9 Fluorescence In Situ Hybridization (FISH)

Within complex mixtures of microorganisms, the FISH method can target both general groups of microorganisms (such as methane-producing organisms) and specific degrading speciesThe lowest taxonomic rank, and the most basic unit or category of biological classification.(www.biology-online.org) of interest such as DehalococcoidesDehalococcoides is a genus of organohalide-respiring bacteria (for example, bacteria that use chlorinated solvents as metabolic electron acceptors) within the phylum Chloroflexi, in the domain Bacteria, and currently represented by a single species, Dehalococcoides mccartyi (Dhc). This species is the only one known with strains that dechlorinate dichloroethenes (DCEs) and vinyl chloride (VC) to ethene and inorganic chloride. mccartyi (Dhc). Further, FISH can provide some information about the general activityRefers to when a microorganism performs a specific function (e.g., sulfate reduction, metabolism of benzene) of the microbial populations of interest. FISH does not require cultivation of the organisms or any technology-based signal amplification. This method also allows for study (visualization) of whole cells from their natural environment, typically containing unknown or nonculturable microorganisms. The FISH technique requires the insertion of a probe inside a microbial cell (in situ) that recognizes a specific DNA sequence and allows direct counting of the number of cells that are degrading the contaminant of interest. Therefore, FISH avoids cultivation issues, DNA extraction efficiency concerns, and PCR amplification biases (such as failure of the PCR reaction by the presence of PCR inhibitors, lack of primer specificity, and variable number of 16S rRNAA subunit of the ribosome composed of ribonucleic acid (RNA).  The RNA sequence is used to classify and identify microorganisms (e.g. genus and species). operons (see Section 10.8).

FISH is the method of choice over other EMDs when other EMDs are not technically feasible (for instance, if qPCR primersShort strands of DNA that are complementary to the beginning and end of the target gene and thus determine which DNA fragment is amplified during PCR or qPCR. are not available) or when information on mixed microbial communities structure is necessary to evaluate the occurrence of biodegradationA process by which microorganisms transform or alter (through metabolic or enzymatic action) the structure of chemicals introduced into the environment (USEPA 2011)..

Visualization of whole cells by FISH can provide information on the following:

• abundance of microorganisms or genes of interest
• cell morphology and growth characteristics
• spatial distributions and associations with other microorganisms
• microbial community structure and architecture

These characteristics can help to interpret microbially-mediated processes in soils, sediments, or groundwater. For example, some microorganisms may exhibit differential geneA segment of DNA containing the code for a protein, transfer RNA, or ribosomal RNA molecule (based on Madigan et al. 2010). expression when part of bioflocs (or biofilms) versus when they are in a planktonic existenceFree floating microorganisms that are not associated with particles, sediments or biofilms.. Spatial distribution may be an important considerations for the functioning of syntrophic populationMicroorganisms that are associated or mutually dependent upon one another. of microorganisms, including contaminant degrading consortia; for instance, such associations may facilitate the transfer of electron donors and metabolites between microorganisms (Duhamel and Edwards 2007). Such information cannot be gained from the analysis of genes alone (i.e., PCR). FISH is best used when combined with other EMD tools, which by themselves may not always provide straight-forward or definitive information about contaminant degradation processes, or to provide additional resolution to understanding of a contaminated site.

In general, detection limits of 100 cells or lower in a sample can be achieved with FISH analyses (Moreno et al. 2011). In general, sampling procedures for FISH analysis are straightforward and are readily integrated into existing monitoring protocols.

The basic FISH procedure includes:

1. fixation and permeabilization
2. hybridization
3. washing
4. microscopy (for counting and visualization) or flow cytometryA method whereby cells or particles move in a liquid stream past a laser or light beam and a sensor detects the relative light scattering and fluorescence of the particles. (for high speed counting)

In certain types of samples the FISH procedure may be preceded by a cell isolation (such as in sediments) or concentration method (low biomass samples). This basic principle and the steps involved in FISH are shown in Figure 9.1 and described in detail in Section 9.4.1.

Additional introductory information is available in the FISH Fact Sheet.

