Transcript Slide 1

Using Microbial Ecology to Teach Experimental
Design and Sampling Methods
Mary E. Allen, Hartwick College
Ruth A. Gyure, Western CT State University
American Society for Microbiology
MicrobeLibrary
POWERPOINT SLIDES TO SUPPLEMENT
EXERCISE 2:
Microbial Community Structure and Organization
SEM of Bacteria in Mouse Ileum. Source: Ken Rozee et al., Microbe Library, ASM
PART I
Introductory material
that will help instructor
prepare and execute
the exercise
REVIEW
CONSIDERATIONS WHEN SAMPLING
MICROBES IN THE NATURAL
ENVIRONMENT:
1. Detection and visualization
2. Definition and differentiation of taxa
3. Cultivation issues and ecological relevance
4. Interdependence (consortia)
5. Adherence (biofilms)
WHAT IS A
PROKARYOTIC
SPECIES???
Current, genetic view of species-level difference:
DNA-DNA hybridization rate of about 70%
16s rDNA similarity of >98%
Figures from Ogunseitan, Microbial Diversity
These isolates are all in the
same genus.
Why so different?????
THEY CHANGE
MORPHOLOGY DEPENDING
ON THE CULTURE MEDIUM!
CAN I ISOLATE?
IS THE ISOLATE ECOLOGICALY RELEVANT?
Photo credit: D. J. Patterson, L. Amaral-Zettler and V.
Edgcomb. Courtesy of micro*scope.
There is a wide diversity of organisms in the marine sample illustrated above.
However, when plated on standard growth media in the lab, very few of these
species will grow. Even if an isolate is obtained, how do we know it is one
that plays an important role in the system?
MICROBIAL
CONSORTIA
Example:
AMETHOX
This pictures was made using "Fluorescent in Situ Hybridization" (FISH). The
organisms in red represent the anaerobic methanotrophic Archaea (ANME) and
the green organisms are sulfate reducing bacteria (SRB).
Picture made by C. Jagersma at the Max Planck Institute Bremen.
See notes that accompany this image.
BIOFILMS
•Elude traditional sampling and visualization methods
•Cells tightly clumped and difficult to separate, isolate, identify
•Phenotypic variation difficult to assess after sampling
S. Lowry—University of Ulster—Stone/Getty Images
Diversity: Species composition, i.e. how
many different species are there, and
how are the numbers distributed among
them
Structure: How are these individuals
distributed and organized in the
environment?
These simplistic diagrams illustrate some possible ways in which organisms
may be distributed in the environment.
Actual distribution will show combinations of these patterns, and will also differ
depending upon the scale at which you observe, sample and and measure it.
Terrestrial environment:
Soil
MARINE “SNOW”
‘Hot spots’ of bacterial
concentration and
Crumb
activity, contributing to
global cycling of carbon
and nutrients
Photo and Diagram from Brock
Biology of Microorganisms,
Azam and Long, Nature, 2001
Madigan and Martinko
These examples illustrate that microbes will organize themselves in response to
environmental conditions and interactions at very small scale; However, such
interactions may not be of interest when larger scale systems are being studied
SCALING – At what scale does one need to sample,
observe, and measure in order to answer the question
posed?
Pictured here – from global scale (oceans) to regional and
local scales; intercellular scale (symbiont community in a
protozoan population)
BRAINSTORM: HOW DOES ONE MAKE
DECISIONS ABOUT SAMPLING?
1. Size of population, community or system, area of interest
2. Scope of study?
3. Budget?
4. Variability (standard deviation, error)? depends on heterogeneity,
abundance, distribution, both spatial and temporal, method, etc. (See link
below for great discussion about statistical considerations )
5. Technological ability (Can one directly observe organisms or cells? Can one
target individuals or groups with specificity? How precise are the units of
measurement?)
6. Experimental approach
7. SCALE! (many issues)
STATISTICAL METHODS AND PRIMER, ENVIRONMENTAL APPLICATIONS:
http://epa.gov/bioindicators/statprimer/
Sampling soil microbes in a relatively
static soil community
Even here – there are challenges of
scaling, and dealing with localized
heterogeneity that could mask larger
scale changes
Isopleth - A of interest.
