Welcome to the CMEIAS v.1.

28 Tutorial Worksheet
for the CMEIAS v. 1.28 Training Tutorial Macro
Prepared by Frank Dazzo, Hassan Hammoud & Jose Zurdo
Center for Microbial Ecology, & Dept. of Microbiology & Molecular Genetics
Michigan State University, East Lansing, Michigan 48824 USA
Figure. 1. Three community images used in this training tutorial.
CoccusRegularRodFilament.tif

Community-A.tif Community-B.tif
 Table 1. CMEIAS / ImageTool Object Analysis of
CocciRodFilament.tif
Object Length Width WLR WLR (a-z)
Mean 3.81 0.85 0.30 0.30
Std. Dev. 4.06 0.38 0.17 0.17
#1 5.40 1.20 0.23 0.01
#2 3.70 1.30 0.35 0.03
#3 7.30 1.80 0.25 0.03
#4 5.50 1.20 0.21 0.08
#5 3.50 1.20 0.34 0.09
#6 43.20 0.60 0.01 0.09
#7 3.20 0.50 0.14 0.10
#8 3.40 0.90 0.26 0.11
#9 4.10 1.10 0.28 0.12
#10 1.30 0.40 0.32 0.12
#11 1.10 0.90 0.81 0.13
#12 4.20 0.50 0.12 0.13
#13 1.10 0.90 0.81 0.13
#14 4.10 1.10 0.27 0.13
#15 4.50 1.20 0.27 0.14
#16 1.30 0.40 0.31 0.14
#17 5.50 1.10 0.19 0.14
#18 1.50 1.10 0.73 0.14
#19 6.60 1.10 0.17 0.15
#20 0.70 0.50 0.75 0.15
#21 3.20 1.20 0.36 0.16
#22 4.90 0.90 0.18 0.16
#23 1.20 0.40 0.29 0.17
#24 2.00 0.70 0.33 0.17
#25 3.20 0.40 0.11 0.17
#26 3.40 1.00 0.29 0.17
#27 4.80 0.50 0.10 0.17
#28 5.70 1.00 0.17 0.17
#29 4.00 1.10 0.27 0.18
#30 1.10 0.40 0.34 0.18
#31 2.50 0.70 0.27 0.18
#32 1.10 0.90 0.81 0.18
#33 3.20 0.80 0.26 0.18
#34 6.70 0.60 0.08 0.18
#35 2.30 0.40 0.17 0.18
#36 3.70 0.70 0.20 0.18
#37 4.40 1.20 0.28 0.18
#38 2.30 0.50 0.23 0.19
 Fig. 2. Length - Width Size Distribution CocciRodFilament.tif
2.5

1.5
Cell Width (um)

0.5

0
0.0 10.0 20.0 30.0 40.0 50.0
Cell Length (u m)
 Fig. 3 Distribution of Width/Length ratios for the cells in
the CoccusRodFilament.tif image
0.90

0.80

0.70
Cocci
Cell Width / Length Ratio

0.60

0.5 W/L
0.50

Regular Rods
0.40

0.30

0.20

0.10 0.0625 W/L

Unbranched Filaments
0.00
0 50 100 150 200
Sorted Cell Number
Fig. 4. New image showing cocci, regular rods, and unbranched filaments pseudocolored by
an IT/CMEIAS-1 classification of their Width:Length ratio.
 Table 2. Shape classification of cells in the
CoccusRodFilament.tif image using the IT/CMEIAS-
1 object classifier