Table 9-1. Example FISH Applications

Title	General information	Contaminants	EMDs used	Project life cycle stage
Test Area North, Idaho National Laboratory, ID (see description below)	Anaerobic	Chlorinated solvents (TCE)	FISH, qPCR, EAPs	Remediation Monitoring and Optimization
Ft. Lewis, WA (see description below)	Anaerobic	Chlorinated solvents (TCE)	FISH, qPCR	Feasibility/Research
Cherokee former manufactured gas plant, IA (see description below)	Aerobic and Anaerobic	PAH, coal-tar	FISH 14CO2 generation Note 1	Remediation Selection (Natural attenuation)
Pesticide degradation in soils; Lodi, Italy (see description below)	Aerobic	Pesticides (Simazine)	FISH	Feasibility/Research
Bitterfeld, near Leipzig, Germany (see description below)	Groundwater megasite; aerobic and anaerobic	Chlorobenzenes	FISH	Feasibility Studies/Research (Bioaugmentation/ Biostimulation)

Note 1:¹⁴C radiolabeled compounds biodegradation was confirmed in a microcosmA sample that is regarded as a small but representative portion of something larger. In environmental studies microcosm are typically small samples of soil, sediment, or water incubated in enclosed containers under laboratory conditions. in which ¹⁴CO₂ was generated.

In situ bioremediationThe treatment of environmental contamination through the use of techniques that rely on biodegradation. Bioremediation has two essential components: biostimulation and bioaugmentation. was conducted at the Test Area North site at the Idaho National Laboratory. FISH analyses were used to monitor the progress of the remediation, along with EAPs and qPCR (ICP 2007, Wymore et al. 2007, M. H. Lee et al. 2008). The EAP results are discussed in Section 8.2.

FISH was used to identify, and quantify, key microbial populations including Dhc species and methanogens during implementation of enhanced anaerobic bioremediation for a chlorinated solvent source area located at Landfill 2, Joint Base Lewis McCord, Washington. FISH was used to track microbial population dynamics prior to injection of bioremediation amendments (whey powder and bicarbonate buffer), and during and after amendment injection and bioaugmentationThe introduction of cultured microorganisms into the subsurface environment for the purpose of enhancing bioremediation of organic contaminants (USEPA 2011) over the course of approximately nine months. FISH was used to correlate changes in quantities of these populations with geochemical changes to verify controlling parameters, such as groundwater pH, and to understand competitive relationships between Dhc and methanogens. FISH data were also compared to similar data collected using quantitative polymerase chain reaction (qPCR)A laboratory analytical technique for quantification of a target gene based on DNA. targeted for Dhc and methanogens. Conclusions from this comparison were that FISH was redundant with qPCR and generally more labor intensive and expensive. Also, methods were not yet developed for evaluating mRNA for tceA, bvcA, and vcrA and/or other strains of Dhc. FISH, however, was much better for evaluating methanogenic populations (Macbeth and Sorenson 2011).

This feasibility study used a combination of FISH on aquifer sediment samples, laboratory microcosm studies, and analysis of aqueous geochemistry to determine the natural attenuation of PAH’s in coal-tar DNAPL-impacted groundwater. Groundwater sampling at the site indicated anaerobic conditions predominate downgradient of the coal-tar DNAPL source area as indicated by the presence of ferrous iron, manganese (II) and hydrogen sulfide. Laboratory microcosms showed degradation of naphthalene and phenathrene under aerobic conditions as well as under anaerobic conditions, although at a slower rate. PAHs were shown to degrade in the laboratory microcosms at a slower rate in the sulfate- and nitrate-reducing microcosms.

FISH analysis on sediment samples showed that PAH-contaminated sediments contained three orders of magnitude higher concentrations of microorganisms as compared to uncontaminated sediments.  FISH analysis of the sediment microbial community indicated that β- and γ-ProteobacteriaA broad phylum of gram negative bacteria that is categorized into six groups, involving many genera, based on 16s rRNA differences, Actinobacteria, and Flavobacteria were dominant in the aerobic sediments (similar to results found in the laboratory microcosm studies). FISH results also showed that sulfate-reducing bacteria dominated (>37%) the microbial community in the sediments of the sulfidogenic region of the aquifer (Rogers, Ong, and Moorman 2007). This study provided evidence of natural attenuation of PAHs in the aquifer sediments and indicated which groups are abundant in microbial communities involved in PAH degradation.

