1 Laboratory of Microbial Ecology and Technology

Advances in Advances in microbial analysismicrobial analysis

Prof. dr. ir. W. VerstraeteDr. ir. N. Boon

Laboratory of Microbial Ecology and Technology (LabMET)

Faculty of BioengineeringGhent UniversityLabMET.Ugent.be

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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

Methods to examine microbial populationsMethods to examine microbial populations

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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

Methods to examine microbial populationsMethods to examine microbial populations

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1.1. IntroductionIntroduction What is known about the microbial diversity?

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1.1. IntroductionIntroduction

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

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1.1. IntroductionIntroduction Microbial detection: historical overview

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1.1. IntroductionIntroduction Van Leeuwenhoeks microscope:

First observation by a microscopein 1674: “animalcules”

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1.1. IntroductionIntroduction Microscopy:

– Quick– Low specificity

Confocal fluorescence microscope (µm)

Light microscope(mm-µm)

Electron microscope(nm)

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1.1. IntroductionIntroductionActivity measurements:

– Microbial processes: respiration,nitrification, dehydrogenase and phosphatase

Information about bacterial activityNothing known about microbial composition

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1.1. IntroductionIntroduction Koch-1882 and Petri-1887:

Culturing micro-organisms on media

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1.1. IntroductionIntroduction Culturing based methods:

– Isolation and enumeration of microbial cells on specific nutrient agars

– currently most used approach

Sample Plating Counting

Results after min 48 h

Total Count, coliforms, E. coli, Legionella pneumophila, Clostridia, Salmonella, Aeromonas, ...

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1.1. IntroductionIntroduction

The great plate count anomaly (Amann, 1990)

Habitat Cultivable (%)

Seawater 0.001-0.1

Fresh water 0.25

Mesotrophic lake 0.1-1

Tap water 0.1-3

Activated sludge 1-15

Sediments 0.25

Soils 0.3

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1.1. IntroductionIntroduction

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

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Culture-independend methods are requiredCulture-independend methods are required

Advanced techniques:Advanced techniques:- Immunology- Immunology- Molecular microbiology- Molecular microbiology

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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

Methods to examine microbial populations

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2.2. Immunological methodsImmunological 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?

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Coating with primary

antibodies

Addition of a sample

with an antigen

2.2. Immunological methodsImmunological methods

ELISA

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Spectrofotometry

Addition of an enzyme

linked to a secondary antibody

Addition of enzyme substrate

2.2. Immunological methodsImmunological methods

ELISA

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2.2. Immunological methodsImmunological methods Enzyme-linked immunosorbent assay (ELISA)

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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

Methods to examine microbial populations

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3.3. Molecular detection methodsMolecular detection methods

Molecular microbiology:

Use the genetic material of bacteria

Bacterial cell

Chromosome

(DNA)

Ribosomes, containing rRNA

rRNA

Proteins and enzymes

mRNA

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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

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3.3. Molecular detection methodsMolecular detection methods

DNA/RNACells

Extraction Lysis of cells:

• enzymatical (lysozym)

• physical (beat beating)

• chemical (SDS, fenol,…)

Methodology

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3.3. Molecular detection methodsMolecular detection methods

DNA extraction: DNA visualization on agarose gel

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3.3. Molecular detection methodsMolecular detection methods

Amplification based techniques:

– Polymerase chain reaction (PCR)

– ‘Real Time’ quantitative PCR

– Fingerprinting techniques

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3.3. Molecular detection methodsMolecular 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

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3.3. Molecular detection methodsMolecular detection methods

DNA/RNACells

Extraction PCR amplification

Amplified fragments

Amplification

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3.3. Molecular detection methodsMolecular detection methods

Amplification based techniques:

– Polymerase chain reaction (PCR)

– ‘Real Time’ quantitative PCR

– Fingerprinting techniques

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30 to 40 times repeated 109 copies of the target-DNA

3.3. Molecular detection methodsMolecular detection methodsPCR: Enzymes will double a part of a DNA in one

PCR cycle (temperature program)

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Agarose gel analysis

Endpointmeasurement after 40 cycles

3.3. Molecular detection methodsMolecular detection methodsPolymerase Chain Reaction (PCR)

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3.3. Molecular detection methodsMolecular detection methodsPolymerase Chain Reaction (PCR):

– In principle: detection of one m.o. is possible within 3 hours

– But: Only presence/absence analysis no quantification!

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3.3. Molecular detection methodsMolecular detection methods

Amplification based techniques:

– Polymerase chain reaction (PCR)

– ‘Real Time’ quantitative PCR

– Fingerprinting techniques

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‘CROSS OVER’ POINT

3.3. Molecular detection methodsMolecular detection methods ‘Real Time’ quantitative PCR: fluorescence signal

corresponds with the amount of application product

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High copy number

Low copy number

3.3. Molecular detection methodsMolecular 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

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3.3. Molecular detection methodsMolecular detection methods

Unknown sample

number of copies/µL

Cyc

le n

um

ber

Standard curve, based on known

DNA concentrations

Standard Curve

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3.3. Molecular detection methodsMolecular detection methodsBenefits 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

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3.3. Molecular detection methodsMolecular detection methods

Amplification based techniques:

– Polymerase chain reaction (PCR)

– ‘Real Time’ quantitative PCR

– Fingerprinting techniques

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3.3. Molecular detection methodsMolecular detection methods

DNA/RNA3 types of cells

ExtractionPCR

amplification

Amplified fragments

Fingerprinting techniques:– Allows a separation of DNA fragments based

on their sequence– Different sequence = different species

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3.3. Molecular detection metodsMolecular detection metods

Agarose Fingerprinting

Fingerprinting techniques: comparison of separation techniques

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•Stress responses•Stability of reactors•Microbial community analysis

3.3. Molecular detection methodsMolecular detection methodsApplication of fingerprinting techniques

– Monitoring mixed microbial communities– One band = one species

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Case study: environmental monitoringCase study: environmental monitoringherbicide usage in agricultureherbicide usage in agriculture

Control: Manual weed removal

Herbicide: Atrazine (0.75 kg/ha)Metachlor (2 kg/ha)

Can both sites be separated, based on their microbial population?