Location of Proposed Phytormediation Cells
th
N or
Stre
et
59.70 ft
L ow M e r c ur y I s opltth (1-20 ppm)
Low Mercury Concentration
A1
A3
Ctl
mer A
High M e r c ur y I s opltth (96-320 ppm)
200 cm
100 cm
115.22 ft
200 cm
A6
W
ate
r
Ctl
wt
B ro
A5
ok
Di
rec
tio
A7
A8
A9
wt
Ctl
Barnum C
o
urt
n
mer A
Plot Key (Enlarged to show detail)
Plot ID
A1
Approximate soil sampling location
A1-1
mer A = presence of mer A gene
Ctl = No Plants
}
Di
rec
tio
n
53.13 ft
A1-2

Populus deltoides type
wt = Wild Ty pe
W
ate
r
187.46 ft
(Isopleth areas are approximate)
nz a
A4
mer A
a
Koh
wt
I nte r me diate M e r c ur y I s opltth (29-50 ppm)
A2
Reference Position
WT
A1-3
A1-4
43.58 ft
17.91 ft
80.60 ft
20.30 ft
81.19 ft
EXERCISE 2:
Let’s “sample” some diagrammatic representations
of microbial communities!
In this exercise, sampling and measuring diversity
alone is not the goal. We would also like to sample
in a way that informs us about the distribution of
organisms present.
HERE IS AN
EXAMPLE OF
ONE
COMMUNITY
THAT MAY BE
ASSIGNED
BEFORE YOU RECEIVE A COMMUNITY, YOU
MUST DECIDE ON A PLOT SAMPLING SCHEME.
1. STEP 1: Trace your 3 large plots onto the
transparency after deciding on a placement plan. When
finished, ask instructor for your sample community.
2. STEP 2: Record the number of individuals for each
species observed in each plot on your data sheet.
3. STEP 3: Discuss the following questions as a group, as
a class – or later as a homework assignment.
a. How is this community different from one that might exist in the water
column of a lake, for example?
b. If you were to imagine a habitat that this simulated diagram might
represent, what would it be?
c. When sampling a community of this sort, does the scale of the area of
investigation matter? In what ways will it affect your sampling approach?
d. By taking data from 9 small plots instead of the 3 relatively large plots
you just did (adding up to same comparable area) – which do you feel
would give a more realistic or accurate picture of the actual community?
What are the advantages and disadvantages of each approach? Do you
think each method would give the same calculated Simpson’s index?
Why or why not?
e. Finally, would a line transect approach be appropriate for sampling this
community? Why or why not?
STEP 4: Repeat the sampling this time tracing 9
small plots rather than 3 larger ones. You must
use same rules for the placement scheme!
Record data as before
STEP 5: Draw 3 transect lines and and determine
a placement scheme. Record data.
STEP 6: Calculate Simpson’s index of diversity for
each of the 3 sampling approaches.
PART II
Post-exercise
discussion and review
In the next 3 slides you will see what the 4
communities actually looked like, and an example of
how the 3 sampling schemes might be applied to
Communities C and D (with a sample data set for
class discussion)
A
B
C
D
v
v
v
C
D
EXAMPLE OF HYPOTHETICAL DATA
Site
Number if individuals (count)
C
Small plots
1
2
0
0
0
0
0
0
0
0
Species
A
B
C
D
E
F
G
H
I
J
K
L
M
3
0
0
0
0
4
3
5
1
1
5
0
0
0
0
6
0
0
0
0
7
1
0
0
0
8
0
0
0
0
9total
0
0
0
0
Large plots
1
4.0
0
5.0
0
1.0
0
1.0
0
0.0
8
0.0
18
0.0
2
0.0
1
0.0
0.0
0.0
0.0
0.0
2
0
0
0
0
0
0
0
0
11.0
Site
Number if individuals (count)
D
Small plots
1
2
8
0
3
2
2
2
1
3
1
4
1 16
2
1
0
1
0
0
0
0
0
0
0
0
0
0
Species
A
B
C
D
E
F
G
H
I
J
K
L
M
3 4
0 0
1 0
1 1
0 0
0 0
0 1
6 0
4 0
5 0
0 0
0 0
5 0
3 11
5
0
0
1
0
0
0
0
2
0
0
0
14
10
6
0
0
0
0
0
0
0
0
2
1
2
0
0
7
1
1
2
1
0
0
0
4
0
0
0
0
0
8
0
0
0
