Introduction Bacteria defined as microscopic single celled organism that can penetrate into healthy tissues & start multiplying into vast numbers. e These are unicellular, free living small microorganism which are visible under the light microscope. © Those are belongs to kingdom prokaryotae (Monera). © They occur in water, soil, air & all natural environments._ EE °The size & shape vy, i ary between the dimensions of 0.75 to 4.0 um, ° The cocci diameter near about 1 pm & bacilli are 1to8 um. °They are found in spherical shape ie coccoid forms or as cylindrical form i.e rod shaped forms.shape of Bacteria On basis of shape, Nau anawin bacteria are classified as follows... Cocci . Bacilli Vibrios . Spirilla . Spirochetes . Actinomycetes . MycoplasamsCocci Cocci aresmall, spherical or oval in shape © In greek ‘kokkos’ means berry © E.g. Micrococcusee Bacilli © They are rod in shapes. © It is derived from greek word ‘Bacillum’ meaning stick. e Some of the bacilli the length of the cell may be equal to width those are called coccobacilli E.g. BracellaSpirilla ° Those are longer rigid rods with several curves or coils. ° Those are helical in shape & rapid bodies. ° E.g. Spirillum ruprem.Acetinomycetes © These are branching filamentous bacteria. © The characteristics shape due to the presence of rigid cell wall. © E.g. Streptomyces species. ; yb JMycoplasma © Those are cell wall deficient bacteria © So that, they do not have stable morphology. e They occur as round or oval bodies with h interlacing fillaments.Denccmentot ceria Coccoid cells to exhibit growth in aggregates. Accordingly that assembly they again exist in following five manners: ° As pairs or diplococci. ° As group of four systematically arranged in a cube or sarcinae. © As unorganized array like a bunch of grapes or staphylococci. ° Aschain like a string of beads or streptococci. © In that cocci divided into two planes & remain in group of four that is tetrads.ARRANGEMENTS OF COCCI coccus diplococei Staphylococel O CO J § 88 streptococci sarcina tetrad-s Some bacilli species are found in chain like structure those called streptobacilli e.g Bacillus subtilis. = ser * Hoo® Some bacilli species are found in chain like structure but have much large area of contact between the adjacent cells those are called trichomes e.g. Saprospira species- Structure of Bacterial cel Prokaryotic Coll StructureFlagella e Flagella are long, slender, thin-hair like structure. © Flagella attached with cytoplasm. e They play important role in bacteria for motility. © They have 0.01 to 0.02 pm in diameter. © They have 3 to 20 :m in length. © Flagella found in both Gram-positive & negative bacteria. © Few coccal forms, most bacilli & almost all of the spirilla & vibrios are motile by flagella. ° They can be seen by compound microscope with special staining technique & can be seen easily under electron microscope & dark filled microscope.igella seen in bacterial species in different manners.. © Monotrichous : Single polar flagella e.g. Vibrio cholera ° Lophotrichous : two or more flagella at only one end e.g Pseudonomas fluorescens © Amphitrichous : single flagella or more flagella at both end e.g Alcaligenes fecales © Peritrichous ; several flagella present all over the surface e.g. Salmonella typhiParts of flagella Three main parts present in flagella those are... ° Filament © Hook ° Basal bodybasal bodyie Fimbriae = © Fimbriae are similar structure like flagella but not involved in motility. © It is shorter than flagella (3 pm). © Fimbriae can be distributed over the entire surface of the cell. © Fimbriae act primarily as adhesions & allow to microorganism toattach to surface. © They responsible for haemagglutination & cell clumping in bacteria.- Pili © Pilis are morphologically & chemically similar to fimbriae. ° But they are present in small in numbers compatibly fimbriae. ¢ Pilis joins to the bacterial cell for transfer of DNA (bacterial conjugation) from one cell to another cell. ® So pili also called as sex pili or fertility pili (F-pili).Capsules & Slime layer ° Many bacteria secrets EPS (extracelluar polysaccharides) that are associated with the exterior of the bacterial cell. ° The EPS contains 2% carbohydrate & 98% water so, they produce gummy exterior to the cell. © Morphologically two extreme forms exist... i. Capsules ii, Slime © Capsules: which forms rigid, tightly & closely associated with cell © Slimes: which are loosely associated with cell.eS Function of canals Bethe ° They protect from desiccation. e They provide a protection barrier against the penetration of biocides. e They protect against engulfment by phagocytes & protozoa. © They may promote the stability of bacterial suspension by preventing the cells from aggregation & settling. © They may promote attachments of bacteria to surface.Cell wall © Cell wall gives definite shape to the bacteria. © Cell wall situated between the capsule & cytoplasmic membrane. © It is about 20- 30 nm in thickness, eIn the cell wall contains diaminopimelic acid (DAP), murmaicacid & teichoic acid. ° These substance joined together to formed a complex polymer structure known as peptidoglycan or murein or mucopeptide.Peptidoglycan is a large macromolecules containing glycan (polysaccharide) chains that are cross-linked by short peptide bridge. © The glycan chain act as a backbone to peptidoglycan. ° Those short peptide bridge composed of alternating residues of N-acetyl muramic acid (NAM) & N-acetyl glucosamine (NAG). e Each molecule of NAM attached a tetrapeptide. © Tetrapeptide consisting of the amino acids L-alanine, D- alanine, D-glutamic acid & lycine or diaminopimelic acid (DAP).This glycan tetrapeptide repeat unit is cross -linked to adjacent glycan chain. © This adjacent glycan chain occurs through a direct peptide linkage or a peptide interbridge. ° The type & number of cross linking amino acids vary from organism to organism.° On the staining technique bacteria are divided into two large groups...i. Gram-positive ii, Gram- negative ¢ This staining technique are called as Gram staining technique. eIn that gram staining technique, the bacterial film treating with crystal violet & iodine solution & then washed with alcohol solution.~ © After washing with alcohol solution the gram negative Organism cells appears the colourless while, gram Positive organisms are retain the dye. ° When both gram positive & negative cells are treated with different colour dye e.g carbol fuchsin (red in colour). © That time, gram negative cells appears red & gram positive appears purple. © On that it reflects that both have different cell wall structure.— neplesitahveteelanelliseatle tts ¢ Gram positive bacterial cell wall consist of a single type of molecules. * Cell wall thick near about 20 to 80 nm. ¢ In that present of 60 to 80 % peptidoglycan. « Gram positive walls frequently contains acidic polysachrides are called teichoic acids. © Teichoic acid are either ribitol phosphate or glycerol phosphate molecules that are connected by phosphodiester bridge.In some gram positive bacteria glycerol-teichoic acids are bound to lipids membrane and termed as lipoteichoic acid. © Those lipoteichoic acid create infection by killing bacteria & shows inflammation.=negative cell wall simu Gram negative cell wall are multilayered & complex typ structure. Gram negative cell wall consist 10 to 20 % peptidoglycan. In that second layer found outside the peptidoglycan layer This layer is asymmetrical & contains protein lipoproteins, phospholipids & lipopolysaccharide (LPS).» This outer layer is attached to peptidog! / fixed in the outer membrane. © Inthe inner leaf of the outer layer conatins phospholipids & it’s outer layer composed with LPS (lipopoysaccharide), a polysaccharide-lipid molecule. e In gram negative cell, the LPS is an important molecule because it determine the antigenicity & it is extremely toxic to animal cell. © In the LPS molecules contains three regions i lipid A ii. Core polysaccharide iii. O-specific polysaccharide© Lipid A linked to core KDO (ketodeoxyoctoni polysaccharide is the O-5 e In the O-polysaccharide six-carbon sugars as we O-antigen 90090 g0aeer f 00e @ | sugars such as abequose. $ Q noo | RA, Lipid A Core EIT —_— weet ae reeein the lipid A components are gives toxic & pathogenic properties to the gram-negative bacteria. © Gram negative bacterial outer membrane is relatively permeable to small molecules but not for enzymes or large molecules. © The region between the outer surface of the cytoplasmic membrane & the inner surface of the outer membrane is called the periplasm.Cell wall structure of Gram-negative bacteria Outer membrane Peptidoglycan ——~— Inner membrane { ——— Membrane protein PorinGytoplasmic Membrane “ ° Cytoplasmic membrane is thin near about 5 to 10 nm. ® Biochemically, the cytoplasmic membrane is fragile, phospholipid bilayer with proteins distributed randomly throughout. ° In the phospholipids bilayer most of the proteins are tenaciously held & are called integral proteins.‘Other proteins are loosely attache ose are called peripheral proteins. © The phospholipids molecules are arranged in two parallel rows, called a phospholipid bilayer. ° Each phospholipid molecule contains a polar head & tail. © Polar head composed of a phosphate group & glycerol. ° The non-polar tails are interior of the bilayer. e Prokaryotic plasma membrane are less rigid than eukaryotic due to lack of sterols.