Modern cell theory concepts (3)
-all living things made up of cells (basic unit of life)
-all cells arise from cell division of pre existing cells
-energy flows within cells (metabolism)
general cell composition
75-80% water
10-20 proteins
nucleic acids
inorganic material
carbs
!! solutes and solvents together form COLLOIDAL SYSTEM
Law of Driesch
the volume of a cell is approx constant for each type in the same species and independent of the organism size
Law of Levi
EXCEPTION TO DRIECH’S LAW:
larger the size of the animal the larger the size of corresponding cells (which aplies to nerves muscles and lens fibers)
what is the factor determining max possible size of cells
SA:V RATIO
-cells need to stay small enough that they have a high SA:V ratio and uptake via diffusion in feasible
what is the relevance of the nucleus: cytoplasm
-needs to be maintained constant
-achieved through cell division and cell adaptations
what is the general process of specimen preparation
- tissue fixation (killing of sample to preserve morphology)
- tissue inclusion (use paraffin to harden sample for slicing)
- cut tissue (using microtomes)
- stain tissue (reagents able to absorb diff wavelengths)
2 types of fixation and why it is necessary
- PHYSICAL: exposure to very low/high temps
- CHEMICAL: exposure to chemical reagents
PURPOSE:
-stabilise tissue elements
-preserve topographic relationships
-inhibition of autolysis
-resistance to manipulation
types of chemical fixation
- IMMERSION: embed in a fixing reagents
- IN VAPORS: exposure to vapours produced by heating fixative solutions (eg. formaldehyde - high volatility)
- PERFUSION: fixative passed through vascular system of animal models to distribute fixative throughout tissues
preparation of a sample POST FIXATION
- DEHYDRATION: tissue passed through increasingly concentrated alcohol solutions, removing water
- CLEARING: immersion in solvent used for inclusion
- INFILTRATION: placed in inclusion agent until it is completely infiltrated
- EMBEDDING: in paraffin or pure resin to make ti hard enough to cut
- CUTTING: form serial sections (ribbons) of tissue using microtome
- DEWAXING + REHYDRATION: dewaxed by xylene, rehydrated with decreasing alcohol concs (relaxes sample and removes paraffin)
- STAINING AND MOUNTING: adding stain and placing coverslip over sample
how is the mounting of a blood smear different than a usual tissue
due to liquid state a second slide is used to smear the blood across the slide to thin it out
dye usually used for blood smears
giemsa
2 types of dyes based on character
- ACIDIC: stain cytoplasm and collagen fibers (acidophillic) EOSIN: negatively charged
- BASIC: stain ground substance, nucleic acids, RER (basophilic) HEMATOXYLLIN: positively charged
what parts of the stain dont stain with either eosin or hematoxylin
- fluids present in tissues or interstitial spaces (blood/lymph)
- lipids and fat droplets
PAS+ staining use
-stains carbs and carb rich macromolecules
-glycogen, basement membranes
Sudan staining use
-detects lipid rich structures of cells (bcos its lipid soluble)
Osmium staining use
-based on tissue lipid oxidation
-forms black or dark brown substances
immunofluorescence purpose + eg
uses antibodies with fluorescent dyes to detect antigens in tissues
!! DAPI STAIN
immunohistochemistry def
enxymes are used to catalyse a reaction that is colour producing
ultramicrotomy for electron microscopes process
-method of cutting tissue into extremely thin slices of 50-100nm for TEMs
-sections cut are placed on grid
-sections are cross colored via heavy metal salts to make sample electron dense
-observation under TEM
Magnification equation
M=image size/actual size
resolving power def
ability of microscope lens or optical system to produce separate images of closely positioned objects
resolution def
the smallest distance between two distinct objects that can be visualised as two different points
resolution of light and electron microscope
light: 0.2 micrometers
electron: 0.2 nanometers
!! smaller resolutions are better
