Unit 2 Microbiology Test

taxonomy

science of classifying organisms
provides universal names for organisms

taxon

subdivisions used to classify organisms
e.g.: kingdom, phylum, order etc

who developed the binomial naming system?

Linneus

Phylogeny

study of evolutionary history of organisms

in 1978, two types of prokaryotic cells were found by

rRNA sequencing

domain

a taxonomic classification based on rRNA sequencing, above the kingdom level

rRNA sequencing

determination of the order of nucleotide bases in rRNA

if an organism has close rRNA sequencing they are determined to be

closely related

3 domain names

Eukarya, Archaea and Bacteria

Characteristics of Domain: Bacteria

cell type: prokaryotic
cell wall: contains peptidoglycan
Membrane lipids: straight chain; ester linkages
DNA:1-2, mostly circular
histones: NO
reproduction: binary fission

Characteristics of Domain: Archaea

cell type: prokaryotic
cell wall: contains NO peptidoglycan, varies in composition
Membrane lipids; branched chains, ether linkages
DNA:1-2, mostly circular
histones: YES
reproduction: binary fission

Characteristics of Domain: Eukarya

cell type: eukaryotic
cell wall: varies in composition, contains CARBOHYDRATES
Membrane lipids: straight chain; ester linkages
DNA:linear
histones: YES
reproduction: Mitosis

endosymbiotic theory: origin of eukaryotes

similarities in the rRNA sequencing indicates that prokaryotes gave rise to mitochondria and chloroplasts in eukaryotic cells

Binomial Nomenclature

not applicable to viruses
used worldwide
consist of Genus species

eukaryotic species

a group of closely related organism that breed among themselves and produce fertile offspring

prokaryotic species

a population of cells with similar characteristics

culture

grown in laboratory media

clone

population of cells derived from a single cell or CFU

strain

genetically different cells within a clone or species

how are strains made up of genetically different cells?

1) Mutation
2) Recombination: transformation, transduction or conjugation

viral species

population of viruses with similar characteristics occupying a particular environmental niche.

Viruses are not placed in kingdom or domain because:

1) not composed of cells
2) cannot grow without a host cell

Kingdoms in the Domain Eukarya

Animalia
Plantae
Fungi
Protista

Animalia

multicellular, no cell walls, chemoheterotropic (uses organic molecules as source of carbon and energy)

Plantae

multicellular, cellulose cell walls, usually photoautrophic (uses light as energy source, CO2 as carbon source)

Fungi

chemoheterotrophic, unicellular or multicellular, cell walls contain chitin, develop from spores or hyphal fragments

Protista

a catchall kingdom for eukaryotic organisms that do not fit into other kingdoms, grouped in clades

clade

group of organisms that share particular common ancestor, based on rRNA

classification

placing organisms in groups of related species, lists of characteristics of known organisms by rRNA sequencuing

identification

matching characteristics of an "unknown" to a list of known organisms

dichotomous key

an identification scheme based on successive paired questions: answer one question leads to another pair of questions until an organism is identified

cladogram/phylogenetic tree

a branching diagram that shows the evolutionary relationships among organisms; based on rRNA sequences

Bergey's Manual of Determinative Bacteriology

provides identification schemes for identifying bacteria and Archaea based on phenotype and biochemical patterns

types of methods used to based off of determinative manual

morphology
differential staining
biochemical testing

Bergey's Manual of Systematic Bacteriology

provides evolutionary history information on bacteria and archaea based on rRNA sequencing

types of identification used in a clinical lab

morphology
differential staining
biochemical testing

biochemical tests

determines presence of bacterial enzymes and metabolic products
only works for pure cultures

Serological testing

used to differentiate between microorganism species and strains of the same species
involves reactions of microorganisms with specific antibodies

serology depends on

binding of antigens and antibodies

types of serological testing

slide agglutination
ELISA
Western Blot

slide agglutination

method of identifying an antigen by combining it with a specific antibody on a slide, similar to blood typing

