BIOL 308 Chapter 1 and 2

what domain is most closely related to eukaryotes

Archaea

6 dimensions of life

metabolism, reproduction, differentiation, communication, movement, evolution

von leuowneck

late 1600s, created microscope to visualize microbes

pasteur

1865, disproved spontaneous generation, helped create germ theory

used flasks with sterile broth and subjected to various conditions and observed growth (closed, open, open with swan neck)

Koch's Postulates

1. The suspected pathogen must be present in all cases of the disease and absent from healthy animals.

2. The suspected pathogen must be grown in pure culture.

3. Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal.

4. The suspected pathogen must be reisolated and shown to be the same as the original.

Koch also developed

solid media via additon of thickening agent

chemolithotrophy

microbes can oxidize inorganic molecules to generate ATP

Prokaryotic carbohydrates

hundreds of primary monosaccharides (instead of 10)

carbons range from 3-12

7/8C sugars almost exclusively found in prokaryotic organisms

Lipopolysaccharide

only found in gram negative bacteria

core sugars: KDO and L-glycero D-manno heptose

Teichoic acid

carbohydrate characteristic of gram-positive bacteria

structural component of cell wall

Archaea lipid diversity

have ether linkages between FA tails and glycerol (vs ester linkages for bacteria/eukarya)

-->less fluid

also can have phytanyl side chain (branching on FA)

-->found in really cold enviro to keep membrane fluid

also can have phospholipid monolayer (2 heads, 1 FA section without break)

-->found in really hot enviro for stability

prokaryote lipid diversity

have different FA chains associated with the membrane vs eukaryotes (depending on bacterial enviro)

head groups of glycerol in membrane is very different for prokaryotes vs eukaryotes

hopanoids

sterol like molecules unique to prokaryotic microbes

serves similar function to cholesterol in eukaryotes

provides fluidity and stability to membrane

LPS

lipopolysaccharide

endotoxin

lipid A (very hydrophobic FA tails, allows to form bilayer)-->found in outer membrane of gram neg bacteria

attached to KDO

inner and outer core (same in all gram negative bacteria)

O antigen-->varies between bacteria based on different sugar groups

L-amino acids

protein synthesis

d-amino acids

special functions (Mainly cell wall structure)

microbe amino acids

22 total (2 more than eukaryotes)

selenocysteine and pyrrolysine

also diaminopimelic acid (not an AA but similar)

Selenocysteine

has selenium instead of sulfur

antioxidant defense mechanism

Pyrrolysine

unique cyclic structure

some archaea and a few bacteria

methane metabolism

diaminopimelic acid

found in bacteria and plants

not incorporated into any proteins

key cell wall component for bacteria

precursor to biosynthesis of lysine

Bond between phosphate and pentose sugars in nucleic acids

phosphodiester bond

Microbial DNA

in both eukaryotes and prokaryotes DNA is antiparallel and doublestranded

eukaryotes have one/multiple linear chromosomes

prokaryotes have a singular circular chromosome with all genetic info

viruses can have DS-DNA, SS-DNA, SS-RNA, or DS-RNA

5 kingdoms

Monera (includes bacteria and archaea), Protista, Fungi, Plantae, Animalia

germ theory of disease

idea that infectious diseases are caused by microorganisms

prokaryotic cells appeared

3-4 billion years ago

eukaryotic microbes evolved approximately

2 billion years ago

eukaryotes and prokaryotes branch from

common ancestor

all prokaryotes are

unicellular

eukaryotes can be

unicellular or multicellular

microbes and nutrient balance

decomposition and nitrogen fixation

major discoveries

microscopy

scientific method

medical microbiology development

microbiological techniques

applications for biotech

metabolism

uptake of nutrients from the environment, their transformation within the cell, and elimination of wastes into the environment

cell is an open system

reproduction/growth

chemicals from the environment are turned into new cells under the direction of preexisting cells

differentiation

formation of a new cell structure such as a spore

communication

cells communicate or interact primarily by means of chemicals that are released or taken up

evolution

cells contain genes and evolve to display new biological properties

microbes compose

the majority of biomass on earth

microbe roles in agriculture

n2 fixation, nutrient cycling, animal husbandry (rumen breakdown of cellulose)

microbe roles in energy/environment

biofuels--fermentation of corn to ethanol

bioremediation

microbial mining

chemoautotrophy

The oxidation of inorganic compounds to yield energy used to reduce carbon dioxide.

chemolithotrophy

h2s-->s0-->s04-2

nh3-->No2 (-) --> no3 (-)

nitrogen fixation

n2 + 6H-->2nH3

5 categories of bacteria

1. chlamydias and spirochetes

2. gram positive bacteria

3. endospore producers

4. gram-negative bacteria

5. cyanobacteria

3 groups of archaea

methanogens, halophiles, thermophiles

microbes can generate energy via

chemoorganotrophy

chemolithotropohy

phototrophy

hyperthermophile

extreme heat-adapted organism

psychrophile

bacteria that prefer cold, thriving at temperatures between 0 C and 25 C.

carbohydrates can have a

aldehyde or ketone group

example of hopanoid

bacteriohopanetetrol

purines

Adenine and Guanine

double ring structure

pyrimidines

cytosine, thymine, uracil

deoxyribose vs ribose

deoxyribose lacks an oxygen atom on the second carbon in the ring (h vs OH)

bonds between nitrogenous bases

hydrogen bonds

six Is of microbiology

inoculation, incubation, isolation, inspection, information gathering, and identification.

inoculation

placing some of a sample with microbes into media that will allow microbes to grow

3 methods of inoculation

1. inoculating loop and spread on solid media (allows for colony isolatioN0

2. place sample into broth culture

3. inoculating cells 9eg. chicken egg)—>viruses only grow within other cells

isolation

isolating individual colonies

a singular cell will undergo binary fission to form a macroscopic colony

each colony is clonal and every cell in colony is genetically identical

techniques for isolation

streak plating and pour plate (loop dilution)

streak plating

A technique for isolating individual colonies from a mixed culture by spreading a diluted sample over the surface of an agar plate using an inoculating loop.

