Transcript Isolation, characterisation and applications of 3

Advances in microbial
analysis
Prof. dr. ir. W. Verstraete
Dr. ir. N. Boon
Laboratory of Microbial Ecology and Technology
(LabMET)
Faculty of Bioengineering
Ghent University
LabMET.Ugent.be
1
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
2
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
3
Laboratory of
Microbial Ecology and Technology
1. Introduction

What is known about the microbial diversity?
4
Laboratory of
Microbial Ecology and Technology
1. Introduction

What kind of information do we want to
obtain?
– In general:
• Diversity: what kind of bacteria are present?
• Function: are the essential players present?
are there unwanted species?
– Practical:
•
•
•
•
Speed: less than 1 day analysis time (online ?)
Accuracy: be sure of the results
Sensitivity: also the less abundant species...
High-throughput: many samples and many
organisms
5
Laboratory of
Microbial Ecology and Technology
1. Introduction

Microbial detection: historical overview
6
Laboratory of
Microbial Ecology and Technology
1. Introduction

Van Leeuwenhoeks microscope:
First observation by a microscope
in 1674: “animalcules”
7
Laboratory of
Microbial Ecology and Technology
1. Introduction

Microscopy:
– Quick
– Low specificity
Light microscope
(mm-µm)
Confocal fluorescence
microscope (µm)
8
Electron microscope
(nm)
Laboratory of
Microbial Ecology and Technology
1. Introduction

Activity measurements:
– Microbial processes: respiration,
nitrification, dehydrogenase and phosphatase
 Information about
bacterial activity
 Nothing known about microbial composition
9
Laboratory of
Microbial Ecology and Technology
1. Introduction

Koch-1882 and Petri-1887:
Culturing micro-organisms on media
10
Laboratory of
Microbial Ecology and Technology
1. Introduction

Culturing based methods:
– Isolation and enumeration of microbial
cells on specific nutrient agars
– currently most used approach
Sample
Counting
Plating
Results after
min 48 h
Total Count, coliforms, E. coli, Legionella pneumophila, Clostridia,
Salmonella, Aeromonas, ...
11
Laboratory of
Microbial Ecology and Technology
1. Introduction
The great plate count anomaly (Amann, 1990)
Habitat
Seawater
Fresh water
Mesotrophic lake
Tap water
Activated sludge
Sediments
Soils
Cultivable (%)
0.001-0.1
0.25
0.1-1
0.1-3
1-15
0.25
0.3
12
Laboratory of
Microbial Ecology and Technology
1. Introduction

Limitations of culturing techniques:
– The exact growth-conditions are unknown:
• Vitamins
• Spore-elements
• Redox potential
– The bacteria grow very slow
– The bacteria do not grow on solid agar
surfaces
– ‘Dormant cells’ do not multiply
– Some organisms can not be cultivated as
single species  e.g. symbiosis
13
Laboratory of
Microbial Ecology and Technology
Culture-independend methods are required
Advanced techniques:
- Immunology
- Molecular microbiology
14
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
15
Laboratory of
Microbial Ecology and Technology
2. Immunological methods

Antibody based detection: ENZYME-LINKED
IMMUNOSORBENT ASSAY (ELISA)
– Very specific
– Purified antibodies
– Cultured cells are needed for antibody
construction
 no detection of uncultivable bacteria
– Sufficient variation in the cell wall?
16
Laboratory of
Microbial Ecology and Technology
2. Immunological methods
ELISA Coating with primary
antibodies
Addition of a sample
with an antigen
17
Laboratory of
Microbial Ecology and Technology
2. Immunological methods
ELISA
Addition of an enzyme
linked to a secondary antibody
Addition of enzyme substrate
Spectrofotometry
18
Laboratory of
Microbial Ecology and Technology
2. Immunological methods