ELISA (enzymes linked immunosorbent assay)

group of serological tests that use enzyme reaction as indicators of the presence of a bacterial antigen in a sample

Western blot

a test that detects antibodies and confirms the results of earlier EIA tests, used for HIV and Lyme disease

phage typing

method of identifying bacteria using specific strains of bacteriophages

DNA fingerprinting

genetic similarities determined by \# and sizes of DNA fragments produced by RE digests
used to determine profiles of organisms

DNA sequencing

the process of determining the precise order of nucleotides within a DNA molecule; can determine % G + C content and compare species

Southern blotting

uses nucleic acid hybridization to identify unknown microorganisms using DNA probes

Ribotyping

rRNA sequencing

Organisms with similar rRNA sequences you can conclude?

they are closely related and share a common ancestor

PCR

used to amplify small amount of microbial DNA in a sample, strand are separated and primers hybridize

Flow cytometry

-microbial suspension forced through small orifice with a laser light beam
-movement of microbe through orifice impacts electric current that flows through orifice
-instances of disruption of current are counted
-specific antibodies can be used to determine size and internal complexity

nucleic acid hybridization

strands can be separated and the base pairing of one strand of a nucleic acid to a complementary sequence on another strand

DNA Chip

is composed of DNA probes and is used to quickly detect a pathogen in a host or environment, sample is labeled with fluorescent dye, sample hybridizes to chip

FISH

fluorescent dye labeled RNA or DNA probes to identify both cultured and uncultured microbes which hybridize to the probe

Fatty Acid Methyl Ester (FAME) Analysis

compares altered membrane fatty acids by gas chromatography to references to identify bacteria

Urease Test

determines the ability of microorganisms to degrade urea by means of the enzyme urease.

Voges-Proskauer Test

Measures the production of the neutral end product acetoin by fermentation of glucose

Simmons Citrate Agar

medium used to determine whether an organism can utilize citrate as its sole carbon source, bromothymol blue will turn blue in the presence of metabolic products

Triple Sugar Iron Agar (TSI)

differential
identifies gram-negative enteric bacilli (enterobacteriaceae)
Tests for:
1. the fermentation of glucose, lactose or sucrose (yellow)
2. the production of hydrogen sulfide (H2S) black
3. the production of gas (bubbles)

Oxidase test

identifies bacteria that have cytochrome oxidase (e.g., Pseudomonas), turns blue if positive

SIM agar

Sulfur Indole Motility
black ppt, red ring, movement from stab

Indole test

Tests organisms ability to breakdown Tryptophan to indole. Utilized Kovac's reagent. Positive - Red, Negative- No color change

Hemolysis on blood agar

no change is gamma, bacteria didn't break down RBC's; 'greenish' tint is alpha-partial destruction of RBCs in blood; clear zone is beta, complete destruction of RBC's

MacConkey Agar

a selective and differential agar.
selective for gram negatives,
Differential for lactose and non-lactose fermenters

Nitrate Reduction Test

mostly gram negative contains nitrate reductase for anaerobic respiration
-red color after reagents = nitrate -\> nitrite, +
-no color after reagents = reqs Zn
-no color after Zn = nitrate -\> nitrite -\> other, +
-red after Zn = no nitrate reduction, -

metabolism

sum of all the chemical reactions,
combining anabolic and catabolic reactions = metabolism

enzyme

speed up chemical reactions without being altered; biological catalysts; act only on specific substrates and lower activation energy; are NOT changed in reactions

oxidation

removal of electrons

reduction

gain of electrons

ATP

(adenosine triphosphate) main energy source that cells use for most of their work

Catabolism

Metabolic pathways that break down molecules, releasing energy.

anabolism

Metabolic pathways that construct molecules, requiring energy.

