MCB 2610 Exam 1

definition of life

• cells and organization

  • genetic info flow

• response to environmental change

  • has to be able to react

• growth and development

• subject of evolution

  • genetic code doesn’t evolve

• energy and metabolism

• homeostasis and regulation

• reproduction

microbiology definition

study of microbes: how they interact with humans, food, and are used by humans (biotechnology)

importance of microorganisms

* most populous and diverse group of organisms
* found everywhere on planet
* play major role in recycling essential elements
* source of nutrients and some photosynthesize
* benefit society (production of food, beverages, antibiotics, and vitamins)
* some cause disease in people, plants, animals

prokaryotic cells

lack true membrane-delimited nucleus (not absolute)

eukaryotic cells

* membrane enclosed nucleus
* more complex morphologically

evolution of classification

* microorganisms divided into 3 domains based on ribosomal RNA comparison: Bacteria (true bacteria), Archaea, Eukarya (eukaryotes)
* rRNA determines relatedness not time of divergence

Domain Bacteria

* usually single-celled
* most lack membrane-bound nucleus
* cell wall made of peptidoglycan
* ubiquitous and some live in extreme env.
* cyanobacteria produce sig. amt of oxygen

Domain Archaea

* unique rRNA sequence
* lack peptidoglycan
* unique membrane lipids
* many live in extreme env.

Domain Eukarya

* protists
* algae
* protozoa
* slime molds
* water molds
* fungi
* yeast
* molds and mushrooms

acellular infectious agents

• viruses

  • smallest of microbes, require host cell to replicate, cause range of disease/cancer

• viroids

  • composed of RNA

• satellites

  • nucleic acid enclosed in protein shell, must coinfect host cell w/ virus

• prions

  • infectious proteins

  • ex. mad cow disease

Stanley Miller experiment

• formed organic molecules from primordial soup

• simulated conditions of primitive earth to determine how organic molecules formed

ribozymes

• RNA molecules that form peptide bonds

• perform cellular work and replication

• believed to be earliest molecule

• ability to catalyze reactions

• dual purpose: catalysis and genetic info storage

• lipid membrane (liposomes) formed around ribozyme

RNA’s evolution

• cellular pool of RNA in modern day cells exist in ribosome (rRNA, tRNA, mRNA)

• catalytic in protein synthesis

• may be precursor to double stranded DNA

• ATP is energy currency and is ribonucleotide

• can regulate gene expression

endosymbiotic hypothesis

primitive prokaryotic microbes ingested other microbes, starting a symbiotic relationship, forming first basic eukaryotes

• ingested microbes could use oxygen for respiratory process to produce chemical energy > mitochondria

• ingested microbes could fix CO2 into organic molecules using light energy > chloroplasts

evidences for endosymbiotic hypothesis

• SSU rRNA genes show bacterial lineage

• genome sequences closely related to proteobacteria and Prochloron

strain

descendants of a single, pure microbial culture

• may be biovars, morphovars, serovars, and pathovars

• Bacteria and Archaea referred to as this since they do not reproduce sexually

microbial species binomial nomenclature

Genus species

• Genus capitalized, species lowercase, both italicized or underlined

phylogeny

natural relatedness b/w groups of organisms

evolution

• all new species originate from preexisting species

• closely related organism have similar features since they evolved from common ancestral forms

earliest metabolism

early energy sources under harsh conditions - inorganics

• cyanobacteria - photosynthesis

• stromatolites - mineralized layers of microorganisms

primary producers

convert inorganic molecules to organic molecules

photosynthesis

• oxygenic: plants - splitting water using photons to produce O2

• non-oxygenic (light > pool of electrons)

chemotrophy

use chemical energy sources

phototrophy

uses light as energy source

chemoorganotrophs

broken down by microbes to harness chemical energy (ATP)