Enzyme-linked immunosorbent assay (ELISA)
19
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
20
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Molecular microbiology:
Use the genetic material of bacteria
Ribosomes,
containing rRNA
Proteins and
enzymes
mRNA
rRNA
Chromosome
(DNA)
Bacterial cell
21
Laboratory of
Microbial Ecology and Technology
1 aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa
61 gtcgaacggt aacaggaaga agcttgctct ttgctgacga gtggcggacg ggtgagtaat
121 gtctgggaaa ctgcctgatg gagggggata actactggaa acggtagcta ataccgcata
181 acgtcgcaag accaaagagg gggaccttcg ggcctcttgc catcggatgt gcccagatgg
241 gattagctag taggtggggt aacggctcac ctaggcgacg atccctagct ggtctgagag
301 gatgaccagc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg
361 gaatattgca caatgggcgc aagcctgatg cagccatgcc gcgtgtatga agaaggcctt
421 cgggttgtaa agtactttca gcggggagga agggagtaaa gttaatacct ttgctcattg
481 acgttacccg cagaagaagc accggctaac tccgtgccag cagccgcggt aatacggagg
541 gtgcaagcgt taatcggaat tactgggcgt aaagcgcacg caggcggttt gttaagtcag
601 atgtgaaatc cccgggctca acctgggaac tgcatctgat actggcaagc ttgagtctcg
661 tagagggggg tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg aggaataccg
721 gtggcgaagg cggccccctg gacgaagact gacgctcagg tgcgaaagcg tggggagcaa
781 acaggattag ataccctggt agtccacgcc gtaaacgatg tcgacttgga ggttgtgccc
841 ttgaggcgtg gcttccggag ctaacgcgtt aagtcgaccg cctggggagt acggccgcaa
901 ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt
961 cgatgcaacg cgaagaacct tacctggtct tgacatccac ggaagttttc agagatgaga
1021 atgtgccttc gggaaccgtg agacaggtgc tgcatggctg tcgtcagctc gtgttgtgaa
1081 atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag cggtccggcc
1141 gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga cgtcaagtca
1201 tcatggccct tacgaccagg gctacacacg tgctacaatg gcgcatacaa agagaagcga
1261 cctcgcgaga gcaagcggac ctcataaagt gcgtcgtagt ccggattgga gtctgcaact
1321 cgactccatg aagtcggaat cgctagtaat cgtggatcag aatgccacgg tgaatacgtt
1381 cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa gaagtaggta
1441 gcttaacctt cgggagggcg cttaccactt tgtgattcat gactggggtg aagtcgtaac
1501 aaggtaaccg taggggaacc tgcggttgga tcacctcctt a
22
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Methodology
Lysis of cells:
Extraction
• enzymatical (lysozym)
• physical (beat beating)
Cells
DNA/RNA
• chemical (SDS, fenol,…)
23
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

DNA extraction: DNA visualization on
agarose gel
24
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Amplification based techniques:
– Polymerase chain reaction (PCR)
– ‘Real Time’ quantitative PCR
– Fingerprinting techniques
25
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

DNA amplification
Copy machine for
books, papers,....
1 to 50 copies
Copy machine for genes (DNA)
1.000.000.000 copies (109)
= PCR machine
26
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Amplification
PCR
amplification
Extraction
Cells
DNA/RNA
27
Amplified fragments
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Amplification based techniques:
– Polymerase chain reaction (PCR)
– ‘Real Time’ quantitative PCR
– Fingerprinting techniques
28
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
PCR: Enzymes will double a part of a DNA in one
PCR cycle (temperature program)
30 to 40 times repeated  109 copies of the target-DNA
29
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Polymerase Chain Reaction (PCR)
Endpoint
measurement after
40 cycles
Agarose gel analysis
30
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Polymerase Chain Reaction (PCR):
– In principle: detection of one m.o. is
possible within 3 hours
– But: Only presence/absence analysis
 no quantification!
31
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Amplification based techniques:
– Polymerase chain reaction (PCR)
– ‘Real Time’ quantitative PCR
– Fingerprinting techniques
32
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

‘Real Time’ quantitative PCR: fluorescence signal
corresponds with the amount of application product
‘CROSS OVER’
POINT
33
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

