Methods Sampling

1
Methods Sampling Enumeration Activity Comm
unity Structure
Medium?? Air, Water, Soil, Sediment What For??
- All, 1 type, metabolic types Characteristic
s 1) Representative (Jonas, Buckley) 2) Not
Contaminate 3) Not Injure (Klein and Wu) 4)
Not grow (Zobell) Medium Zobells, GASW
2
Sampling Water Surface Jar, Bucket Deep
Pump Bottle (Niskin, Kemmerer, Butterfly)
Decompression issues - barophylic
3
(No Transcript)
4
Sampling Water Surface Jar, Bucket Deep
Pump Bottle (Niskin, Kemmerer, Butterfly)
Decompression issues - barophylic
5
(No Transcript)
6
Sampling Sediment Grab Corer Box Corer
Sampling Surfaces Swab Washing
(picture) Rodac Plate Hard Surface Corer
(Image)
7
Sampling Sediment Grab Corer Box Corer
Sampling Surfaces Swab Washing
(picture) Rodac Plate Hard Surface Corer
(Image)
8
Sampling Sediment Grab Corer Box Corer
Sampling Surfaces Swab Washing
(picture) Rodac Plate Hard Surface Corer
(Image)
Microbial-Vac System (M-Vac) is a hand-held,
non-destructive, wet-vacuum surface sampling
device that allows a revolutionary new approach
to acquiring microbial samples.
9
Sampling Sediment Grab Corer Box Corer
Sampling Surfaces Swab Washing
(picture) Rodac Plate Hard Surface Corer
(Image)
10
Coral Sampling by SS Corer Lee Stocking
Island Photos by R.B. Jonas
11
Sampling Air Anderson Sampler
(Image) Membrane Filter on agar on broth
(w pad) Air Plate Sorbant surface
toilettogram
Sampling Food and Tissues Macerate in diluent
e.g. oyster Swab surfaces
Sampling Air/Water Interface why?? Adsorb
teflon sheet Contact slides Capillary Tubes (w
bait)
12
Anderson Sampler
13
Sampling Air Anderson Sampler
(Image) Membrane Filter on agar on broth
(w pad) Air Plate - pix Sorbant surface
toilettogram
Sampling Food and Tissues Macerate in diluent
e.g. oyster Swab surfaces
Sampling Air/Water Interface why?? Adsorb
teflon sheet Contact slides Capillary Tubes (w
bait)
14
Sampling Air Anderson Sampler
(Image) Membrane Filter on agar on broth
(w pad) Air Plate - pix Sorbant surface
toilettogram
Sampling Food and Tissues Macerate in diluent
e.g. oyster Swab surfaces
Sampling Air/Water Interface why?? Adsorb
teflon sheet Contact slides Capillary Tubes (w
bait)
15
Sample Storage and Processing 1) Direct Count
preserve w 2 formaldehyde gluteraldehyde Dur
ation months at 4oC BUT note literature and
Stand. Methods
2) Viable Count Hold at in situ
temperature lt 6 hours Special Types may
refrigerate or hold on ice for some time (up to
24 hours) E. coli yes, Vibrio no (probably)
16
Dilution - Difficult Choice Chemical
Composition pH Temp. Water dw, di, Tap, Site
(Ches. Bay) Saline buffer (Physiological,
PO4) Peptone
17
Anaerobes non-fastidious vs strict Strict
Purge Diluents w N2, CO2 - serum vials
Anaerobic Hood (Image) Reducing medium fluid
thioglycollate Corer design maintains anoxic
conditions
syringe
Serum vial cap
H2S in Blue Holes
18
(No Transcript)
19
Anaerobes non-fastidious vs strict Strict
Purge Diluents w N2, CO2 - serum vials
Anaerobic Hood Reducing medium fluid
thioglycollate Corer design maintains anoxic
conditions
syringe
Serum vial cap
H2S in Blue Holes
20
Direct Count Procedures Epifluorescent
Acridine Orange Hobbie, Daley, Jasper
1977 FITC fluorescein isothiocyanate DAPI
(4,6-diamido-2-phenylindole) Hoechst 33258
bisbenzimide surfaces that bind AO Tabor and
Neihoff
21
Petroff Hauser Counter
22
Live Dead direct count green alive, red
dead (green in all) (red penetrates only CM not
intact)
green-fluorescent SYTO 9 stain
red-fluorescent propidium iodide stain
23
Direct viable count INT-INT Formazan (Image
CTC) Reduced by ETS (20 min) Respiring bacteria
deposit RED spots (2-iodophenyl-3-nitrophenyl-5ph
enyl tetrazolium chloride) Autoradiography Label
w 3H glucose (or thymidine) Thin photographic
emulsion Silver grains AODC to see Invert
Inactive
Active
BUT cells too small size of silver grain
24
(No Transcript)
25
Direct viable count INT-INT Formazan (Image
CTC) Reduced by ETS (20 min) Respiring bacteria
deposit RED spots (2-iodophenyl-3-nitrophenyl-5ph
enyl tetrazolium chloride) Autoradiography Label
w 3H glucose (or thymidine) Thin photographic
emulsion Silver grains AODC to see Invert
Inactive
Active
BUT cells too small size of silver grain
26
Fluorescent Antibody Technique W fluorescein
isothiocyanate bound to AB study individual
organisms.
