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The first simple forms of life appeared on earth more then 3 billion years ago. Microscopic forms of life are present in vast numbers in nearly every environment like soil, water, food, air , etc.

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  1. 1. Nursing Path www.drjayeshpatidar.blogspot.com
  2. 2. INTRODUCTION  The first simple forms of life appeared on earth more then 3 billion years ago.  Their descendants have changed & developed into the several million type of animals , plants & microorganisms are recognized.  Microscopic forms of life are present in vast numbers in nearly every environment like soil, water, food, air , etc. Nursing Path 6/23/2016 2www.drjayeshpatidar.blogspot.com
  3. 3. Microbiology means…..  this is the science dealing with the study of microorganisms. Microbiology is the science of living organisms that are not directly visible to necked eye but only under the microscop. Nursing Path 6/23/2016 3www.drjayeshpatidar.blogspot.com
  4. 4. Historical events…..  The credit for observation & description of bacteria goes to a biologist Antony van leeuwenhoek.  Scientific development of microbiology was ushered by Louis Pasture , perfection on microbiologycal studies occurred by Robert Koch & Lord Lister introduced the antiseptic surgery. Nursing Path 6/23/2016 4www.drjayeshpatidar.blogspot.com
  5. 5.  Louis Pasteur (1822-1895) was trained chemist. His studies on fermentation of wine led him to take interest in microbiology. He is known as the founder or father of modern microbiology .  Joseph Lister was a professor of surgery. He applied Pasteur’s work & introduced antiseptic technique in surgery for killing bacteria in wounds & in the air with carbolic acid. He is known as father of antiseptic surgery. Nursing Path 6/23/2016 5www.drjayeshpatidar.blogspot.com
  6. 6.  Robert Koch(1843-1910),German general practitioner , perfected bacteriological techniques & introduced methods for isolation of pure strains of bacteria. He introduced staining techniques.  He is known as father of medical microbiology. Nursing Path 6/23/2016 6www.drjayeshpatidar.blogspot.com
  7. 7. Branches of microbiology 1. Medical microbiology 2. Industrial microbiology 3. Food microbiology 4. soil microbiology 5. Plant microbiology Nursing Path 6/23/2016 7www.drjayeshpatidar.blogspot.com
  8. 8.  also we connected with medical microbiology. It studied under following headings: a. Parasitology deals with the study of parasites causing disease in human being. b. Mycology deals with the study of fungus causing disease in human being. c. Immunology is connected with mechanism involved in the development of resistance by body to infectious disease. d. Bacteriology deals with the study of becteria. e. Genetics is the study of heredity & variations. f. Virology is the study of viruses. Nursing Path 6/23/2016 8www.drjayeshpatidar.blogspot.com
  9. 9. Scope of microbiology  1.Diagnostic isolation & identification of causative organism from pathological lesions. Widal’s test – typhoid fever 2. Prognosis of disease in widal’s test rising titer signifies active disease & ineffective treatment . falling titer means effective treatment & curing of disease. Nursing Path 6/23/2016 9www.drjayeshpatidar.blogspot.com
  10. 10.  3. Guidance in treatment by culturing the organisms in pure form & then performing drug sensitivity test that can suggest the effective drug for the treatment of that particular infection. 4. Source of infection in sudden outbreak of infectious disease we can find out the source of infection. Nursing Path 6/23/2016 10www.drjayeshpatidar.blogspot.com
  11. 11.  Bacteria are unicellular free living organisms without chlorophyll having both DNA and RNA.  They are capable of performing all essential processes of life, e.g. growth, metabolism and reproduction.  They have rigid cell wall containing muramic acid. They were originally classified under plant and animal kingdom.  This being unsatisfactory a third kingdom PROTISTA was proposed by Hackel in 1866 for them.  Protista is again divided into 2 groups: A) Prokaryotes B) Eukaryotes Nursing Path 6/23/2016 11www.drjayeshpatidar.blogspot.com