A laboratory study was conducted to determine the following:

• degradation of the pesticide simazine (a triazine herbicide) in the absence and presence of urea (used to represent co-application of nitrogen fertilizer with herbicides)
• the changes overall microbial community during simazine biological degradation
• the abundance of ammonia oxidizing bacteria.

Microbial community abundance was determined by FISH methods and the study quantified total bacteria and the α, β, and γ-Proteobacteriasubgroups as well as ammonia oxidizing bacteria. Microcosms were setup in the laboratory and contained soils historically treated with simazine and fertilized with nitrogen. Herbicide degradation was observed according to the SETAC guidelines for assessing the environmental fate of pesticides in laboratory and soil degradation studies (SETAC 1995). The results of this study showed that simazine half-life was approximately 39 days when urea was absent and 32 days when urea was added to the treatment. FISH results indicated that bacterial abundance increased during simazine degradation with or without the presence of urea, although the presence/absence of simazine and urea affected the relative abundance of different groups. Additionally FISH results indicated that ammonia oxidizing bacteria may be involved in the degradation of simazine due to changes in abundance of the bacteria (Caracciolo et al. 2005).

This feasibility study was undertaken to evaluate biological degradation of chlorobenzene, and 1,2- and 1,4-dichlorobenzene by various Pseudomonas putida microorganisms. A set of microcosms were developed in the laboratory containing groundwater from the contaminated site, one of three P. putida microorganisms previously shown to degrade chlorobenzenes, and additional concentrations of the three contaminants. Concentrations of the three organisms over time were monitored by FISH and the total microbial community was evaluated using single-strand polymorphism (SSP) analysis of the 16S rRNA gene from the total microbial community. The results showed that bioaugmentation with two cultures P. putida GJ31 and a genetically modified microorganism P. putida F1ΔCC were capable of degrading 30 mg/L of chlorobenzene, 2 mg/L of 1,2 dichlorobenzene, and 2 mg/L of 1,4-dichlorobenzene to less than 1 mg/L. Further the study showed that these organisms were capable of degrading the chlorbenzenes under both aerobic and nitrate oxidizing conditions (Wenderoth et al. 2003; Wenderoth et al. 2002).

Included in Table 9-2 below is information that should be provided in laboratory reports of FISH data including common laboratory report information, recommended information about the FISH method, and desirable information about the FISH method and results.

Additional information regarding sample handling and collection can be found in Section 10.4 and Section 10.5.

Table 9-2. Recommended and desirable information in FISH laboratory report

Report information	Typical information or acceptable ranges
Common Laboratory Report Information
Site Identifier
Sample type or matrix	Groundwater, sediment, soil
Sample storage/transportation	4°C, overnight shipping, chain of custody forms
Sample handling methods	Filtering methods, filters used, sediment washing, volume of water filtered, cell separation methods
Specific FISH Information
Recommended
Results	Total cell and active cells counts including standard deviations of multiple fields; spatial distribution; biofilm, floc, or planktonic state
Reporting Units	Cells per volume of water or mass of soil
Typical Detection or Reporting Limits	100 cells per sample volume or mass (e.g., filtered groundwater); 104 cells/ml (e.g., unfiltered wastewater sample)
Limit of Detection	Varies, should be adequate for the application
FISH target	Ribosomal gene (e.g., 16S rRNA), functional geneA segment of DNA that encodes an enzyme or other protein that performs a known biochemical reaction. For example, the functional gene tceA encodes the reductive dehalogenase enzyme that initiates reductive dechlorination of TCE. Other genes can code for RNA entities which can regulate the activity of other DNA target sequences. name, probe sequence
FISH probe fluorescent marker	Fluorescein, Alexa Fluor®
Sample fixation method	Chemicals used for fixation (e.g., paraformaldehyde)
Hybridization conditions	Hybridization time, hybridization solution composition (e.g., probe concentration), and temperature
Method of quantification	Manual Counts of cells (30 to 200 cells per field) including number of fields counted (e.g., minimum of 20 fields), equipment used for quantification (epi-fluorescent microscope used and filter sets) Automatic flow cytometry conditions and instrumentation (e.g., post processing software)
QA/QC information	Laboratory positive and negative control results (non-sense probe)
Narrative of analyses	Indicating interferences or quality control issues encountered
Desirable
Photomicrographs and graphs from flow cytometry analyses
Number of laboratory duplicates and replicate results

