Acid Fast Staining

• The Gram staining method, developed by Hans Christian Gram in 1882, is crucial in microbiology and is typically the first test for identifying bacteria
• The primary stain in the Gram's method is crystal violet, which can be substituted with methylene blue
• Microorganisms that retain the crystal violet-iodine complex appear purple or brown under microscopic examination and are classified as Gram-positive or Gram-negative
• Gram-positive cells retain the crystal violet-iodine complex and appear purple or brown, while Gram-negative cells do not retain the stain and appear red
• In the AFB staining procedure, acid-fast organisms like Mycobacteria have cell walls that can withstand strong acid decolorizers due to the presence of long-chain waxy mycolic acids
• Non-acid-fast organisms are easily decolorized with acid alcohol and take up the counterstain
• Acid-fast organisms are hard to stain and require heat or solvents to drive the stain into the cell wall
• Sample collection for AFB staining includes early morning sputum as the preferred specimen
• Preparation of the sputum smear involves transferring an appropriate portion to a labeled slide, making an oval translucent smear, air-drying, fixing, and staining
• AFB staining procedure includes steps like flooding the slide with carbol fuchsin, steam heating, decolorizing with acid alcohol, counterstaining with methylene blue, and air or blot drying
• Microscopy for AFB staining involves reading the slide from left to right and then from right to left, recording results, and identifying acid-fast organisms as red and non-acid-fast organisms as blue
• To see bacteria under a microscope, staining reagents are necessary to apply color
• Stained bacteria can be observed and studied with respect to their shape, size, and arrangement
• Preparation of a stained bacterial smear involves several steps:

  • Place a bacterial colony on a glass slide and "fix" it with heat
  • Apply a basic stain for simple staining, which carries a positive electrical charge
  • The negatively charged surface and cytoplasm of bacteria attract the stain, leading to staining
• Direct Smears from swab:

  • Roll the swab across the slide to avoid destruction of cellular entities
• Direct Smears from concentrated centrifuged specimens:

  • Prepare smears using a sterile, plastic transfer pipette
• Smears from Cultures (Subculture Smears):

  • Spread a thin film of bacteria on a clean glass slide
  • Place a loopful of culture in the square without spreading unless growth is exceptionally heavy
• Smears from Blood Culture Bottles:

  • Use one slide per specimen
  • Decontaminate the bottle top with an alcohol prep
  • Puncture the top with a safety venting subculture unit and allow a drop to fall onto the slide, spreading it until the liquid is about the size of a nickel
• Smears from Solid Media:

  • Place a small drop of water in the square using a 1-mL tuberculin syringe
  • Emulsify a colony by rubbing it into the dry glass slide and mixing with water until turbid
• Simple Staining:

  • Using a single stain to color a bacterial organism
  • Common dyes for simple staining include methylene blue, basic fuchsin, and crystal violet
  • Staining times are relatively short, usually from 30 seconds to 2 minutes
  • Useful for determining basic morphology and the presence or absence of certain granules
• Bright Field Microscopy:

  • Examines the detail of a microorganism by transmitted light
• Phase Contrast Microscopy:

  • Uses contrast enhancement technique to examine unstained specimens
• Fluorescence Microscopy:

  • Specimens are stained with fluorochrome or fluorescent dye
• Care for the Microscope:

  • Keep the area clean and organized
  • Keep lenses clean using lens paper after each use
  • Turn off the microscope after use and do not keep the light on all day
  • Return the objective lenses to the lowest magnification and set the stage at its lowest part after use
  • Always use the revolving nosepiece in changing objective lenses
  • Hold the microscope with two hands; one on the arm and the other on the base
• How to Use the Microscope:

  • Turn on the microscope and adjust light intensity
  • Switch to 10x objective, place the specimen on the stage and focus using the coarse adjustment knob
  • Adjust the condenser and iris diaphragm until the desired light focus and area are acquired
  • Switch to other objectives, ensuring the specimen is focused before moving to another objective
  • If using oil immersion objective, apply the necessary oil before moving
• Terms in Microscopy:

  1. Magnification: The ratio of the apparent size of an object as seen through the microscope to the actual size of the object

  2. Resolution/Resolving Power: The ability of the lens to clearly separate or distinguish two points or lines individually in the image

  3. Numerical Aperture: A measurement of the ability of the condenser and the objective lens to gather light

  4. Focal Length: Thickness of the object that may be seen at one time under focus

  5. Working Distance: Distance between the front lens of the objective lens and the top of the cover glass when the specimen is in focus

  6. Parfocal: Refers to the quality of the objectives and eyepiece where practically no change in focus has to be made when the objective is substituted for another

  7. Refractive Index: Bending of light rays away from the objective lens when light passes from the glass of the microscope slide to the air
• Types of Objective Lenses:

  • Low Power Objective:
  • Resolving Power: 1.34um
  • Numerical Aperture: 0.25NA
  • Magnifying Power: X10
  • Field of View Diameter: 2.00mm
  • Working Distance: 7.20mm
  • High Power Objective:
  • Resolving Power: 0.52um
  • Numerical Aperture: 0.65NA
  • Magnifying Power: X40
  • Field of View Diameter: 0.40mm
  • Working Distance: 0.60mm
  • Oil Immersion Objective:
  • Resolving Power: 0.26um
  • Numerical Aperture: 1.30NA
  • Magnifying Power: X100
  • Field of View Diameter: 0.20mm
  • Working Distance: 0.20mm
• Magnification:

  • Total Magnification = Eyepiece x Objective
  • Example: Eyepiece magnification = 10x, Objective magnification = high power field (40x), Total Magnification = (10x) (40x) = 400x total magnification
• Magnification:

  • Magnification = Image size / Actual size
  • Example: Image size measured = 20 mm → 20,000 um, Actual size measured = 100 um, Magnification = 20,000um / 100um = 200X
• Formula for Microorganism Size:

  • Actual size (um) = Image size (um) / Magnification
  • Example: Oil Immersion magnification: 100, Image size measured = 1000um, Actual size = 1000um / 100 = 10um