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Bacteriology Basics Morphology, Classification, Staining Methods

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Bacteriology Basics Morphology, Classification, Staining Methods

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Bacteriology Basics Morphology, Classification, Staining Methods

  1. 1. Bacteriology Basics Morphology, Classification, Staining Methods Dr.T.V.Rao MD
  2. 2. Introduction: • Microorganisms – several classes of living beings • Based on the organization of their cellular structures, all living cells can be divided into two groups: eukaryotic and prokaryotic – Eukaryotic cell types - Animals, plants, fungi, protozoans, and algae – Prokaryotic cell types - bacteria & blue green algae Dr.T.V.Rao MD 2
  3. 3. Antioni van Leeuwenhoek • Leeuwenhoek is called "the inventor of the microscope" • Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 • Could reach magnifications of over 200x with simple ground lenses Dr.T.V.Rao MD 3
  4. 4. How a Microscope Works with.. Ocular Lens (Magnifies Image) •Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again). Dr.T.V.Rao MD Body Tube (Image Focuses) Objective Lens (Gathers Light, Magnifies And Focuses Image Inside Body Tube) 4
  5. 5. The Light Microscope • many types Dr.T.V.Rao MD –bright-field microscope –dark-field microscope –phase-contrast microscope –fluorescence microscopes • compound microscopes –image formed by action of 2 lenses 5
  6. 6. The Compound Microscope • The Optical System – Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece. Dr.T.V.Rao MD • Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus. – Eyepiece or Ocular Lens: the lens closest to the eye. • Monocular: a microscope having only one eyepiece 6 • Binocular: a microscope having two eyepieces.
  7. 7. Phase Contrast Microscopy • light rays through objects of different change in phase, not intensity • special ring-shaped condenser diaphragm • special glass disc in objective – change phase differences to intensity differences – can view transparent objects as dark on light background (without staining) • Right; human brain glial cells Dr.T.V.Rao MD 7
  8. 8. The Bright-Field Microscope • Produces a dark image against a brighter background • Has several objective lenses Dr.T.V.Rao MD –par focal microscopes remain in focus when objectives are changed • total magnification – product of the magnifications of the ocular lens and the objective lens 8
  9. 9. Fluorescence Microscopy • Illuminate specimen with UV visible fluorescence (filter removes harmful UV) • View auto-fluorescent objects (e.g., chloroplasts) • Stain with specific fluorescent dyes, which absorb in region 230-350 nm & emit orange, yellow or greenish light • Images appear coloured against a dark background Dr.T.V.Rao MD 9
  10. 10. Schematic of typical animal (eukaryotic) cell, showing subcellular components. Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles Dr.T.V.Rao MD 10
  11. 11. Background Information Prokaryotes • Prokaryotes represent two domains, bacteria and archaea. • Archaea live in Earth’s extreme environments. • Bacteria are the most abundant and diversified organisms on Earth. Dr.T.V.Rao MD 11
  12. 12. Eukaryotes have organelles • Much larger; more complex than prokaryotes • Processes compartmentalized into organelles – – – – – – Nucleus Protein synthesis (ribosomes, RER, Golgi) Mitochondria; chloroplasts Lysosomes Plasma membranes have different modifications Cytoskeleton Dr.T.V.Rao MD 12
  13. 13. Differences between prokaryotic & eukaryotic cells Character Eukaryotes Nuclear membrane Absent Present Nucleolus Nucleus Prokaryotes Absent Present Chromosome One circular Cell division Binary fission Cytoplasmic Structure and fluid phospholipid membrane Composition bilayer, lacks sterols Function Incapable of endocytosis (phagocytosis and pinocytosis) and exocytosis Dr.T.V.Rao MD One or more paired and linear Mitosis fluid phospholipid bilayer containing sterols Capable of endocytosis and exocytosis 13