Interpretation of FISH data varies depending on the site microbiology, the degradation pathways present, and the contaminants. The following overview illustrates the selection of an appropriate analysis and integration of the subsequent results with site monitoring plans. Each bioremediation strategy (e.g., monitored natural attenuation, biostimulationA remedial technique which provides the electron donor, electron acceptor, and/or nutrients to an existing subsurface microbial community to promote degradation., and bioaugmentation) will be discussed as well as specific information given for common contaminants.

FISH data are used to determine the presence, spatial relationship and (in some cases) activity of microbes of interest in a sample. Visualization of whole cells by FISH can provide information on cell morphology and growth characteristics, spatial distributions and associations with other microorganisms, and microbial community structure and architecture—which may be important for interpreting microbially-mediated processes in soils, sediments, or groundwater. FISH signals can provide some information about activity of the target organisms, although no rate information can be obtained. Presence of various microorganisms or genes of interest should be used in combination with other data (e.g., contaminant concentrations) to provide a lines of evidence argument regarding biodegradation potential and activity. To illustrate interpretation of FISH results, each question relevant to FISH in Table 2.3 is discussed.

9.3.2.1 Site Characterization

A) Are contaminant-degrading microorganisms present?

FISH results can be used to determine the presence of contaminant- degrading organisms in water samples and in some cases in soil and sediment samples. Depending on which FISH probesShort sequences of single stranded DNA carrying a fluorescent label. When the probe binds to the target DNA/RNA sequence of the microorganism(s) of interest in an environmental sample, the target cell will fluoresce and can be visualized and counted using a specialized microscope or a flow cytometer. are used, FISH data can quantify total bacteria, organisms with specific metabolic capabilities (such as sulfate reducers) or concentrations of organisms known to degrade contaminants (such as chlorinated solvent degraders or naphthalene degraders).

For example, at a site where the shallow aquifer is contaminated with heating oil and low concentrations of oxygen are present in the plume. Monitored natural attenuation parameters were collected (groundwater monitoring data, geochemical characterization) and an evaluation of the potential for biological attenuation performed. FISH was performed to determine the abundance of both sulfate reducers and methanogens during concomitant degradation of petroleum hydrocarbons. Sulfate reducers can degrade and grow on petroleum hydrocarbons (Kleikemper 2002). Methanogens do not directly degrade petroleum hydrocarbons in low oxygen systems, rather they facilitate the fermentation of petroleum hydrocarbons by making the conditions conducive to growth and activity of petroleum hydrocarbon fermentors (i.e., hydrogenotrophic methanogens keep H₂ and CO₂ low and acetoclastic methanogens degrade end products of fermentation) (Kleikemper 2005).

FISH analytical results included cells/ml groundwater or cells/g aquifer material of the following microorganisms:

Total microorganisms

• Total bacteria
  • Sulfate reducing bacteria
• Total ArchaeaMicroorganisms that are genetically distinct from bacteria. Methanogens are an example of archaea (www.biology-online.org, accessed online, 2013).
  • Methanogens

FISH results showed that sulfate reducers consisted of a relatively large percentage of the bacteria present (45%), and the concentration of methanogens in groundwater was greater than the concentrations extracted from soil as normalized by total microorganisms.

B) Are contaminant degrading microorganisms active?

With emerging approaches, such as quantifying the amount of mRNA for a degradation genes with FISH, one can examine the activity of microorganisms performing important biodegradation functions. If the mRNA detected by FISH correlates to a functional gene for a biodegradation process, then biodegradation of a contaminant is potentially occurring in the contaminated matrix. Additionally, the percentage of the total versus active microbial population containing the functional gene could also potentially be quantified.