Width / Cell Count

Mean Length Class 56.67
Std. Dev. Ranges 82.78
Unbranched Filaments < 0.06 3
Regular Straight Rods 0.06 - 0.50 152
Cocci > 0.50 15
 Table 3. Cell Size (Area) Community Object Analysis
Cell Size (Area) Unsorted (um2) Cell Size (Area) Ascending Sort (um2)
Object Number Community-A Community-B Community-A Community-B
Mean 3.75 2.93 3.75 2.93
Std. Dev. 2.97 3.54 2.97 3.54
#1 18.01 0.94 0.31 0.94
#2 6.30 0.71 0.32 0.16
#3 7.25 2.25 0.35 0.16
#4 8.98 6.23 0.35 0.18
#5 10.24 35.44 0.36 0.19
#6 5.70 5.59 0.38 0.22
#7 2.51 7.51 0.39 0.26
#8 4.18 6.97 0.40 0.31
#9 1.57 8.13 0.42 0.35
#10 7.28 2.62 0.42 0.35
#11 1.15 9.71 0.42 0.37
#12 3.65 0.48 0.43 0.38
#13 4.40 0.45 0.44 0.42
#14 1.10 0.49 0.44 0.43
#15 0.98 4.30 0.48 0.45
#16 1.65 0.22 0.48 0.48
#17 5.08 5.42 0.50 0.48
#18 6.48 3.00 0.66 0.48
#19 4.29 0.53 0.79 0.49
#20 4.57 0.26 0.80 0.49
#21 12.55 6.23 0.82 0.50
#22 2.96 5.30 0.83 0.50
#23 6.17 9.23 0.84 0.50
#24 0.31 0.42 0.89 0.50
#25 3.87 8.40 0.98 0.50
#26 1.46 0.62 0.98 0.50
#27 0.48 0.48 0.98 0.50
#28 3.24 3.09 0.98 0.53
#29 2.09 4.25 0.98 0.53
#30 1.00 2.08 0.98 0.53
#31 4.18 0.50 0.99 0.53
#32 4.25 0.50 1.00 0.53
#33 0.48 0.53 1.00 0.54
#34 5.64 0.61 1.00 0.56
#35 7.16 0.50 1.00 0.58
#36 3.52 1.59 1.00 0.60
#37 0.44 0.53 1.01 0.61
#38 4.11 0.53 1.05 0.62
#39 1.24 0.85 1.07 0.64
 Fig. 5. Cell Size (Area) Distribution -- Communities A and B

35

30

25
Cell Area (um2)

20
Community-A
Community-B
15

10

0
1
9
17
25
33
41
49
57
65
73
81
89
97
105
113
121
129
137
145
153
161

Sorted Cell Number 169

 Table 4. Optimized bin increments and their use in size
classification of cells in Communities A and B

Comparison of Cell Size (Area) Distribution in

Community-A.tif and Community-B.tif Images using the
IT/CMEIAS-1 Object Classifier and Cell Area
Measurements
Area Range Cell Count per Area Bin Class
0.5 (um2) Community-A Community-B
0.98 Mean 10.63 10.63
1.15 Std. Dev. 8.82 11.12
1.79 -0.5 17 19
3.35 0.50 - 0.98 12 33
3.87 0.98 - 0.15 16 9
4.4 1.15 - 1.79 11 19
4.6 1.79 - 3.35 32 37
5.71 3.35 - 3.87 7 9
6.48 3.87 - 4.40 13 12
7.52 4.40 - 4.60 6 2
8.13 4.60 - 5.71 26 6
10.24 5.71 - 6.48 11 4
12.55 6.48 - 7.52 7 9
15.51 7.52 - 8.13 2 4
8.13 -10.24 5 4
10.24 -12.55 1 2
12.55 -15.51 2 0
15.51 - 2 1

Community-A object area range = 0.31 to 18.01. Steps

to optimize upper bin class limits: Plot ascending sort,
move cursor to obvious breaks in data, register x,y
coordinates, use Y coordinate.

This calibration sequence is saved as:

OptimizedAreaBins.ocd
 Fig. 6A. Distribution of Cell Sizes (Area)
100%

15.51 -
90%
12.55 -15.51

10.24 -12.55
80%
8.13 -10.24

70% 7.52 - 8.13

6.48 - 7.52
% Total Cell Areas

60%
4.60 - 5.71

4.40 - 4.60
50%
3.87 - 4.40

40% 3.35 - 3.87

1.79 - 3.35
30%
1.15 - 1.79

0.98 - 0.15
20%
0.50 - 0.98

10% -0.5

0%

Community-A Community-B
 Cell Abundance

0
5
10
15
20
25
30
35
0. 40
50 -0.5

17
0. - 0.

