Techniques in experimental microbiology

Question 1

Give five examples of considerations when sampling

Answer 1

Representing the whole field, sample type, equipment, sterility and cross-contamination, changing the microenvironment

Question 2

Give four examples of equipment used for soil/sediment sampling, shallowest to deepest

Answer 2

Trowel, corer/auger (specified depth), mechanical coring rig, Jenkin surface mud sampler (substrate in water)

Question 3

What can be a problem with hand pumps to collect groundwater from boreholes?

Answer 3

Difficult to keep sterile

Question 4

What can be used for the sterile collection of water and what are its limitations?

Answer 4

Johnson-ZoBell water sampler
Cannot be used for open water sampling

Question 5

What can be used to collect water at greater depths?

Answer 5

Niskin bottles
Lid closes at the specified depth
No cross contamination

Question 6

What else can be used to sample microbes?

Answer 6

Minerals

Question 7

What sensors can be used to collect in situ measurements for metadata in water?

Answer 7

CTD (conductivity, temperature, depth) diver
Other sensors for chlorophyll fluorescence, pH, O2 conc

Question 8

What sensor can be used to collect in situ measurements for metadata in gas?

Answer 8

GasClam for O2, CO2, H2S, temperature, pressure

Question 9

What sensor can be used to collect in situ measurements for metadata in sediment?

Answer 9

Handheld XRF for pH and Eh probes

Question 10

How are water samples processes if the cell numbers are low?

Answer 10

Concentration by centrifugation or filtration
Pore size of filters can be used to target different groups

Question 11

What is aimed to be preserved in soil sampling?

Answer 11

3d structure because it shows symbiosis

Question 12

What does the technique for sample storage depend on?

Answer 12

How it will be analysed

Question 13

How are samples stored in order to preserve specific cellular components?

Answer 13

Frozen at -20 to -70°C in liquid nitrogen

Question 14

How are samples stored for cell counts?

Answer 14

Fixative solution, e.g. formaldehyde or alcohol
Can introduces changes in microbial community

Question 15

When should samples not be frozen?

Answer 15

Cultivation or activity is being measured
Should be measured asap after sampling
Storage at 4°C
Microbial communities may have changed

Question 16

Give three methods of measuring microbial numbers

Answer 16

Direct count (gives highest number), biomass estimation, culturing

Question 17

Give three methods of direct counting of microbes

Answer 17

Light microscopy, fluorescent dyes, flow cytometry

Question 18

Give four examples of chemical assays (potential targets) of biomass estimation

Answer 18

ATP (not stable: active biomass), cell envelope components e.g. lipopolysaccharide, chitin, total phospholipid fatty acids

Question 19

Give two benefits and a limitation of culturing

Answer 19

Can target specific organisms and get an indication of activity
Underestimates diversity

Question 20

What are the two main methods of counting from culturing?

Answer 20

Plate counts and MPN (most probable number) counts (in liquid culture)

Question 21

Describe the process of plate counts of cultured samples

Answer 21

Samples are diluted is there are large numbers of bacteria, diluted sample is spread on agar plates, then incubated and the colonies are counted

Question 22

What are the two types of media used in culturing?

Answer 22

Selective and diagnostic

Question 23

Give three methods of measuring microbial activities

Answer 23

Direct chemical measurements, specific metabolic inhibitors, radiotracers

Question 24

Describe direct chemical measurements for microbial activities

Answer 24

Quantifying changes in concentration of biologically transformed chemical species
e.g. de/nitrification, metal reduction/oxidation
Measures potentials, not in situ rates
Limited sensitivity

Question 25

Describe the use of specific metabolic inhibitors for measuring microbial activities

Answer 25

Added to the sample and effect on process, e.g. sodium reduction, methanogenesis, measured

Question 26

Describe the use of radiotracers for measuring microbial activities

Answer 26

Uptake of radiolabeled compounds measured

Question 27

Summarise the limitations of culture-based sampling techniques

Answer 27

90+% of microbes cannot be isolated in the lab

Question 28

Give three benefits to using 16S gene in analysis

Answer 28

Found in all prokaryotes, can be isolated, differentiates between bacteria

Question 29

Give four limitation of 16S rRNA gene sequencing

Answer 29

Taxonomy only, limited resolution, contamination, relative proportions (not quantitative)