Week 2 - Techniques for environmental microbiology

3 components of the geo-bio interface

Cellular physiology

Aqueous environment

Mineralogy

Why take samples

Difficult to measure a whole ecosystem

Heterogeneity makes replication important

Downside of samples

Microenvironment is changed during, so components behave differently

Sampling sediments/soil

Topsoil with trowel

Deeper with boreholes/corers

In lakes with a Jenkin sampler

Sampling water

Johnson-ZoBell → sucks water up into the bottle via a glass tube, maintaining sterility with a rubber stopper to create a vacuum

Groundwater pump

Niskin bottle for deeper water and larger samples, sealing airtight at desired depth

CTD diver measures Conductivity, Temperature and Depth in situ

How do you concentrate biomass in a water sample

Filtration and centrifuge

Methods of sample storage

Freeze between -20 → -70C using liquid nitrogen/dry ice for direct measurement of cellular components

Fixative solution (formaldehyde, alcohol, commercial) for cell counts (cant freeze due to ice crystals rupturing)

Cultivation or activity measurements as soon as possible after sampling or after storage at 4^oC

Geochemical analysis of samples

pH probe

Redox probe

Ion Chromatography (IC)

Inductively Coupled Plasma - Atomic Emission Spectrometry

(ICP-AES)

GC

TIC/TOC

Electron microscopy

Xray techniques

3 ways to measure microbial numbers

Direct counts

Biomass estimation

Culturing

Direct counts

Use a microscope

Can be difficult to distinguish between living and dead cells and debris (aided by fluorescent dyes)

Good for larger cells

Overestimates by up to 1-2 orders of magnitude

Biomass estimation

Measures biomass and total numbers

Can target ATP cell envelope components

Culturing

Isolation of selected media

Targets specific organisms

Plate counts

Underestimates diversity

Methods of measuring microbial activity

Radiotracers → uptake of radiolabelled compounds, degredation of C14 measured

Discoveries in the molecular revolution

rRNA gene isolated

PCR for DNA amplification