19
98 98

12
1. - 0. 33
15 15

16

9
1. - 1.
79 79

11
3. - 3.

19
35 35
32

3. - 3.
37

87 87

7
9
4. - 4.
40 40

13
4. - 4.

12
60 60

2
5. - 5.
71 71
26

6
6. - 6.
48 48 11

4
7. - 7.
52 52
7
9
8. - 8
13 .1

2
10 - 1 3 4
.2 0.2
Fig. 6B: Cell Size (Area) Distribution

4
5

12 -1 4
4

.5 2.5
Community-B
Community-A

5
1
-1 5
5.
5
2 2

15 1
0

.5
1
-
2
1
Fig. 7 Hierarchy outline of microbial morphotypes classified by CMEIAS
(Fig. 28 of the CMEIAS 1.27 Operator Manual)
 Table 5. Object Analysis of Cells in Community-A.tif using all the Shape Measurement
Features Required for a CMEIAS-2 Morphotype Classification
Object Elongation Roundness Compactness Max Curv. WLR ABR FD0 FD1 FD2 FD3 FD4 FD5 FD6 FD7

Mean 4.13 0.56 0.59 0.02 0.29 0.91 0.48 0.04 0.04 0.12 0.02 0.01 0.01 0.04
Std. Dev. 2.20 0.21 0.16 0.01 0.18 0.11 0.19 0.06 0.06 0.04 0.02 0.01 0.01 0.01
#1 1.08 0.50 0.81 0.01 0.21 0.56 0.22 0.34 0.19 0.14 0.12 0.03 0.05 0.03
#2 4.95 0.47 0.50 0.01 0.19 0.97 0.57 0.01 0.02 0.13 0.01 0.01 0.01 0.05
#3 3.91 0.53 0.54 0.01 0.22 0.90 0.50 0.01 0.02 0.13 0.01 0.01 0.01 0.04
#4 14.39 0.10 0.25 0.03 0.03 0.69 0.87 0.03 0.01 0.14 0.10 0.06 0.06 0.05
#5 14.09 0.06 0.21 0.04 0.02 0.49 0.91 0.31 0.31 0.10 0.06 0.02 0.01 0.06
#6 6.62 0.29 0.42 0.02 0.11 0.92 0.68 0.19 0.18 0.13 0.02 0.03 0.02 0.05
#7 3.58 0.58 0.59 0.02 0.27 0.96 0.46 0.00 0.00 0.14 0.01 0.00 0.00 0.04
#8 4.45 0.45 0.52 0.02 0.18 0.93 0.54 0.14 0.13 0.13 0.00 0.01 0.01 0.05
#9 10.36 0.24 0.35 0.05 0.09 0.98 0.78 0.06 0.06 0.13 0.06 0.01 0.01 0.05
#10 3.66 0.53 0.56 0.01 0.22 0.91 0.49 0.03 0.03 0.14 0.01 0.01 0.01 0.03
#11 3.10 0.68 0.64 0.03 0.36 1.01 0.40 0.03 0.03 0.13 0.01 0.00 0.00 0.03
#12 5.03 0.45 0.50 0.02 0.18 0.99 0.57 0.01 0.01 0.14 0.02 0.01 0.01 0.05
#13 2.85 0.69 0.64 0.01 0.34 0.93 0.38 0.00 0.00 0.11 0.01 0.00 0.00 0.02
#14 3.00 0.70 0.65 0.03 0.34 1.00 0.39 0.02 0.02 0.13 0.01 0.00 0.00 0.03
#15 1.00 1.02 1.02 0.01 0.81 0.81 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01
#16 3.09 0.63 0.61 0.02 0.30 0.90 0.43 0.01 0.02 0.13 0.01 0.00 0.00 0.02
#17 3.38 0.59 0.59 0.01 0.27 0.93 0.44 0.03 0.03 0.13 0.01 0.00 0.00 0.03
#18 4.27 0.52 0.52 0.01 0.22 0.92 0.53 0.01 0.02 0.13 0.01 0.01 0.01 0.04
#19 3.42 0.60 0.59 0.01 0.28 0.95 0.45 0.01 0.01 0.13 0.01 0.01 0.00 0.03
#20 5.03 0.22 0.42 0.03 0.08 0.69 0.72 0.31 0.28 0.10 0.02 0.05 0.04 0.05
#21 3.32 0.56 0.58 0.01 0.25 0.88 0.45 0.01 0.01 0.13 0.00 0.01 0.00 0.03