  12. 12.  Bacteria and green algae (photosynthetic and possess chlorophyll which can exhibit gliding movement like photosynthetic bacteria) are prokaryotes while fungi, algae, slime moulds and protozoa are eukaryotes.  It is worth mentioning to enumerate the differences between prokaryotic and eukaryotic cell. Nursing Path 6/23/2016 12www.drjayeshpatidar.blogspot.com
  13. 13. Bacterial Anatomy  The outer layer or cell envelope of bacterial cell consists of two components  A) A rigid cell wall  B) underlying cytoplasmic membrane (plasma membrane)  The cell wall encloses the protoplasm comprising of cytoplasm (ribosomes inclusion granules mesosomes and nuclear body) and single circular chromosome of DNA. Nursing Path 6/23/2016 13www.drjayeshpatidar.blogspot.com
  14. 14. Nursing Path 6/23/2016 14www.drjayeshpatidar.blogspot.com
  15. 15. Some bacteria in addition may possess additional structure such as protective gelatinous covering outside the cell wall known as capsule. When it is too thing it is known as microcapsule Apart from this some bacteria possess filamentous appendages flagella and fimbria which protruded from cell surface. Nursing Path 6/23/2016 15www.drjayeshpatidar.blogspot.com
  16. 16. capsule  It is an outer covering of thick, jelly-like material that surrounds the bacterial cell.  It contain: a) 0.2 µm width b) 98% water c) 2% solids  Capsules which are much narrower than true capsule and cannot be demonstrated by light microscope are called microcapsules. E.g. N. meningititidis; H. influenzae. Nursing Path 6/23/2016 16www.drjayeshpatidar.blogspot.com
  17. 17. Function:  Capsule serves as protective covering against anti bacterial substance such as bacteriophage, phagocytes and enzymes.  it enhance bacterial virulence  Capsular antigen is hapten in nature and specific for the bacteria. Capsulated organisms:  H. Enfluenza, S. pneumoniae, bacillus anthracis Demostration:  The capsule is best seen in pathological specimence like pus, blood, sputum & exudate.  In artificial culture medium, the size of capsule is reduced and finaly inlatter stages of growth the capsule may disappear due to accumulation of capsule degrating enzymes or due to carbon or energy starvation. Nursing Path 6/23/2016 17www.drjayeshpatidar.blogspot.com
  18. 18. Demonstration  A) negative staining with India ink: in this procedure, bacterial bodies and space in between are filled with India ink and capsule is seen as halo around cell.  B)Special capsule staining technique using copper salt as moderant.  C) Serological method: If suspension of capsulated bacterium is mixed with its specific serum and examine under microscope, capsule becomes prominent and appears swollen due to increase in refractivity (Quellung reaction). Nursing Path 6/23/2016 18www.drjayeshpatidar.blogspot.com
  19. 19. Cell wall  Cell wall is a tough & rigid structure surrounding the bacterium like a shell.  The cell wall is 10-20nm in thickness & confers rigidity upon bacteria in addition to giving protection to the cell against osmotic damage.  It 20 to 25% of the dry weight of the bacterial cell.  It is elastic & porous also freely permeable to solute molecules of less than 10,000 molecular weight. Nursing Path 6/23/2016 19www.drjayeshpatidar.blogspot.com
  20. 20.  The cell wall takes part in cell division by forming an ingrowths from the cell wall.  The mucopeptide component of cell wall possess target site for antibiotics , lysozymes & bacteriophages. Nursing Path 6/23/2016 20www.drjayeshpatidar.blogspot.com
  21. 21. Structure of cell wall  The chemical structure of cell wall of gram positive & gram negative bacteria is different.  The rigid part of the cell wall is peptidoglycan.  A) peptidoglycan (murein) it is the principal structural component of the cell wall . It is present in both gram positive & negative bacteria. I ) lipoprotein layer- it connects the outer membrane to peptidoglycan. Nursing Path 6/23/2016 21www.drjayeshpatidar.blogspot.com
  22. 22.  Peptidoglycan structure- the peptidoglycan polymers consist of alternating stands of N-acetylmuramic acid & N-acatylglucosamine . The N-acetylmuramic acid links the disaccharides to an oligopeptide chain consisting of four amino acids.  Peptidoglycan cross-linking involves the cross- linking between the terminal residue of the peptide side chain with the penultimate residue of an adjoining side chain. Nursing Path 6/23/2016 22www.drjayeshpatidar.blogspot.com
