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    1. 1. 1 Chapter 2 :Techniques in Histology
    3. 3. CHAPTER 2 TECHNIQUES IN HISTOLOGY Chapter 2 :Techniques in Histology 3
    4. 4. HISTOLOGICAL TECHNIQUE A. Histology involves the preparation of tissues for examination with a microscope. 1. Basic methods of histological preparation of tissues. a. Fix tissue (e.g. 4 % paraformaldehyde + buffer) b. Dehydrate tissue (alcohol series followed by toluene) c. Embed tissue in “hard” medium (e.g. wax) d. Section embedded tissue on a microtome e. Mount sections on a supportive structure (e.g. slide) that can be placed on a microscope stage f. Usually remove the embedding medium g. Stain tissue (e.g. hematoxylin-eosin) h. Examine tissue with microscope. Chapter 2 :Techniques in Histology 4
    5. 5. Artifacts 1. Preparative techniques are often “harsh” and can traumatize and change the natural structure of a tissue. 2. As a result, what you see is not always real ! 3. Tissue/cellular structures that are “created” by preparative techniques for histological specimens are called “artifacts.” Chapter 2 :Techniques in Histology 5
    6. 6. Fixation   Need to prevent autolysis of tissue samples by enzymes/bacteria present Fixation may be chemical or less frequently, physical methods   chemical fixatives stabilize or cross-linking agents  need small to get complete penetration of tissue – intravascular perfusion  routine fixative is 4% formalin, gluteraldehyde – interact with amine groups of proteins  EM requires additional fixation – gluteraldehyde and OsO4 (stains lipids and proteins) physical fixation – freezing sample Chapter 2 :Techniques in Histology 6
    7. 7. TISSUE FIXATION     Fixation is a complex series of chemical events that differ for the different groups of substance found in tissues. Main purpose: to preserve material in a life-like manner. The aim of fixation: 1- To prevent autolysis and bacterial attack. 2- To fix the tissues so they will not change their volume and shape during processing. 3- To prepare tissue and leave it in a condition which allow clear staining of sections. 4- To leave tissue as close as their living state as possible, and no small molecules should be lost. Fixation is coming by reaction between the fixative and protein which form a gel, so keeping every thing as their in vivo relation to each other. Chapter 2 :Techniques in Histology 7
    8. 8. The fixation solutions used for microscopy intended to:     Penetrate rapidly to prevent post mortem changes in the cells Coagulate the cell contents into insoluble substances Protect tissues against shrinkage and distortion during subsequent processing Allow cell parts to become selectively and clearly visible when stained. Chapter 2 :Techniques in Histology 8
    9. 9. Factors affect fixation: - PH. - Temperature. - Penetration of fixative. - Volume of tissue. According to previous factors we can determine the concentration of fixative and fixation time. Types of fixative: Acetic acid, Formaldehyde, Ethanol, Glutaraldehyde, Methanol and Picric acid. Chapter 2 :Techniques in Histology 9
    10. 10. Why fix tissue? Preserve structure. Essentially to make the structural components of the tissue more durable so that the tissue can be manipulated in various ways. • Fixed material is dead. You want to preserve the structure (chemical and morphological) of the living material so that it appears the same as it was in life. • It will never be exactly the same. Important to choose fixative that does the best job. Fixative used will depend on type of tissue to be fixed. Why dehydrate the fixed tissue Most fixatives are water soluble, most embedding media are nonpolar and are not miscible with water. So, you have to move the tissue from a polar (water-based) medium to a non-polar medium (e.g. toluene) that is miscible with the embedding medium. Chapter 2 :Techniques in Histology 10
    11. 11. TISSUE PROCESSING The aim of tissue processing is to embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut , and yet soft enough not to damage the knife or tissue. Stages of processing: 1- Dehydration. 2- Clearing. 3- Embedding. Chapter 2 :Techniques in Histology 11
    12. 12. Dehydration To remove fixative and water from the tissue and replace them with dehydrating fluid. There are a variety of compounds many of which are alcohols. Several are hydrophilic so attract water from tissue.  To minimize tissue distortion from diffusion currents, delicate specimens are dehydrated in a graded ethanol series from water through 10%-20%-50%-95%-100% ethanol.  In the paraffin wax method, following any necessary post fixation treatment, dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol, progressing through 90%-95% ethanol, then two or three changes of absolute ethanol before proceeding to the clearing stage. Chapter 2 :Techniques in Histology 12
