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    A different notes on bacterial endospore for students

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    A different notes on bacterial endospore for students

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    11 views23 pages

    Imgtopdf 1110231859015

    Uploaded by

    dg4111799
    A different notes on bacterial endospore for students

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    Bacterial spore ones are small oval or spherical structures that are very resistant to high temperatures, radiation, desiccation, and chemical agents. When they are formed - called endospore, The bacterial cell producing spore is called vegetative Bactet acellularly, they Spores have thick walls. They can resist high temperatures, humidity, and other ronmental conditions. The bacteria Clostridia form spores. These spores create the bacteria “e condition called gas gangrene and a type of colitis that is linked to use of Sale Members of the genera Bacillus (e.g.B. anthracis) andClostridium (e.¢..C. tetani orbotulinum) (soil bacteria), are spore formers. Under harsh environmental conditions, such as loss of a nutritional requirement, these bacteria can convert from a vegetative state to adormant state, of spore, The location of the spore within a cell is a characteristic of the bacteria and can + in identification of the bacterium. Bacterial species have different coping mechanisms with selective harsh environmental conditions, One of the most common coping mechanisms for bacteria is forming spores to protect themselves against ecological degrading agents. Bacterial spores are the most dormant form of bacteria since they exhibit minimal metabolism and respiration, as well as reduced enzyme production. Typically, Gram-positive bacteria are best known for producing intracellular spores called endospores as a survival mechanism. Endospores are highly retractile and thick-walled structures formed inside the bacterial cells. It is most common for Bacillus species as well as Clostridium species to create endospores. B. cereus is a member of the Bacillus species and is well-known for its ability to cause foodborne illness as a result of its spores surviving various temperatures. Similarly, C. perfringens spores are acid-soluble proteins that show high resistance to chemicals and heat. Endospores can resist inactivation from ethanol treatment. They also can survive high temperatures for up to 150°C, making specific Gram-positive species heat resistant. Further, bacterial spores can show typical viability signs at temperatures near the absolute zero, Endospores are resistant to the chemical agents, e.g., triphenylmethane dyes, and can even protect the bacterial cells against ultraviolet radiation, extreme pH gradients, drought, and nutrition depletion. Endospores germinate back into vegetative cells (an active bacterial cell that undergoes metabolism) when surrounding environmental conditions favor bacterial growth and reproduction. Several stimulants revert bacterial cells to their active vegetative cells, such as timal close-to-body temperature and diffusion of nutrients and water through bacterial cell through alteration of their surface tension s of spore formation is a multistep process. It starts from replication of the bacterial lowed by the formation of the forespore, which is, by definition, pinching of the ma membrane between the replicated chromosome. Then, a cortex forms between membrane to enclose the © inner and outer membrane by extending the second cellulai the inner and outer membrane by exte 1 pore eoat surrounds the forespore with calcium and dipicolinic acid. Finally, the externa ‘elease. end spore before its Microscopic examination to delineate the morphology of endospores involves differential staining processes such as malachite green and fluorescence staining techniques. Staining dormant bacterial samples with malachite green as the primary stain and safranine as the counteract stain results in the appearance of green oval endospores enclosed inside pink Vegetative bacterial cells. There are different locations of the endospores inside the bacterial cell. For instance, central endospores are located in the middle of the bacterial cell, while the terminal endospore appears at the end. There is also a subterminal type of endospores that appears between the middle and the end of the cell Despite their sturdy and resistant nature to environmental threats, endospores can get affected by certain eradication factors. During the 17th century, John Tyndall, a famous European physicist. discovered Tantalization. The latter is the process of heating liquids and objects at a temperature of 80 to 100°C for 30 minutes; then, the sample is incubated. The procedure is repeated for three consecutive days. The principle behind successive heating for three days is that heating endospores