#22 3.28 0.59 0.60 0.02 0.27 0.91 0.44 0.02 0.01 0.13 0.01 0.01 0.01 0.03
#23 4.08 0.50 0.54 0.01 0.21 0.93 0.51 0.01 0.01 0.14 0.01 0.00 0.00 0.04
#24 2.62 0.82 0.70 0.03 0.39 1.01 0.40 0.02 0.02 0.12 0.01 0.02 0.02 0.03
#25 2.71 0.68 0.66 0.01 0.33 0.93 0.36 0.01 0.02 0.12 0.01 0.00 0.00 0.02
#26 6.54 0.34 0.43 0.04 0.14 0.95 0.68 0.01 0.01 0.14 0.04 0.00 0.00 0.05
#27 4.00 0.57 0.57 0.03 0.26 1.03 0.53 0.05 0.05 0.15 0.04 0.00 0.01 0.04
#28 5.05 0.38 0.45 0.03 0.15 0.82 0.64 0.05 0.05 0.13 0.02 0.02 0.02 0.05
#29 7.35 0.30 0.40 0.04 0.12 0.92 0.71 0.01 0.01 0.13 0.05 0.00 0.00 0.06
#30 1.00 1.02 1.02 0.01 0.81 0.81 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01
#31 3.55 0.58 0.57 0.02 0.26 0.90 0.47 0.02 0.02 0.12 0.01 0.00 0.00 0.03
#32 1.00 0.94 1.01 0.01 0.80 0.80 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
#33 3.88 0.58 0.57 0.02 0.29 0.99 0.53 0.03 0.03 0.14 0.01 0.00 0.00 0.04
#34 4.44 0.46 0.51 0.01 0.19 0.92 0.55 0.02 0 0.14 0.01 0.01 0.01 0.05
#35 5.58 0.41 0.47 0.01 0.16 0.96 0.6 0.02 0 0.13 0.03 0 0 0.05
#36 2.54 0.72 0.67 0.01 0.37 0.90 0.35 0.01 0.02 0.11 0.01 0.00 0.01 0.02
#37 3.35 0.64 0.62 0.02 0.31 1.02 0.48 0.01 0.01 0.14 0.02 0.00 0.00 0.05
#38 5.02 0.43 0.49 0.02 0.17 0.96 0.58 0.03 0.03 0.14 0.02 0.01 0.01 0.05
#39 2.67 0.66 0.65 0.02 0.32 0.88 0.39 0.02 0.02 0.12 0.01 0.02 0.01 0.02
 Fig. 9 A-B. CMEIAS Classification Result Image of
Community-B.tif Before Reassignment of a Coccus to a
Regular Rod (arrows)

B
 Fig. 9 C-D. CMEIAS Classification Result Image of Community-
B.tif After Reassignment of a Coccus to a Regular Rod (arrows)

D
 Cell Count for Each Morphotype Class
Table 6.
Community B Community B
Community A before edit after edit
Morphotype classification classification
Coccus 14 62 61
Spiral 5 22 22
Curved Rod 11 7 7
U-shaped Rod 5 5 5
Regular Rod 135 51 52
Unbranched Filament 0 6 6
Ellipsoid 0 4 4
Club 0 5 5
Prosthecate 0 5 5
Rudimentary Branched Rod 0 1 1
Branched Filament 0 2 2
Other-1 0 0 0
Other-2 0 0 0
Other-3 0 0 0
Other-4 0 0 0
Other-5 0 0 0
Total Number of Cells 170 170 170
 Table 7. Descending Sort of Ranked Abundance -
Microbial Morphotypes in Communities A vs. B
Morphotype Community A Community B
Regular Rod 135 52
Coccus 14 61
Curved Rod 11 7
Spiral 5 22
U-shaped Rod 5 5
Unbranched Filament 0 6
Ellipsoid 0 4
Club 0 5
Prosthecate 0 5
Rudimentary Branched Rod 0 1
Branched Filament 0 2
 140 135

