Identification of Bacterial Species by 16S rRNA gene sequencing

16S ribosomal RNA PCR

16S rRNA

• all bacteria contain 16S rRNA

• 9 variable regions interspersed among conserved regions along 16S

• sequence of variable regions can be determined → identification of species

Application of 16S rRNA gene sequencing

• clinical diagnosis

• NIH Human Microbiome Project

  • characterise microbial communities found at multiple human body sites

  • look for correlations between changes in microbiome and human health

• metagenomics: bacterial diversity in environment

Sterilisation

Heat

• Steam sterilisation

  • performed in autoclave

  • used for sterilising agar media or other equipment

  • 121°C for 15-30 min at 15 psi

  • ╳ solutions containing organic solvent

  • autoclave tape is used to monitored autoclaving condition

    • white: before sterilisation

    • black: after sterilisation

  • saturated steam under pressure→ denature protein and enzyme at high temp

• Dry-heat sterilisation

  • performed in oven

  • used for sterilising metalware

  • ╳ suitable for plasticware (melt)

  • 160°C for 3 hr; 170°C for 1 hr; 180°C for 30 min

Filtration

• pore size of filter: 0.22μm (smaller than most micro-organism) → retain microbes on membrane

• used for heat labile (不穩定) or flammable reagent

• ╳ applicable to viruses as too small

UV irradiation

• DNA or RNA damage → inactivate and inhibit growth of bacteria

• wavelengths: 200-280 nm

• performed in a biological safety cabinet

Gram stain

Purpose

• classify bacteria into Gram positive, Gram negative and Gram variable bacteria

• visualise the shape and arrangement of the bacterial cells

Principle

Differences between Gram positive and Gram negative

• Gram positive bacteria

  • single membrane

  • thick peptidoglycan layer

  • lower lipid content of cell wall

• Gram negative bacteria

  • double membrane

  • thin peptidoglycan layer

  • higher lipid content of cell wall

Peptidoglycan

• a carbohydrate backbone of alternating units of NAG (N-acetylglugosamine) and NAM (N-acetylmuramic acid) residues linked to peptide

• peptide cross bridges linking the tetra peptide on a NAM to an amino group of a terapeptide on a neighbouring NAM

• → forming a larger polymer network

Principle of Gram stain

• stain with crystal violet dye

• then add Gram’s iodine solution (iodine & potassium iodide)to form a complex between crystal violet and iodine

  • complex is a larger molecule than iodine and crystal violet and insoluble in water

• use ethyl alcohol or acetone to decolorize the sample

  • dehydrate the peptidoglycan layer → shrinking and tightening the layer

• large crystal violet-iodine complex: ╳penetrate the tightened peptidoglycan layer

  • → trapped in cell in Gram positive bacteria

• outer layer of Gram negative bacteria is degraded

  • → thinner peptidoglycan layer ╳ retain the complex→ colour lost

• counter stain using weakly water soluble safranin (red)

  • Gram positive bacterial cells remain purple (safranin is lighter than crystal violet)

  • Gram negative bacterial cells are stained red

Using light microscope

• magnification: 10X (eyepiece) & 100X (objective)= 1000X

• oil immersion lens

  • oil has the same refractive index as glass

  • more light is gathered→ brighter image and higher resolution

  • only suitable for specific objective lens (e.g. 100X)

Morphology of bacteria

Shape

• coccus (sphere)

• bacillus (rod)

• spiral

  • vibrio

  • spirillum

  • spirochete

Arrangement

• diplo-

• strepto- (forming a line)

• tetrad

• sarcinae

• staphyl- (a cluster)

Determination of nucleic acids concentration by spectrometry

Beer-Lambert Law

Formulas

• T = I/I₀

• A (Absorbance or extinction or optical density) = log(1/T) = log(I₀/I) = ε_λcℓ

  • T = transmittance

  • I₀ = intensity of incident light

  • I = intensity of transmitted light

  • ε_λ= molar absorbance coefficient at wavelength λ( M^-1·cm^-1 or dm^3·mol^-1·cm^-1)

    • 0.02 for double stranded DNA

  • ℓ = path length of sample

    • 1cm for a cuvette

    • 0.05 cm for microplate ot μDrop plate

  • c = concentration of absorbing solution ( M or mol·dm^-3)

Deviation

• Beer-Lambert law only valid for low concentration

• higher concentrations → association of molecules (they have different light absorption characteristics)

Determining nucleic acid concentration

• measure absorbance at 260 nm, 280 nm and 230 nm

  • determine A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratio to check purity of nucleic acid

• purity of nucleic acid

  • A₂₆₀/A₂₈₀

    • A₂₆₀/A₂₈₀ ≈ 1.8 for pure DNA

    • A₂₆₀/A₂₈₀ ≈ 2.0 for pure RNA

    • lower A₂₆₀/A₂₈₀ indicates protein contamination

    • ratio also depend on sample pH, wavelength accuracy and nucleic acid composition

  • A₂₆₀/A₂₃₀

    • A₂₆₀/A₂₃₀ > 2.0 for pure DNA or RNA

    • A₂₆₀/A₂₃₀ < 2.0 → possible contamination with phenol, guanidium salt