Micro Exam 4

Taxonomy

science of organizing organisms into two hierarchal groups based on shared characteristics

Microbial genomics

study of structure, function and evolution of microbial genomes

clinical microbiology

branch of microbiology focused on identifying and understanding microbes that cause human disease

immunology

study of immune system components and how they detect and respond to microbes

Taxa

hierarchal groups of organisms formed through classification

Identification

process of characterizing an organism using morphology, physiology and genotype

morphology

observable physical features of an organism -

physiology

functional traits of an organism, such as metabolism or biochemical capabilities

genotype

genetic makeup of an organism

classification

organization of organisms inot groups based on identified similiarities

nomenclature

formal system used to assign standardized names to organisms

species (prokaryotic)

group of closely related strains that share high genetic similarity and differ distinctly from other groups

strain

population of cells derived from a single ancestor that accumulates minor genetic differences over time

prokaryotic taxonomic hierarchy

ordered system of ranks used to classify prokaryotes from species up to domain

16S rRNA gene sequence similarity

molecular comparison used to assess relatedness in prokaryotes

genotpic characteristics

identification features based on an organism’s genetic sequence, often used to determine evolutionary relationships

Phenotype-dependent characteristics

observable traits such as morphology or metabolism used for practical identification

Algorithm/Dichotomous key

step-by-step series of yes/no questions used to narrow down microbial identity

microscopic morphology

size, shape, arrangement, and staining traits observed under a microscope

culture characteristics

• observable colony traits such as color, texture and growth patterns that appear on culture media

  • req. pure culture

metabolic capabilities

• biochemical traits that reveal how a microbe uses specific nutrients or produces metabolic by-products

  • req. pure culture

serology

identification based on antigen-antibody interactions involving microbial surface structures

protein profile

characteristic pattern of cellular proteins, often measured by MALDI-TOF MS, used for rapid microbial identification

• req. pure culture

acid-fast stain

• describes bacteria such as mycobacterium species, that retain certain dyes due to waxy mycolic acids in their cell walls

mycolic acid

long, waxy lipids found in the cell walls of acid-fast bacteria, responsible for their staining behavior and resilience

Auramine O

• fluorescent dye used in modern acid-fast staining to detect organisms like mycobacterium tuberculosis

colony morphology

• visible structure and appearance of a microbial colony, including shape, elevation and pigmentation

selective media

culture media that inhibit some microbes while allowing others to grow

differential media

media containing indicators that reveal metabolic differences between microbes through color or pattern changes

MacConkey agar

selective and differential medium used to isolate G- bacteria and detect lactose fermentation

blood agar

differential medium used to detect hemolysis patterns produced by certain bacteira

B-hemolysis

complete lysis of RBCs around a colony, producing a clear zone

biochemical tests

lab tests that detect specific metabolic processes, often through visible color or gas changes

catalase test

test that detects the enzyme catalase by observing the breakdown of H2O2 into O2 and water

sugar fermentation

metabolic process in which bacteria breaks down sugars to produce acidic end products, often altering the pH of the medium

Phenol Red

pH indicator commonly used in biochemical tests that turns yellow in acidic conditions and remains red or pink when no acid is produced

Urease

enzyme that hydrolyzes urea into ammonia and CO2, raising the pH of the surrounding medium

urea breath test

diagnostic method for H. pylori that detects carbon-labeled CO2 produced when urease breaks down labeled urea

commercial kits

• preassembled biochemical test systems that allow multiple metabolic reactions to be assessed at once

ELISA (enzyme-linked Immunosorbent Assay)

• biochemical test that uses antibodies and enzyme-linked color changes to detect antigens or antibodies

Sandwich ELISA

• an ELISA format that detects microbial antigens using antibody-coated wells to capture the pathogen’s surface structures

  • traps them b/w 2 specifc antibodies

  • detects antigen → current infection

Indirect ELISA

• an ELISA format that detects host antibodies produced during infection rather than microbial antigens themselves

  • detects antibody → current/recent past infection

antigen

molecule, often found on microbial surfaces, that triggers antibody binding

antibody

protein produced by the immune system that specifically recognizes and binds antigens

anti-pathogen antibody

• antibody designed to recognize and bind a specific microbial antigen

  • used as the ‘capture’ antibody in Sandwich ELISA

Enzyme-linked detection

method in which an antibody-associated enzyme produces a color change to indicate a pos reaction

pathogen-specific antibody

antibody produced by the immune system that binds to a distinct microbial antigen

Point-Of-Care (POC) test

diagnostic test performed at or near the site of patient care, providing rapid, actionable results

lateral flow assay

• rapid immunochromatographic test that detects antigens or antibodies using capillary movement across a membrane