For example, at a site that contains a coal-tar impacted groundwater aquifer naphthalene is one contaminant. Microbial metabolism of naphthalene begins with dioxygenase-mediated transformations and is encoded for by naphthalene dioxygenase genes in Pseudomonas putida. Groundwater samples from the site could be evaluated for the presence of naphthalene dioxygenase mRNA by FISH (Bakermans and Madsen 2002; Wilson, Bakersman, and Madsen 1999). Detection of naphthalene dioxygenase mRNA by FISH would suggest that microorganisms are biologically degrading naphthalene. For example, samples could be analyzed for the following targets and reported as cells/ml groundwater:

Total microorganisms

• Total active microorganisms
  • Total bacteria
    • Naphthalene dioxygenase mRNA

The FISH results could show:

1. the percentage of cells that were bacteria versus Archaea in the groundwater sample
2. if mRNA encoding for naphthalene dioxygenase is present at the site and how it changes with time
3. what percentage of the native microbial population contains mRNA encoding for naphthalene dioxygenase by comparing total active microorganisms by acridine orange counts as compared to naphthalene dioxygenase mRNA counts.

C) Are the microorganisms capable of complete degradation?

See Question A.

9.3.2.2 Remediation

H) Are numbers of contaminant-degrading microorganisms and/or genes changing?

FISH can be used to track the number of contaminant-degrading microorganisms and/or genes changing in groundwater, soil, or water environmental samples. For example, a site has s-triazine contaminated soil (i.e., simazine) that is undergoing natural attenuation. FISH could be used to determine the concentration of the atzB gene which has been linked to biological degradation of s-triazines by hydrolytic deamination over time. These atzB genes have been found in soils that have been historically exposed to triazines, but not in soils that have not been exposed previously. Additionally, the atzB gene has been found to be correlated to the mineralization rate of simazine (Martin et al. 2008). Therefore detection of the atzB gene in the soils at the area contaminated with triazines could provide another line of evidence that biological degradation of triazines is occurring.

I) Is the remediation strategy affecting the numbers or types of contaminant-degrading microorganisms?

FISH can be used to track the number of contaminant-degrading microorganisms and genes changing in groundwater, soil, or water environmental samples. For example, a site is contaminated with chlorobenzenes such as 1,2-dichlorobenzene, 1,4-dichlorobenzene, and chlorobenzene. The site has undergone bioaugmentation with a mixed culture ofPseudomonas putida GJ31 and Pseudomonas putida F1DCC. After bioaugmentation, one portion of the site is sparged with air and the other site is left under anaerobic conditions. Over time, degradation of chlorobenzenes is observed and concentrations of the P. putida species are monitored by FISH. Concentrations of P. putida as measured by FISH could show an increase in concentration with time during the simultaneous degradation of chlorobenezenes as has been shown previously (Wenderoth, et al. 2003). However, less significant growth of P. putida could be observed under aerobic conditions and could correlate with decreased rates of chlorobenzene degradation as compared to the portion of the plume undergoing bioremediation under anaerobic conditions. Therefore, FISH results when used in combination with traditional groundwater monitoring methods could reveal which bioremediation strategies resulted in optimal growth of bioaugmentation cultures and suggest which approach resulted in increased chlorobenzene degradation rates.

J) Is there a biological basis for intermediates accumulating?

FISH can be used to determine if the right microorganisms are present to completely degrade contaminants to non-toxic byproducts. For example, FISH could be used to determine if appropriate dehalogenating communities are present at a site which is contaminated with chlorinated ethene and their approximate concentrations. Only Dhc has been shown to completely degraded chlorinated ethenes (e.g.  PCE) to non-toxic byproducts (i.e., ethene). Therefore FISH probes for Dhc (such as Dhc1259t) could be used in combination with other probes for total bacteria and total Archaea to determine the abundance of this important dechlorinating community. However if Dhc is not present, but the total biological community is fairly robust, then degradation of PCE may be stalling at cis-DCE or be degrading by different mechanisms. Currently FISH methods cannot adequately distinguish between Dhc that degrade PCE to ethene and other strains in the genusA category of organism classification (taxonomy). A particular genus is a group of related species. For example, Pseudomonas is a genus of bacteria. which do not completely degrade chlorinated solvents. Continued research may develop probes specific to the various Dehalococcoides , thus discriminating between the various contaminant degrading organisms and degradation pathways.