fermentation

doesn’t need oxygen but doesn’t yield much energy for microbes

aerobic respiration

requires oxygen but yields much more energy

microbial ecology

relationship of organisms with their environment

• inorganic molecules cycled to organic molecules and back

• live in diverse groups in nature > microbial community

microbial biotechnology

• can mass-produce molecules

• ex. human insulin production by E. coli

• been around since Egyptians and mummies

medical microbiology

diseases of humans and animals

public health microbiology

control and spread of communicable diseases

immunolgy

how immune system protects host from pathogens

agricultural microbiology

impact of microorganisms on food production

food microbiology

used to make food and beverages, spoilage microbes

industrial microbiolgy

penicillin and other antibiotics, vaccines, steroids, alcohols and other solvents, vitamins, amino acids, enzymes, and biofuels

microbial physiology

studies metabolic pathways of microorganisms

microbial genetics

nature of genetic info and its regulation of development and function of cells

synthetic microbiology

microbes are model system of genomics

colony

mound of cells grown in container, consists of one species

pure culture

single species growing in container

mixed culture

multiple species growing in container

contaminants

unknown or unwanted microbes

coccus

spherical shape

bacillus

rod

coccobacillus

very short and plump

vibrio

gently curved, half moon shape

spirillum

helical, comma, twisted rod

spirochete

spring-like

pleomorphism

each cell of species has slightly diff shape

cocci arrangements

• singles

• diplococci - in pairs

• tetrads - groups of four

• irregular clusters - “staphylo-”

• chains - “strepto-”

• cubical packets - sarcina

bacilli arrangements

• diplobacilli - two rods side by side

• chains - “strepto-”

• palisades

magnification

ability to enlarge objects, extent of enlargement

resolving power

ability to show detail, how well you can distinguish between 2 diff objects

focal point

focus light rays at this specific place

• bending of light hits diff angles

focal length

distance b/w center of lens and focal point, strength of lens related to this

refraction

bending of light when passing from one medium to another, each medium bends light different

refraction index

measure of how greatly a substance slows velocity of light

compound microscope

has more than one lense, all modern microscopes

bright-field microscope

• produces dark image against a brighter background

  • source of light comes from beneath

• both stained and unstained

  • allows us to see live specimen (unstained)

• has several objective lenses

purpose of staining

increases contrast

• stained mo are killed

• most mo are colorless

parfocal microscope

more than one objective lense

total magnification

product of the magnifications of ocular and objective lenses

resolution

ability of lens to distinguish small objects that are close together

how wavelength affects resolution

shorter wavelength results in greater resolution

numerical aperature

ability of lens to gather light

• ranges from 0.1 to 1.25

how numerical aperature affects resolution

larger numerical aperature provides better resolution

purpose of immersion oil

oil has different refraction index, so more light reaches the lens, increasing numerical aperature, so better resolution

working distance

distance b/w surface of lens and surface of cover glass or specimen when it is in sharp focus

how working distance affects resolution

smaller working distances give better resolution - can better separate close objects bc light spreads out more

• high mag causes short working distance, can’t collect a lot of light so immersion oil used

dark-field microscope

• image is formed by light reflected or refracted by specimen

• produces bright image of object against a dark background

• used to observe living, unstained preparations

• light comes from 2 diff directions

  • will only see objects that bounce light perpendicularly

phase-contrast microscope

• uses slight differences in refractive index and cell density

  • diff structures in cells will bend light differently

• uses hollow cone of light - polarize light so each compound reflects light differently

• cone of light passes thru specimen some is retarded (out of phase)

• light passes thru phase plate bringing it back into phase, excellent way to observe unstained living cells

differential interference contrast microscope (DIC)

• creates image by detecting differences in refractive indices and thickness of diff parts of specimen

• use two beams of polarized light to create 3D image of specimen

• live, unstained cells appear brightly colored and 3D

fluorescence microscope

• exposes specimen to ultraviolet, violet, or blue light

  • shine one wavelength > get diff wavelengths back

• specimens usually stained with fluorochromes (dyes)

