Observing microbes, functional anatomy Flashcards
Who came up with the tree of life and is thought to be the father of modern taxonomy?
Carl Linnaeus (1775)
Botanist, physician and zoologist
Father of modern taxonomy
Five domains of life
Binomial nomenclature
Common ancestor – Animals, Plants, Bacteria , Fungi, and monera
Explain Binomial Nomenclature
Genus and species: Escherichia coli
- Staphylococcus aureus*
- Staphylococcus epidermidis*
- Bacillus cereus:* Bacillaceae family
- Clostridium* sp.
(Kingdom phylum class order family genus species)
What is the tree of life?
What was the difference between Linneasus’ classification and Carl Woeses?
Linnaeus 1770s
Phenotypic classification
Five domains of life
Binomial nomenclature
Carl Woese 1977
used sequencing or rRNA and came up with 3 domains of life
Genotypic classification
Based on comparative
rRNA gene sequencing
The Tree of Life - Woese
Carl Woese (1977)
Based on comparative 16S rRNA gene sequencing to create a phylogenetic tree starting from LUCA (last universal common ancestor)
Three domains of life
Describe Microbial evolution
A summary of life on Earth through time and origin of the cellular domains
(a) Cellular life was present on Earth by 3.8 billion years ago (bya).
Cyanobacteria began the slow oxygenation of Earth about 3 bya, but current levels of O2 in the atmosphere were not achieved until 500–800 million years ago.
Eukaryotes are nucleated cells and include both microbial and multicellular organisms.
(b) The three domains of cellular organisms are Bacteria, Archaea, and Eukarya.
Archaea and Eukarya diverged long before nucleated cells with organelles (“modern eukaryotes”) appear in the fossil record.
LUCA, last universal common ancestor
Compare the contribution of Linneause and Carl Woese
- Linnaeus
Five domains of life
Binomial nomenclature
- Carl Woese
Based on comparative
rRNA gene sequencing
Three domains of life
Eukaryotic vs. Prokaryotic Cell
Pro means before Karyote means nucleus Eu means with
Describe Symbiogenesis (or endosymbiotic theory)
- 1910 by Russian Botanist Konstantin Mereschkowski
- Theory that eukaryotic cells evolved from symbiosis of single celled prokaryotes about 1.5 billion years ago
- Molecular and biochemical evidence suggests this is true:
- Mitochondria – proteobacteria
- Chloroplasts - cyanobacteria
Life at the Microscopic Scale
At X10,000 can see intricate external surface of bacteria
What Microbial cells can be seen under different microscopes?
- Naked eye (up to 100μm- human egg)
- Light microscope (up to 200nm- bacteria, mitochondria, flu virus)
- (Cryogenic) Electron microscope (up to 0.1 nm- atom, lipids)
What are the parts of a light microscope?
Observing Bacteria: Light microscopy
What is the max magnification and resolution?
Magnification: how much the apparent size of an object has been enlarged
Max magnification of 1000x (10 x eye piece, 100x objective)
Resolution: separation of objects ( max resolution=
0.2µm)
What is the function of oil immersion?
Reduces light scattering (improves resolution)
Electron Microscopy: Transmission (TEM)
- Sample sectioned
- Freeze fracture
- Prepare 60nm slices
100,000x = max magnification
0.2nm= max resolution (1000 fold additional resolution)
Electron Microscopy: Scanning (SEM)
100,000x
Shows surface only
Observing Bacteria: Generic Staining
- Increasing contrast for bright-field microscopy
- Generic stains:
- Methylene blue
- Crystal violet
Observing Bacteria: Differential Stains
Gram-stain preparation
- Preparation of smear
- Heat-fixing on slide
- Staining
- Microscopy
Preparing the Smear
DON’T OVERLOAD SLIDE – touch on the hand (multiple times)
- Drop of sterile water on slide
- Touch loop to colony
- Put loop in sterile water
- Using bunsen burner pass slide backwards and forwards through the blue flame
Describe gram staining
- Apply crystal violet (purple)
rinse with water to remove excess stain
- Apply an iodine mordant to form dye-iodine complex which decreases its solubility within the cell
- Drip on ethanol or acetone, gram negative bacteria decoluorise, gram positive bacteria remain purple and retain crystal violet
excress ethanol washed off with water
- Counterstain using safronin turn gram negative pink
rinse with water and dry with filter paper
- when viewed under a microscope gram positive=purple and gram negative=pink because gram staining correlates with cell wall stucture
Bacterial Cell Wall
- Gram positive bacteria have a thick peptidoglycan layer around cytoplasmic membrane. Tend to be spherical and have a smooth outer layer
- Gram negative membrane have an outer membrane then a thin peptidoglycan layer then another membrane. Tend to be rod shaped and have a rough outer layer
Gram +ve vs Gram -ve
Explain the Major Cell Morphologies
Cocus- spherical
Bacillus- rod shaped
Spirillium- spiral
Spirochete, budding and appendaged baceria and filamentous bacteria
Explain Bacterial cell arrangements
What is the binomial nomenclature for E.Coli?
Kingdom: Eubacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Enterobacteriales
Family: Enterobacteriacea
Genus: Escherichia
Species: E.Coli
Why must fresh bacterial cultures be used in the gram staining process?
As they get older they loose the ability to retain the stain