9.3.2.3 Monitoring

O) Does the microbial community compositionDescription of the types or identities of microorganisms present in a sample. support the remediation strategy?

Based on the combination of probes used, the FISH method can indicate which specific microorganisms are present, whether functional genes associated with biodegradation are present, or the percentage that larger groups of microorganisms constitute of the total biomass. For example, at a site where groundwater is contaminated with uranium and is undergoing bioremediation to reduce soluble U(VI) to insoluble U(IV). Specifically, Desulfotomaculum sp., a sulfate reducer, is using U(VI) as the sole electron acceptorA chemical compound that accepts electrons transferred to it from another compound (based on USEPA 2011). and precipitating U(IV). FISH can be used to determine the concentration of sulfate reducing bacteria present in the groundwater during the precipitation of uranium.

P) Do contaminant-degrading microorganisms continue to be sufficiently abundant?

See Question A.

Q) Are contaminant-degrading microorganisms remaining active?

See Question B.

R) Is there a biological basis for intermediates accumulating?

FISH can be used to determine if the right microorganisms are present to completely degrade contaminants to non-toxic byproducts. For example, FISH could be used at a site contaminated with chlorinated ethenes to determine if appropriate dehalogenating communities are present and their approximate concentrations. Only Dhc has been shown to completely degraded chlorinated ethenes (e.g.  PCE) to nontoxic byproducts (i.e., ethene). Therefore FISH probes for Dhc (e.g., Dhe1259t) could be used in combination with other probes for total bacteria and total Archaea to determine the abundance of this important dechlorinating community. However if Dhcis not present, but the total biological community is fairly robust, then degradation of PCE may be stalling at cis-DCE.

9.3.2.4 Closure

Some variability of closure requirements exists among states and programs. However, in many situations, EMD data could serve as an additional line of evidence for understanding what processes are important in reducing concentrations and reaching the applicable closure levels. The evidence provided by EMD data would reveal whether biodegradation processes are occurring, have sufficiently proceeded or are likely to continue.

W) Is contaminant degradation likely to continue?

FISH can be used to track the concentrations of microorganisms of interest over time. In particular the concentration of microorganisms known to biodegrade contaminants could be monitored over several sampling events to show the sustained presence of contaminant degrading microorganisms. Demonstrating steady state concentrations of biodegrading organisms at a site could provide additional lines of evidence, along with geochemical analyses and downward trends in contaminant concentrations, that biological natural attenuation processes may continue and that residual contamination will not pose a threat to human health or the environment following closure.

A study conducted by Robertson et al. in 2002 highlighted some potential issues associated with using FISH counts of organisms of interest for bioremediation activities. First, the team found that although the sulfate reducing organism (Desulfosporosinus meridiei) associated with aromatic hydrocarbon biodegradation under anaerobic conditions in the laboratory was present in groundwater at the Eden Hill site, there were no differences in the concentration of the organism inside or outside the plume. The team also found that the organism was not correlated with hydrocarbon concentrations or with indications of sulfate reduction. Second, the team found that autofluorescence of the sample and poor nutritional state of the groundwater environment lead to problems with quantification of the microorganism of interest. Poor nutritional state of the environment will lead to lower concentrations of 16S rRNA as this gene is only present in high numbers in actively replicating microorganisms, which requires a nutritionally rich environment. The poor fluorescence of the samples was further confounded by the presence of autofluorescent particles in the samples.

The authors concluded that FISH may not be an appropriate method for quantifying bacteria in nutritionally poor environments or when the organism is slow growing (Robertson et al. 2002). Many of the issues raised by the Robertson, et al. (2002) study regarding the FISH method can be addressed using newer, more sensitive detection techniques such as those described below in Section 9.4.2.1(CARD-FISH). Others have suggested that FISH can be used to monitor dynamic temporal changes in intrinsic biodegradation activity when specific probe sets are used and where the target bacteria has been definitively linked to contaminant degradation at the site of interest (Rogers, Ong, and Moorman, 2002; Yang and Zeyer 2003). When specific probes are not available, FISH can be used in combination with isotopic techniques (CSIA, SIP, or MAR-FISH; see Section 9.4 below) to show which organisms are actively degrading contaminants of interest.