for the first-time results in reverting them into vegetative cells killed through repetitive heat in the second and third days. Anthrax Bioweapon (Bioterrorism) B. anthracis is a gram-positive spore-forming bacteria that are commonly found in the soil of endemic areas. Many factors make B. anthracis a good bioweapon. Its endospores can be placed into food, water, powder, and sprays, spreading the anthray infection without anyone's knowledge, as Spore Can suvvive adverse conaitione for yours 4 Core - DNA. Ribosomes Giyeaytie Enzymes Cytoplasmic ‘membrane. Spore Wall Normal pepticogiycan Cortex Thick layer of Joss cross-linked peptidoglycan y, Keratin Spore Coat Protein endospores are microscopic, a) ae oe Bacterial Spo a A > a 4 > structure, types, sporulation and germination + Spore is metabolically dormant structure produced during unfavorable condition by the process called sporulation + Sporulation occur during late log phase or early stationary phase = Under favorable condition spores germinate to give vegetative cell. + Size: 0.2 pm + Spore are resistant to nutrition starvation, temperature, extreme pH, antibiotics ete Structure of Core DNA ANA, Frowins, SASFS, DPA, Cat Inner membrane | Lipid/Protein Germ cellwat Pepiidogiyean Corex Modified peptidoglycan ‘Gutermembrene | Lipid/Protein Toner spere coats | Proteins Suter spore coats | Proteins Exosporium Prataine endospore: An endo: ore has following layers 1. Exosporium . Spore coat 3. Cortex 4. core Exosporium: + Itisthe outermost layer made up of protein that encloses spore coat. In some bacterial spore, exosporium is made up of polysaccharide and lipid. Spore coat: Itis thick double layered covering that encloses cortex Spore coat consists of spore specific protein, mainly contains cysteine and hydrophobic amino acids. Due to presence of these aminoacids,spore are resistant to adverse environmental condition Cortex: Inside the Spore coat, there is cortex made up of loosely arranged peptidoglycan layer. Inner layer: comprises about 20% of peptidoglycan, it is tightly arranged Outer layer: it is loosely arranged, it can be hydrolysed during spore germination.it comprises alalnine (55%), tetra-peptide (15%) and muramic lactum (30%). Core: It is the innermost part of spore Itis also known as spore protoplast Core consists of core wall, cytoplasmic membrane, cytoplasm, nucleoid, ribosomes and other cellular materials. Core contains (10-25%) water so, the cytoplasm is gel like 9 It contains high amount of calcium and dipicolinic acid in the form of calcium dipicolinate (10-15% by dry weight). Core also contains high percentage of small acid soluble protein (SASP). SASP is synthesized during sporulation and it binds to DNA in core and protect it from potential damage caused by UV radiation, desiccation and drying, In addition, SASPs also provides nutrition and energy for spore germination. Types of bacterial spore 1. Endospore: It is produced within the bacterial cell. Bacteria producing endospore ar Bacillus, Clostridium, Sporosarcina etc. 2. Exospore: It is produced outside the cell Bacteria producing exospore: Methylosinus Sporulation Bacterial cell undergoes spore formation in nutritionally deprived conditions, and this process is called sporulation, Spore develops from a portion of protoplasm (forespore) near one end of the cell. The remaining part of the cell is called sporangium. Spore-forming bacilli form endospores during unfavorable conditions (especially when carbon and nitrogen become depleted or unavailable). During unfavorable condition, vegetative cell converts into spore by the process known as sporulation Sporulation can be divided into several stages. In Bacillus subtilis, entire process of sporulation takes 8 hours to complete from stage 0 to stage VII Stages of sporulation: + [stages ona. 5) eee a Exper Stage 0 + Normal conditions of a vegetative cell ge L: Avial filament formation stage + bacterial chromosome become thread like known as axial filament In this stag Axial filaments attached to cytoplasmic membrane by mesosome Elongation of cell take places PHBA is the reserved food material in Bacillus spp is utilized in sporulation. Stage U: forespore formation + Asymmetric cell division occurs + Cell membrane forms septum near one end which encloses a small portion of DNA forming forespore ge III: enguifment of forespore + Mother cell membrane grow around the forespore engulfing it + Fore spore now has two membrane layer .ge IV: synthesis of exosporium + Chromosome of mother cell disintegrates + Exosporium synthesis occurs + Forespore starts Forming primodial cortex between two membrane. + Dehydration of cell Stage V: synthesis of dipicolonie cacid * Production of SASPs and dipicolinic acid occurs + Incorporation of calcium ions with dipicolonie acid occur forming calcium dipicolonate + Further dehydration of cytoplasm * Formation of coat layer Stage Viz maturation * Maturation of endospore Stage VIL: release of endospore * Cell lysis and release of endospore Spore germination + Endospore remains dormant for years. But under favorable conditions each endospore germinates to give rise to a vegetative cell pore germination involves 3 process. 