    13. 13. Types of dehydrating agents: Ethanol, Methanol, Acetone. • Duration of dehydration should be kept to the minimum consistent with the tissues being processed. Tissue blocks 1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick require up to 90 minutes or longer in each change. Tissues may be held and stored indefinitely in 70% ethanol without harm Chapter 2 :Techniques in Histology 13
    14. 14. Clearing Replacing the dehydrating fluid with a fluid that is totally miscible with both the dehydrating fluid and the embedding medium.  Choice of a clearing agent depends upon the following: - The type of tissues to be processed, and the type of processing to be undertaken. - The processor system to be used. - Intended processing conditions such as temperature, vacuum and pressure. - Safety factors. - Cost and convenience. - Speedy removal of dehydrating agent . - Ease of removal by molten paraffin wax . - Minimal tissue damage . Chapter 2 :Techniques in Histology 14
    15. 15. • Some clearing agents: - Xylene. - Toluene. - Chloroform. - Benzene. - Petrol. Chapter 2 :Techniques in Histology 15
    16. 16. Embedding  Is the process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified will provide sufficient external support during sectioning.  Paraffin wax   properties : Paraffin wax is a polycrystalline mixture of solid hydrocarbons produced during the refining of coal and mineral oils. It is about two thirds the density and slightly more elastic than dried protein. Paraffin wax is traditionally marketed by its melting points which range from 39°C to 68°C. The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax: - improve ribboning. - increase hardness. - decrease melting point - improve adhesion between specimen and wax Chapter 2 :Techniques in Histology 16
    17. 17. Precaution while embedding in wax • The wax is clear of clearing agent. • No dust particles must be present. • Immediately after tissue embedding, the wax must be rapidly cooled to reduce the wax crystal size. Chapter 2 :Techniques in Histology 17
    18. 18.  There are four main mould systems and associated embedding protocols presently in use : 1- Traditional methods using paper boats 2- Leuckart or Dimmock embedding irons or metal containers 3- the Peel-a-way system using disposable plastic moulds and 4- systems using embedding rings or cassette-bases which become an integral part of the block and serve as the block holder in the microtome. Chapter 2 :Techniques in Histology 18
    19. 19. Tissue processing Embedding moulds: (A) paper boat; (B) metal bot mould; (C) Dimmock embedding mould; (D) Peel-a-way disposable mould; (E) base mould used with embedding ring ( F) or cassette bases (G) Chapter 2 :Techniques in Histology 19
    20. 20.  General Embedding Procedure 1- Open the tissue cassette, check against worksheet entry to ensure the correct number of tissue pieces are present. 2- Select the mould, there should be sufficient room for the tissue with allowance for at least a 2 mm surrounding margin of wax. 3- Fill the mould with paraffin wax. 4 Using warm forceps select the tissue, taking care that it does not cool in the air; at the same time. 5- Chill the mould on the cold plate, orienting the tissue and firming it into the wax with warmed forceps. This ensures that the correct orientation is maintained and the tissue surface to be sectioned is kept flat. 6- Insert the identifying label or place the labeled embedding ring or cassette base onto the mould. 7- Cool the block on the cold plate, or carefully submerge it under water when a thin skin has formed over the wax surface. 8- Remove the block from the mould. 9- Cross check block, label and worksheet. Chapter 2 :Techniques in Histology 20
    21. 21. Chapter 2 :Techniques in Histology 21
    22. 22. Why embed? a. Tissue will be sectioned. Needs to be durable enough to withstand the sectioning process. Also, want components of tissue to remain in their natural positions. Don't want them to be moved to new positions. b. Embedding in wax or plastic immobilizes structural components of tissue. Holds them in place as sectioning is done. Chapter 2 :Techniques in Histology 22
    23. 23.   ORIENTATION OF TISSUE IN THE BLOCK Correct orientation of tissue in a mould is the most important step in embedding. Incorrect placement of tissues may result in diagnostically important tissue elements being missed or damaged during microtomy. elongate tissues are placed diagonally across the block  tubular and walled specimens such as vas deferens, cysts and gastrointestinal tissues are embedded so as to provide transverse sections showing all tissue layers  tissues with an epithelial surface such as skin, are embedded to provide sections in a plane at right angles to the surface (hairy or keratinised epithelia are oriented to face the knife diagonally)  multiple tissue pieces are aligned across the long axis of the mould, and not placed at random Chapter 2 :Techniques in Histology 23
    24. 24. Processing methods and routine schedules  Machine processing  manual processing Chapter 2 :Techniques in Histology 24