—— EEE membrane ° They including in transportation of nutrients, © It provides mechanical strength to the bacterial cell. ° It helps in DNA replication. © It contains the enzymes involved in the biosynthesis of membrane lipids & various macromolecules of the bacterial cell wall.‘beructure of cytoplasmic membrane WAWK nya Peripheral : membrane protein Integral membrane proteins¢ In the bacterial cytoplas is a type of suspension, in that contains organic, inorganic solute ina viscous water. e It contains the nucleus, ribosomes, proteins & other water soluble components & reserve material. © The cytoplasm bacteria differ from that of higher eukaryotic microorganisms in not containing endoplasmic reticulum, golgi apparatus, mitochondria & lysosomes. e In most of the bacteria also contains extrchromosomal DNA (i.e DNA are not connected to chromosome) is also present.= Ribosomes e Ribosomes are most important structure in bacterial cytoplasm. ° They involved in protein synthesis. ° Ribosomes numbers varies with the rate of protein synthesis. e If greater the number of ribosomes then the greater the protein synthesis. ® They have 200 A° in diameter. ° They are characterised by their sedimentation properties.“© These bacterial ribosomes are called as 70 S ribosomes. — S= svedberg unit..unit of sedimentation. © After sedimentation carried in ultra-centrifuge & then placed in low concentration of magnesium that time 70 S ribosomes dissociated into 50 S & 30 S particles. ° Each 50 S particles contain...one molecule of 23 S RNA, one molecule of 5 S RNA & 32 different proteins. © And, each 30 S particles contains...one molecule of 16 S RNA & 21 different proteins. © During protein synthesis these ribosomes are associated with the m-RNA & such association are called polysomes.Mesosomes I e In most of the bacteria, particularly in Gram-positive bacteria the growth condition depending upon the membrane appears to be infolded at more than one point. © Such infoldings are called mesosomes. © Mesosomes presents in two types... Incentral (septal) mesosomes & peripheral (lateral) mesosomes.“= Central mesosomes present deep into the cytoplasm & locate near the middle of the cell. © These are involved in the DNA segregation & in the formation of cross walls during cell division. © The peripheral mesosomes are not present at central location & are not associated with nuclear material. © Mesosomes are also called as chondroids & are visible only under electron microscope. ° Larger numbers of mesosomes have a higher respiratory activity e.g. Azotobacter.° Nucleus appears oval or elongated bodies & generally present one per cell. © The genome consists of a single molecule of double stranded DNA arrangement in acircle. ° It may open under certain conditions to form a long chain about 1000 pm in length. e In bacterial nucleus does not contains nuclear membrane, nucleous & deoxyribonucleoprotein. © The bacterial chromosome is haploid & replicated by simple fission instead of mitosis as in an eukaryotic cell.a Spores ° Many bacterial species produce spores inside the cell & outside the cell. ° Inside the spores are called endospores & outside the spores are called exospores. E.g Bacillus anthracis, Bacillus subtilis ete. e Spores are extremely resistant to desiccation, staining, radiation, disinfecting chemicals & heat. ¢ Each bacterial spore on germination forms a single vegetative cell. © They remain viable for long time & help bacteria to survive for long period under unfavourable condition.dospores are thic —produced one per cell. © All the endospores contain large amount of DPA (dipicolinic acid). © It occurs in combination with large amount of calcium, which is present in central part of the spore (core). © That calcium & DPA complex play important role in the heat resistant of endospores. © Endospores consists of a core or envelope or protoplast. © In the core or protoplast consist of DNA & ribosomes, t-RNA & enzymes. © The spore envelop consist of the inner membrane, outer membrane, cortex & spore coat. © In some species have the outer layer called exosporium which bears ridges & fold.INNER SPORE MEMBRANE SPORE COAT EXOSPORIUM OUTER SPORE MEMBRANESPORULATION F ° The process of endospore formation is known as sporulation it may take 4 to 8 hrs in a vegetative cell. ¢ Sporulation process... ¢ Firstly..a newly replicated bacterial chromosomes & small portion of cytoplasm are isolated by an ingrowth of the plasma membrane called a spore septum. e The situated septum derived from the cytoplasmic membrane is then formed by a process of invagination which divides into a forespore & sporangium, e The forespore is subsequently encircled by a dividing septum as a double layered membrane. © Between the two layers is laid a spore cortex & outer layer is transformed into spore coat which consists of several layer.