1. Activation 2. Germination 3. Outgrowth Activation of endospore: + The g unles: ermination of bacterial spore do not occur even when the environment is favorable itis firs activated + At first the Spore coat must be damaged by hieating for several minutes Germination: The activated spore initiates germination after binding of effector molecules. + Binding of effectors molecules activates autolysis that destroy peptidoglycan of cortex. + After destruction of peptidoglycan, water is taken up and calcium dipicolinic acid is released. Outgrowth: + After uptake of water swelling of spore occurs. + Along with swelling, synthesis of DNA, RNA and proteins also occurs. + A small germ cell emerges out after breaking the Spore coat and begins to grow into vegetative cell Resistance Bacterial spores are highly resistant to ordinary boiling. disinfectants, and heating, but spores of all medicelly important bacteria are destroyed by autoclaving. Bacterial spores are highly resfstant to: ¢ Heat,Dehydration. Radiation and Chemicals. The following factors/constituents play major roles in the resistance of bacterial spore: Calcium dipicolinate in core.Keratin spore coatNew enzymes (i.e. dipicolinie acid synthetase, heat-resistant catalase), Increases or decreases in other enzymes. A mature endospore contains a complete set of genetic material (DNA) from the vegetative ribosomes, and specialized enzymes. Mature endospores are released from the vegetative cell to become free ender wis are placed in an environment that supports growth, the endospores wi ren ‘tative cells in a process called germination. It should be noted that unlike the process 0! binary elative cells. endospore formation is not 2 reproductive process but a process of differentiation that provides the bacteria with a mechanism for survival. sion observed with ve Demonstration of Endospore Endospores cannot be stained by ordinary methods, such as simple and Gram staining, because dyes do noi penetrate the endospore wall. But endospores can be seen as an unstained refractile body within the cell. in gram-stained smears. or unstained preparations. Without a special stain, it’s hard to differentiate endospores from inclusions of stored material J Zighl-Neelsen method can be used for endospore staining, the most commonly used endospore stain is the Schaetfer-Fulton Posi ions of Bacterial Spores Fhe shape and the position of spores vary in different species and can be useful for classification and identification purposes. The position of the spores can be seen in the smear using dospore staining method. Lndospores may be located in the middle of the bacierium (central). at the end of the bacterium (terminal), near the end of the bacteria (subterminal), and maybe spherical or elliptical 1. Central endospores are located within the middle of the vegetative cell, 2. Terminal endospores are located at the end of the vegetative cell. 3. Sub-terminal endospores are located between the middle and the end of the cell. * Central or equatorial, giving the bacillus a spindle shape (eg. Clostridium bifermentans) Sub-terminal, the bacillus appearing Club shaped (eg. Clostridium perfringens) Oval and terminal, resembling a tennis racket (eg. Clostridium tertium) erical and terminal, giving a drumstick appearance (Clostridium terani) Important features of Spores Spores are highly resistant to heating: spores are not killed by boiling (100°C) but are killed at 1c. Spores are highly resistant to many chemicals, includ disinfectants. most Spores can survive for many years in soil and other inanimate objects. Spores do not exhibit measurable metabolic activity. Spores are formed only when nutrients are insufficient. Medical Importance of Bacterial Spores Medical Implications Medical supplies must 21°C for at least 15 minutes to be sterilized. The only solution designated as sporicidal will kill spores. Wounds contaminated with soils can be infected with spores and cause diseases such as tetanus and gas gangrene. Antibiotics spores. are ineffective Spores are not often found at the site of infection because nutrients are not limited. be heated to against — Tynes we lecawbwlesy ae £9 Singl- 17 Stains and Staining Technique 17.1. STAINS AND STAINING TECHNIQUE Microorganisms.are omnipresent but their visualization in living state is extremely difficult not only because they are minute but also because they are transparent and practically colourless and usually motile when suspended in an aqueous medium. Also, the microorganisms often possess the same refractive index as the aqueous solution in which they are suspended and this makes them nearly invisible. Their