Techniques in experimental microbiology Flashcards
Give five examples of considerations when sampling
Representing the whole field, sample type, equipment, sterility and cross-contamination, changing the microenvironment
Give four examples of equipment used for soil/sediment sampling, shallowest to deepest
Trowel, corer/auger (specified depth), mechanical coring rig, Jenkin surface mud sampler (substrate in water)
What can be a problem with hand pumps to collect groundwater from boreholes?
Difficult to keep sterile
What can be used for the sterile collection of water and what are its limitations?
Johnson-ZoBell water sampler
Cannot be used for open water sampling
What can be used to collect water at greater depths?
Niskin bottles
Lid closes at the specified depth
No cross contamination
What else can be used to sample microbes?
Minerals
What sensors can be used to collect in situ measurements for metadata in water?
CTD (conductivity, temperature, depth) diver
Other sensors for chlorophyll fluorescence, pH, O2 conc
What sensor can be used to collect in situ measurements for metadata in gas?
GasClam for O2, CO2, H2S, temperature, pressure
What sensor can be used to collect in situ measurements for metadata in sediment?
Handheld XRF for pH and Eh probes
How are water samples processes if the cell numbers are low?
Concentration by centrifugation or filtration
Pore size of filters can be used to target different groups
What is aimed to be preserved in soil sampling?
3d structure because it shows symbiosis
What does the technique for sample storage depend on?
How it will be analysed
How are samples stored in order to preserve specific cellular components?
Frozen at -20 to -70°C in liquid nitrogen
How are samples stored for cell counts?
Fixative solution, e.g. formaldehyde or alcohol
Can introduces changes in microbial community
When should samples not be frozen?
Cultivation or activity is being measured
Should be measured asap after sampling
Storage at 4°C
Microbial communities may have changed
Give three methods of measuring microbial numbers
Direct count (gives highest number), biomass estimation, culturing
Give three methods of direct counting of microbes
Light microscopy, fluorescent dyes, flow cytometry
Give four examples of chemical assays (potential targets) of biomass estimation
ATP (not stable: active biomass), cell envelope components e.g. lipopolysaccharide, chitin, total phospholipid fatty acids
Give two benefits and a limitation of culturing
Can target specific organisms and get an indication of activity
Underestimates diversity
What are the two main methods of counting from culturing?
Plate counts and MPN (most probable number) counts (in liquid culture)
Describe the process of plate counts of cultured samples
Samples are diluted is there are large numbers of bacteria, diluted sample is spread on agar plates, then incubated and the colonies are counted
What are the two types of media used in culturing?
Selective and diagnostic
Give three methods of measuring microbial activities
Direct chemical measurements, specific metabolic inhibitors, radiotracers
Describe direct chemical measurements for microbial activities
Quantifying changes in concentration of biologically transformed chemical species
e.g. de/nitrification, metal reduction/oxidation
Measures potentials, not in situ rates
Limited sensitivity
Describe the use of specific metabolic inhibitors for measuring microbial activities
Added to the sample and effect on process, e.g. sodium reduction, methanogenesis, measured
Describe the use of radiotracers for measuring microbial activities
Uptake of radiolabeled compounds measured
Summarise the limitations of culture-based sampling techniques
90+% of microbes cannot be isolated in the lab
Give three benefits to using 16S gene in analysis
Found in all prokaryotes, can be isolated, differentiates between bacteria
Give four limitation of 16S rRNA gene sequencing
Taxonomy only, limited resolution, contamination, relative proportions (not quantitative)