  14. 14. Differences between prokaryotic & eukaryotic cells Character Cytoplasm Prokaryotes Eukaryotes Mitochondria Absent Present Lysosomes Absent Present Golgi apparatus Absent Present Endoplasmic reticulum Absent Present Vacuoles Absent Present 70 S 80 S Ribosomes Dr.T.V.Rao MD 14
  15. 15. Differences between prokaryotic & eukaryotic cells Character Prokaryotes Eukaryotes Cell Wall Present Animals & Protozoans – Absent Plants, Fungi & Algae Present Peptidoglycan – complex carbohydrate Cellulose or chitin Flagella Flagella/ Cilia Composition Locomotor organelles Dr.T.V.Rao MD 15
  16. 16. Prokaryotic Cells • Much smaller (microns) and more simple than eukaryotes • Prokaryotes are molecules surrounded by a membrane and cell wall. • They lack a true nucleus and don’t have membrane bound organelles like mitochondria, etc. • Large surface-to-volume ratio : nutrients can easily and rapidly reach any part of the cells interior Dr.T.V.Rao MD 16
  17. 17. Size of Bacteria • Unit of measurement in bacteriology is the micron (micrometre, µm) • Bacteria of medical importance –0.2 – 1.5 µm in diameter –3 – 5 µm in length Dr.T.V.Rao MD 17
  18. 18. Eukaryotic cell Prokaryotic cell Gram + (e.g. animal) Rough endoplasmic reticulum Nucleus Cell membrane Flagellum Nucleoid Cell wall Gram Pili Granule Cytoplasm Mitochondria Capsule Cell (inner) membrane Outer membrane Dr.T.V.Rao MD Ribosomes 18 Cell wall
  19. 19. Shapes of Bacteria • • • • • • Cocci – spherical/ oval shaped major groups Bacilli – rod shaped Vibrios – comma shaped Spirilla – rigid spiral forms Spirochetes – flexible spiral forms Actinomycetes – branching filamentous bacteria • Mycoplasmas – lack cell wall Dr.T.V.Rao MD 19
  20. 20. Bacteria Have One of Three Cellular Shapes • Rods (bacilli) • Coccoid-Shaped • Spirilla Dr.T.V.Rao MD 20
  21. 21. Arrangement of bacteria: Cocci Coccus Cocci in pair – Diplococcus Tetrad – groups of four Cocci in chain - Streptococci Cocci in cluster - Staphylococci Dr.T.V.Rao MD Sarcina – groups of eight 21
  22. 22. Reproduction • Prokaryotic cell division is binary fission. – Single DNA molecule that first replicates. – Attaches each copy to a different part of the cell membrane. – Cell begins to pull apart. – Following cytokinesis, there are then two cells of identical genetic composition. Dr.T.V.Rao MD 22
  23. 23. Arrangement of bacteria: Bacilli Dr.T.V.Rao MD 23
  24. 24. Other shapes of bacteria Comma shaped Spirilla Spirochetes Dr.T.V.Rao MD 24
  25. 25. Anatomy of a Bacterial Cell Dr.T.V.Rao MD 25
  26. 26. Anatomy of A Bacterial Cell • Outer layer – two components: 1. 2. Rigid cell wall Cytoplasmic (Cell/ Plasma) membrane – present beneath cell wall • Cytoplasm – cytoplasmic inclusions, ribosomes, mesosomes and nucleus • Additional structures – plasmid, slime layer, capsule, flagella, fimbriae (pili), spores Dr.T.V.Rao MD 26
  27. 27. Structure of Bacteria • All cells have 3 main components: – DNA (‘nucleoid”) • genetic instructions – surrounding membrane (“cytoplasmic membrane”) • limits access to the cell’s interior – cytoplasm, between the DNA and the membrane • where all metabolic reactions occur • especially protein synthesis, which occurs on the ribosomes • Bacteria also often have these features: – cell wall • resists osmotic pressure – flagella • movement – pili • attachment – capsule • protection and biofilms Dr.T.V.Rao MD 27
  28. 28. Typical shapes of bacteria Most bacteria retain a particular shape; a few are pleiomorphic Dr.T.V.Rao MD 28
  29. 29. Typical prokaryotic structures Working from the outside in… Dr.T.V.Rao MD 29
  30. 30. Characteristic grouping (or not grouping) Dr.T.V.Rao MD 30
  31. 31. The Cell Envelope Gram Positive Dr.T.V.Rao MD Gram Negative 31
  32. 32. GRAM POSITIVE Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane Cytoplasm GRAM NEGATIVE Porin Outer Membrane Lipopolysaccharide Braun lipoprotein Inner (cytoplasmic) membrane Cytoplasm Dr.T.V.Rao MD 32
  33. 33. Gram-positive and gram-negative bacteria