  • bright objects on dark background

  • non-living organisms

  • if organism is autofluorescent (green color), doesn’t have to be killed to be observed

recombinant staining

attach fluorescent protein to other proteins (chimera protein)

immuno-fluorescence

can attach fluorochrome to antibodies which then attach to bacterial cell

confocal microscope

• creates sharp, composite 3D image of specimens by using laser beam, aperture to eliminate stray light and computer interface

  • laser allows us to focus on depths

• specimen usually fluorescently stained

wet mounts/hanging drop mounts

• allow examination of characteristics of live cells

• mix sample with water > put on slide

preparation and staining of specimens

• increases visibility of specimen

• accentuates specific morphological features

• preserves specimens

heat fixation

• routinely used with bacteria and archaea

• expose glass to high heat, melts sugars, fixes sample to glass

• preserves overall morphology but not internal structures

chemical fixation

• used with larger, more delicate organisms

• protects fine cellular substructure and morphology

basic dyes

• have positive charges

• bind to negatively charged molecules: nucleic acids, many proteins, surfaces of bacterial and archaeal cells

• will stain living organisms well

acidic dyes

• have negative charges

• bind to positively charged cell structures

• will not stain living organisms well

simple stains

• can use any stain with positive charge

  • outer membranes of cell are negative which attract positive

• can determine size, shape, and arrangement of bacteria

negative stain

• stains everything but cells

• creates shadows (neg. picture)

differential staining

• divides mo into their groups based on their staining properties - at least 2 results

• ex. gram stain - majority of organisms divided by membrane contents

• ex. acid-fast stain - not stained well with gram stain, know what the cells have in their cell walls

gram staining steps

1) crystal violet for 1 min, water rinse - cells stain purple

2) iodine (mordant) for 1 min, water rinse - cells remain purple

3) alcohol (decolorizer) for 10-30 sec, water rinse - gram + cells remain purple, gram - cells become colorless (differential step)

4) safranin (counterstain) for 30-60 sec, water rinse, blot dry - gram + cells remain purple, gram - cells appear red

acid-fast staining

• high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics

• + red cells - cluster bacilli

• - blue cells

capsule staining

• capsules may be colorless against a stained background

• may be indicative of virulent

• capsule: most external layer of bacteria

• negative staining

flagella staining

• visualize flagella

• mordant applied to increase thickness of flagella

endospores staining

• endospores stain green

• spores have complex structure > hard for dye to penetrate

electron microscopy

• electrons replace light as illuminating beam

• more precise

• allows us to see smaller things like viruses

• wavelength of electron beam much shorter causing higher resolution

Transmission Electron Microscope (TEM)

• electrons scatter when they pass thru thin sections of specimen > used to produce clear image

• denser regions in specimen scatter more electrons and appear darker

• stream of electrons directed by electron magnets

advantages of TEM

• much better resolution

• more precise

disadvantages of TEM

• electrons can only penetrate very thin specimens

• usually gives only 2D image

• must be viewed under high vacuum

• specimens are dead

negative staining

• heavy metals do not penetrate specimen but render dark background

• used for study of viruses and cellular microbes

shadowing

• coating specimen with thin film of heavy metal on only one side

• 3D results

• useful for virus particle morphology, flagella, DNA

freeze-etching

• sample freezed > cracked > exposes diff structures

• can see 3D light structures

major differences b/w light and electron microscopes

• light

  • highest mag: 1k to 1.5k

  • best res: 0.2 um

  • radiation source: visible light

• electron

  • highest mag: over 100k

  • best res: 0.2 nm

  • radiation source: electron beam

scanning electron microscope

• uses electrons excited from surface of specimen to create detailed image

• produces realistic 3D image

• can determine actual in situ location

• can take natural samples - dried samples coated with thin film of metal

electron cryotomography

rapid freezing technique using slices and depths creates better resolution