MICROSCOPY AND STAINING CHAPTER 3
Metric Units Microbe sizes – μm to nm
Light Properties (λ) Wavelength Electromagnetic spectrum Shorter wavelength Higher energy More resolution
Light Properties (RP) Resolving Power (Resolution) Distance between 2 points that can be distinguished
Light Travel Reflection Waves are bounced off some materials Transmission Waves pass through Absorption Energy modification
Light Travel Refraction Altered angle of movement
Compound Light Microscopy Condenser Iris diaphragm Objective lenses Ocular lens(es) Stage Focusing knobs Total Magnification
Light Microscopy Types Bright Field Dark Field Spirochaetes T. pallidum B. burgdorferi
Dual Beam Microscopy 2 light beams Phase Contrast Interference creates areas of contrast Internal cellular structures Nomarski Higher resolution Shallow depth of field
Fluorescence Microscopy Ultraviolet light Excited electrons emit light after UV absorption Natural fluorescence versus flouorochromes FAb staining Antibodies tagged with flourescent dyes Specific for molecules
Imaging Techniques Confocal Microscopy (UV) Digital Microscopy
Electron Microscopy (EM) Uses X-rays Focused by electromagnets Transmission (TEM) Thin tissue slices Heavy metal staining Scanning (SEM) Gold/Carbon coating Profiles or fractures
EM Images
EM Images
Light Microscope Specimen Preparation Wet Mounts Thick liquid preparation Smears Placement of cells Air drying Heat fixation
Staining Principles Acidic (-ve) / Basic (+ve) Simple Stains Differential Stains Special Stains
Gram Stain Differential Stain CV-I complex Alcohol wash 2 colors for Gram +ve and Gram –ve Developed by Hans Gram Distinguishes microbes based on peptidoglycan content in cell wall CV-I complex Crystal violet + iodine forms a large molecular complex Alcohol wash Cannot wash out of the Gram +ve cell wall Gram –ve wall is easily disrupted by alcohol wash Saffranin counterstain Gram +ve cells stay purple Gram –ve cells stain pink Diagnostic importance Peptidoglycan is a target for many antibiotics when present in a thick layer Penicillins Monobactams Carbapenems
Appearance After Each Step Crystal violet Addition of Iodine Alcohol rinse step Safranin Counterstain
Ziehl-Neelsen Acid Fast Stain Acid Fast Bacteria Mycobacterium genus identification Waxy lipid mycolic acid repels most dye chemicals Genus is one of few that stain pink Diagnosis Tuberculosis Leprosy
Negative (Capsule) Stain Visualizes the Capsule Thick glycocalyx around cell Negatively charged dyes Repelled by –ve charge on glycocalyx Halo effect is seen Cells are counterstained with positively charged dye Virulence factor Repels phagocytic WBC May prevent antibiotic entry into cells
Endospore Stain (Schaefer-Fulton) Endospores are hard to see with Gram stain Waxy dipicolinic acid Visualizes pathogens Clostridium tetani C. botulinum C. perfringens Bacillus anthracis Vegetative cells vs. Endospores Sterilization challenge Heat and dessication-resisitant