  34. 34. Gram-Negative Bacteria Difference Between Gram-Negative and Gram-Positive Bacteria Gram-Positive Bacteria More complex cell wall. Simple cell wall. Thin peptidoglycan celll wall layer. Thick peptidoglycan celll wall layer. Outer lipopolysaccharide wall layer. No outer lipopolysaccharide wall layer. Retain safranin. Retain crystal violet/iodine. Appear pink/red. Appear blue/purple. Dr.T.V.Rao MD 34
  35. 35. Cell Envelope • The cell envelope is all the layers from the cell membrane outward, including the cell wall, the periplasmic space, the outer membrane, and the capsule. – All free-living bacteria have a cell wall – periplasmic space and outer membrane are found in Gram-negatives – the capsule is only found in some strains Dr.T.V.Rao MD 35
  36. 36. Structure & Function of Cell Components
  37. 37. CELL WALL • Outermost layer, encloses cytoplasm 1. Confers shape and rigidity 2. 10 - 25 nm thick 1. Composed of complex polysaccharides (peptidoglycan/ mucopeptide) - formed by N acetyl glucosamine (NAG) & N acetyl muramic acid (NAM) alternating in chains, held by peptide chains. Dr.T.V.Rao MD 37
  38. 38. • Cell Wall Cell wall – 4. Carries bacterial antigens – important in virulence & immunity 5. Chemical nature of the cell wall helps to divide bacteria into two broad groups – Gram positive & Gram negative 6. Gram +ve bacteria have simpler chemical nature than Gram –ve bacteria. 7. Several antibiotics may interfere with cell wall synthesis e.g. Penicillin, Cephalosporins Dr.T.V.Rao MD 38
  39. 39. Transport Across the Cell Membrane • Basic rule: things spontaneously move from high concentration to low concentration (downhill). This process is called diffusion. – Getting many molecules into the cell is simply a matter of opening up a protein channel of the proper size and shape. The molecules then move into the cell by diffusing down the concentration gradient. Passive transport, or facilitated diffusion. • To get things to move from low to high (uphill), you need to add energy: the molecules must be pumped into the cell. Pumps are driven by ATP energy. Dr.T.V.Rao MD Active transport. 39
  40. 40. Outer Membrane Gram negative bacteria • major permeability barrier • space between inner and outer membrane –Periplasmic space store degradative enzymes • Gram positive bacteria • no Periplasmic space Dr.T.V.Rao MD 40
  41. 41. Gram positive cell wall The Gram-positive cell wall is composed of a thick, multilayered peptidoglycan sheath outside of the cytoplasmic membrane. Teichoic acids are linked to and embedded in the peptidoglycan, and lipoteichoic acids extend into the cytoplasmic membrane Dr.T.V.Rao MD 41
  42. 42. Gram negative cell wall The Gram-negative cell wall is composed of an outer membrane linked to thin, mainly single-layered peptidoglycan by lipoproteins. The peptidoglycan is located within the periplasmic space that is created between the outer and inner membranes. The outer membrane includes porins, which allow the passage of small hydrophilic molecules across the membrane, and lipopolysaccharide molecules that extend into extracellular space. Dr.T.V.Rao MD 42
  43. 43. Cell Wall Dr.T.V.Rao MD 43
  44. 44. Summary of the differences between Gram positive & Gram negative bacteria Property of bacteria Gram Positive Gram Negative Thickness of wall 20-80 nm 10 nm Number of layers in wall 1 2 Peptidoglycan content >50% 10-20% Teichoic acid in wall + - Lipid & lipoprotein content 0-3% 58% Protein content 0% 9% Lipopolysaccharide 0 13% Sensitive to penicillin Yes Less sensitive Digested by lysozyme Yes Weakly Dr.T.V.Rao MD 44
  45. 45. Cytoplasmic (Plasma) membrane • Thin layer 5-10 nm, separates cell wall from cytoplasm • Acts as a semipermeable membrane: controls the inflow and outflow of metabolites • Composed of lipoproteins with small amounts of carbohydrates Dr.T.V.Rao MD 45
  46. 46. Other Cytoplasmic Components • • Ribosomes – protein synthesis Mesosomes – 1. Multilaminated structures formed as invaginations of plasma membrane 2. Principal sites of respiratory enzymes 3. Coordinate nuclear & cytoplasmic division during binary fission 4. More prominent in Gram +ve bacteria • Intracytoplasmic inclusions – reserve of energy & phosphate for cell metabolism e.g. Metachromatic granules in diphtheria bacilli Dr.T.V.Rao MD 46
