Dehan, Rodney

Huth, William

Launder, Ariel

Nocker, Andreas

Rogerson, Andrew

Snyder, Richard A.

English

Aquatic Commons

Microbiological indicators of water 
quality in submerged karst caves of 
Wakulla Springs
Richard A. Snyder1,  Ariel Launder1, Andreas Nocker1, Andrew 
Rogerson2, William Huth1, Rodney Dehan3
1Center for Environmental Diagnostics and Bioremediation
University of West Florida, Pensacola, FL
2Nova Southeastern University, Oceanographic Center
8000 N Ocean Dr., Dania, FL  
3Florida Geological Survey
903 West Tennessee St., Tallahassee, FL
CEDB
Dr. Todd Kincaid of Hazlett-Kincaid Inc.
Syringe samples taken by divers of cave sediment from walls, nooks and floor
Molecular analysis Isolation of Amoebae spp
Photo: WKPP team
Glass slides in 
acrylic racks 
affixed to
quadpod 
meter supports 
for future 
retrieval
Photo: WKPP team
Photo: WKPP team
Photo: David Sweetin
http://www.gue.com/sites/wkp/overview.shtml
Water samples taken at 4 time points
•Bacterial direct count
• Plate counts
• Enterococcus 
• Total Coliforms and E. coli
• R2A and TSA
•Molecular analysis
•Elemental analysis of CN
•Major ionic constiuents
Well tubes purged for 
minimum 3x the void 
volume prior to 
sample collection.
Sample water 
collected as a 5L 
composite.
Photo: Andreas Nocker

Cave amoeba 1. 
Eruptive vahlkampfiid, about 17 µm in length
Cave amoeba 2. 
Active, non-eruptive. Usually 10 – 12 µm.
Cave amoeba 3. 
Limax, and mildly eruptive, 17 to 25 µm in length
Cave amoeba 7.
Large, steady limax amoeba. 68 µm in length.
Cave amoeba 6. 
Slow oving with occasional eruptive ‘bulges’. 
Probably Rhizamoeba.  Up t  34 
Micrographs: Andrew Rogerson
Code Identity Size (mean)
Am 1 unidentified 11 µm
Am 2 Sacchamoeba 26 µm
Am 3 Vannella 14 µm
Am 4 unidentified testate 10 µm
Am 5 unidentified 8.5 µm
Am 6 Acanthamoeba 20 µm
Am 7 vannellid 34 µm
Am 8 vahlkampfiid 17 µm
Am 9 Dactylamoeba-like 10 µm
Am 10 unidentified 24 µm
Am 11 vahlkampfiid 10 µm
Am 12 Rhizamoeba 34 µm
Am 13 Sacchamoeba 68 µm
Am 14 unidentified filose 8.5 µm
Am 15 vannellid 20 µm
Am 16 Gephyramoeba-like 60 µm
Am 17 vahlkampfiid 20 µm
Am 18 Vexillifera 25 µm
Am 19 unidentified 9 µm
Sample event 15 Nov 2003. 
B tunnel: Am 3, 8, 9, 10, 11, 12, 13
C tunnel: Am 1, 7 
Sample event 5 Jan 2004
A-D tunnel: Am 1, 2, 4
A-D tunnel: Am 1, 2, 3, 5, 6
A-D tunnel: Am 1, 2
Sample event 7 Feb 2004
K tunnel 1: no amoebae
Ktunnel 2: Am 8
D tunnel: Am 4, 6
D tunnel : Am 1
A-K tunnel : Am 2, 9, 19
A-K tunnel : Am 2, 3, 15, 16, 17, 18
Number of Species in each sample
Number of shared Species
B C D A/D K A/K
B 7 0 0 1 1 2
C 2 1 1 0 0
D 3 3 0 0
A/D 6 0 2
K 1 0
A/K 9
Sorenson's Similarity Coefficient
B C D A/D K A/K
B 100% 0.00% 0.00% 15.38% 25.00% 25.00%
C 100% 20.00% 25.00% 0.00% 0.00%
D 100% 66.67% 0.00% 0.00%
A/D 100% 0.00% 26.67%
K 100% 0.00%
A/K 100%
DAPI fluorochrome bacterial direct counts
D tunnel
10 Apr 04, 1750 x
Emerald Sink
K tunnel
A/K tunnel
100
1000
104
105
106
107
108
109
B C D
A
/
D K
A
/
K
R2A CFU 29 Feb 04
C
F
U
 
m
l
-
1
100
1000
104
105
106
107
108
109
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
E
m
e
r
a
l
d
 
t
u
b
e
R2A CFU 11 Apr 04
C
F
U
 
m
l
-
1
100
1000
104
105
106
107
108
109
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
R2A CFU 14 Mar 04
C
F
U
 
m
l
-
1
104
105
106
107
108
109
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
E
m
e
r
a
l
d
 
t
u
b
e
R2A CFU 28 Mar 04
C
F
U
 
m
l
-
1
100
1000
104
105
106
107
108
109
Feb/29 Mar/7 Mar/14 Mar/21 Mar/28 Apr/4 Apr/11
R2A
B
C
D
A/D
K
A/K
Emerald well
C
F
U
 
m
l
-
1
100
1000
104
105
106
107
108
109
Feb/29 Mar/7 Mar/14 Mar/21 Mar/28 Apr/4 Apr/11
R2A
Ames Cherokee Emerald (sw)
C
F
U
 
m
l
-
1
100
1000
104
105
106
107
108
B C D
A
/
D K
A
/
K
Tryptic Soy Agar (TSA) 29 Feb 04
C
F
U
 
m
l
-
1
A
100
1000
104
105
106
107
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
Tryptic Soy Agar (TSA) 14 mar 04
C
F
U
 
m
l
-
1
A
100
1000
104
105
106
107
108
109
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
E
m
e
r
a
l
d
 
w
e
l
l
Tryptic Soy Agar (TSA) 11 Apr 04
C
F
U
 
m
l
-
1
A
100
1000
104
105
106
107
B C D
A
/
D K
A
/
K
A
m
e
s
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
 