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Tested microbialTested microbial indicators indicators Soil activity

– Respiration– Nitrification– Bacterial growth

Plating– Total count– Lactobacilli

Molecular fingerprinting– All bacteria– Ammonium oxidizers– Actinomycetes– Acidobacterium

He3 un iK22He3 u ni K32He3 uni K12He3 uni K11He3 u n iK1He3 uni K21He3 u ni K2He3 u ni K31He3 uni A2He3 uni A12He3 uni A21He3 uni A31He3 uni A11He3 uni A1He3 u ni A22He3 uni A32Ref 2Ref 3Ref 1

100959085

Controle

Herbicidebehandeld

NO

DIFFERENCES

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Luckily we had the Luckily we had the methane oxidizersmethane oxidizers......

CH4 CO2

• Autotrophic bacteria• Oxidise 20-60 million ton methane/year!• Kyoto: methane capture 20 x more heat than CO2

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Clear effect of the herbicide treatmentClear effect of the herbicide treatment

Control

Herbicide treated

Possible indicator?

Seghers et al., 2003, FEMS Microbiol. Ecol.

Fingerprinting analysis Fingerprinting analysis of methane oxidizersof methane oxidizers

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Methanotrophic bacteria influenced by fertiliser treatments?

Soil treatments

C: control soil (no fertiliser)

R: soil with manure ORGANIC

M: soil with mineral fertiliser

CONVENTIONAL

G: soil with GFT-compost ORGANIC

C R M G

Seghers et al., 2003 Environ. Microbiol.Also the fertiliser has a clear effect!Also the fertiliser has a clear effect!

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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

Methods to examine microbial populations

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Gram+, Gram-

Live/Dead cells

In situ identification:

DNA probes, hybridization

4.4. Whole cell analysisWhole cell analysis

Direct counts by fluorescent staining

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Fluorescent Fluorescent in situin situ Hybridisation Hybridisation(FISH)(FISH)

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3’-TCCGCCACGCGATTGGGC-5’ Dye

---AGGCGGUGCGCUAACCCG--- ----TCCGAATCCGGGTTCCTAA----

16S rRNA

4.4. Whole cell analysisWhole cell analysisFluorescent in situ hybridisation (FISH)

DNA-probes: fluorescent labeled desoxyoligo-nucleotides specific for the target organism

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16S rRNA

MATCH NO MATCH

4.4. Whole cell analysisWhole cell analysisFluorescent in situ hybridisation (FISH)

DNA-probes: fluorescent labeled desoxyoligo-nucleotides specific for the target organism

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Sample with bacteria

Permeabilization of cell wall

Addition of probes(green and yellow label)

Hybridisation to the complementary rRNA

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Sample with bacteria

Permeabilization of cell wall

Addition of probes(green and yellow label)

Hybridisation to the complementary rRNA

After washing:

target organisms are green or yellow

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Fluorescence microscopy

4.4. Whole cell analysisWhole cell analysis FISH: analysis of the

samples with…

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Pseudomonas

E.coli

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Activated sludge floc: localization of ammonium oxidisers

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0

0,5

1

1,5

2

2,5

3

3,5

Day 0 Control NotBioprotected

Bioprotected

% a

mm

oniu

m o

xid

isers

Good nitrification

No nitrification

Boon et al., 2003

4.4. Whole cell analysisWhole cell analysis FISH-applications:

– Study of bacterial groups• Ammonium oxidisers (AO)

• Nitrite oxidisers (NO)

– Quantification• Combining universal and specific probes

• % Area ratio

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4.4. Whole cell analysisWhole 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

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4.4. Whole cell analysisWhole cell analysis

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Injector Tip Sheath

fluid

Fluorescent Fluorescent detectiondetection

e.g. fluorescent antibodies (cfr. ELISA) to detect pathogens

4.4. Whole cell analysisWhole cell analysis

Fast analysis by flow cytometry Principle: fluorescent stained cells are detected as

single events by a laser

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0 mol/L SphingosineNo EtOH0 mol/L SphingosineEtOH25 mol/L SphingosineEtOH50 mol/L SphingosineNo EtOH150 mol/L SphingosineEtOH

LIVE INJURED

DEAD

Possemiers S. & Bolca S.

4.4. Whole cell analysisWhole cell analysis

Flow cytometry: live dead staining

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Grate et al. Analytica Chimica Acta, 478:85

4.4. Whole cell analysisWhole cell analysis Application flow cytometry

– Portable detection systems:Portable flow cytometers withautomated sample preparation

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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

Methods to examine microbial populations

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5.5. Conclusions and PerspectivesConclusions and PerspectivesPerspectives and benchmarking1. 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

• Flow cytometrie: On-site analysis (on-line in the future) Very fast detection (1-3 hours)

50 € / analyse

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Take home messageTake home message Cultivation based analysis of micro-

organisms 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

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LabMET workshop LabMET workshop

August 2005August 2005

Introduction to molecular techniques for Introduction to molecular techniques for monitoring and detection of micro-monitoring and detection of micro-

organisms in the environmentorganisms 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.