  47. 47. Nucleus • No nucleolus • No nuclear membrane • Genome – –single, circular double stranded DNA. –Haploid –Divides by binary fission Dr.T.V.Rao MD 47
  48. 48. Additional Organelles 1. Plasmid – – – – – – Extra nuclear genetic elements consisting of DNA Transmitted to daughter cells during binary fission May be transferred from one bacterium to another Not essential for life of the cell Confer certain properties e.g. drug resistance, toxicity Dr.T.V.Rao MD 48
  49. 49. Additional Organelles 2. Capsule & Slime layer – – – Viscous layer secreted around the cell wall. Polysaccharide / polypeptide in nature a) Capsule – sharply defined structure, antigenic in nature • Protects bacteria from lytic enzymes • Inhibits phagocytosis • Stained by negative staining using India Ink • Can be demonstrated by Quellung reaction (capsule Dr.T.V.Rao MD swelling reaction) 49
  50. 50. Additional Organelles 3. Flagella – – Long (3 to 12 µm), filamentous surface appendages – Organs of locomotion – Chemically, composed of proteins called flagellins – The number and distribution of flagella on the bacterial surface are characteristic for a given species - hence are useful in identifying and classifying bacteria – Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) – Presence shown by motility e.g. hanging drop preparation Dr.T.V.Rao MD 50
  51. 51. Types of flagellar arrangement Polar/ Monotrichous – single flagellum at one pole Lophotrichous – tuft of flagella at one pole Amphitrichous – flagella at both poles Peritrichous – flagella all over Amphilophotrichous – tuft of flagella at both ends Dr.T.V.Rao MD 51
  52. 52. Cocci do not have flagella Peritrichous monotrichous (or amphi, or lophotrichous Dr.T.V.Rao MD 52
  53. 53. FLAGELLA • • • • Some bacteria are motile Locomotory organelles- flagella Taste environment Respond to food/poison – chemo taxis Dr.T.V.Rao MD 53
  54. 54. • Flagella – embedded in cell membrane – project as strand – Flagellin (protein) subunits – move cell by propeller like action Dr.T.V.Rao MD 54
  55. 55. Additional Organelles 4. Fimbriae/ Pili – – Thin, hairlike appendages on the surface of many Gramnegative bacteria – 10-20µ long, acts as organs of adhesion (attachment) - allowing bacteria to colonize environmental surfaces or cells and resist flushing – Made up of proteins called pilins. – Pili can be of two types –   Common pili – short & abundant Sex pili - small number (one to six), very long pili, helps in conjugation (process of transfer of DNA) http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/yespili.html http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/nopili.html Dr.T.V.Rao MD 55
  56. 56. Additional Organelles 5. Spores – – Highly resistant resting stages formed during adverse environment (depletion of nutrients) – Formed inside the parent cell, hence called Endospores – Very resistant to heat, radiation and drying and can remain dormant for hundreds of years. – Formed by bacteria like Clostridia, bacillus Dr.T.V.Rao MD 56
  57. 57. The cycle of spore formation and germination At the beginning of spore formation, a septum forms, separating the nascent spore from the rest of the cell and all of the genetic material of the cell is copied into the newly-forming cell. The spore contents are dehydrated and the protective outer coatings are laid down. Once the spore is matured it is l released from the cell. On germination, the spore contents rehydrate and a new bacterium emerges and multiplies. Dr.T.V.Rao MD 57
  58. 58. Shape & position of bacterial spore Oval central Spherical central Non bulging Oval sub terminal Oval sub terminal Oval terminal Bulging Spherical terminal Free spore Dr.T.V.Rao MD 58
  59. 59. Spores • Some bacteria can form very tough spores, which are metabolically inactive and can survive a long time under very harsh conditions. –Allegedly, some bacterial spores that were embedded in amber or salt deposits for 25 million years have been revived. These experiments are viewed skeptically by many scientists. Dr.T.V.Rao MD 59