0
1
0
1
0
0
0
0
0
0
9total
0
9.0
2
9.0
0
9.0
0
5.0
0
6.0
5
23.0
0
10.0
0
11.0
0
7.0
0
1.0
0
2.0
0
19.0
1
25.0
136.0
Transects
3total
1
0
0.0
0
0
0.0
0
0
0.0
0
0
0.0
0
0
8.0
0
0
18.0
0
0
2.0
0
0
1.0
0
0.0
0.0
0.0
0.0
0.0
2
0
0
0
0
0
0
0
0
3total
0
0
0
0
0
0
0
0
29.0
Large plots
1
6
5
4
1
1
12
4
1
1
0
7
8
3
2
5
6
1
3
2
9
2
0
0
0
5
9
1
Transects
3total
1
8
19.0
1
4
15.0
2
6
11.0
0
1
5.0
0
2
5.0
0
10
31.0
1
4
10.0
3
0
1.0
2
0
1.0
5
1
1.0
0
8
20.0
0
6
23.0
1
2
6.0
0
148.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2
1
1
0
1
0
0
0
0
1
1
2
0
0
3total
0
0
0
0
0
0
1
2
1
0
0
2
3
2.0
3.0
0.0
1.0
0.0
1.0
4.0
4.0
7.0
1.0
2.0
3.0
3.0
31.0
Some examples from the literature to
review and discuss concepts of
diversity, scaling, distribution
Drop-size soda lakes (Qvit-Raz , Genetics, 2008)
This is an amzing study showing actual differences in complex communities that go
through successional stages in tiny drops that form from dew and exudates of the
Tamarax tree which releases salty compounds.
An ecosystem where large diversity at very small-scale was
observed, relating to microgradients of physical and chemical
variables.
GUT MICROBIAL COMMUNITY
Ley et al., 2006
Is a community with a large number of closely related species more diverse
than one with fewer numbers of more distantly-related ones?
Here we notice that although the gut community is known to be “very diverse,”
microbial mats in nature tend to show far greater diversity when taxa are
differentiated at higher group levels rather than species level.
Our view or estimate of diversity will depend upon how we decide to define it…
BIOGEOGRAPHY:
IS EVERYTHING
EVERYWHERE???
Fenchel and Findlay’s work,
as reported in Science, 2005
See notes that accompany
this slide
PNAS, 2007
What types of environments on earth are “most diverse?” Why do we care?
(This study suggests that generally speaking, at a global level, salty
environments are more diverse than freshwater or soil)
Findlay 1982:
Both approaches yield similar estimates
of abundance, but larger scale sampling
tends to give poor representation of
distribution/patchiness
WHAT ABOUT MOLECULAR ANAYSIS OF MICROBIAL COMMUNITIES?
Ranjard et al. 2003 tested effect of SIZE of soil sample used in molecular
community analysis.
For bacteria, SIZE OF SAMPLE did not affect ability to distinguish unique
communities from one another. For fungi, samples <1g may not be adequate.
HOWEVER, in larger samples, technique of DNA extraction is biased toward
dominant organisms and may not accurately assess diversity.
FOR BACTERIA: Many subsamples are often taken, and it is the selection of
the sampling location that will give best indicator of larger scale abundance
and diversity
When is use of transects appropriate?
•One example: when one hypothesizes a gradient relationship and wishes to
test it using regression analysis, see below!
•In this study, researchers looked for change in numbers of fecal indicator
organisms across various transects in relation to water’s edge.
•Keep in mind, most aquatic depth sampling is, in effect – transect sampling
Desmarais 2002
FINAL THOUGHTS:
•The question being asked drives the experimental design.
•The practical limitations of sampling limit the type of question that
can be asked – and answered!
•Pilot sampling is always essential to assess the nature of the
system!!
Students will better understand scientific process when
we appreciate the considerations that go into every
scientist’s sampling and measurement plan!