    25. 25. Sectioning    Use a microtome to section embedded samples 1-10 µm thick Float on surface of warm water to remove wrinkles and load on to slide Frozen samples are cut with a cryostat  use this method when wanting to study enzyme activity which can be damaged by fixation Chapter 2 :Techniques in Histology 25
    26. 26. CUTTING • using the microtome Chapter 2 :Techniques in Histology 26
    27. 27. • A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination. Most microtomes use a steel blade and are used to prepare sections of animal or plant tissues for histology. The most common applications of microtomes are : Chapter 2 :Techniques in Histology 27
    28. 28. 1- Traditional histological technique: Tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 25 micrometers thick. From there the tissue can be mounted on a microscope slide, stained and examined using a light microscope Chapter 2 :Techniques in Histology 28
    29. 29. 2- Cryosection: Water-rich tissues are hardened by freezing and cut frozen; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs. 16 hours) and are used in operations to achieve a quick diagnosis. Cryosections can also be used in immunohistochemistry as freezing tissue does not alter or mask its chemical composition as much as preserving it with a fixative. Chapter 2 :Techniques in Histology 29
    30. 30. 3- Electron microscopy: After embedding tissues in epoxy resin, a microtome equipped with a glass or diamond knife is used to cut very thin sections (typically 60 to 100 nanometers). Sections are stained and examined with a transmission electron microscope. This instrument is often called an . 4- Botanical microtomy: ultramicrotome   hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin a regular microtomy. Microtome blades are extremely sharp, and should be handled with great care. Safety precautions should be taken in order to avoid any contact with the cutting edge of the blade. Chapter 2 :Techniques in Histology 30
    31. 31. Microtome knives • STEEL KNIVES • NON-CORROSIVE KNIVES FOR CRYOSTATS • DISPOSABLE BLADES • GLASS KNIVES • DIAMOND KNIVES Chapter 2 :Techniques in Histology 31
    32. 32. Why section? a. Allows you to see internal structure of tissue. b. Allows stains, or specific markers such as antibodies to more easily infiltrate the tissues. c. Allows light to pass through tissue making structure visible. d. While sectioning is useful in many instances, in some cases tissues are stained and examined without sectioning. Chapter 2 :Techniques in Histology 32
    33. 33. STAINING Chapter 2 :Techniques in Histology 33
    34. 34. Purpose 1. Add contrast to the image – separation of bacteria in terms of morphological characteristics and cellular structures. 2. Identify chemical components of interest (visualize) 3. Locate particular tissues, cells or organelles (differentiation) Chapter 2 :Techniques in Histology 34
    35. 35. Staining   Need to stain samples to impart contrast to various structures, without hard to identify tissues Dyes will stain more or less selectively – usually acids or bases and form electrostatic linkages with tissue components   basic dyes – attach to acid components, basophilic  toluidine blue, methylene blue and hematoxylin acid dyes – attach to basic components, acidophilic  orange G, eosin, acid fuchsin Chapter 2 :Techniques in Histology 35
    36. 36. Stains  Hemotoxylin and Eosin – (H&E) most common stain for general morphology and structures    nucleus, RNA-rich areas in cytoplasm and matrix of hyaline cartilage is blue while cytoplasm and collagen are pink Trichromes – additional dyes that will differentiate other structures Counterstains – use to give general structure when doing immunohistochemistry – do not want to overpower the complex attached to what we are looking for Chapter 2 :Techniques in Histology 36
    37. 37. Hematoxylin and Eosin (H & E) H & E is a charge-based, general purpose stain. Hematoxylin stains acidic molecules shades of blue. Eosin stains basic materials shades of red, pink and orange. H & E stains are universally used for routine histological examination of tissue sections. Chapter 2 :Techniques in Histology 37
    38. 38. Staining machine Chapter 2 :Techniques in Histology 38
    39. 39. Why stain the tissue? a. Creates higher contrast that allows observation of structure that is not visible in unstained tissue. b. May reveal differences in chemical nature of regions of the tissue. Chapter 2 :Techniques in Histology 39
    40. 40. Staining Dyes create contrast by imparting a color to cells or cell parts     Basic dyes – cationic, positively charged chromophore Acidic dyes – anionic, negatively charged chromophore Positive staining – surfaces of microbes are negatively charged and attract basic dyes Negative staining – microbe repels dye, the dye stains the background Chapter 2 :Techniques in Histology 40 40
    41. 41. Staining reactions of dyes Chapter 2 :Techniques in Histology 41 41