Allows for dilution of number of cells and space in between individual colonies.

3 total spreads with flames in between

pour plate/loop dilution

A method for isolating colonies by diluting a sample in molten agar and pouring it into a petri dish, allowing colonies to grow throughout the medium.

2 dilutions

inspection

The process of examining cultures for characteristics such as colony morphology, color, and growth patterns

macroscopic characteristics

shape, color, edges, size, consistency, height

if there is a second colony type confined to one area—>contamination

if the 2 species are spread throughout the plate, suggests original sample was mixed culture

microscopy types

bright field, dark field, phase contrast, fluorescent, confocal, scanning EM

phase contrast

good for cells that are more transparent (eg. animal/plant)

fluorescent microscopy

involves dye that recognizes structure on bacterial surface

uses special microscope

confocal microscopy

cross-sectional images of whole specimesn

scanning EM

surface view of bacteria

good for biofilms—>impact in environmental and healthcare settings

Staining

1. simple/negative stains

2. differential stains

3. structural staining

simple staining

using one dye to observe cells after fixing with heat or chemicals

eg. crystal violet—>peptidoglycan binds well

negative stain—>doesn’t bind bacteria bc of charge interactions; bacteria appear clear against dark background

differential staining

involves using multiple dyes to distinguish between different types of bacteria

gram, acid fast, endospore staining

gram staining results

positive is purple, red/pink is negative

acid fast staining

acid fast = red

non acid fast = blue

mycobacterium cannot be stained with gram, but can be with acid fast

structural stains

used to visualize specific structures within cells, such as endospores or capsules.

capsular stains

capsules are made of thick carb layers which prevent stain entry

stain generates halos around cellsf

flagellar stains

detect motility

Gram Stain steps

1. crystal violet stain

2. gram’s iodine (mordant)

3. alcohol (decolorizer)

4. Safranin

CV stain

binds to peptidoglycan

gram’s iodine (mordant)

locks CV into cells

alcohol (decolorizer)

penetrates outer membrane of gram neg, cell loses CV

safranin

a counterstain that colors gram-negative bacteria pink after decolorization.

gram stain preparation

take loop from broth culture and smear on slide OR take loop of water, spread on slide, sterilize loop, touch to colony, and spread into water

let slide air dry

fix microbes to slide (pass through flame)

gram neg vs pos

gram + have thick layer of peptidoglycan vs thin layer for gram neg

gram negative also have outer membrane which has LPS

information gathering

biochemical differences

media types

enriched, differential, selective

enriched media

has something extra added to support growth of organisms that need external supply

eg. blood or specific AA

differential media

bacteria show different appearance and color when grown on media

eg. MacConkey media—>bacteria that use lactose turn pink/red

selective media

only allows certain bacteria to grow (eg. selecting for antibiotic resistance to verify plasmid incorporation)

chocolate agar

used for fastitidious bacteria

blood is added to hot agar which lyses RBCc and releases heme

eg. haemophilus influenzae

doesn’t have ability to synthesizie porphyrin ring (needed for heme—energy metabolism)

needs to have from enriched media such as chocolate agar

fastidious bacteria

have certain nutrient requirements and require enriched media

Immunological gathering

staining or agglutination

immunological staining

antibody molecules bind to specific regions of bacterial cells (epitope)

antibody is labeled with fluorescent molecule for visualization

immunology agglutination

antibody binds to antigen on bacterial cell surface and agglutinates many bacteria together—>clumping in tube

synthetic media

components are highly specified and defined (doesn’t occur in nature)

complex media

exact chemical composition not defined

most general purpose media is complex

blood agar

enriched

can show zones of clearing around bacteria (some bacteria secrete enzymes to lyse RBCs)

Beta is complete hemolysis

alpha is incomplete lysis—>shows greenish tinge around cells

gamma is no lysis of RBCs by bacteria

some plates can have media that is selective and differential

EMB plate

inhibits growth of gram + bacteria

differentiates gram negative as well

lactose fermenters are purple with green sheen

coliforms are red

gram negative that aren’t coliforms look clear

carbohydrate fermentation media

contains sugars that can be fermented, converted to acids, and a pH indicator to show this reaction

fermentation media ex

liquid growth media with only 1 sugar (has smaller tube inverted within)

add phenyl red dye—>red at neutral/alkaline conditions, yellow at acidic

if bacteria ferment sugar and make acid, this pH change will cause media to turn yellow

if CO2 is produced during sugar processing—>gas bubbles appear in small tube

can repeat for all monosaccharides

identification using

Bergey’s model of Determinative Bacteriology

cell characteristics

reproduction, heredity/DNA, growth and development, responsiveness, transport, metabolism and enzymes