27
FISH Fluorescent In Situ Hybridization
See Sect 11.6 Brock 10th
Live/Dead FISH Bact vs Virus
28
Pseudomonas in milk FISH CTC
29
Scanning Electron Microscope 3
dimensional electron dense (even unstained)
30
Coulter Counter electronic particle
counter estimate number in size class Issue
biological vs non-biological
The Coulter method of sizing and counting based
on measurable changes in electrical
resistance produced by nonconductive particles
suspended in an electrolyte.
31
Flow Cytometry sort and laser characterize
http//flowcytometry.uchc.edu Good description of
how this works
Measures light scattered or fluorescence
emission from (purified) cells in suspension
focused to single cell wide column.
32

• Viable Count Procedures
• Plate Count
• 1 cell 1 colony (CFU)
• Spread Plate
• Pour Plate
• Selects by medium and incubation conditions
• pH, salt, nutrients, Temp., gas, pressure
• Agar, silica gel solidifying agents
• Selective, Differential
• TCBS Vibrio
• EMB Coliforms
• SS Salmonella
• Phytoplankton extract, cellulose, chitin
• Fungi - ltpH, antibiotics

33
2) Anaerobic Roll Tubes
3) Most Probable Number less precise than plate
count Dilution Extinction Poisson Distribution
(PIX) Liquid Medium - or only (growth, no
growth) Multiple Tube 3, 5, 10 specific
carbon source radiolabelled compounds gas
production Nitrifiers, Denitrifiers
diphenylamine NO3, NO2 blue color
34
Poison Distribution Describes random
occurrences of objects (bacteria) in space
(media) or events in time.
Where µ is population mean of Occurrences in
space or time.
35
2) Anaerobic Roll Tubes
3) Most Probable Number less precise than plate
count Dilution Extinction Poisson Distribution
(PIX) Liquid Medium - or only (growth, no
growth) Multiple Tube 3, 5, 10 specific
carbon source radiolabelled compounds gas
production Nitrifiers, Denitrifiers
diphenylamine NO3, NO2 blue color
36
Atlas but Shrug!
Not for water Maybe not most
Sediment
37
Mixing Time Effect on CFU estimates for Soil
38
Number versus Biomass and Activity
5 36
4 5 x 10-12 g/cell
10 20
39
Number Biomass
West East
Seaward
Decline Same Same ?? 2 60
40
Biochemical Methods Estimate s/biomass Criteri
a 1) direct correlation between amount of
biochemical and organisms 2) Biochemical
ONLY in Microbes Rarely met absolutely
ATP Adenosine triphosphate LPS lipopolysacchar
de Muramic Acid
41
ATP (and total adenylates) 1) all contain ATP
2) in relatively constant proportion to cell
carbon 3) Lost rapidly when die Therefore (ATP)
Living Cells or biomass Method boil in TRIS
buffer at pH 7.75 stops ATPase,
phosphorylases Measure by Luciferin/luciferase
red Luc. O2 ? Oxid. Luc. LIGHT
Luciferase Mg2 ATP Lite is Proportional
to ATP Conversion 250 x ATP cell carbon in
Aquatic Orgs 120 x ATP cell carbon in Soil
Orgs.
