Microbiology Exam 1 (Dr. Rebecca Riggs)

Acid-fast stain

carbolfuchsin used to stain Mycobacterium species

Spore Stain

malachite green used to detect spores of Bacillus and Clostridium

Negative Stain

colors the background, which makes capsules more visible

Gram Stain

Hans C. Gram

1) Slide w/ H20 and sample

2) Crystal Violet

3) Gram's Iodine

4) EtOH

5) Safranin

Robert Koch

- founder of scientific method

- demonstrated chain of infection

POSTULATES

1) Microbe found in all cases of diseased but none healthy

2) Microbe can be isolated and grown in pure culture

3) When microbe is introduced to healthy host, same disease occurs

4) The same strain of microbe can be obtained from newly diseased host

1&3 false if immunity

2 false because we can't grow all bacteria

if 1-3 are true, 4 is likely true

Angelina and Walther Hesse

created solid medium using agar

Julius Petri

double dish container

Sergei Wingradsky

- discovered lithotrophs ( get nutrients from rocks and minerals)

- developed enrichment cultures

- Winogradsky column

Winogradsky column

a wetland model ecosystem containing regions of enrichment for microbes of diverse metabolism. Each uses the others' waste

Francesco Redi

Experiment : Covered and uncovered meat

Conclusion: Discounted spontaneous generation because covered meat did not have maggots

Argument against: Meat jar was sealed

Lazzaro Spallazani

Experiment: Covered & uncovered broth

- Conclusion: concluded that broth became contaminated with microorganisms from the air entering the flask, not spontaneous generation

- Opposition: people thought that since the sealed flask was cut off from vital air, their would not be any microorganisms

Louis Pasteur (pasteurizing weakens things)

- Experiment: swan neck flask. Disproved spontaneous generation because flask was open to the air but did not grow bacteria. Bacteria cannot climb s-curve. Proved something in the air causes bacterial growth.

- Proposed biogenesis

- Developed first vaccines based on weakened strains. Fowl cholera/rabies.

Florence Nightingale

Stastistician who showed correlation of battle wounds and infectious diseases

Mr. Onesimus

A slave from West Africa. Introduced smallpox variolation in American colonies.

Lady Mary Montagu

Introduced Variolation to Europe from Turkey

Edward Jenner

First to vaccinate - infected patients with matter from cowpox lesions

Lynn Margulis

- Proposed symbiotic theory

- Mitochondria came from proteobacteria (maybe Typhus bacterial DNA)

- Chloroplasts came from cyanobacteria (Similar DNA)

Oswald Avery (A plus)

discovered that the transforming material between bacteria is DNA

Frederick Griffith (bad teacher)

Discovered transformation in bacteria. Horizontal gene transfer where some bacteria take up DNA (not known yet) from environment.

Rosalind Franklin

Used X-ray diffraction to discover the double-helical structure of DNA.

Watson, Crick, and Maurice Wilkins

Discovered DNA's complementary bases and antiparallel-ness.

Ernst Haeckel

- microbes are neither plant nor animal, a third kind of life called monera

Herbert Copeland (into cups)

- divided monera into

1) Eukaryotic - nucleus. protists. protozoa and algae.

2) Prokaryotic - no nucleus. monera. bacteria.

Robert Whittaker

Added fungi as 5th kingdom of eukaryotes

Robert Hooke

- built 1st compound microscope

- published micrografia (drew what he saw)

- coined the term "cell"

Antoine van Leeuwenhoek

- built single-lens magnifiers

- first to observe single-celled microbes

- called them "small animals"

Carl Woese

- discovered archaea

- difference between archaea and bacteria is rDNA

- proposed three domains of life (Archaea, Bacteria, Eukarya)

Ignaz Semmelweis (hitler)

Ordered doctors to wash hands with chlorine. Mortality rates of women in birth fell.

Joseph Lister

Developed carbolic acid (antiseptic)

Alexander Fleming (flame- big deal)

Discovered penicillum mold.