9.3.3.1 Limitations

The FISH method is not widely available commercially. Currently, only specialized research laboratories are performing these analyses. Validated probes and procedures are not available for a wide range of organisms. While several databases provide access to over 2600 rRNA targeted oligonucleotide probes (probeBase, SILVA rRNA database project), there are numerous additional 16S rRNA or functional gene sequences of environmental remediation significance that could be used for FISH probe design, yet have not been developed and validated. The lack of validation for these sequences may be related to the expense of the FISH method and associated with the expertise and labor needed for direct microscopic counting. FISH can be automated to some extent by using flow cytometry to count target cells more efficiently, thus reducing the analysis costs, but information regarding spatial relationships among and between the cells in the sample is lost in the process. Finally, standard protocols for sample collection, storage and analysis have not yet been developed for many degradation processes.

9.3.3.2 Ribosomal content

As a majority of FISH applications target the ribosomes of microbial cells, variations in the concentration of ribosomes in a cell can affect the sensitivity of a FISH method. For example the average number of ribosomes in a microbial cell can change over time. During exponential growth, E. coli can contain upwards to 72,000 ribosomes. In contrast after reaching a stationary phase of growth, E. coli cells have been shown to contain only 6,800 ribosomes (Bremer and Dennis 1996). In relatively smaller microorganisms (for instance, Dhc with cell diameters of 0.5 um) there are significantly fewer ribosomes, a few hundred, due to physical space restrictions. It is difficult to detect smaller cell with fewer ribosomes by fluorescently labeling. However, new methods to detect cells with fewer ribosomes have overcome some of these limitations include most commonly, CARD-FISH. This method includes the use of horseradish peroxidase-labeled oligonucleotide probes, in combination with catalyzed reported deposition (CARD) of fluorescently labeled tryamides (Fazi et al. 2008; Hoshino et al. 2008). Multiple tryamides react with the peroxidase enzyme to amplify the fluorescent signal and increase the sensitivity of hybridization method (lower detection limit).

Several new technologies are emerging that will advance the detection of even individual genes in bacteria by FISH and automate this method (for reviews see Amann and Fuchs 2008; Czechowska et al. 2008; Lee et al. 2011).

This method has been used for the identification, quantification and characterization of microbial communities or their degradation associated genes of interest in complex environmental samples.

The basic principle of FISH is to bind (hybridize) a target reporter (fluorescently labeled oligonucleotide probe) to a sequence of interest (such as 16S rRNA) inside a microorganism and visualize or count the resulting fluorescent signal by micrsoscopy or other method. The basic FISH procedure includes: 1) fixation and permeabilization; 2) hybridization; 3) washing;and 4) microscopy (for counting and visualization) or flow cytometry (for high speed counting). In certain types of samples the FISH procedure may be preceded by a cell isolation (e.g., sediments) or concentration method (e.g., low biomass samples). This basic principle and the steps involved in FISH are shown in Figure 9.1 and described in detail below. Independent of the specific FISH approach applied, only cells that contain the target DNA are recognized by the probe and will be fluorescently labeled when visualized with appropriate techniques.

Fixation and Permeabilization. Fixation and permeabilization of microbial cells is required to 1) preserve the integrity and shape of all cells; 2) prevent cell loss through lysis; 3) allow penetration of the fluorescent FISH probes into the cell; and 4) protect the target gene (typically RNA) from degradation during storage and analysis. Typically a sample, such as groundwater or other water sample, is settled on membrane filters and covered with a fixing agent or the sample itself is mixed with fixing agent. Formaldehyde and ethanol are typically used for fixation of cells (Roller et al. 1994). The fixing agent serves to permeabilize as many cells as possible to allow the large labeled oligonucleotides entry to the cells and subsequent diffusion of the probes to their intracellular rRNA targets. After fixation and several steps to remove residual fixative, the sample is transferred to a microscope slide and the microorganisms are dehydrated by washing with ethanol. 