    42. 42. Staining    Simple stains – one dye is used; reveals shape, size, and arrangement Differential stains – use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain) Structural stains – reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains Chapter 2 :Techniques in Histology 42 42
    43. 43. Microbiological stains Crystal Violet Methylene Blue Gram Stain Capsule Stain Acid Fast – Red Fast, blue non Flagella Stain Spore Stain green Chapter 2 :Techniques in Histology 43 43
    44. 44. Simple Stains 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. Simple stains use one dye that stains the cell wall. The cells are then visible against a light background. Steps: 1. Place the slide on the staining rack. 2. Flood the slide with a basic stain: either crystal violet (1 min.), Safranin (2 min.), or Methylene blue (2 min.). 3. Wash the stain off the slide with deionized water. 4. Blot the slide with bibulous paper. Chapter 2 :Techniques in Histology 44
    45. 45. Differential Staining  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).  The most common differential stain used in microbiology is the Gram Stain.  Basic stains, due to their positive (+) charge will bind electrostatically to negatively charged molecules such as many polysaccharides, proteins and nucleic acids.  Acid stains ( - ) bind to positively charged molecules which are much less common, meaning acidic stains are used only for special purposes.  Some commonly encountered basic stains are crystal violet, safranin (a red dye) and methylene blue.  Basic stains may be used alone (a simple stain) or in combination (differential stain) depending on the experiment involved. Chapter 2 :Techniques in Histology 45
    46. 46. Mounting sections • Wet mounts – to observe fresh specimens 1. Isolate the specimen 2. Place the specimen in the small droplet of the relevant fluid (fresh water, seawater) on a microscope slide. 3. Gently lower a coverslip onto the droplet, using forceps of two needles with absorbent paper. 4. Remove any excess water on or around the coverslip with absorbent paper. Chapter 2 :Techniques in Histology 46
    47. 47. Mounting sections • Temporary mounts -involve wet mounting in a mountant with a short useful life – for identification purposes. Chapter 2 :Techniques in Histology 47
    48. 48. Mounting sections • Permanent mounts – protect sections during examination and allow storage without deterioration. Procedure 1. Apply little mountant to a coverslip of appropriate size. 2. Turn the coverslip over and place on its edge to one side of the sections. 3. Lower the coverslip slowly down onto the sections so as to displace all the air and sandwich the sections between the slide and the coverslip. 4. Press firmly form the centre outwards to distribute the mounting medium evenly. 5. Allow the solvent to evaporate – best results come from slow drying when time allows, but many synthetic mountant will tolerate brief heating when speed is essential. Chapter 2 :Techniques in Histology 48
    49. 49. The Gram Stain   Developed by Christian Gram in 1884 A staining procedure that differentiates between bacteria based on the structure of their cell walls.   Thick Cell Wall  Penicillin and derivatives of penicillin are used on these bacteria because it attacks peptidoglycan (cell wall) synthesis. Thin Cell Wall with Outer Membrane  Penicillin is ineffective on these bacteria because they have a lipid membrane outside of their cell wall Chapter 2 :Techniques in Histology 49
    50. 50.      Gram Staining The Gram Stain is a differential stain. Four different reagents are used and the results are based on differences in the bacterial cell wall. Gram Positive bacteria have a relatively thick cell wall composed of a special carbohydrate called Peptidoglycan. Gram Negative bacteria have a much thinner cell wall composed of the same carbohydrate, Peptidoglycan, but with certain chemical differences, such as the presence of Lipopolysaccharides (LPS). Notice that both Gram Positive and Gram Negative bacteria have a cell wall composed primarily of Peptidoglycan. Gram Staining Steps 1. 2. 3. 4. 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. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall). 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.) Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization. Chapter 2 :Techniques in 50 Histology
    51. 51. Gram Staining Procedure Chapter 2 :Techniques in Histology 51