structural details can also not be observed clearly because of lack of diagnostic purposes. To solve this problem, therefore, a variety of stains and staining procedures in conjunction with light microscopy have been devised, and which are now the major tools in microbiological studies. q 11.1.1. What are Stains ? A stain is any colouring organic compound that, when combined with another substance, imparts a colour to that substance. The terms ‘dyes’ and ‘stains’ are often used interchan ; : geably by biolo same. The term ‘dye’ is used to refer to 2 colouring agent that-is used for heel eee ey me not Oe ‘stain’ is used 1 refer to that dye which is used for biological fhereas the term: : : purposes. Most ain : for bacteria, are aniline dyes, so called because their derivation from aniline eaeen eligi ec used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin ee. St COmMONY 11.1.2. Purpose of Staining ‘Staining is done for any or all of the following three purposes: (@ Tosee organism better. Staining enables to see th i 'e Organism better in contrast Sea with background, () To differentiate one organism from another. Some microorganisms take colour ae given 301 Jins and Staining Technique __ staining conditions, some do not. Such differences are particularly evident in staining procedures which are therefore called “differential stains’, the most common differential stains being the Gram- stain and the acid-fast stain. (©) To determine particular structures. There are special stains which react only with certain structures of the organism, ¢.g., spores, cell wall, nuclei, or’others. This is why an organism stained with a cell wall-stain shows only the presence or absence of its cell wall. 17.1.3. Structural Components (Nature) of Stains Stains (dyes) usually have complex molecular structure and are chiefly benzene derivatives. A stain consists of three constituents: the organic compound containing a benzene ring, the chromophore, and the auxochrome. ‘Thus a stain (Fig. 17.1) may be defined chemically as an organic compound containing both chromophore and auxochrome groups linked to its benzene 8 Benzene Organic colourless solvent Coloured compound not | + a stain (chromogef) | Chromophore Chemical group that imparts colour to + benzene { Auxochrome Chemical group that intensifies the colour by Stain conveing the property -of ionization to the chromogen and enabling it to form salts and. binding to the biological substance FIG. 17. Asian (dye). The ability of a stain to bind macromolecular cellular components such as proteins or nucleic acids depends on the electrical charge found on the chromogen portion, as well as on the cellular component to be stained. For convenience, when benzene of an organic colourless solvent binds to the nitro group of chromophore, it results in a yellow coloured compound called crinitrobenzene in wiich three hydrogen atoms in the benzene molecule are replaced by three nitro groups. Trinitrobenzene is a chromogen but not a stain. If however, another hydrogen atom is replaced by an auxochrome’ group, such as OH, the compouid known. as picric acid (trinitrohydroxybenzene) is formed. The picric acid is capable of ionization or electrolytic dissociation to form sali that binds to opposite-charged biological substance (Fig. 17.2). Thus the picric acid, which is yellow in colour, is a stain (dye). The colour of picric acid is due to the chromophoric nitro group (NO,), and its staining property is due to the auxochromic hydroxyl group (OH), which imparts the molecule the ee of ionization or oe dissociation. i i OH 4 ON. No, foization no Beans > Be orate ° H H Electolytic 4 H 1 issociation a NO, Benzene _Nitro group Trinitrobenzene Hydroxyl group Picrié acid (C2louriess) (Chromephore) chromogen, | (auxochrome) _(rinirohycroxy- yellow in colour benzene), due to the presence ‘the yellow stain of chromophores Fic. 17.2. Conversion of benzene ino trintrobenzene (2 chromagen) hen it combines with the nitro group (chromophore) ‘and the conversion of tinitrobenzene chromogen into picric acid rae) the yefow stain, when added with hydroxyl group (auvochrome). Microbiology 302 es 17.1.4. Chromophore and Auxochrome Chromophore (Gk. chroma = colour; ph0P@s = 10 bear) is such a group with multiple bond that is associated with a compound and produces colout " that compound. It was Otto N. Witt (1876) who first designated the chromophore, Chromophore containing Molecule is called chromogen. ‘The most effective chromophores are nitroso (NO), nitro (NO,), azo (N = N)s P-Quinoid, o-Quinoid, ete, The chemical structure of a chromophores is given in Fig, 17.3. The presence of SY One of the chromophores in a molecule is usually sufficient to produce colour. Thus nitrobenzene is pale-green, azobenzene is orange-red, p-quinones are yellow, and o- quinones are orange or red ° ° 0 ene =weo

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