(
s
w
)
E
m
e
r
a
l
d
 
w
e
l
l
Tryptic Soy Agar (TSA) 28 mar 04
C
F
U
 
m
l
-
1
A
100
1000
104
105
106
107
108
109
Feb/29 Mar/7 Mar/14 Mar/21 Mar/28 Apr/4 Apr/11
TSA
B
C
D
A/D
K
A/K
Emerald well
100
1000
104
105
106
107
108
109
Feb/29 Mar/7 Mar/14 Mar/21 Mar/28 Apr/4 Apr/11
TSA
Ames Cherokee Emerald (sw)
C
F
U
 
m
l
-
1
08
16
24
32
40
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
Enterococcus 29 Feb 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
8
16
24
32
40
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
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l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Enterococcus 14 Mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
8
16
24
32
40
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Enterococcus 28 mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
8
16
24
32
40
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Enterococcus 11 Apr 04
C
F
U
 
1
0
0
 
m
l
-
1
A
02
4
6
8
10
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Enterococcus Geomeans
E
n
t
e
r
o
c
o
c
c
u
s
 
C
F
U
 
1
0
0
 
m
l
-
1
0
5
10
15
20
25
30
35
Feb/22 Mar/3 Mar/13 Mar/23 Apr/2 Apr/12
Sheet1
B Tunnel
C Tunnel
D Tunnel
A/D Tunnel
K Tunnel
A/K Tunnel
Emerald Tube
Emerald SW
Ames
Cherokee
E
n
t
e
r
o
c
o
c
c
u
s
 
1
0
0
 
m
l
-
1
A
0120
240
360
480
600
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue Total coliforms 11 Apr 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
120
240
360
480
600
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue Total coliforms 14 mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
120
240
360
480
600
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
 
T
u
b
e
E
m
e
r
a
l
d
 
S
W
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue Total coliforms 28 mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
6
12
18
24
30
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
E
m
e
r
a
l
d
(
s
w
)
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue E. coli 14 mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
6
12
18
24
30
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
E
m
e
r
a
l
d
(
s
w
)
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue E. coli 28 mar 04
C
F
U
 
1
0
0
 
m
l
-
1
A
0
6
12
18
24
30
B
 
T
u
n
n
e
l
C
 
T
u
n
n
e
l
D
 
T
u
n
n
e
l
A
/
D
 
T
u
n
n
e
l
K
 
T
u
n
n
e
l
A
/
K
 
T
u
n
n
e
l
E
m
e
r
a
l
d
E
m
e
r
a
l
d
(
s
w
)
A
m
e
s
C
h
e
r
o
k
e
e
Coliblue E. coli 11 Apr 04
C
F
U
 
1
0
0
 
m
l
-
1
A
http://www.millipore.com/catalogue.nsf/docs/M00PMCB24
AK-tunnel
B-tunnel
C-tunnel
D-tunnel
050
100
150
200
250
300
350
400
D
A
/
D B C
A
/
K K
A
M
E
S
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
Particulate Carbon
27 Feb 04
13 mar 04
μ
M
 
 
C
a
r
b
o
n
0
5
10
15
D
A
/
D B C
A
/
K K
A
M
E
S
C
h
e
r
o
k
e
e
E
m
e
r
a
l
d
Particulate Nitrogen
27 Feb 04
13 mar 04
μ
M
 
 
N
i
t
r
o
g
e
n
-0.2
0
0.2
0.4
0.6
0.8
0 0.2 0.4 0.6 0.8 1
Principle Components of Ion Composition
     
     Blue = 28 Feb 04
     Red = 13 Mar 04
     Green = 27 Mar 04
B tunnel
C tunnel
D tunnel
A/D tunnel
K tunnel
A/K tunnel
B tunnel
C tunnel
D tunnel
A/D tunnel
K tunnel
A/K tunnel
B tunnel
C tunnel
D tunnel
A/D tunnel
K tunnel
A/K tunnel
A
-0.2
0
0.2
0.4
0.6
0.8
1
0 0.2 0.4 0.6 0.8 1
Principle Components of Ion Composition
     
     Red = 13 Mar 04
     Green = 27 Mar 04
    
Cherokee
Emerald
Ames
Emerald Tube
Cherokee
Emerald
Ames
A
Conclusions
•Conduits have different microbial floras 
o Undescribed species of Amoebae were found
o Amoebae distribution associated with organic enrichment
o R2A counts higher than TSA counts
o TSA counts more reflective of seasonal change
•Organic enrichment increased with the onset of Spring
•Little to no contamination with fecal bacteria
•Potential for using bacterial community structure as 
water quality indicator
Acknowledgements
GUE Divers
Sandy Cook & crew
Wakulla Springs State Park
James McClean, FL DEP


Microbiological indicators of water

quality in submerged karst caves of

Wakulla Springs

http://aquaticcommons.org/760/1/microbiol_indicators_of_wq_wakulla_rsnyder.pdf

Microbiological indicators of water quality in submerged karst caves of Wakulla Springs

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