Hybridization. During hybridization, a target reporter such as a fluorescently labeled probe is bound to the sequence of interest. Only organisms containing the target genes incorporate the fluorescent label, so they can be directly visualized and counted using a microscope or other technique. In environmental microbial ecology studies, FISH applications have targeted ribosomal RNA (rRNA), particularly the 16S gene, with oligonucleotide probes. The rRNA molecule is targeted for identification of microorganisms (Bacteria and Archaea) as all cells required ribosomes for translationThe second step of gene expression where messenger RNA (mRNA) produced by transcription is decoded by the cell to produce an active protein. and growth. Since each cell contains many ribosomes, there can be 100 to 100,000 targets per cell. Additionally, the rRNA of each species of microorganism contains short signature sequences that are unique to each group of microorganisms (for example, Dhc specific 16S rRNA sequences). Oligonucleotide probes are molecules composed of 15 to 30 nucleotides and are covalently linked to a fluorescent dye. Other types of cell labeling techniques that are used less commonly than oligonucleotide probes include combinations of reporter molecules (dioxygenin), fluorescent antibodies (horseradish peroxidase) and enzymatic signal amplification (tryamide), or polyribonucleotide probes.

Hybridization of probes to the target sequence must be carried out under stringent conditions to ensure proper annealing of the probe to the target sequence. During this crucial step of FISH, preheated hybridization buffer containing fluorescently labeled probes complementary to the target gene is applied to the sample and incubated in a dark, humid chamber at exact temperatures for 30 minutes to several hours.

Washing. During the washing step the microscope slides containing the fixed and hybridized cells are briefly rinsed to remove unbound probe that would interfere with quantification of the target microorganisms. The slides are then dried, mounted with an anti-fading agent and visualized.

Visualization or counting. Hybridized cells are visualized or counted by epifluorescence microscopy or by flow cytometry. Fluorescence miscroscopy uses a high intensity light to illuminate a sample, which excites fluorescence species bound to the olidonucleotide probes, resulting in an emission of a longer wavelength light. The image magnified in an epifluorescence microscope is actually an image of the light emanating from the fluorescent molecules bound to the oligonucleotide probes rather than illuminated light as in light microscopy. Cells are enumerated by the laboratory technician or by an automated counting programs (Pernthaler, Pernthaler, and Amann 2003; Selinummi et al. 2005). More efficient counting of labeled cells is achieved with flow cytometry (Porter et al. 1997; Vives-Rego, Lebaron and Nebe-von Caron 2003). In flow cytometry, labeled cells are diluted so that individual cells pass through a laser beam, which detects and counts fluorescently labeled cells. Various cell staining procedures are sometimes combined with FISH probes to allow quantification of all microorganisms (see Table 1, FISH Fact Sheet).

9.4.2.1 CARD-FISH

As mentioned previously, this method is used to increase the FISH signal intensity thus allowing the quantitation of low activity microbial assemblages or organisms with low ribosomal contents (Fazi et al. 2008).

9.4.2.2 MAR-FISH

This technique involves the uptake of radioactively labeled substrates into cells, which can be detected by microautoradiography (MAR) with simultaneous identification of the cells by FISH. Unlike with stable isotopeTwo atoms with the same number of protons but a different number of neutrons. probing (see Section 7), where the labeled substrates contain non-radioactive isotopes, MAR-FISH requires the use of radioactive isotopes such as¹⁴C, ³²P or ³H (Wagner et al. 2006). This technique has been widely used (Wagner et al. 2006) and recently has been automated (Alonso and Pernthaler 2005; Cottrell and Kirchman 2003).

9.4.2.3 Raman-FISH

This technique involves the uptake of stable isotope tracers into microbial cells, determination of uptake of the stable isotopesForms of an element that do not undergo radioactive decay at a measureable rate. by Raman microspectroscopy, and identification of the microbial cells by FISH (Huang et al. 2007). This method overcomes the equipment costs and reduces the resolution that has limited SIMS and MAR-FISH applications (Amann and Fuchs 2008).

9.4.2.4 NanoSIMS-FISH

Nano-scale secondary-ion mass spectrometry (SIMS) allows multiple-isotope imaging in cells and identification of the microbial cells by FISH with resolutions down to 50 nm. The advantage of this technique is that one can potentially detect metabolic activities, such as contaminant degradation, in single cells (Musat et al. 2012).