- c__» Vegetative Cell Sree ep = Atgmemetete Cot Piven __> Stage III Vern pare Kagetiment eo Stages IV+V Spore Cortes and Case Syaemente Stages VI+VII Apere Materation ama Mather Cem EyerBacterial reproduction & growth kinetics 1.Multiplication & divisional cycle ¥ Many bacterial cell multiply by binary fusion. ¥ Means, each individual cell increase in size until it is large enough to divide into two identical daughter cells. ¥ At that time of separation each daughter cell must be capable of growth & reproduction. ¥ While each daughter cell will automatically contain mRNA, tRNA, ribosomes, enzymes, cytochromes etc. from mother cell.uring DNA replication cterial proce: chromosome which is circular in shape that attached to the cytoplasmic membrane where, it is able to uncoil the DNA. ¥ The process of DNA replication proceeds at a fixed rate & it’s depend on temperature, Therefore the time taken to copy of an entire chromosome depend on the number of base pairs within it & the growth temperature. ¥ eg Escherichia coil growing at 37°C will take 45 mints for replication of chromosome.ese copies of chromosome must segregates to opposite sides of the cell before cell division can proceed. ¥ Cell division occurs differently in Gram-positive & Gram- negative bacteria. ¥ In Gram positive cells have rigid cell wall in that develop cross wall that divides the cell into two equal halves. ¥ Construction & cross-wall formation takes approximately 15 mints. to complete. ¥ In Gram negative cells do not have rigid cell wall so that divide bya process of construction followed by membrane fusion.“In DNA replication, chromosome segregation (C-phase) & cell divison (D-phase) occur sequentially in slow-growing cells with generation times of greater than 1 hr & are the final events of the bacterial cell. ¥ Cells are able to replicate faster than once every hour by initiating several rounds of DNA replication at a time. Thus partially replicated chromosomes become segregated into the newly formed daughter cells.~ 2. Population growth ¥ Population growth means an orderly increase in all cellular constituents, v Increase of mass May not really reflects growth because there is only increase in the size & weight of the cell. “All actively growing cells mainly multiply by the binary fission. “In the binary fission one specific cell undergoes division to give rise to the formation of two cells.us, population growth increases, geometri 1323 2 23> 24... 29 Where, n = number of generations. Assuming that there is no cell death at all, each succeding generation shall give rise to double its population. Thus, the total population N at the end of aspecific given time period is expressed as.... N=1x2" But, under practical condition, it is difficult to inoculate only one bacteria so formula as.. N=N,x2" N, is the number of bacteria inoculated at time zero.Taking logarithm on log N = log N, + nlog2 n= logN—log N, log 2 n= log N-logN, 0.301 n = 3.3 (log N - log N,) If we know the initial population & the population after growth then we can calculate the number of generation by using the above formula.Growth curve of bacteria ati, neseen nf ah Le mwtnntn abantnnd Le. 220..1. ain.thordecline phase: ¥ Inthat phase, the number of bacterial cells decreases exponentia essentially the inverse of growth during the log phase. j responsible are depletion of nutritic ~ A variety of conditions re products etc. ¥ Atthis phase the log of number of c® decline the line. te Asynochronous or synochronized culture is a microbial culture or cell culture that are all in the same growth stage. ° Thus, the entire population is kept uniform with respect to growth & division. © But practically it is not possible to determine a single bacterial cell to obtain the information about growth behavior. © Synchronous culture provides the entire cell crop in the same stage. ¢ Synchronized culture provides information on measurement made on such culture are equivalent to the measurement made on individual cells.