Exam 1: Special Techniques Flashcards
Electron microscopy (EM)
Higher resolution than light microscopy
Requires specialized equipment and technical expertise
Very expensive
Different fixatives –> Glutaraldehyde
•Short life span, must be in fridge
Types of EM
Transmission EM
Scanning EM
Transmission EM
2D image - looks flat
Electrons go through stuff
Used to see details of cell/organism
Can see nuclear membrane, granules, etc.
Scanning EM
3D image
Electrons bounce off
Used to see structure
•like spherical cells
Target detection
Looking for specific things in cells
Types of target detection
Antibody - detect proteins
•ihc
Probe/primer - detect DNA/RNA
•ish
Immunohistochemistry (ihc)
Affinity of antibodies for specific antigens (proteins)
Ab-Ag bond (specific)
•both are proteins
•Ab - immune response
Identification - cell type, tissue/cell component, pathogens
ihc - where do antibodies come from
Immune system
•Rodents and Rabbits
•Goats/Horses
Immortal cells (myeloma)
•Cell cultures
•Myeloma - tumor of antibody producing cells
ihc - polyclonal and monoclonal
Polyclonal - antibodies to multiple epitopes
Monoclonal - antibodies to a single specific epitope
Epitope - part of protein that antibody attaches to
ihc - direct and indirect
Direct ihc - primary (and only) antibody is tagged
Indirect ihc - Secondary antibody (targeting primary antibody) is tagged
•Secondary antibody attaches to primary and gives the ability to see primary antibody
Nucleic acid detection: PCR
Primer/Probe
•Matching DNA sequence to DNA or RNA
•Double each cycle
PCR - amplify DNA to get bigger sample size
Need probe specific to the DNA sequence
PCR types
Conventional - gel
Fluoresce
Gel PCR
Semi-quantitative
Must for sequencing
Can only look at it at the end
Fluoresce PCR
Quantitative
Standard: ‘known’
Attach fluorescent probe
Can look at it at every cycle
In situ hybridization, ish
Split DNA with temperature, apply probe/color, if DNA present will see color
Can be used for anything that has DNA
Can make probe for mRNA to find products being produced in specific cells
IHC vs ISH
ISH is more specific than IHC because ISH will only detect the DNA you are looking for
Blepharitis Conjunctivitis
Affects house finches
Caused by Mycoplasma gallisepticum
Use IHC to see the mycoplasma as dark brown area
Mycoplasmosis
Smallest self replicating prokaryote
Sunny side up egg colony
House finch and spread of mycoplamsosis
East and west populations
Birds brought to east - colony came from a few birds - not a lot of genetic variation –> started seeing conjunctivitis a lot
Western population not affected as much - more genetic variation
Cytology
Dry and stain, fixing optional
Impression smears, FNA, cyto spins
Don’t have structure of tissue
Cell biology history
Robert Hooke - term “cell”
Schwan and Shleiden - “cellular theory”
Virchow - “cellular pathology”
What makes you alive (9 things)
Irritability Conductibility Contractibility Absorption Secretion Excretion Assimilation Growth Reproduction
Irritability
Ability to detect and respond to a stimulus
Cell type - neutrophil
Conductibility
Ability to conduct a stimulus
Cell type - neuron
Contractibility
Ability to change shape
Cell type - muscle cell
Absorption
Ability to introduce materials into the cell
May be through phagocytosis (solids) or pinocytosis (liquids)
Secretion
Release of useful products
Cell type - goblet cell
Excretion
Release of waste
Assimilation
Construction of new cellular elements
Growth
Increase in cellular mass keeping the nuclear-cytoplasm ratio
Reproduction
Produce a daughter cell while transferring genetic material
Cell type - ova, sperm