  60. 60. Spores • Spores can also survive very high or low temperatures and high UV radiation for extended periods. This makes them difficult to kill during sterilization. • Anthrax • Spores are produced only by a few genera in the Firmicutes: • Bacillus species including anthracis (anthrax) and cereus (endotoxin causes ~5% of food poisoning) • Clostridium species including tetani (tetanus), perfringens (gangrene), and botulinum (botulism: food poisoning from improperly canned food) Dr.T.V.Rao MD 60
  61. 61. Pleomorphic & Involution forms • Pleomorphism – great variation in shape & size of individual cells e.g. Proteus species • Involution forms – swollen & aberrant forms in ageing cultures, especially in the presence of high salt concentration e.g. plague bacillus • Cause – defective cell wall synthesis Dr.T.V.Rao MD 61
  62. 62. Bacterial Taxonomy • Includes three components: 1. Classification : orderly arrangement 2. Identification of an unknown unit 3. Nomenclature : naming the units Dr.T.V.Rao MD 62
  63. 63. Bacterial Taxonomy: Classification • Orderly arrangement : Kingdom – Division – Class – Order – Family – Tribe – Genus – Species  Phylogenetic classification – represents a branching tree like arrangement. One characteristic being used for division at each branch or level  Molecular or Genetic classification – based on the degree of genetic relatedness of different organisms  Intraspecies classification – based on biochemical properties (biotypes), antigenic features (serotypes), bacteriophage susceptibility (phage types) Dr.T.V.Rao MD 63
  64. 64. Bacterial Taxonomy: Nomenclature • Two kinds of name are given to bacteria –Casual / common name – for local use, varies from country to country e.g. “typhoid bacillus” –Scientific / International Name – same all over world, consists of two words (in Italics) e.g. Salmonella typhi, Staphylococcus Dr.T.V.Rao MD 64
  65. 65. Microscopy helps to Measure and Observe the Bacteria • Measurement – Microorganisms are very small – Use metric system – Metre (m) : standard unit – Micrometre ( m) = 1 x10-6 m – Nanometre (nm) = 1 x10-9 m – Angstrom (Å) = 1 x10-10 m Dr.T.V.Rao MD 65
  66. 66. Why we should be Stain Bacteria Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. Dr.T.V.Rao MD 66
  67. 67. What is a Stain • A stain is a substance that adheres to a cell, giving the cell color. • The presence of color gives the cells significant contrast so are much more visible. • Different stains have different affinities for different organisms, or different parts of organisms • They are used to differentiate different types of organisms or to view specific parts of organisms Dr.T.V.Rao MD 67
  68. 68. Simple staining • Methylene blue, Basic fuchsin • Provide the color contrast but impart the same color to all the organisms in a smear • Loffler's ethylene blue: Sat. solution of M. blue in alcohol - 30mlKoH, 0.01% in water 100mlDissolve the dye in water, filter. For smear: stain for 3’. For section: stain Dr.T.V.Rao MD 68
  69. 69. Simple Staining Easier to Perform But has Limitations • Simple easy to use; single staining agent used; using basic and acid dyes. • Features of dyes: give coloring of microorganisms; bind specifically to various cell structures Dr.T.V.Rao MD 69
  70. 70. Differential Stains Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Both techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness). Dr.T.V.Rao MD 70
  71. 71. Gram staining • Named after Hans Christian Gram, differentiates between Grampositive purple and Gram-negative pink stains and is used to identify certain pathogens. Dr.T.V.Rao MD 71
  72. 72. Gram staining - Principles • Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics. • Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria Dr.T.V.Rao MD 72
  73. 73. Gram Staining Steps 1. Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria. 2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall). 3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.) 4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization Dr.T.V.Rao MD 73
