; and the coat and exosporium, lying outside of the facing
membranes. In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a
set of unique spore constituents. Sporulation requires only about 10 hours.
Germination is a transformation of dormant spores into active vegetative cells. It takes 4-8 hours in average. The
germination process occurs in 3 stages: activation, initiation, and outgrowth.
1. Activation-Even when placed in favorable environment (such as a nutritionally rich medium), bacterial spores
will not germinate unless first activated by one or another agent that damages the spore coat. Among the agents that can
overcome spore dormancy are heat, abrasion, acidity, and compounds containing free sulfhydryl groups.
2. Initiation (germination). Once activated, a spore will initiate germination if the environmental conditions are
favorable. This process is characterized by spore swelling, rupture or absorption of the spore coat, loss to heat and other
factors, release of dipicolinate calcium and increase in metabolic activity.
3. Outgrowth-Degradation of the cortex and outer layers results in the emergence of a new vegetative cell
consisting of the spore protoplast with its surrounding wall. A period of active biosynthesis follows; this period, which
terminates in cell division, is called outgrowth. Outgrowth requires a supply of all nutrients essential for cell growth.
Because spores are impermeable to most strains, they often are seen as colorless areas in bacteria treated with
aniline dyes. To visible spores the special methods are used (by Anjesky, Peshkov, Bitter, Schaeffer-FuIton). One of
them is Anjesky`s method.
Anjesky's staining.
1. A thick smear is dried in the air, treated with 0.5 per cent sulphuric acid, and heated until it steams.
2. The preparation is washed with water, dried, fixed above the flame
3. The smear is stained by the Ziehl-Neelsen’s technique as it is prescribed later.
Spores stain pink-red, the cell appears blue.
2. Acid-fast bacteria.
The cell wall of acid-fast bacteria is generally composed same cell wall of Gram-positive microorganisms.
However, their cell walls have very high lipid content and contain waxes with 60 to 90 carbon mycolic acids (fatty acids).
The presence of mycolic acids and other lipids outside the peptidoglycan layer makes these microorganisms acid-fast.
Important for medicine acid-fast rods are species of genus of Mycobacteria, which cause tuberculosis in human, animals
and birds.
Acid-fast microorganisms are not stained by either simple methods or Gram`s method. They are appeared by
special method, which was provided by Ziehl-Neelsen. The smear is heat with a mixture of basic fuchsin and phenol.
Once dye has penetreated with the aid of heat and phenol, acid-fast cells are not decolorized by an acid-alcochol wash and
remain red. Non-acid-fast bacteria are decolorized by acid-alcochol and thus are stained by counterstain methylene blue.
Ziehl-Neelsen’s staining.
1. Dried and fixed smear is covered with phenol fuchsin solution through the slip of filter paper
(one can use filter paper saturated with a dye and then dried).
2. The covered smear is heated over the flame till steam rises. Repeat heating 2-3 times..
3. Cooled smear is rinsed with water.
4. The specimen is washed off with 5% solution of sulphuric acid until bleached (3-5 sec)
5. It is rinsed with water several times
6. The smear are counterstained with Löffler`s methylene blue solution for 3-5 min.
7. The preparation is rinsed with water and is dried.
Upon staining by Ziehl-Neelsen technique acid-fast bacteria acquire a bright red colour, while the remaining
microflora is stained light-blue
II. Students` practical activities:
1. To examine a stained smear of a sputum of a patient with tuberculosis by Ziehl-Neelsen’s technique. Sketch
the image.
2. Prepare smear from culture of spore-producing microorganisms and stain it by Ziehl-Neelsen technique.
Microscopy the preparation and sketch it.
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