‘Real Time’ quantitative PCR, ‘Cross over’ point:
– Number of cycles where the fluorescence signal is
stronger then the background
– Depends of the original amount of target DNA
High copy number
Low copy number
34
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Standard Curve
Standard curve,
based on known
DNA
concentrations
Unknown
sample
35 copies/µL
Laboratory of
number of
Microbial Ecology and Technology
3. Molecular detection methods

Benefits of Real-Time PCR:
– Accurate and reproducible nucleic acid
quantification
– Large dynamic range of detection
– Closed-tube chemistries
– No electrophoresis
– No post-PCR processing
– High Sample throughput
 Mostly used for the detection of
pathogens
36
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Amplification based techniques:
– Polymerase chain reaction (PCR)
– ‘Real Time’ quantitative PCR
– Fingerprinting techniques
37
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods

Fingerprinting techniques:
– Allows a separation of DNA fragments based
on their sequence
– Different sequence = different species
PCR
amplification
Extraction
3 types of cells
DNA/RNA
38
Amplified fragments
Laboratory of
Microbial Ecology and Technology
3. Molecular detection metods

Fingerprinting techniques: comparison
of separation techniques
Agarose
Fingerprinting
39
Laboratory of
Microbial Ecology and Technology
3. Molecular detection methods
Application of fingerprinting techniques
– Monitoring mixed microbial communities
– One band = one species
•Stress responses
•Stability of reactors
•Microbial community analysis
40
Laboratory of
Microbial Ecology and Technology
Case study: environmental monitoring
herbicide usage in agriculture
Herbicide:
Atrazine (0.75 kg/ha)
Metachlor (2 kg/ha)
Can both sites be separated,
based on their microbial population?
Control:
Manual weed removal
41
Laboratory of
Microbial Ecology and Technology
Tested microbial indicators

Soil activity
– Respiration
– Nitrification
– Bacterial growth

Plating
NO
– Total count
– Lactobacilli

DIFFERENCES
Molecular fingerprinting
–
–
–
–
All bacteria
Ammonium oxidizers
Actinomycetes
Acidobacterium
85
90
95
100
42
nH
u
e3
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
u
e3
H
ni
u
e3
H
u
e3
H
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
i
n
u
e3
H
u
e3
H
i
n
u
e3
H
ef2
R
ef3
R
ef1
R
K
2
2
3
K
2
1
K
1
K
1
K
1
2
K
2
K
1
3
K
2
A
2
1
A
1
2
A
1
3
A
1
A
1
A
2
A
2
3
A
Controle
Herbicide
behandeld
Laboratory of
Microbial Ecology and Technology
Luckily we had the
methane oxidizers...
CH4
CO2
• Autotrophic bacteria
• Oxidise 20-60 million ton methane/year!
• Kyoto: methane capture 20 x more heat than CO2
43
Laboratory of
Microbial Ecology and Technology
Fingerprinting analysis
of methane oxidizers
Herbicide treated
Control
Clear effect of the herbicide treatment
Possible
indicator?
Seghers et al., 2003, FEMS Microbiol. Ecol.
44
Laboratory of
Microbial Ecology and Technology
Methanotrophic bacteria influenced by
fertiliser treatments?
C
Soil treatments
R
M
G
C: control soil (no fertiliser)
R: soil with manure
ORGANIC
M: soil with mineral fertiliser
CONVENTIONAL
G: soil with GFT-compost
ORGANIC
Also the fertiliser has a clear effect!
45
Seghers et al., 2003
Environ. Microbiol.
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
46
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

Direct counts by fluorescent staining
Live/Dead cells
Gram+, Gram-
In situ identification:
DNA probes,
hybridization
47
Laboratory of
Microbial Ecology and Technology
48
Fluorescent in situ Hybridisation
(FISH)
4. Whole cell analysis
Fluorescent in situ hybridisation (FISH)
DNA-probes: fluorescent labeled desoxyoligonucleotides specific for the target organism
3’-TCCGCCACGCGATTGGGC-5’
---AGGCGGUGCGCUAACCCG---
----TCCGAATCCGGGTTCCTAA----
16S rRNA
49
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis
Fluorescent in situ hybridisation (FISH)
DNA-probes: fluorescent labeled desoxyoligonucleotides specific for the target organism
MATCH
NO MATCH
16S rRNA
50
Laboratory of
Microbial Ecology and Technology
Sample with bacteria
Permeabilization of cell wall
Addition of probes
(green and yellow label)
Hybridisation to the
complementary rRNA
51
Laboratory of
Microbial Ecology and Technology
Sample with bacteria
Permeabilization of cell wall
Addition of probes
(green and yellow label)
After washing:
Hybridisation to the
complementary rRNA
target organisms are
green or yellow
52
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