© A synchronc physical sepz by forcing a condition by © physical sep FRESH MEDIUM by periodic | CeLLs WASHED AWAY conditions populations, NEW BACTERIAL CELLS © The synchror GROWTH AND DIVISION CYCLE FIG. 19.4, Helmsterer. Cumming technique of obtaining aynehronous euituren. rated either by e of division or 1, physiological on, filtration or environmental dividing cell seneration.e In that techn log phase o1 environment. © It is necessary process. © This techniqu ¢ In this appara controlled rat © The rate of gi rates. Hence any constant i FIG. 19.5, The chemestat. a conbnuous culture system. atained in the in a constant ch & industrial ystate growth. h chamber at a ing the inflow nite growth at———————————————— oe e Three major process involved in genetic exchange... ¥ Transformation ¥ Transduction ¥ Conjugation_____ Transformation © The early work of Fred Griffith in 1928 on the transfer of virulence in the pathogen Streptococcus pneumoniae . ° The stage for the research that first showed that DNA was the genetic material. © Griffith found that if he boiled virulent bacteria and injected them into mice, the mice were not affected and no pneumococci could be recovered from the animals. © When he injected a combination of killed virulent bacteria and a living nonvirulent strain, the mice died; moreover, he could recover living virulent bacteria from the dead mice. © Griffith called this change of nonvirulent bacteria into virulent pathogens transformation.© Defined as: a phenomenon causes genetic recombination in bacteria wherein DNA is carried from one specific bacterium to another by a bacteriophage. © There are group of viruses are called bacteriophage. Bacteriophage have bacterial cells as their hosts. © These bacteriophage inject viral DNA into the bacterial cell & after that viral DNA is then replicated & transcribed at the expense of the host & assembled into new viral particles. Normally the host cell lysed in order release the viral progeny but in exceptional conditions they enter in a replication cycle of the viral DNA & become incorporated by recombination into the chromosome of the bacterium. © This is known as temperate phage.© Then, viral DNA forms part of the bacterial chromosome & will be copied to all daughter cells. © As well as temperate phage will be active once again at a low frequency & phasing between temperate & lytic forms ensures the long-term survival of the virus.Conjugation is a natural process representing the early stages in a true sexually reproductive process. In that transcribed to produce singular viral elements, which cannot assemble or lyse the host cell, Such DNA strand are known as plasmids. Plasmids are circular & can either be integrated into the main chromosome, in which case they are replicated along with chromosome & passed to daughter cells or they are separate from it & can replicate independently. The simplest form of plasmid is F-factor (fertility factor). This can be transcribed at the cell membrane to generate F-pilus within the cell envelop & cells containing an F-factor are designed F*e The F-pilus is a hollow appendage that is capable of transferring DNA from one cell to another. © In its simplest form an unassociated F-factor will simply transfer a copy to a recipient cell & such a transfer process is known as cojugation.Bacteria required the nutrition’s, pH, oxygen & temperature for growth & multiplication process. So, for cultivation of microorganism required elements such as sodium, potassitim, magnesium & iron. ¢ As well as in media required contains of source of carbon, nitrogen, hydrogen, oxygen & phosphorus. © Bacteria can be classified depending upon nutritional requirements...such as carbon, energy, electron etc.of energy: poe Energy obtained from sunlight are called phototrophs bacteria e.g. Rhodospirillum rubrum. Energy obtained from chemical reaction those called chemotrophs bacteria e.g. Escherichia coli or E-coli. ° Source of electrons: All bacteria required electrons for metabolism. Lithotrops : In that type of bacteria species use the inorganic compounds as electron donor e.g pseudomonas pseudoflava. Organotrophs : In that type of bacteria species use the organic compounds as electron donor e.g Escherichia coli or E-coli.Bee 2 sume | pee inorganic compound (H2S) as source of electron. e.g. Chromatium okenii. Photoorganotrophs: some phototropic bacteria use organic compound such as fatty acids & alcohols as electron donors @.g Rhodospirillum rubrum. Chemolithotrophs: some chemotrophic bacteria use inorganic compound as source of electron. e.g. Nitrosomonas europaea. Chemoorganotrophs: some chemotrophic bacteria use organic compound such as sugar &amino acids as electron donors e.g Escherichia coli or E-coli.