1. MICROSCOPY AND STAINING
CHAPTER 3

2. Metric Units
Microbe sizes – μm to nm

3. Light Properties (λ) Wavelength Electromagnetic spectrum
Shorter wavelength
Higher energy
More resolution

4. Light Properties (RP) Resolving Power (Resolution)
Distance between 2 points that can be distinguished

5. Light Travel Reflection Waves are bounced off some materials
Transmission
Waves pass through
Absorption
Energy modification

6. Light Travel
Refraction
Altered angle of movement

7. Compound Light Microscopy
Condenser
Iris diaphragm
Objective lenses
Ocular lens(es)
Stage
Focusing knobs
Total Magnification

8. Light Microscopy Types
Bright Field
Dark Field
Spirochaetes
T. pallidum
B. burgdorferi

9. Dual Beam Microscopy 2 light beams Phase Contrast
Interference creates areas of contrast
Internal cellular structures
Nomarski
Higher resolution
Shallow depth of field

10. 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

11. Imaging Techniques
Confocal Microscopy (UV)
Digital Microscopy

12. 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

13. EM Images

14. EM Images

15. Light Microscope Specimen Preparation
Wet Mounts
Thick liquid preparation
Smears
Placement of cells
Air drying
Heat fixation

16. Staining Principles Acidic (-ve) / Basic (+ve) Simple Stains
Differential Stains
Special Stains

17. 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

18. Appearance After Each Step
Crystal violet
Addition of Iodine
Alcohol rinse step
Safranin Counterstain

19. 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

20. 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

21. 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