Eukaryote Identification Notes

• These notes cover identification of eukaryotes, bacterial morphologies and arrangements, microscope skills, staining techniques, and bacterial characteristics

Identifying Eukaryotes Under a Microscope

• Schistosoma mansonni (Helminth) at 40X magnification
• Enterobius (Parasitic Worm) at 10X magnification
• Anabena (Cyanobacteria) at 40X magnification
• Peridinium (Dinoflagellate) at 40X magnification
• Spirogyra (Algae) at 10X magnification
• Diatoms (Algae) at 40X magnification
• Paramecium (Protozoa) at 40X magnification
• Trichomonas vaginalis (Parasitic Protozoa)
• Trypanosoma gambiensae (Parasitic Protozoa mixed with RBCs), viewable under 100X magnification
• Penicillium (Fungi, Mold type with purple slide) at 40X magnification
• Rhizopus (Fungi, Mold type with pink slide) at 40X magnification
• Aspergillus (Fungi, Mold type with green slide) at 40X magnification
• Saccharomyces cerevisiae (Fungi) at 100X magnification
• Culex (Vector) at 4X magnification
• Pulex (Vector) at 4X magnification

Identifying Bacterial Morphologies and Arrangements

• Coccus bacterial morphology at 1000X magnification
• Spirillum bacterial morphology at 1000X magnification
• Rod/bacillus bacterial morphology at 1000X magnification
• Staph bacterial arrangement at 1000X magnification
• Strep bacterial arrangement at 1000X magnification

Distinguishing Prokaryotes from Eukaryotes

• Know diseases that each organism can cause, if any result from infection

Microscope Skills

• Identifying microscope parts
• Using all objective lenses, including the oil immersion lens
• Locating and focusing specimens using oil immersion techniques

Resolution vs. Magnification

• Resolution is the ability to distinguish two points
• Magnification is the ability to enlarge an object

Oil Immersion

• Use oil with the 100x objective lens (total 1000x magnification)
• Immersion oil enhances resolution by preventing light loss and having the same refractive index as glass

Maximum Microscope Resolution

• Maximum resolution is 0.2um with a 100x objective

Total Magnification

• Determine total magnification by 10x multiplied by the objective lens number

Microscope Preparation

• Before storing, switch to 4x (or 40x) and lower the stage

Improving Lighting

• If cells appear washed out, increase light, use oil immersion with the 100x lens, adjust the diaphragm, or use coarse and fine focus

Microscope Parts

• Condenser: focuses light into the specimen
• Diaphragm: controls the amount of light
• Objective lenses: 10x, 40x, 100x (low, high, dry, oil immersion)
• Ocular lens: magnifies image 10x
• Eyepiece: part you look through
• Coarse adjustment: moves stage up and down for focusing
• Fine adjustment: moves stage slightly to sharpen the image
• Stage: holds the microscope slide in place
• Mechanical stage adjuster

Light Path

• Trace the light path through a microscope, starting with the light course, diaphragm, condenser, stage, objectives, revolving nose piece, body tube, and ocular lens

Eukaryotes and Prokaryotes

• Identify eukaryotes and prokaryotes
• If transitioning from 40x to 100x and the cell disappears; center the specimen, open the diaphragm, use fine focus, clean lenses, and ensure the lens is clicked into place

Cellular Morphology

• Cellular morphology describes the shape, size, arrangement, and staining reaction of cells
• Includes coccus, spirillum, and bacillus shapes

Field of Vision

• The field of vision is smaller at higher powers; center objects to avoid losing them
• Use the mechanical stage adjuster to adjust

Refractive Index

• The amount that light bends

Parfocal Lenses

• Focus under lower power before moving to higher power

Gram Stain

• Know how to perform, prepare an A+ smear, what the stain helps identify

Preparing a Smear

• Spread cells evenly, avoid clumps, mark smear location, label slide, and heat fix

Focusing Stained Slides

• Focus stained slides under oil immersion

Heat Fixing

• Heat fixing adheres the specimen to the slide for staining

Drying Smears

• Air dry smears before heat fixing to prevent lysis

Staining Techniques

• Simple Stain: observe basic external structures; uses methylene blue
• Gram Stain: identify cell wall type; uses crystal violet, iodine, ethanol, safranin. Gram-positive have thicker peptidoglycan with no outer membrane. Gram-negative have thinner peptidoglycan and an outer membrane
• Acid-Fast Stain: identify mycolic acids in Mycobacterium; uses cabolfuchsin, acid alcohol, methylene blue

Role of Iodine

• Iodine fixes crystal violet to the cell wall

Gram Stain Reactions

• Gram-positive organisms maintain the crystal violet-iodine complex due to their cell wall structure

Gram Stain Reagents

• Crystal violet (primary stain): colors the cell purple
• Iodine (mordant): fixes crystal violet to the cell wall
• Ethanol (decolorizer): removes crystal violet from gram-negative cells
• Safranin (counterstain): stains gram-negative bacteria pink

Mordant and Counterstain

• Mordant improves stain adherence
• Counterstain provides contrasting color

Acid-Fast Staining Importance

• Acid-fast staining is important for identifying TB and leprosy to diagnose Mycobacterium tuberculosis

Acid Fast Cell Walls

• A high lipid content

Acid-Fast Stain

• Carbolfuchsin (primary stain, red); lipid soluble
• Acid alcohol (decolorizer): removes unbound carbol fuchsin
• Methylene blue (counterstain): contrasts non-acid fast cells
• Cannot penetrate mycolic acid;

Bacteria and Endospores

• Bacillus and Clostridium produce endospores, a survival mechanism under harsh conditions

Disease and Sporulating Bacteria

• Endospores allow bacteria dormancy, resistant to heat, UV, desiccation, and chemicals
• Sporulating bacteria cause Enterocolitis, Pseudomembranous colitis from Clostridium difficile (C.diff), and Clostridium Tetani

Gram Stain Identification

• Identify gram-positive cocci and rods, and gram-negative rods

Identifying Acid Fasts

• Identify acid-fast positive and negative bacteria

Explore microscope techniques for identifying various eukaryotes. Learn to distinguish between helminths, algae, protozoa, fungi, and cyanobacteria. Also review bacterial morphologies, staining, and key characteristics for accurate identification.