Fig. 10A. Ranked Abundance of Morphological Diversity

in Microbial Communities A and B
120

100
Abundance

80

61
60
52
Community A
40 Community B

22
20 14
11
7 5 5 5 6 5 5
4
0 0 0 0 0 1 0 2
0

od
s

od

te
b
l

t
od

id
od
ra

en

en
cu

lu

a
o

R
i
rR

ec
C
m

m
Sp

ps
oc

ed
d

ed

la

la
h
a

li
ve
C
ul

st
Fi

Fi
El

h
p
ur

nc
eg

ha

o
ed

ed
Pr
C

ra
-s
R

ch

h
B
U

nc
n

y
ra

ra
ar
nb

B
t
en
U

im
ud
R
 Fig. 10B. Morphotype Diversity of Communities A vs. B
100%

90%
Branched
Filament
80% Rudimentary
Branched Rod

Prosthecate
70%

Club
% of total abundance

60%
Ellipsoid

50% Unbranched
Filament

U-shaped Rod
40%

Spiral
30%
Curved Rod

20% Coccus

Regular Rod
10%

0%
Community A Community B
 Table 8. Community diversity indices computed from
CMEIAS morphotype classification data
in EcoStat (Trinity Software)
Parameter Community A Community B
Morphotype Richness = 5 11
Total cell abundance = 170 170
Simpson Dominance (l) = 0.641 0.241
Simpson Diversity (D) = 0.359 0.759
Max of D = 0.805 0.914
Simpson evenness = 0.446 0.830
Inverse dominance (d) = 1.560 4.152
Max of d = 5.121 11.692
d evenness = 0.305 0.355
Log used for H' = 2.718 2.718
Shannon Diversity (H') = 0.773 1.726
H'max = 1.609 2.398
H' evenness(J%) = 0.480 0.720
Brillouin diversity = 0.728 1.622
Brillouin max = 1.551 2.351
Brillouin evenness = 0.469 0.690
Hill's N1 = 2.167 5.618
Hill's N2 = 1.560 4.152
Hill's evenness = 0.720 0.739

Comments on the parameters reported in Tables 8 and 9:

All parameters are computed in EcoStat (Trinity Software)
from inputs of CMEIAS data on the number of different
morphotypes (substitute for species) found and the
distribution of abundance among each of them. The indices of
community characteristics include measures of morphotype
richness, diversity, dominance, and evenness. Community
similarity analyses compare the similarity and dissimilarity in
morphotype diversity among the two communities. Some
parameters are significantly influenced by sample size, and
the 2 communities analyzed each contain equal (170 cells)
sample sizes. Consult Howard Towner's Ecostat manual for
mathematical details.
 Table 9. Community similarity
analyses comparing morphotype
diversity in communities A vs. B

Prop. similarity (%) = 48.824 Note this 'bottom line" computation!

Jaccard coefficient = 0.455
Sorenson coefficient = 0.625
Morisita index = 0.634
Horn index = 0.758
Sneath/Sokal distance = 0.663
Bray/Curtis distance = 0.512
Chord distance = 0.769

CMEIAS analysis indicates

that communities A and B
have 48.82% proportional
similarity in morphological
diversity, with Community
B being 2.2-fold higher in
morphotype richness and
diversity indices and 1.5-
fold higher in distribution
of morphotype evenness.

These features illustrate how CMEIAS can

strengthen the microscopy-based approaches that
compliment other methods (e.g., 16S rDNA-based,
nutritional versatility, etc) of polyphasic analysis to
characterize the structure and function of complex,
growing microbial communities in situ without
cultivation. Enjoy CMEIAS !