Problems Physiological State Other than
Micro-organisms Bound ATP
42
LPS Lipopolysaccharide in cell wall of Gram
LAL Limulus Amoebocyte Lysate - coagulen LPS
LAL Turbid Solution proportional to LPS
(nephelometry) LPS content Constant
proportion of cells estimate IF G- dominant
e.g. Marine, estuarine (inc BG algae)
http//www.mbl.edu/animals/Limulus/blood/bang.html
43
Muramic Acid cell wall peptidoglycan ONLY in
bacteria MA ? Lactate released (assay
colorimetrically)
Acid hydrolysis cleave PDG Column chromatography
MA Alk. Hydrolysis assay
Alkaline hydrolysis
G 44 ug MA/mg C G- 12 ug MA/mg C
13 ug MA/mg C 35 ug MA/mg C
44
(No Transcript)
45
Low ?? High
EC ATP ½ ADP / ATP ADP AMP
46
Biomass vs Numbers Microscopic - and
size Weight ATP LPS MA Fungal Measure hyphal
strands, Chitin Algal - Pigments
Conversions 10 fg C / cell cell 10-12 g
live wt
47
Microbial Metabolism Natural Rates 1)
Heterotrophic Potential Technique 2) Tracer
Technique 3) Respiration 4) O2 Consumption
48
(No Transcript)
49
Lineweaver Burke Transformation
LOW v
LOW (S)
(Y MX B straight line eq)
50
V rate (mgC/m3/h) c radioactivty taken
up (DPM) f correction for isotope (1.05) Sn
natural S conc. A added concentration C
DPM from 1 uCi Subs u quantity added 14C
(Cu added DPM) t incubation time hours
Wright and Hobbie Lineweaver Burke
Transformation of S and P
Cu t / c (Kt Sn) / Vmax A / Vmax
(1 / Vmax A) ((Kt Sn) / Vmax) Y
MX B
51
F fraction taken up
52
(No Transcript)
53
2) True Tracer Technique If add only ONE
Substrate Concentration at level lt 10 of Sn
Then NOT affect Turnover Time (Tt)
or Turnover Rate (Tr)
If add 100 molecules of glucose Incubate 0.5
h Find that 25 molecules taken up Then Tr
(25/100) / 0.5 50 / h And Tt 2 hours
Tt t / f (hours) t is incub. Time f is
fraction taken up (c/Cu) Tr f / t ( / h)
54
Bacterial (2o)Production NOT Tracer -
Saturation DNA Produced is Proportional to
biomass/cell Growth Rate Measure DNA
production by Incorporation of Thymidine 3H
methyl thymidine 3H-TdR Plus cells Treat with
cold TCA (Trichloroacetic acid) Collect
macromolecules (cells) on filter 1) only
bacteria take up exogenous thymidine 2) All
bacteria use 3) Goes to DNA only 4) Specific
Activity not affected by ambient Thymidine 5)
DNA 25 Thymidine 6) Measure amount of TdR per
cell produced 2 x 1018 cells / mole Thymidine
55
1) CO2 gas analyzer IR absorb 2) Trap CO2 in
Alkali and titrate 3) Trap 14CO2 in hyamine
hydroxide phenethylamine e.g. 14C
cellulose, pesticides, lignin 4) Respirometry
Warburg manometer U tube with side arm
flask (with well) CO2 absorbed reduced
pressure
56
4) Closed Bottle (short term) O2
Consumption Essentially short-term BOD, incubate
in dark
5) Energy Charge - Measure of metabolic
state Active 0.8 0.95 Stationary
0.6 Resting - lt0.5
EC ATP ½ ADP / ATP ADP AMP
6) Enzyme Assays Dehydrogenase
triphenyltetrazolium chloride ?
triphenylformazan (extract and assay by
spec.) Cellulases measure sugars Chitinases
measure aminosugars Rubisco ribulose
bispohosphate carboxylase
57
Nitrogen Cycle Enzymes
Nitrogenase 15N (NN) ? 15NH4 Mass
Spec HCCH ? C2H4 GC-FI ethylene acety
lene Nitrification NH3 ? NO3
chemolithotrophs O2 NO3
coupled uncoupled Dentrification - NO3 ? N2O ?
N2 -O2 analyses 15N, or chemical by
acetylene blockage at N2O level
58
1 hr
2 hr
4.5 hr
5.5 hr
Acetylene blockage
59
Nitrogen Fixation
Control
Sewage amended