Germ Theory of Disease,Microscopy & Staining

Nestor T. Hilvano, MD, MPH

Learning Objectives You should be able to: 1. List Koch’s postulates and describe its function. 2. Define microscopy, magnification, and resolution. 3. Contrast simple and compound microscopes. 4. Contrast transmission electron microscopes with scanning

electron microscopes. 5. Explain the purpose of a smear, heat fixation, and chemical

fixation in the preparation of a specimen for microscopic viewing.

6. Describe the simple, Gram, acid-fast, and endospore staining procedures.

7. List the types of special stain and their purpose. 8. List the hierarchy of taxonomy and define binomial

nomenclature. 9. Describe the three domains proposed by Carl Woese.

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.

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)

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

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

Preparing specimen for staining

Steps in Smear Preparation:1.Sterilize Inoculating loop/wire2.Dip into culture of organism3.Spread a thin film over the glass slide4.Air dry to dessicate5.Pass over flame6.Staining What is the purpose of fixation?

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 stainsAcid dyes – eosin; nigrosin; acid fuchsinBasic dyes – methylene blue; crystal violet; safranin O; malachite green

*____ stain – use of single basic dye; soaking smear in dye for 30-60 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

Gram StainSteps: (VIAS)1. Crystal violet for 1 min., and rinse2. Iodine solution for 1 min., and rinse3. Decolorized with alcohol (ethanol and acetone)

for 10 – 30 sec., and rinse w/ water4. Safranin for 1 min., and rinse w/ waterResults : 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

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) 1. Cover smear w/ tissue paper to retain dye2. Carbolfuchsin several mins. while warming over steaming

water; remove paper, cool slide3. Decolorized w/ acid (HCL) and alcohol solution4. Counterstain w/ methylene blue• Results :

pink/red cells = AFB; blue cells = non-AFB Exercises:___ primary stain in AFS.___ decolarizing agent___ counterstain

Endospore Stain

• Endospores – found in Bacillus and Clostridium sp.; dormant, resistant cell forms inside the cytoplasm of bacteria

• Schaeffer-Fulton endospore stain: 1. uses heat (5 min) to drive the primary stain, malachite

green, into the endospore. 2. Cool the slide and decolorized w/ water.3. Counterstain with safranin.• Results: green= endospores; red= vegetative cells• Diseases= Anthrax, gangrene, and tetanus

produce endospores

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)

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 Bacteria2. domain Arachaea3. domain Eukarya

Homework 1. Define terms: taxonomy, binomial nomenclature, heat

fixation, mordant, smear, magnification, resolution, simple stain, differential stain, negative stain, gram stain, and acid-fast stain.

2. What is germ theory of disease?3. Describe the steps of smear preparation.4. Describe the steps in doing a gram’s stain. 5. Discuss how pulmonary tuberculosis can be proven

according to Koch’s postulates. 6. What type of microscope is used to examine

submicroscopic structures (organelles), viruses, and small microbes)?

7. How does light (compound) microscope operates?