  23. 23. B)autolysins- all bacterial cell wall possess associated enzymes called autolysins which can hydrolyze their own cell wall substance . The capacity of autolysins to dissolve the peptidoglycan in essential for cell growth, cell septation, sporulation, & in transformation. Nursing Path 6/23/2016 23www.drjayeshpatidar.blogspot.com
  24. 24. Gram-positive cell wall I ) The peptidoglycan layer of gram positive bacteria is much as 50% of the dry weight of the cell wall & 10% of the total cell much thicker(15-25nm) than in gram negative bacteria(10-15nm) & it is consist of multiple layers.  The periplasmic space is absent & the peptidoglycan is closely associated with the cytoplasmic membrane. II)Special components: cell wall contain…… significant amount of teichoid & teichuronic acids as. Nursing Path 6/23/2016 24www.drjayeshpatidar.blogspot.com
  25. 25.  They are water soluble polymers containing ribotol or glycerol polymers & maintain the level of divalent cations outside the cell membrane.  The teichoic acids constitute major surface antigens of grampositive bacteria. III) Other components: certain gram positive cell walls also contain antigens such as the polysaccharide & protein. Nursing Path 6/23/2016 25www.drjayeshpatidar.blogspot.com
  26. 26. Gram negative cell wall  This cell wall is a complex structure & thinner than that of gram-positive cell with a bimolecular layer of peptidoglycan. Outer membrane: it is an additional outer membrane & much thicker than the single peptidoglycan layer . It lies above the peptidoglycan layer . it is made of phospholipid bilayer proteins lipopolysaccharide (LPS) Nursing Path 6/23/2016 26www.drjayeshpatidar.blogspot.com
  27. 27. Bacteria with defective cell wall  Removal of cell wall result in lyses of bacteria.  1) gram positive bacteria- complete removal of cell wall of gram + bacteria result in the formation of is protoplast . it lyses unless it is osmotically stabilised . the protoplast constituted by the cytoplasmic membrane & bacterial contents 2)Gram negative bacteria- the complexity of cell wall result in resistance to enzymatic destruction of cell wall . that gram- bacteria with damaged cell wall become spheroplast. Nursing Path 6/23/2016 27www.drjayeshpatidar.blogspot.com
  28. 28. Difference between gram pos.& gram Neg. No. Contain Gram positive Gram negative 1 Thickness 15-25 10-15 2 Varity of amino acid Few several 3 Aromatic & sulfur containing amino acids Absent Present 4 Lipids Low 2 to 4% High 15 to 20% 5 Teichoid acids Present Absent 6 Periplasmic space Absent Present 7 Result of enzyme digestion protoplast spheroplast Nursing Path 6/23/2016 28www.drjayeshpatidar.blogspot.com
  29. 29. Demonstration  When intact bacteria are placed in a solution of very high solute concentrations & osmotic pressure ,protoplast shrinks thus retracting the cytoplsmic membrane from cell wall . The process is called plasmolysois & is useful in demonstrating cell wall.  Cell wall may also be demonstrated by a special technique is called micro dissection.  Reaction with specific antibody is also a way to study cell wall.  Electron microscope gives detailed structural information of even very particles like parts of cell wall. Nursing Path 6/23/2016 29www.drjayeshpatidar.blogspot.com
  30. 30. Function  1) protection of internal structure  2)Gives shape to the cell  3) confers rigidity & ductility  4) role in division of bacteria  5) offers resistance to harmful effect of environment.  6) contains receptor sites for phages & colicin  7)Provide attachment to complement Nursing Path 6/23/2016 30www.drjayeshpatidar.blogspot.com
  31. 31. Cytoplasmic membrane  Definition: “thin semipermiable membrane which lies just beneath the cell wall that is called as cytoplasmic membrane” The whole bacterial cytoplasm is bound peripherally by very thin , elastic and semipermiable cytoplasmic membrane also known as cell membrane. It is 5-10nm in width Electron microscope shows the presence of three layer constituting a unit membrane structure. Nursing Path 6/23/2016 31www.drjayeshpatidar.blogspot.com
  32. 32.  Chemically the membrane consist of phospholipid with small amount of protein.  DEMONSTRATION: The separation of membrane from cell wall is achieved by readily in gram negative bacteria when they are suspended in medium of high osmotic tension. such phenomenon is called as plasmolysis. Electron microscope. Nursing Path 6/23/2016 32www.drjayeshpatidar.blogspot.com
