1. Germ Theory of Disease, Microscopy & Staining
Nestor T. Hilvano, MD, MPH

2. Learning Objectives You should be able to:
List Koch’s postulates and describe its function.
Define microscopy, magnification, and resolution.
Contrast simple and compound microscopes.
Contrast transmission electron microscopes with scanning electron microscopes.
Explain the purpose of a smear, heat fixation, and chemical fixation in the preparation of a specimen for microscopic viewing.
Describe the simple, Gram, acid-fast, and endospore staining procedures.
List the types of special stain and their purpose.
List the hierarchy of taxonomy and define binomial nomenclature.
Describe the three domains proposed by Carl Woese.

3. Germ Theory of Disease
Disease is caused by infections of pathogenic microorganisms (Pasteur, Koch, etc.)
Robert Koch – identified anthrax, TB, cholera bacterium, dormant endospores, and used agar plates
Koch’s postulates:
1. The suspected pathogen must be present in
every case of the disease.
2. That pathogen must be isolated and grown in
pure culture.
3. The cultured pathogen must cause the disease
when it is inoculated into a healthy, susceptible
experimental host.
4. The same pathogen must be reisolated from
the diseased experimental host.

4. Microscopy Unit of measurement - metric units (meter)
1/1000 m = 1 millimeter (mm)
1/1000 mm = 1 micrometer (um)
1/1000 um = 1 nanometer (nm)
Microscopy - use of light or electrons
- Magnification
- Resolution = dependent on wavelength of electromagnetic radiation and numerical aperture of lens
(0.2um)

5. Light Microscope Light microscope a. simple b. compound
- light source, condenser, glass lenses
- magnification 1000X; resolution of 0.2 um
Dissecting microscope
- light focused, condenser lens built into light fixture, glass lenses
- magnification low (25X); cheaper than LM

6. Electron Microscope
Transmission EM study details of _____ of cells.; setup upside down compared to LM; vacumn; Magnification to 100,000 – 250,000X; resolution 0.2 nm
Scanning EM observed the______ of cells.; setup analogous to DM; magnification 30,000X; cheaper than TEM
a. Surface structure b. internal structure

7. Preparing specimen for staining
Steps in Smear Preparation:
Sterilize Inoculating loop/wire
Dip into culture of organism
Spread a thin film over the glass slide
Air dry to dessicate
Pass over flame
Staining
What is the purpose of fixation?

8. Principle of Staining Most microorganisms = colorless, thin, invisible
Dissecting Microscope = no stain, large samples
TEM = metal stains (osmium tetraoxide, uranyl acetate, lead citrate)
SEM = metal stains (gold palladium coating)
Light Microscope = organic stains
Acid dyes – eosin; nigrosin; acid fuchsin
Basic dyes – methylene blue; crystal violet; safranin O; malachite green
*____ stain – use of single basic dye; soaking smear in dye for secs, then rinse w/ water; determinesSize, shape, and arrangement of cells.
* ____ stain – use of more than one dye; ex. gram stain; acid-fast stain, and endospore stain.
a. differential stain b. simple stain

9. Gram Stain Steps: (VIAS) Crystal violet for 1 min., and rinse
Iodine solution for 1 min., and rinse
Decolorized with alcohol (ethanol and acetone)
for 10 – 30 sec., and rinse w/ water
4. Safranin for 1 min., and rinse w/ water
Results : purple = gram + (thick CW); pink = gram – (thin CW)
Cocci gram (+), except genera Neisseria, Veillonella sp.
Rods gram (-), except genera Bacillus, Nocardia, Corynebacterium, Clostridium sp.
Exercises:
___ primary stain of gram’s staining.
___ use as mordant, substance which binds to a dye & make it less soluble, cells
remain purple.
___ decolarizing agent, allow stain & mordant to be washed out in gm. – organisms
___ counterstain, provide contrast color
a. Alcohol b. safranin c. crystal violet d. iodine

10. Acid –Fast Stain: Zeil- Neelsen Method
Used for cells with waxy cell walls; cells are colorfast w/ acid (acid can’t penetrate waxy CW, retain red color of AFB)
Steps: (CAM)
Cover smear w/ tissue paper to retain dye
Carbolfuchsin several mins. while warming over steaming water; remove paper, cool slide
Decolorized w/ acid (HCL) and alcohol solution
Counterstain w/ methylene blue
Results :
pink/red cells = AFB; blue cells = non-AFB
Exercises:
___ primary stain in AFS.
___ decolarizing agent
___ counterstain

11. Endospore Stain
Endospores – found in Bacillus and Clostridium sp.; dormant, resistant cell forms inside the cytoplasm of bacteria
Schaeffer-Fulton endospore stain:
uses heat (5 min) to drive the primary stain, malachite green, into the endospore.
Cool the slide and decolorized w/ water.
Counterstain with safranin.
Results:
green= endospores; red= vegetative cells
Diseases= Anthrax, gangrene, and tetanus
produce endospores

12. Special stains
Negative stain – acidic dyes (eosin) used to reveal the negatively charged capsules (ex. Klebsiella pneumoniae)
India ink test – Cryptococus neoformans (fungal infection), identify capsule as halo
Flagellar stain – pararosanaline and carbolfuchsin, and mordants (tannic acid and K alum) in a series of steps (ex. Proteus vulgaris)

13. Linnaeus Taxonomic Categories
Taxonomy – science of classifying and naming of organisms
Binomial nomenclature- scientific name, Genus + specie; in italics when typed, underlined when hand written
Ex: Homo sapiens
3 Domains based on r-RNA sequencing (Carl Woese)
1. domain Bacteria
2. domain Arachaea
3. domain Eukarya

14. Homework
Define terms: taxonomy, binomial nomenclature, heat fixation, mordant, smear, magnification, resolution, simple stain, differential stain, negative stain, gram stain, and acid-fast stain.
What is germ theory of disease?
Describe the steps of smear preparation.
Describe the steps in doing a gram’s stain.
Discuss how pulmonary tuberculosis can be proven according to Koch’s postulates.
What type of microscope is used to examine submicroscopic structures (organelles), viruses, and small microbes)?
How does light (compound) microscope operates?