    52. 52.        Gram Staining Results When reporting the results of the Gram stain you indicate the type of stain used, the reaction, and the morphology of the cells observed. Round (spherical), purple (or dark blue) cells are reported as Gram positive cocci (GPC). Rod-shaped, purple (or dark blue) cells are reported as Gram positive bacilli (GPB). The standard abbreviations for the four types of Gram stain and morphology are: 1. 2. 3. 4. Gram Positive Cocci (GPC) Gram Positive Bacilli (GPB) Gram Negative Cocci (GNC) Gram Negative Bacilli (GNB) Notice that both Gram Positive and Gram Negative bacteria have a cell wall composed primarily of Peptidoglycan. Spiral-shaped bacterial cells do not Gram stain well and are usually observed using dark-field microscopy. There are no standard abbreviations for Gram stain reactions of the spirilla of medical importance. Certain other types of bacteria may not Gram stain well, such as, Acid-fast Mycobacteria (Mycobacterium tuberculosis). Chapter 2 :Techniques in 52 Histology
    53. 53. Bacterial Cell Shapes  Bacteria can have several different shapes, but the primary shapes we will be observing are: 1. 2. 3.  Spherical or round cells – cocci (plural) or coccus (singular) Rod shaped – bacilli (plural) or bacillus (singular) Spiral shaped – spirilla Some bacteria have characteristic clustering or arrangements, usually due to how the cells divide and whether they remain attached together when they divide.    Diplococci – divide in one plane and remain attached together after cell division. Streptococci – divide in one plane and form long chains of attached cells. Staphylococci – divide in many planes and remain attached together forming a “grape-like cluster” Chapter 2 :Techniques in Histology 53
    54. 54. The Microscope Key characteristics of a reliable microscope are:  Magnification – ability to enlarge objects   Anybody have cheap telescope or binoculars? Resolving power – ability to show detail Chapter 2 :Techniques in Histology 54 54
    55. 55. Magnification in most microscopes results from interaction between visible light waves and curvature of the lens    Angle of light passing through convex surface of glass changes – refraction Depending on the size and curvature of the lens, the image appears enlarged Extent of enlargement – magnification Chapter 2 :Techniques in Histology 55 55
    56. 56. Light Microscopy   Mechanical and optical components make up microscope 3 systems of lenses    condenser – collects and focuses light onto object objective – enlarge and project the illuminated image in direction of eyepiece eyepiece – further magnification and project to the retina or detector Chapter 2 :Techniques in Histology 56
    57. 57. Chapter 2 :Techniques in Histology 57 57
    58. 58. Principles of Light Microscopy  Magnification occurs in two phases –    The objective lens forms the magnified real image The real image is projected to the ocular where it is magnified again to form the virtual image Total magnification of the final image is a product of the separate magnifying powers of the two lenses power of objective x power of ocular = total magnification Chapter 2 :Techniques in Histology 58 58
    59. 59. Chapter 2 :Techniques in Histology 59 59
    60. 60. Resolution Resolution defines the capacity to distinguish or separate two adjacent objects – resolving power  Function of wavelength of light that forms the image along with characteristics of objectives  Visible light wavelength is 400 nm–750 nm  Numerical aperture of lens ranges from 0.1 to 1.25  Oil immersion lens requires the use of oil to prevent refractive loss of light  Shorter wavelength and larger numerical aperture will provide better resolution  Oil immersion objectives resolution is 0.2 μm  Magnification between 40X and 2000X Chapter 2 :Techniques in Histology 60 60
    61. 61. Variations on the Optical Microscope    Bright-field – most widely used; specimen is darker than surrounding field; live and preserved stained specimens Dark-field – brightly illuminated specimens surrounded by dark field; live and unstained specimens Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures Chapter 2 :Techniques in Histology 61 61
    62. 62. Three views of a basic cell Bright Field Dark Field Phase Contrast Chapter 2 :Techniques in Histology 62 62
    63. 63. Phase Contrast and DIC Microscope  The differential interference microscope is similar to the phase contrast but has more refinements Chapter 2 :Techniques in Histology 63 63
    64. 64. Phase-Contrast and Differential Interference Microscopy  Use phase-contrast for viewing images of transparent samples   light changes speed when passing thru cellular and extracellular structures with different refractive indices use to view living cells Chapter 2 :Techniques in Histology 64
    65. 65. 2 Types of Electron Microscopes   Transmission electron microscopes (TEM) – transmit electrons through the specimen. Darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts. Scanning electron microscopes (SEM) – provide detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it. Chapter 2 :Techniques in Histology 65 65
    66. 66. Transmission E.M. Electrons pass through the sample Chapter 2 :Techniques in Histology 66 66
    67. 67. Scanning EM Scanning looks at surface only Chapter 2 :Techniques in Histology 67 67
    68. 68. NEXT CLASS: Chapter 3 TECHNIQUES OF MICROBIOLOGY THANK YOU Chapter 2 :Techniques in Histology 68