Microbiology Lab: PCR, Molecular Phylogeny, and ESKAPE Pathogens

Vocabulary practice flashcards covering Polymerase Chain Reaction (PCR) components, thermal cycling parameters, 16S rRNA molecular phylogeny, and ESKAPE pathogens with their safe relatives.

Polymerase Chain Reaction (PCR)

A molecular biology technique used to exponentially amplify specific target DNA sequences through repeated thermal cycles of denaturation, primer annealing, and enzymatic extension.

Thermal Cycler

The laboratory instrument engineered to automatically cycle through precise temperature changes required for the successive stages of PCR amplification.

16S rRNA

A universally conserved ribosomal RNA component utilized as a genetic marker gene for comparing microorganisms, determining taxonomic relationships, and performing sequence-based microbial identification.

Phylogenetic Tree of Life

A universal branching model depicting evolutionary relationships among organisms, classifying all cellular life into three primary domains: Bacteria, Archaea, and Eucarya.

Taq Polymerase

A thermostable enzyme derived from a thermophilic bacterium that catalyzes the polymerization of free dNTPs into a nascent complementary DNA strand during PCR.

dNTPs

Deoxynucleotide triphosphates; the equimolar mixture of nucleotide bases that serve as the chemical building blocks for synthesizing new DNA strands in PCR.

Tris-HCl (in PCR Buffer)

A buffering agent present in the PCR master mix that maintains a stable, optimal pH environment required for Taq polymerase catalytic activity.

EDTA (in PCR Buffer)

A chelating agent incorporated into the PCR reaction mixture that binds divalent cations to inhibit nucleases and keep the DNA template intact.

MgCl2 (in PCR Buffer)

A chemical additive that supplies magnesium ions ($\text{Mg}^{2+}$), which act as an essential enzymatic cofactor for Taq polymerase function.

KCl (in PCR Buffer)

A salt component of the PCR buffer that neutralizes negative electrostatic charges along the DNA backbone, thereby facilitating primer annealing.

Triton (in PCR Buffer)

A non-ionic surfactant included in the PCR buffer to stabilize the DNA molecules during thermal cycling.

Primers (PCR)

Short, single-stranded oligonucleotide sequences designed to flank and locate specific target DNA fragments, providing a starting point for polymerase synthesis.

Denaturation (PCR Step)

The reaction phase performed at approximately $95^\circ\text{C}$ that breaks hydrogen bonds between bases, causing double-stranded DNA to separate into single template strands.

Annealing (PCR Step)

The reaction step carried out at approximately $55^\circ\text{C}$ to $56^\circ\text{C}$ for $30\text{ s}$, allowing forward and reverse primers to bind complementary target sequences on single-stranded DNA.

Extension (PCR Step)

The enzymatic elongation step performed at $72^\circ\text{C}$ where Taq polymerase builds the new complementary DNA strand.

Master Plate

An agar plate culturing unique bacterial isolates grown in isolated, numbered colonies that serves as the stock source for transfer onto screening plates.

ESKAPE Pathogens

A group of clinically significant, opportunistic bacterial species commonly associated with multidrug-resistant nosocomial infections: Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species.

ESKAPE-Safe Relative

A non-pathogenic or reduced-virulence bacterial counterpart used safely in undergraduate laboratory settings to test the antimicrobial potential of soil isolates.

Enterococcus raffinosus

The designated ESKAPE-safe relative utilized as a laboratory surrogate for Enterococcus spp.

Staphylococcus epidermidis

The designated ESKAPE-safe relative used as a surrogate organism for the pathogen Staphylococcus aureus.

Escherichia coli (as an ESKAPE Relative)

The designated ESKAPE-safe surrogate organism used in place of the clinical pathogen Klebsiella pneumoniae.

Acinetobacter bayly

The designated ESKAPE-safe relative used as a non-pathogenic model organism for Acinetobacter baumanni.

Pseudomonas putida

The designated ESKAPE-safe relative utilized as a surrogate for testing against Pseudomonas aeruginosa.

Enterobacter aerogenes

The designated ESKAPE-safe relative utilized as a model organism representing Enterobacter species.

Bacterial Lawn

A uniform, confluent layer of bacterial growth created across an entire agar plate, typically prepared using 3-way swabbing.

Pasteurization

A heat-treatment process that destroys heat-sensitive pathogenic microorganisms and decreases spoilage bacteria without sterilizing the product completely.

Broad Spectrum Antibiotic

An antimicrobial agent capable of inhibiting or killing a wide range of bacterial taxa, typically spanning both Gram-positive and Gram-negative groups.

Narrow Spectrum Antibiotic

An antimicrobial compound specifically active against a select group or family of microorganisms.

Human Microbiome

The collective community of microorganisms that normally inhabit the surfaces and cavities of the human body, contributing to host health rather than causing obligatory harm.