  33. 33. Function  Transport: (1) Active transport: it is site of numerous enzymes (oxidase, polymerase, permease) involved in the active transport of selective nutrients. It is impermeable to macromolecules & ionised substances. (2) Passive transport: It is act as semipermiable membrane through inward and outward passage of water and passive transport of molecule lipid soluble solutes take place by diffusion. Nursing Path 6/23/2016 33www.drjayeshpatidar.blogspot.com
  34. 34. (2) Concentration: it is also concentration sugar, amino acids and phosphate so that a 300-400 fold gradient exists across osmotic barrier. (3) Enzymatic function: it also contain cytochrome oxidase, enzyme of tricarboxylic acid cycle and polymerizing enzyme necessary for synthesis of cell wall. Nursing Path 6/23/2016 34www.drjayeshpatidar.blogspot.com
  35. 35. Cytoplasm  The bacterial cytoplasm is suspension of organic & inorganic solutes in viscous watery solution.  It dose not exhibit protoplasmic streaming (internal mobility) .  It lacks endoplasmic reticulum or mitochondria.  It contains ribosomes, mesosomes, inclusions,& vacuoles. Nursing Path 6/23/2016 35www.drjayeshpatidar.blogspot.com
  36. 36. Ribosome  Ribosome appear as small granules & pack the whole cytoplasm.  The ribosomal particles become linked up & travel along the mRNA stand.  Ribonucleoprotein granules measure 10 to 20nm units in diameter & their sedimentation coefficient is 70 Svedberg units. The 70s ribosome is composed of two smaller units of 50s &30s.  These are strung together on stands of mRNA to from polymers.  The code of mRNA is translated into peptide sequence at this place. the ribosomal particles become linked up & travel along the mRNA stand . it determines the sequence of amino acids brought to the site on tRNA molecules & build up the polypeptide.  FUNCTION-they are site of protein synthesis. Nursing Path 6/23/2016 36www.drjayeshpatidar.blogspot.com
  37. 37.  Polysomes- they are group of ribosome's linked together like beads of chain by messenger RNA .  Mesosomes- they are vesicular , convoluted , or multilaminated structure.  They are prominent in gram-positive bacteria.  Two types- 1) septal mesosomes 2)lateral mesosomes 1) The septal mesosomes is attached to bacterial chromosome & is involved in DNA segregation & the formation of cross walls during cell division. They are also called as chondroids & are visualized under electron microscop. 2) The lateral mesosomes in lateral site. Function- 1) They are the sites of respiratory enzymes in bacteria. 2) Coordinate nuclear & cytoplasmic division during binary fission. 3) Responsible for compartmenting DNA at sporulation. Nursing Path 6/23/2016 37www.drjayeshpatidar.blogspot.com
  38. 38. Nucleus  It is a long filament of DNA rightly coiled inside the cytoplasm.  the bacterial nucleus is not surrounded by nuclear membrane.  The nuclear DNA dose not appear to contain some basic protein .  Nucleus cannot be demonstrated under direct light microscope. It appears as oval or elongated body , generally one per cell. Nursing Path 6/23/2016 38www.drjayeshpatidar.blogspot.com
  39. 39.  The genome consist of a single molecule of double stranded DNA arranged in the form of circle.  It may open under certain conditions to from long chain about 1000 µ in length.  The bacterial chromosome is haploid & replicates by simple fission .  Genes are arranged along the length of chromosome in fixed order.  Bacteria may sometimes have extra nuclear genetic material these are called plasmid or episomes.  Plasmids are not essential for the life of cell. they may confer certain properties like toxigenecity virulence & drug resistance. Nursing Path 6/23/2016 39www.drjayeshpatidar.blogspot.com
  40. 40. Flagella Nursing Path 6/23/2016 40www.drjayeshpatidar.blogspot.com
  41. 41. Flagella  Parts- These are long, sinnous contractile filamentous appendages known as flagella. These are organs of locomotion . ex:-Escherichia coli salmonella, vibrio , pseudomonas, etc. The number of flagella varies up to 10 to 20 per cells according to species of bacteria. These are extremely thin (diameter)12 to 30 nm, helical shaped structure of uniform diameter throughout their length . these are 3 to 20 nm long. Nursing Path 6/23/2016 41www.drjayeshpatidar.blogspot.com
