Lecture 7: Sampling and Enumeration

Question 1

What are the components of a quality assurance plan?

Answer 1

1. Sampling
   - Number, type, location, timing
   - Replication
2. Methods
   - Specific techniques, etc.
3. Storage
   - Preservation (refrigeration, freezing, fixation)
   - Duration issues

Question 2

What are the challenges in sampling?

Answer 2

• Replication: can be difficult to ensure it’s the same
• Little to no baseline data
• Controls: need to find something that doesn’t have the thing you’re researching
• Little to no data in acceptable ranges

Question 3

What is the function of transects?

Answer 3

You can just sample along the transects instead of the entire sample area
- Ex. a lake, sampling along even x amount of meters

Question 4

What are some issues to consider in soil/sediment sample collection?

Answer 4

• Contamination: hard to decontaminate such a large drill
• Compaction: a portion of sediment might clog your piston
• Water content: sometimes too much water

Question 5

How are soil/sediment samples collected?

Answer 5

• Coring: piston corer (can get nice stratification)
• Van Veen grab

Question 6

Why is aquatic sampling easier than soil/sediment sampling?

Answer 6

• More homogeneous
• Easier to get samples, even remotely

Question 7

What are some difficulties in aquatic sampling?

Answer 7

1. May have to filter/concentrate large volumes of water
2. Viruses require care
   - High volumes
   - Adsorption, elution, reconcentration

Question 8

What are some bottles and nets used in aquatic sampling? When would you use each one?

Answer 8

• Plankton net tows: to isolate microbes of different sizes
• Niskin bottle:
• CTD rosette: measure physical characteristics of water

Question 9

Compare and contrast crossflow filtration and direct flow filtration.

Answer 9

1. Crossflow (aka tangential flow)
   - High permeate rate
2. Direct flow (aka dead-end)
   - Low permeate rate

Question 10

What is eDNA sampling?

Answer 10

• Way to monitor different species in an environment
• Looking at all the DNA in a sample
• Snapshot of total community diversity

Question 11

How is air sampling conducted?

Answer 11

Impaction on agar

Question 12

What is enumeration?

Answer 12

Determining how many microbes there are in a sample

Question 13

What are the difficulties in the environment in enumeration?

Answer 13

• Small size of microbes - hard to see to count
• Hard to determine viability of samples

Question 14

What is the Great Plate Anomaly?

Answer 14

In certain environments, <1% of microorganisms are cultivable (VBNC)

Question 15

List viable count methods (aka standard plate count).

Answer 15

• Plate counts and most probable number
• Selective growth medium

Question 16

What does growth (as in on a plate) represent?

Answer 16

Presence and growth potential
- NOT the environmental potential
- Does not give total count like direct counting

Question 17

Explain serial dilution.

Answer 17

Question 18

How do you get the most probably number (MPN)?

Answer 18

• Serial dilution of a liquid sample
• Growth in successive dilutions allows estimation of numbers in original smaple

Question 19

How is turbidity measured?

Answer 19

Optical density
- Measure how much light passes through the sample
- Use the same media as your blank

Question 20

What tools are used to take direct counts?

Answer 20

1. Microscopic measures
   - Counting chamber
   - Gridded filters
2. Epifluorescence microscopy
   - Fluoresce when you hit them (natural autofluorescence, acridine orange, DAPI)

Question 21

What are fluorochromes?

Answer 21

Molecules that emit light in response to irradiation

Question 22

What is stokes shift?

Answer 22

Emission is always of longer wavelength than excitation

Question 23

What is photobleaching?

Answer 23

Fading of FCs

Question 24

What are the advantages and drawback of direct counts?

Answer 24

1. Advantages
   - Higher numbers (10-100x more than viable counts)
   - Not environment-specific
   - Correlation with biomass
2. Drawbacks
   - Hard to tell live from dead
   - Debris interference
   - Inability to take any further analysis (typically fix cells with formaldehyde)
   - Laborious

Question 25

What are automated direct counts?

Answer 25

Digital image analysis software to count cells - Must be a "clean", clear, bright sample - Expensive

Question 26

What is flow cytometry? When would you use it?

Answer 26

- Analyzes the size, shape, and properties of cells by passing them through a tube single-file and then shooting a laser beam at them - Soil sample, algal blooms, seawater samples for counting phytoplankton

Question 27

What is cryopreservation protocol?

Answer 27

Question 28

Which habitats are easiest to get a representative sample? A. Soil B. Seawater C. Deep subsurface D. Lake sediment

Answer 28

**A. Soil B. Seawater** C. Deep subsurface D. Lake sediment