SIP

• Microbiology


  The study of microorganisms, including bacteria, viruses, fungi, and protozoa
• Importance of Microbiology


  • Helps identify and diagnose infectious diseases
  • Assists in the development of new treatments and vaccines
  • Plays a crucial role in infection prevention and control
• Preparation of Smears and Staining Methods

  1. Use glass slides

  2. Prepare smears

  3. Apply stains (crystal violet, iodine, alcohol, safranin)

  
• Gram Stain


  Primary test to differentiate between gram-positive and gram-negative bacteria

  
• Gram-positive bacteria


  • Retain the crystal violet-iodine complex due to their thick peptidoglycan layer
  • Appear purple or blue
• Gram-negative bacteria


  • Lose the crystal violet-iodine complex due to their thin peptidoglycan layer and outer membrane
  • Appear pink from the safranin stain
• General rules to determine gram-positive or gram-negative

  • All cocci are gram-positive except Neisseria, Veillonella, Branhamella and Morococcus
  • All bacilli are gram-negative except Mycobacterium, Clostridium, Corynebacterium, Bacillus, Listeria monocytogenes, Erysepelothrix insidiosa
  • Spiral forms are hard to stain but those that are stainable are gram-negative
• Gram Staining Procedure


  1. Crystal violet (primary stain)

  2. Gram's iodine (mordant)

  3. Alcohol (decolorizer)

  4. Safranin (secondary stain)
• Culture media for ocular microbiology

  • Blood agar
  • Chocolate agar
  • Thayer Martin agar
  • Gram-negative media (EMB, MacConkey)
  • Sabouraud dextrose agar
  • Anaerobic media (cooked meat, thioglychollate)
• Anaerobic media


  • Solid agar media is better for primary isolation than thioglychollate broth
  • Ideal anaerobic media is PRAS (pre-reduced anaerobically sterilized)
  • Conventional blood agar enriched with vitamin K and hemin can also be used
• Methods for producing anaerobic environment

  • Anaerobic globe
  • Roll tube culture
  • Anaerobic gas jar
  • Disposable anaerobic system (plastic bag, anaerobic generator, catalyst, indicator)
• Kimura spatula


  Used to isolate fungi from the cornea, better than cotton swabs
• Ocular microorganisms


  • Cultural and microscopic characteristics
  • Biochemical tests for identification

  
• Fungi


  Yeasts (single-celled, reproduce by budding)<|>Molds (consist of long, branching filaments called hyphae, asexual or spore reproduction, opportunistic infections)
• Antimicrobial susceptibility


  Determines which medication will help the patient the most to fight the infection
• Laboratories assist in the identification of outbreaks by confirming the organism and detecting unusual organisms and antimicrobial susceptibility patterns
• Clinical microbiology plays an important role in the practice of infection prevention
• Histopathologic technique


  Deals with the preparation of solid tissue coming from different specimen from a living body for microscopic examination

  
• Pathology


  The science of the causes and effects of diseases, especially the branch of medicine that deals with the laboratory examination of samples of body tissue for diagnostic or forensic purposes
• Sources of specimen


  • Surgical source - specimen obtained after biopsy, operation
  • Autopsy specimen - from dead bodies, after doing post-mortem examination
• Fixation


  1. Preservation of tissue to prevent destruction and post-mortem changes

  2. Hardening of the tissue

  3. Killing of tissue properly

  4. Prevent the growth of microorganism

  5. Act as mordant (preparatory for next process)
• Proper fixation requirements


  • Tissue must be cut into small thin pieces at least 0.5 to 1 cm in size
  • Obtain a representative tissue section
  • Tissue must be placed in a wide mouth container for proper fixation
  • Volume requirement: 10-20 as much as the tissue
  • Duration: 1-3 days (24-72 hours)

  
• Formalin


  Routine histopathologic fixative, 10% buffered neutral formalin, fixative for special stain, for frozen section tissue (use also for lipids)
• Decalcification


  Removal of calcium salt or lime salt from hard tissue, by chemical substitution, acids replace the calcium, duration: at least 3-7 days
• Embedding


  1. Dehydration

  2. Clearing

  3. Infiltration

  4. Molding the shape

  5. Blocking (solidification)
• Dehydration


  Removal of water from the tissue using different concentration of ethyl alcohol in ascending concentration
• Clearing


  Dealcoholization, removal of alcohol from the tissue after dehydration to make the tissue transparent for better microscopic study
• Clearing agents


  Xylene, Chloroform, Benzene, Toluene, Dioxane, THF (tetrahydrofuran), Oils - cedarwood oil, oil of wintergreen, methyl salicylate, oil of Bergamot
• Infiltration


  The actual embedding procedure, impregnation, penetration of the tissue with a liquid medium in order to solidify and fill up spaces between the tissues for better cutting and for better preparation
• Infiltration materials


  Waxes - paraffin, Gelatin, Plastic - has poor penetrating action, Vacuum - special material for embedding
• Paraffin infiltration requirements


  Paraffin must be in pure and crystallized paraffin

  Know the melting point (56-58 C)

  Know the freezing point (-5 C)
• Molding the shape


  Making of paper boat
• Sectioning (accurate cutting)


  Cutting of tissue in thin slices measured in microns or millimicron by the use of apparatus known as microtome, 4-6 microns
• Major parts of microtome

  1. Knife - for cutting of tissue block
  2. Tissue holder - holds the tissue block to the microtome
  3. Micrometer - adjust the thinness and thickness of the section 1-15 u
  4. Rotating wheel - moves mechanically to do the cutting procedure
• Rotary microtome


  Routine microtome used, rotation of the wheel will move the tissue holder up and down and the knife is steady
• Sliding microtome


  Rotation of the wheel will move the knife to cut the tissue to and from
• Fixing the ribbon in the slide

  1. Use of floater or floating out bath

  2. Use of adhesive (Mayer's) egg + albumin

  3. Fishing out

  4. Orientation

  5. Deparaffinization
• Floater


  Shallow container with water (temperature is slightly increased) 40-50 C (46-48 C), purpose: ribbon must fall after sectioning, check the accuracy of the ribbon, ribbon will be flattened
• Adhesive


  Mixture of solution or reagent in order that the ribbon will attach to the slide
• Fishing out


  Transferring the ribbon from the floater to slide by rapid movement using camel hairbrush or applicator stick