  42. 42. Each flagellum consist of hook & basal body. It originates in a spherical body located just inside cell wall. These are antigenic & are composed of protein called flagellin which has properties of fibrous protein , keratin , & myosin. The number & arrangement of flagella are characteristic of each bacteria. Nursing Path 6/23/2016 42www.drjayeshpatidar.blogspot.com
  43. 43.  Types :- There are 4 types of flagellar distribution on bacteria. Monotrichous- singal polar flagellum, ex.-cholera vibrios . Amphitrichous- single flagellum attached to each end , ex.- alcaligenes fecales. Lophotrichous- tufts of flagella at the end , ex.- pseudomonas , spirilla. Peritrichous- numerous flagella all over the bacterial body , ex.- typhoid bacilli. Nursing Path 6/23/2016 43www.drjayeshpatidar.blogspot.com
  44. 44. Function It is responsible for bacterial motility. Motility may be observed microscopically or by detecting the spreading growth in semi solid agar medium. Demonstration – Dark ground microscopy. Special staining techniques in which their thickness is increased by mordanting. Electron microscop. Hanging drop preparation. Nursing Path 6/23/2016 44www.drjayeshpatidar.blogspot.com
  45. 45. Fimbria (Pili)  Fimbria are filamentous , short , thin , straight , hair like appendage.  This is 0.1 to 1.5 µ long & less than 4 to 8 nm thick.  They are also called as Pili.  Fimbriae are seen only in some gram negative bacteria.  Each bacterium may have 100 to 500 Fimbriae on all over the body of bacteria.  They project from cell surface as a straight filaments.  They are best developed in freshly isolated strains & in liquid culture.  They are composed of protein known as pillin (molecular weight 18000 Daltons). Nursing Path 6/23/2016 45www.drjayeshpatidar.blogspot.com
  46. 46.  Different forms of fimbria – i)common pili ii)F (fertility) pili iii) Col I (colicin)pili i)common pili- They are numerous, short in size(1.5µ) & Peritrichous in distribution. Ex.- red cell of various animal species. Common pile are of 6 types based on their morphology number per cell, adhesive properties, & antigenic nature. Nursing Path 6/23/2016 46www.drjayeshpatidar.blogspot.com
  47. 47.  TYPE:-  TYPE 1:-It is relatively thick & involved in hem agglutinating activity.  TYPE 2:- They are same as type 1 but they are non- hem agglutinating.  TYPE 3:- They are thin & are mannose resistance. They are confer hem agglutinating activity on parent cell.  TYPE 4:-They are thinner than type 3 & are mannose resistance with hem agglutinating activity on fresh red blood cell.  Type 5:-They are monopolar which have been found only in one species of pseudomonas.  Type 6:-They are very long & few of them are seen in one species of klebsiella. Nursing Path 6/23/2016 47www.drjayeshpatidar.blogspot.com
  48. 48.  F pili:- they are associated with fertility & help in bacterial conjugation process . They are longer (20µ length) than common & Col I pili.  Col I pili:-they are about 2µ in length & associated with colicin factor I.  DEMONSTRATION:-  Electron microscop.  Hem agglutination.  Fimbriated bacteria form pellicle in liquid media. Nursing Path 6/23/2016 48www.drjayeshpatidar.blogspot.com
  49. 49.  FUNCTION:- a) Organ of adhesion. b) Hem agglutination. c) They are antigenic. d) Agglutination & pellicle formation. e) Genetic material is transferred from the donor to recipient cell. Nursing Path 6/23/2016 49www.drjayeshpatidar.blogspot.com
  50. 50. SR NO FLEGELLA FIMBRIAE 1 Size larger & thicker. Smaller & thinner. 2 Arise from the cytoplasm or cytoplasmic membrane but not attached to the cell wall. Attached to the cell wall. 3 Organ of movement. Organ of adhesion & conjugation. 4 They are never straight. They are always straight. 5 No require for conjugation. Required for conjugation. Nursing Path 6/23/2016 50www.drjayeshpatidar.blogspot.com
  51. 51. SPORES “spores are highly resistant dormant stage of bacteria formed in unfavourable environmental condition such as starvation and dessication.” As the spores are formed within the parent bacterial cell so they are also called as endospores. During germination each spore give rise to only one vegetative bacteria. Exospores found in fungi(conidia) formed extracellularly from end of parent cells. Nursing Path 6/23/2016 51www.drjayeshpatidar.blogspot.com