Immunochromatographic assay

• detection method in which antigen-antibody binding produces a visible signal as the sample flows through the test device

sensitivity

• ability of a test to correctly identify true positives

  • often lower in rapid POC tests

IgM detection

identifying early-stage antibodies that indicate a recent infection

IgG detection

• identifying long-lasting antibodies that signal past exposure or late stage infection

False-negatives

test result that incorrectly indicates no infection when the pathogen is actually present

false-positive

test result that incorrectly indicates infection when the pathogen is absent

Co-infection

presence of more that one infectious agent in a patient, which POC tests may overlook

Serovar/Serotype

strain defined by its unique antigenic profile

O antigen

variable polysaccharide portion of LPS used to differentiate G- strains

H antigen

flagellar protein antigen used to distinguish motile bacterial strains

K antigen

capsular antigen used to classify strains with distince protective surface capsules

E. coli O157:H7

• pathogenic strain identified by its O157 and H7 flagellar antigens

E. Coli K12

non-pathogenic lab strain ID’d by its capsule (K) antigen

Immunomagnetic bead (IMB) technology

method that uses antibody-coated magnetic beads to selectively capture and isolate specific microbes from complex samples

Immunofluorescence

• technique using fluorescently labeled antibodies to visualize specific microbes or antibodies under a fluorescence microscope

Direct immunofluorescence

detection method in which fluorescent antibodies bind directly to microbial antigens on a specimen

Indirect immunofluorescence

method in which patient antibodies bind to fixed antigens, followed by a fluorescent secondary antibody to reveal the reaction

fluorochrome-labeled antibody

antibody linked to a fluorescent dye used in immunofluorescence assays

MALDI-TOF MS

rapid, mass-spec technique that identifies microorganisms by measuring the masses of their cellular proteins

mass spectrum

• unique distribution of protein mass signals produced during MALDI-TOF, used as a molecular fingerprint for Identification

specific nucleotide sequences

DNA regions unique to particular microbial groups, used as genetic targets for ID

NAATs (Nucleic Acid Amplification Test)

tests that amplify target DNA sequences to detect microbes with high sensitivity

FISH (Fluorescence in situ hybridization)

method that uses fluorescent DNA probes to identify and visualize microbes based on rRNA or other genetic sequences

rRNA (ribosomal RNA)

highly abundant RNA molecule often targeted in FISH due to its high copy number in bacterial cells

PCR (polymerase chain reaction)

technique that amplifies DNA and visualizes the result on an agarose gel to confirm the presence of a target organism

qPCR (Quantitative PCR)

• fluorescent version of PCR that measures DNA amplification in real time, allowing quantification of microbial load

primers (F&R primers)

short DNA sequences that bind to the target region and guide DNA synthesis

Taq polymerase

heat-stable DNA polymerase from Thermus aquaticus used to synthesize new DNA strands during PCR

dNTPs

nucleotide building blocks used by polymerase to make new DNA

Denaturation

high-temperature step that separates double-stranded DNA into single strands

Annealing

lower-temperature step where primers bind to their complementary sequences, controlling PCR specificity

Extension

step where Taq polymerase synthesizes new DNA strands, enabling exponential amplification

exponential amplificiation

• repeated doubling of target DNA during PCR cycles, producing very large quantities of DNA

Positive control (PCR)

• sample known to contain the target DNA, ensuring the PCR rxn worked as expected

Negative control (PCR)

• rxn lacking template DNA, used to confirm that no contamination or unintended amplification occurred

DNA ladder

• set of DNA fragments of known sizes used as reference markers during gel electrophoresis

SYBR Green

• fluorescent dye that binds to double-stranded DNA and increases in fluorescence as amplification proceeds

TaqMan probe

fluorescent reporter-quencher probe cleaved during DNA synthesis, releasing fluorescence only when the target sequence is amplified

Ct value (cycle threshold)

• PCR cycle at which fluorescence first rises above background, inversely related to starting DNA quantity

reporter dye

fluorescent molecule that emits light once separated from the quencher

Quencher

• molecule that suppresses reporter fluorescence until cleavage occurs

5’ → 3’ exonuclease activity

• ability of Taq polymerase to degrade DNA ahead of it during extension, enabling probe cleavage

background fluorescence

• baseline signal before amplification produces detectable fluorescence

standard curve

• reference graph created by amplifying known quantities of DNA or cells to relate Ct values to actual abundance

Standards

samples containing known number of cells or DNA copies used to build the standard curve

Quantification

• process of estimating how much microbial DNA is present in an unknown sample comparing it to the standard curve

fluorescent tag

• molecule attached to a probe that emits light under fluorescence microscopy

hybridization

• binding of a probe to its complementary nucleotide sequence within intact cells

In situ

“in the original place”

• refers to detecting microbes directly in the sample without culturing

conserved regions

Stretches of DNA that change very little over evolutionary time, allowing universal primer binding

variable regions

segments of the 16S gene that differ among microbial groups and help distinguish taxa