° Source of carbon: microorganism required carbon for synthesizing cell components. Autotrophs: some species use CO2 as the major source of carbon these microorganisms are called autotrophs. e.g. Chromatium okenii. Heterotrops: some species use organic compounds as a source of carbon such species are called heterotrophs. e.g. Escherichia coli or E-coli.trogen: Nitrogen is the major component of protein & nucleic acids, so that bacteria can use nitrogen from the atmosphere or from inorganic compounds such as nitrites, nitrate. © Sulphur: Sulphur is needed for synthesis of aminoacids. © Phosphorus: Phosphorus usually supplied in the form of phosphate is an essential component of nucleotides, nucleic acid etc.It is the major essential nutrient as it account for about 80 to 90% of the total weight of cell. © Mineral salts: Bacteria require salts, particularly the anions such as phosphate & sulphate & the cations as sodium, potassium, magnesium, iron & calcium. These are present in the natural environment or may be added in cultural media.© Cultural or bacteriological media are mainly used for growth, isolation, purification, maintenance & identification of microorganisms. e Nutrient agar is mainly used for growth of many bacterial species. e¢ Common ingredients present in cultural media are.. e Water ° Peptone © Yeast extract e Meat extract e AgarNW Glassification of cultural medial © Depending on physical state (media consistency) 1. Solid media (1.5 to 2.5% agar) e.g. Nutrient agar. 2. Semisolid media (0.2 to 0.5% agar) e.g. Nutrient broth cont. 0.5% agar. 3. Liquid media (absence of agar) e.g. fluid thioglycollate broth. e Depending on oxygen requirement 1, Aerobic media e.g. MacConkey’s broth. 2. Anaerobic media e.g. Robertson's cooked meat medium.ing on chemic. =~ Simple or basal media 2. pS ewavewns Synthetic or defined media Non-synthetic or undefined or complex media Depending on functional type Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media Assay media Storage mediaDepending on chemical composition reatereetel ¢ Simple or basal media: In that type of media peptone water & nutrient broth is added & used in the study of common bacteria. Addition of 2%agar to nutrient broth forms a nutrient agar medium which is solid basal medium. ¢ Complex or non-synthetic or undefined media: In that type of media included biological origin such as blood or milk or yeast those provides cultivation of bacteria. ° Synthetic or defined media: Those media are prepared from pure chemical substance. These media are used for research purpose & used in metabolic studies of specific microorganisms.e Enriched media: In that type of media included of substance such as blood, serum & egg to basal medium. E.g. blood agar (streptococcus). e Enrichment media: specific substance added in the liq.medium which inhibits the unwanted bacteria & favors only wanted bacteria such media are called enriched media. E.g tetrathionate broth. © Selective media : same as enrichment media but only in solid form & added to a solid medium. E.g MacConkey’s agar (E- coli).A Pecies growth in them that & Blair medium. ° Differential media: these media distin © Indicator media: When bacterial s time it change in colour, E.g. Wilson igwish between types of bacteria, E.g. MacConkey's medium shows lactose fermenter as red colonies & non lactose as white or plae colonies. ° Assay media: these type of media used for the assay of antibiotics, amino acids, vitamins & also used for testing of disinfectants. * Storage media: used for help in preservation & storage of bacteria for long time of period. e.g. Dorset’s egg medium.————E= Pecies growth in them that E.g. Wilson & Blair medium, ~ © Indicator media: When bacterial s time it change in colour, ° Differential media: these media distingwish between types of bacteria. E.g, MacConkey’s medium shows lactose fermenter as red colonies & non lactose as white or plae colonies. ° Assay media: these type of media used for the assay of antibiotics, amino acids, vitamins & also used for testing of disinfectants. * Storage media: used for help in preservation & storage of bacteria for long time of period. e.g. Dorset's egg medium.Indicator media: When bacterial species growth in them that time it change in colour. E.g. Wilson & Blair medium. © Differential media: these media distingwish between types of bacteria. E.g. MacConkey’s medium shows lactose fermenter as red colonies & non lactose as white or plae colonies. © Assay media: these type of media used for the assay of antibiotics, amino acids, vitamins & also used for testing of disinfectants. © Storage media: used for help in preservation & storage of bacteria for long time of period. e.g. Dorset's egg medium.