electron transport chain

Electrons are transferred from one electron carrier to another along an electron transport chain (system) on a membrane that releases energy to generate ATP;

substrate-level phosphorylation

The formation of ATP by directly transferring a phosphate group to ADP from an intermediate substrate (organic compound) in catabolism.

oxidative phosphorylation

The production of ATP using energy derived from the redox reactions of an electron transport chain; the third major stage of cellular respiration.

photophosphorlyation

The production of ATP by chemiosmosis during the light reactions of photosynthesis.

carbohydrate catabolism

breakdown of carbohydrates to release energy: aerobic, anaerobic, fermentation

glycolysis

glucose is oxidized to produce pyruvic acid (will also produce some ATP and NADH)
Overall net gain of two molecules of ATP and 2 NADH for each molecule of glucose oxidized

glycolysis: preparatory stage

2 ATP are used
Glucose is split to form two molecules of glyceraldehyde 3-phosphate

glycolysis: energy-conserving stage

The two glyceraldehyde 3-phosphate molecules are oxidized to 2 pyruvic acid molecules
4 ATP are produced
2 NADH are produced

net gain of glycolysis

2 ATP for each molecule of glucose

aerobic respiration

1: glycolysis
2: Kreb cycle; pyruvic acid (from glycolysis) oxidized and decarboxylation (loss of CO2) occurs
3: electron transport chain, requires oxygen

Chemiosmosis

A process for synthesizing ATP using the energy of an proton gradient and the ATP synthase enzyme.

Intermediate/transition step

Pyruvic acid (from glycolysis) is oxidized (produces NADH) and decarboxylation (loss of CO2) occurs. combines with Coenzyme A to form Acetyl CoA

Kreb Cycle (Citric Acid Cycle)

Oxidation of acetyl CoA produces NADH, FADH2, and GTP (which will form ATP), and liberates CO2 as waste

Electron Transport Chain (ETC)

A sequence of electron carrier molecules (membrane proteins) that shuttle electrons during the redox reactions that release energy used to make ATP.

fermentation

metabolic process that follows glycolysis; does not require oxygen Uses organic molecule as final electron acceptor; Produces NAD+ so that glycolysis can continue, does not produce ATP

lactic acid fermentation

the chemical breakdown of carbohydrates that produces lactic acid as the main end product

homolactic fermentation

produces lactic acid only

heterolactic fermentation

produces lactic acid and other compounds

alcohol fermentation

produces ethanol + CO2
glucose oxidized to pyruvic acid; pyruvic acid converted to acetaldehyde and CO2; NADH reduces acetaldehyde to ethanol

phenol red

indicator dye for fermentation tests

oxidation-reduction reaction

In biological systems, electrons and protons are removed at the same time; equivalent to a hydrogen atom
Biological oxidations are often dehydrogenations

chemiosmosis

Electrons (from NADH) pass down the electron transport chain while protons are pumped across the membrane
Establishes proton gradient (proton motive force)
Protons in higher concentration on one side of the membrane diffuse through ATP synthase
Releases energy to synthesize ATP

molecular oxygen (O2)

final electron acceptor in the electron transport chain during aerobic respiration

anaerobic respiration

The final electron acceptor in the electron transport chain is NOT O2, can be sulfates, nitrates, iron
Yields less energy than aerobic respiration

lipid catabolism

break down fatty acids down two different arms: glycerol enters glycolysis, Fatty acids are metabolized by Beta-oxidation which can make acetyl CoA.

protein catabolism

proteins break down in to amino acids and undergo a variety of conversions; deamination, decarboxylation, desulfurization, enters the kreb cycle

light-dependent (light) reactions

conversion of light energy into chemical energy (ATP and NADPH)
Light excites electrons in chlorophyll, electrons travel down ETC, pumps H+ to create proton gradient. H+ flow through ATP synthase by chemiosmosis to create ATP

light-independent (dark) reactions

ATP and NADPH are used to reduce CO2 to sugar (carbon fixation) via the Calvin-Benson cycle
Energy from light reaction is used to make sugar molecules