  74. 74. Structure and Reactivity to Gram Staining. Dr.T.V.Rao MD 74
  75. 75. Dr.T.V.Rao MD 75
  76. 76. Gm+ve cocci & Gm-ve bacilli Dr.T.V.Rao MD 76
  77. 77. GRAM-POSITIVE BACTERIA • GRAM-POSITIVE BACTERIA are characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids. The peptidoglycans which are sometimes also called murein are heteropolymers of glycan strands, which are cross-linked through short peptides. Dr.T.V.Rao MD 77
  78. 78. What are Gram Negative Bacteria • Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counter stain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls. On the other hand, Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution. Dr.T.V.Rao MD 78
  79. 79. Gram negative bacteria • On most Gram-stained preparations, Gramnegative organisms will appear red or pink because they are counterstained. Due to presence of higher lipid content, after alcoholtreatment, the porosity of the cell wall increases, hence the CVI complex (Crystal violet -Iodine) can pass through. Thus, the primary stain is not Dr.T.V.Rao MD retained. 79
  80. 80. Gram Negative Bacteria • Also, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide Dr.T.V.Rao MD 80
  81. 81. ACID FAST STAINING Dr.T.V.Rao MD 81
  82. 82. Acid-Fast Stain • Acid-fast cells contain a large amount of lipids and waxes in their cell walls – primarily mycolic acid • Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia – Therefore, this stain is important to identify Mycobacterium or Nocardia Dr.T.V.Rao MD 82
  83. 83. Ziehl-Neelsen stain • Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures like Gram staining. • The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green. Dr.T.V.Rao MD 83
  84. 84. Acid-Fast Organisms • Primary stain binds cell wall mycolic acids • Intense decolorization does not release primary stain from the cell wall of AFB • Color of AFB-based on primary stain • Counterstain provides contrasting background Dr.T.V.Rao MD 84
  85. 85. AFB Staining Methods • Zeihl Neelsen’s-hot stain • Kinyoun’s-cold stain • Modifications Dr.T.V.Rao MD 85
  86. 86. ALBERT’S STAINING FOR C.diptheria Dr.T.V.Rao MD 86
  87. 87. Diphtheria is Serious Disease When you suspect Diphtheria • In all cases of suspected cases of Diphtheria, stain one of the smears with Gram stain • If Gram stained smear shows morphology suggestive of C.diptheria, proceed to do Albert staining which demonstrates the presence or absence of metachromatic granules. Dr.T.V.Rao MD 87
  88. 88. Appearance of C.diptheria • C.diptheria are thin Gram positive bacilli, straight or slightly curved and often enlarged (clubbing) at one or both ends and are arranged at acute angles giving shapes of Chinese letters or V shape which is characteristic of these organisms (Fig 1). Present in the body of the bacillus are numerous metachromatic granules which give the bacillus beaded or barred appearance. These granules are best demonstrated by Albert’s stain. Dr.T.V.Rao MD 88
  89. 89. Albert staining • Albert stain I • Toluidine blue 0.15 gm Malachite green 0.20 gm Glacial acetic acid 1.0 ml Alcohol(95%) 2.0 ml Distilled water 100 ml • Albert stain II • Iodine 2.0 gm Potassium iodide 3.0 gm Distilled water 300 ml Dr.T.V.Rao MD 89
  90. 90. Albert staining Procedure • Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes. • Wash with water. • Cover the smear with Albert stain II. Let it stand for two minutes. • Wash with water, blot dry and examine. • Dr.T.V.Rao MD 90
  91. 91. How the C.diptheria appear • To demonstrate metachromatic granules in C.diptheria. These granules appear bluish black whereas the body of bacilli appear green or bluish green. • Dr.T.V.Rao MD 91
  92. 92. • Programme created by Dr.T.V.Rao MD from several resources in world wide web, and Thankful for Dr. Ekta www.medmicrobes from basic programme on Bacterial cell • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 92

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