= © Counting chamber method or Haemocytometer mehtod. ¢ Counting method used for determination of cell numbers or count the bacteria. e This is the simple & rapidly counting technique with minimum equipment required, e In that technique take, a minute drop of the culture is placed on Neubar'’s slide. © Neuber’s slide is tiny, shallow, rectangular glass slide with accurately ruled into squares that are 1/400 mm*. e In counting method, cells can be determined by using a phase contrast microscope.Total no. of bacterial cells/mm = No. of cells counted x Dilution area counted x Depth of fluid Cells counted in 5 squares & each square divide into 16 small squares & each square is equal to1/400 mm*. So, area of 5 square = 5 x16 =80 squares. = 80/400 mm? =1/5 mm?It is a special microscopic slide with a counting chamber 1/10 mm deep so that, Volume of lig. Over a one square = 1/10 mm Dilution = 1:200 =200 So formula.. No.of bacterial cells counted = N_x 200 V5 X1/10 =Nx200x50 = Nx 10,000 cells/mm}¢ Identification of microorganism means in that study included for determination of morphological structure of microorganisms. e In the morphological studies depending upon the no.of factors such as, stain studied, nature of culture media, temp. & duration of incubation. ° By using differential staining technique was present on that we can easily determined the nature of microorganism, the size of microorganism, shape of microorganism & also determined their nature of microorganism with using microscope.° Stain is an organic compounds which contains benzene ring with chromophore & auxochrome group. ° Different techniques was used for visualisation, differentiation & separation of bacteria in terms of morphology. © Staining different techniques are... Simple staining Negative staining Gram staining Acid-fast staining Spore staining 6. Capsule staining 7. Other staining Yawn° By using simple staining technique, we can easily determine their morphology & arrangement of bacterial cell. e In the simple staining technique used the single stain e.g. crystal violet, methylene blue, carbol fuchsin safranin etc. e In that used the basic stain with a positively charged chromogen. e When, positively charged chromogen react with bacteria, having a negative charge on nucleic acid & certain cell wall components. © That time, negative charge components strongly binds with positively charged chromogen.e In negative staining technique use the acidic stain e.g. nigrosin or eosin. ° Acidic stain are negative charge so they not penetrate into the cell but, it deposit the around the cell. © So that, unstained cells are easily observed against the coloured background. ° Advantage of that staining tech. compare with simple technique that, it doesn't required heat fixation & in that technique determine the natural size & shape of microorganism can be seen.° Gram staining technique discovered by Dr. Christian Gram in 1884, © By that technique use for not just for determination of morphology but also use for the differentiae in between Gram-positive & Gram negative cell. ° Gram positive cell retain the violet stain. © But gram negative cell decolourised & appears the red colour in some species e.g E-coli, salmonella typhi, vibrio cholerae, klebsiella pneumoniae etc.° Acid fast staining technique used for differentiate between acid-fast & non-acid fast bacteria. ° Mycobacterium species & actinomycetes bacterial species containig mycolic acid & other waxy material in their cell wall. e Such bacterial species do not get stained with ordinary staining technique. So, ZNCF (Zhiel-Neilson Carbol Fuchsin) stain is used with steam heating. © The acid fast bacteria appear pink & non acid bacteria appears blue.Spore staini © Spore staining technique used for detection of spore carrying bacteria & type of spores. ° For spore staining use Dorners method. © In Dorners method, carbol fuchsin is used as primary satin. e After heating the slide with stain for 5 to 10 minutes, wash it & perform negative staining procedure.Capsule stainin ° Capsule staining technique used for determination of capsule outside the cell. For performing, using the positive staining & negative staining technique. © By positive staining the crystal violet is applied & also applied copper sulphate solution (20%) which is created osmotic difference, which causes diffusion of stain towards the outer surface. © After removing stain, the capsular layer as light violet colour against deep violet cell. ° In negative staining, stains the cell & capsule are visible colourless, By negative staining technique it can easily visible.MYCOBACTERIUM-INTRODUCTION Mycobacterium is a genus within the order Actinomyectales that comprises a large number of well characterised species, several