Howard Florey and Ernst Chain (prune flowers. chain reaction)

- purified penicillin and was first commercial antibiotic to save lives.

Carolus Linnaeus (puritan)

- microbial world "chaos"

- father of taxonomy

- microbes do not interbreed ( not like other species)

Gram positive vs negative cell wall

Gram negative has:

- LPS

- Outer membrane

- Much less peptidoglycan

- only peptide bonds in peptide layer because also has outer mem for stability.

- Porins

Gram positive has:

- No LPS

- only 1 membrane

- Much more peptidoglycan (anchored by teichoic acid)

- peptide AND glycosidic bonds in peptide layer

- no porins

Parfocal

Property of microscope which allows objectives to be changed without having to refocus

Light source in compound microscope?

Comes up from below specimen

Gram negative organism stains purple. What was missed?

Decolorizer (EtOH)

Student switches from 40x to 100x objective lens but cannot focus. What should they check?

Immersion oil.

Resolution

smallest distance between objects that two objects can be separated and still distinguished.

Detection

ability to determine presence of object

Refraction

Bends light, slows light speed. Magnifies image.

TEM vs SEM

TEM - 2D internal structures. up to 100,000x

SEM - 3D external structures. up to 10,000x

Bacilli, cocci, and spirilla

Bacilli = rods

cocci = perfect circles

Spirochetes = flexible cell wall

Spirilla = rigid cell wall

bright field microscopy

generates a dark image of an object over a light background

increasing numerical aperture

increases resolution

compound microscope

A light microscope that has more than one lens. Must be parfocal

Ocular lens

10x magnification

Objective Lens

4x, 10x, 40x, 100x

Total magnification

ocular magnification x objective magnification

Ocular lens causes what kind of image

inverted

Simple stain

adds dye to see cell shape. Ex methylene blue.

Differential stain

stains different colors based on cell type. Ex gram or acid fast.

dark field microscopy

specimen is bright, background is dark. Good for thin objects.

Resolution depends on

1) contrast between object and its medium

2) Wavelength smaller than object

3) Magnification

Immersion oil

has different refractive index than air, diminishes the loss of refracted light and improves resolution

Wet mount

Place microbe in drop of water on slide w/ coverslip. Can observe cells in natural state. BUT little contrast and sample may dry out quicker.

Fixation

Cells are made to adhere to a slide in fixed position. Helps with detection and resolution.

Simple Stain procecure

1) Place microbe culture on slide, let dry

2) Add drop of dye (e.g. methanol)

3) Let stain for 1 min

4) Wash off stain with water

5) View under microscope

Phase-contrast microscopy

Reveals difference in refractive index. Used for live cells and organelles. To see internal structures of living cell.

Fluorescence Microscopy

Uses fluorophores to label specimen. Specimen becomes fluorescent. Because emission wavelength > absorbed/excitation wavelength.

Used for test for syphillis (detects antibodies bound to the bacterium)

Light microscope vs electron microscope

Light microscope: inverted

Electron microscope: inverted + rotated

Why is electron microscopy in higher resolution than light microscopy?

Because electron microscopes use a beam of electrons instead of a beam of light. The electrons have a much shorter wavelength than the light which leads to higher resolution.

If forget crystal violet in gram stain

All cells will stain safranin (red)

Endospores/gram stain?

Yes, you can see endospores with gram stain. Cell will be purple with uncolored ball in middle.

cell membrane

thin, flexible barrier around a cell; regulates what enters and leaves the cell. Phospholipid bilayer, transporter proteins, used for eg production.

Characteristics of Prokaryotes

- thick outer envelope

- compact genome (nucleoid)

- no membrane-bound organelles

- no nucleus

- may have flagella

subcellular fractionation

A procedure to separate cell components; often includes ultracentrifugation.

structural analysis of cell parts

imaging through electron microscopy or x ray crystallography

Genetic analysis of cell parts

Determines function of cell part

Ultracentrifugation

Rotation of cells at high speeds to separate cell parts.