FISH: analysis of the
samples with…
Fluorescence microscopy
53
Laboratory of
Microbial Ecology and Technology
Pseudomonas
E.coli
54
Laboratory of
Microbial Ecology and Technology
Activated sludge floc: localization of ammonium
oxidisers
55
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

FISH-applications:
– Study of bacterial groups
• Ammonium oxidisers (AO)
• Nitrite oxidisers (NO)
– Quantification
• Combining universal and specific probes
• % Area ratio
Good nitrification
No nitrification
Boon et al., 2003
56
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

Fluorescent in situ hybridisation (FISH):
– Detection limit is determined by the volume
that is analysed
– Samples can be concentrated:
1 liter sample over a filter  1 propagule/L
– Observation: microscopy
– Counting: flow cytometry (50.000 cells/sec)
– Results can be obtained in 2 to 3 hours
57
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis
58
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

Fast analysis by flow cytometry

Principle: fluorescent stained cells are detected as
single events by a laser
Injector
Tip
Sheath
fluid
e.g. fluorescent antibodies
(cfr. ELISA) to detect
pathogens
Fluorescent
detection
59
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis
LIVE
0 mol/L
Sphingosine
50
mol/L
Sphingosine
150
mol/L
Sphingosine
25
No EtOH
EtOH
INJURED
DEAD
Possemiers S. & Bolca S.
Flow cytometry: live dead staining60
Laboratory of
Microbial Ecology and Technology
4. Whole cell analysis

Application flow cytometry
– Portable detection systems:
Portable flow cytometers with
automated sample preparation
Grate et al. Analytica
Chimica Acta, 478:85
61
Laboratory of
Microbial Ecology and Technology
Methods to examine microbial populations

Introduction
–
–
–
–


Historical overview
Microscopy
Activity measurements
Great Plate Count Anomaly
Immunological methods
Molecular detection methods
– PCR detection
– Real Time PCR quantification
– Microbial fingerprinting

Whole cell analysis
– Fluorescent in situ Hybridisation (FISH)
– Flow cytometry

Conclusions and perspectives
62
Laboratory of
Microbial Ecology and Technology
5. Conclusions and Perspectives
Perspectives and benchmarking
1. Plating: Off-site analysis
Slow method (2-7 days)
15 - 70 € /analysis
2. Molecular fingerprinting: Off-site analysis
Relative fast detection (2 days)
150 € /analysis
3. Real-Time PCR: Off-site analysis
Fast detection (0,5 - 1 day)
100 € / analyse
4. Flow cytometrie: On-site analysis (on-line in the future)
Very fast detection (1-3 hours)
50 € / analyse
63
Laboratory of
Microbial Ecology and Technology
Take home message

Cultivation based analysis of microorganisms is highly biased
 A variety of molecular methods exist to
detect and quantify micro-organisms
 New molecular methods allow to:
– Accurately identify microbes
– Monitor population dynamics
– High throughput of environmental samples
Design and monitor clean-up techniques based
on micro-organisms for contaminated sites
64
Laboratory of
Microbial Ecology and Technology
LabMET workshop
August 2005
Introduction to molecular techniques for
monitoring and detection of microorganisms in the environment
Aim:
• To introduce the theoretical and practical knowledge of molecular
techniques
• To show their strong and weak points
• To discuss the differences between these methods
The course includes both hybridization and PCR based techniques,
discussed by LabMET experts and with open eye to new forthcoming
technology.
65
Laboratory of
Microbial Ecology and Technology