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

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.

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: 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?

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 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 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

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

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

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 Bacteria 2. domain Arachaea 3. domain Eukarya

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?