Theodore Svedberg

invented ultracentrifuge. Developed sedimentation rate equation.

Bacteria vs Archaea Cell Membrane

Bacteria (and eukarya): ester bonds link phospholipid layer. Have pep

Archaea: ether bonds. Allow organisms to handle low temps. Some have lipid monolayer. NO pep

Passive Transport

Molecules move with their concentration gradient

Active transport

Against concentration gradient. Requires ATP.

How will prokaryotes without cell walls maintain membrane strength?

Actively pump out sodium ions to maintain cell pressure. Cell membrane has sterols which may help with structure.

What do antibiotics target?

peptidoglycan layer. Penicillin/vacomycin inhibits enzyme that links the peptides. Resistant strains = MRSA, VRSA

Diagram?

1) Sugar bond between NAG and NAM

2) D-alanine terminal residue of tetrapeptide

3) Diaminopimelic acid

4) NAM

5) NAG

6) Crosslink/peptide bridge

Capsule

Snot-like outer coating on gram positive bacteria (and some gram negative). Protects cell from being destroyed by immune system.

S-layer

- almost all archaea and bacteria

- made of protein

- protects cell from osmotic stress

Lipopolysaccharide (LPS)

- attached to outer membrane

- O-polysaccharide side chain (Antigen)

- Lipid A (endotoxin)

- medically significant

Mycobacterium

Complex cell walls. Difficult to stain. Requires heat and acid-fast stain.

DNA Replication

Copied by DNA polymerase in both directions simultaneously. Supercoiling- to fit DNA into cell. Extra if archaea b/c extremophile.

Replication, Transcription and Translation

Are all coordinated to speed up bacterial division!

Cell division (of bacteria)

Does NOT do mitosis or meiosis (that's Eukaryotes!

Instead

1) DNA replicated

2) Septum forms (septation)

3)Division into 2 cells

How is DNA compacted

Supercoiling. Archaea DNA has to be extra supercoiled for extreme conditions.

Streptococci/Staphylococci/Tetrads, Sarcinae

Streptococci: septation in parallel planes

Staphylococci: septation in random planes

Tetrads & Sarcinae: septation in perpendicular planes

FtsZ

Protein that assembles "Z ring" - drawstring around cell equator to help cell divide. Found in both archaea and baceria

Pilus/Fimbrae/Sex Pilus

Pilus: made of pilin protein. used in twitching motility

Fimbrae: Used for attachment ex. biofilms

Sex Pilus: used for conjugation : transfer of DNA from donor cell (F+) to recipient cell (F-)

Gallionella ferruginea (art gallery)

Bacteria with a stalk that acts as holdfast to environment.

Flagellum

Spiral filament made of flagellin. Propellors.

-Monotrichous: single flagellum

-Ampitrichous: both ends

=Lophotricous: tuft at one end or both ends

-Peritrichous: random

Parts of Flagella

Filament: long part

Hook: attaches filament to basal body

Basal body: anchored in cell wall and cytoplasmic membrane. Motor. Driven by protons moving across gradient in cell's cytoplasm

Gliding motility

Alternative to flagella. Bacteria moves across a film in direction of its long axis.

Chemotaxis

Cell movement- response to chemical gradients.

Attractants: CCW rotation, run

Repellent: CW rotation, tumble

Random walk

Combo of runs and tumbles in chemotaxis.

If attractant conc increases, biased random walk. Causes net movement towards attractants and away from repellents

Twitching motility

pili, drag across surface

Corkscrew-like motion

alternative to pili or flagella

Macronutrients

CHONPS + Mg2+, Ca2+, Fe2+, K+

Micronutrients

Co, Cu, Mn, Zn, Mo, Ni

Heterotroph

use preformed organic molecules and release CO2

Autotrophs

fix CO2 and make into organic molecules

Phototrophs: light energy

Lithotroph: energy from oxidiation of minerals

Phototroph

light is energy source