of which are associated with human and animal disease such as tuberculosis and leprosy. Aerobic bacilli -non spore forming non motile,rod shaped. Cell wall -rich in lipids Acid-fast bacilli Very slow growingCULTURE-m.tuberculosis * Itincludes non-selective and a selective media. * There are three general formulation that can be used for both the media they are: — Semisynthetic agar media — Inspissated egg media — Broth mediaMycobacterium tuberculosis ‘The bacterium that causes tuberculosis. M. tuberculosis has unusually waxy walls, is slow-growing and among the most recalcitrant bacteria to treatment. Thin straight reds-0.4x 3m. On artificial media, coccoid and filamentous forms are seen with variable morphology from one species to another. cannot be classified as either gram-positive or gram-negative The Ziehl-Neelsen technique of staining is employed for identification of acid-fast bacteria.CONSTITUENTS OF TUBERCLE BACILLI © The constituents listed below are found mainly in cell walls. © Mycobacterial cell walls can induce delayed hypersensitivity and some resistance to infection. — Lipids-mycelicacid,waxes,phosphatides — Proteins-induce tuberculin sensitivity — PolysaccharidesPATHOGENSIS + Inhaled aerosols Engulfed by alveolar macrophages: Bacilli replicate Macrophages die * Infected macrophages migrate > 1ocal lymph nodes * Develop Ghon's fOCUS py Primary complex * Cell mediated immune response _ stops cycle of destruction and spread + Viable but non replicating bacilli present in macrophagesAcute pulmonary disease Systemic spread ma symptomati: MILIARY TUBERCULOSIS Pulmonary meningitis@ © © PRIMARY INFECTION TYPE OF TB When a host has first contact with tubercle bacilli, the following features are usually observed: (1) An acute exudative lesion develops and rapidly spreads to the lymphatics and regional lymph nodes, The exudative lesion in tissuc often heals rapidly. (2) The lymph node undergoes massive caseation, which usually calcifies, (3) The tuberculin test becomes positive. This primary infection type occurred in the past, usually in childhood but now frequently in adults who have remained free from infection and therefore tuberculin-negative in early life. In primary infections, the involvement may be in any part of the lung but is most often at the base.REACTIVATION TYPE OF TB © The reactivation type is usually caused by tubercle bacilli that have survived in the primary lesion, © Reactivation tuberculosis is characterized by chronic tissue lesions, the formation of tubercles, caseation, and fibrosis. © Regional lymph nodes are only slightly involved, and they do not caseate. ® The reactivation type almost always begins at the apex of the lung, where the oxygen tension (PO,) is highest. © These differences between primary infection and reinfection or reactivation are attributed to (1) resistance and (2) hypersensitivity induced by the first infection.DIAGNOSIS © Specimens fresh sputum, gastric washings, urine, pleural fluid, cerebrospinal fluid, joint fluid, biopsy material, blood, or other suspected material, © Smears Sputum, exudates, or other material is examined for acid-fast bacilli by ZiehI-Neelsen staining. Stains of gastric washings and urine generally are not recommended, because saprophytic mycobacteria may be present and yield a positive stain. Fluorescence microscopy with auramine-rhodamine stain is more sensitive than acid-fast stain. If acid-fast organisms are found in an appropriate specimen, this is presumptive evidence of mycobacterial infection.TUBERCULIN TESTS © Inan individual who has not had contact with mycobacteria, there is no reaction. © An individual who has had a primary infection with tubercle bacilli develops induration, edema, erythema in 24-48 hours, and, with very intense reactions, even central necrosis. © The skin test should be read in 48 or 72 hours. © Itis considered positive if the injection of 5 TU[Tuberculin units] is followed by induration 10 mm or more in diameter. © Positive tests tend to persist for several days. Weak reactions may disappear more rapidly.Interpretation of Tuberculin Test * Apositive tuberculin test indicates that an individual has been infected in the past. It does not imply that active disease or immunity to disease is present. © Tuberculin-positive persons are at risk of developing disease from reactivation of the primary infection, whereas tuberculin-negative persons who have never been infected are not subject to that risk, though they may become infected from an external source.TREATMENT * Anti-tuberculous drugs ' INAH — Rifampicin Ethambutol — Pyrazinamide © Multi-drug resistant tuberculosis