  52. 52. Spore forming bacteria A. Gram positive bacilli: (1) Obliterate aerobic- genus bacillus. E.g:- B. anthracis, B. subtilis. (2)Obliterate anaerobic : genus clostridia. E.g:-C.tetani,C.welchii,C.botulinum B. Other bacteria: Gram positive coccus (sporosarcina) Gram negative bacilli(coxiella burnetii) Nursing Path 6/23/2016 52www.drjayeshpatidar.blogspot.com
  53. 53. Spore morphology Nursing Path 6/23/2016 53www.drjayeshpatidar.blogspot.com
  54. 54. Sporogenesis  spontaneous sporulation occurs in condition unfavorable condition such as starvation, dessication, presence of disinfectants and in extreme temperature. Nursing Path 6/23/2016 54www.drjayeshpatidar.blogspot.com
  55. 55. Nursing Path 6/23/2016 55www.drjayeshpatidar.blogspot.com
  56. 56. SPOROGENESIS PROCESS Spore formation is initiated by appearance of clear area in portion of protoplasm near one end of bacterial cell and that protoplasm gradually become more opaque that form fore spore The cell membrane grow inwards and undergoes in folding forming double layered membrane structure around the core The inner most layer of spore wall forms spore membrane in future vegetative bacterium will develop. The spore wall synthesis a thick covering layer cortex and multilayered thin but tough outer layer spore coat Nursing Path 6/23/2016 56www.drjayeshpatidar.blogspot.com
  57. 57. Conti…… Spores of some spesis have additional apparently rather loose outer covering called exosporium Spore cortex contains unusual type of peptidoglycan sensetive to lysosomes the spore cot is made up of keratin which is impervious to antibacterial chemical agent Exosporium is a lipoprotein membrane with some carbohydrate residue. Young spores remain attached to parent cell Nursing Path 6/23/2016 57www.drjayeshpatidar.blogspot.com
  58. 58. Shape and position  The young spore remain attached to the parent cell. The precise position and shape and relative size of spores remain constant within a particular bacteria.  Spore may be central , sub terminal or terminal in position.  Its diameter may be same or less than the width of the bacteria.  It may be oval or spherical. Nursing Path 6/23/2016 58www.drjayeshpatidar.blogspot.com
  59. 59. Resistance  Bacterial spores are resistant to ordinary boiling, heating, and disinfectant. They can withstand boiling up to 3 hr, dry heat at 150˚c for 1 hr however they are destroyed by autoclaving at 121˚c for 15-20 min.  The highly impervious spore coat, low water content, low metabolic activity and high concentration of calcium dipicolinate of spore make resistant to drying and heating. Nursing Path 6/23/2016 59www.drjayeshpatidar.blogspot.com
  60. 60. Germination  Definition: “ The process of conversation of spore in to vegetative cell under suitable environment is known as germination” There are three stages of germination……  A. activation  B. initiation  C. outgrowth Nursing Path 6/23/2016 60www.drjayeshpatidar.blogspot.com
  61. 61. 1. Activation  The germination of bacterial spore do not occur even when placed in environment that favours process.  Unless first activated by one or another agent damage the coat of spore such as heat, abrasion and compound containing free sulphydryl groups. Nursing Path 6/23/2016 61www.drjayeshpatidar.blogspot.com
  62. 62. 2.Initiation  The process of initiation is not clear, however the spore will initiate germination in favorable condition.  Different species of bacteria recognizes different effectors as signalling a rich medium such as L-alanine for one species & adenosine for another species. Nursing Path 6/23/2016 62www.drjayeshpatidar.blogspot.com
  63. 63. 3. Out growth  With the swelling of spore wall and disintegration of cortex a single germ cell emerge after breaking open the spore coat.  The new vegetative cell consist of spore protoplast with its surrounding wall.  This is followed by a period of active biosynthesis producing an outgrowth.  Outgrowth is denoted as the stage from germination up to the formation of vegetative cell & prior to first cell division. Nursing Path 6/23/2016 63www.drjayeshpatidar.blogspot.com
  64. 64. Demonstration  By ordinary stain and modified Z-N stain.  Laboratory use: for making sterilization 1. B. stearothermophilus-destroyed at a tem. of 121˚c in 10-20 min. 2. B. subtilis-destroyed at 105˚c in 5 min. Nursing Path 6/23/2016 64www.drjayeshpatidar.blogspot.com
  65. 65. Nursing Path 6/23/2016 65www.drjayeshpatidar.blogspot.com