Are certain members of Dehalococcoides (DHC) more capable of degrading Vinyl Chloride?

Certain members of DHC (Dehalococcoides) are more efficient at converting vinyl chloride to ethene. MI can quantify the functional gene BAV1 R-Dase (bvcA) that is present in a strain of Dehalococcoides (Bav1) that is known to be very efficient at catalyzing the direct dechlorination of VC.

Can culture methods be used to quantify Dehalococcoides?

As Dehalococcoides is a strictly anaerobic organism, it is difficult to grow in laboratory cultures.  qPCR is the best method to quantify the abundance of this organism in a sample.

Can Microbial Insights receive international samples?

Yes.  Microbial Insights has an international soil permit and has received samples without problem from many different countries. Please see international shipment for additional information.

Can Microbial Insights receive samples on Saturday?

Microbial Insights is now open on Saturdays.  However, coolers should be shipped to a FedEx drop location and held where an MI representative will collect them.  It is important to ship to the Saturday delivery address and follow all procedures as indicated under the sample shipment section.  Please note that due to the short hold time associated with RNA and culture, it is recommended that these samples NOT be shipped for Saturday delivery.

Can results between different target groups be compared to one another?

Results based on the CENSUS approach can be compared to each other because a conversion from gene copies to cells is made (based on an average number of gene copies found in that organism or group).

However, results obtained using CENSUS-Expression (RNA) need to be compared based upon what type of gene we analyzed (functional gene vs. taxonomic gene). Results which rely on taxonomic biomarkers or the 16S rRNA are determined based on ribosomal RNA (rRNA) found within the cell whereas results obtained from targeting functional genes are based upon messenger RNA (mRNA).

Do I need to add a preservative to my samples?

For most analyses, the only preservative that is required is that the samples are shipped on ice at 4c for next day delivery.

Samples collected for CENSUS-expression (RNA) must be collected using the bio-flo sampling approach or bio-trap samplers. Following collection, each bio-flo sample MUST BE preserved using a special RNA preservative which is shipped along with your other sampling supplies. Please see the bio-flo sampling protocol for more information about this approach.

Do I need to provide more than one sample to analyze for multiple targets (organisms) from one sampling location?

No, Once the DNA or RNA is extracted from your sample this product can be analyzed for numerous targets.

Following the completion of your analyses, the remaining DNA or RNA is archived for a minimum of 3 years. If you need to analyze this sample for additional targets in the future, it would be available.

How are detection limits determined?

The method detection limit (MDL) was determined by taking the lowest number of cells in ten replicates which could be detected at least 70% of the time. For example, the average number for Dehalococcoides spp. was 10 cells/sample. Our lower detection limit (LDL) was set at 10x this value (i.e. 100 cells/sample) and our practical quantification limit (PQL) was set at 5x our LDL (500 cells/sample).    Quality control samples are also analyzed at LDL concentrations ten times to ensure that it is detected at least 95% of the time.

How are results reported?

Results for CENSUS are reported as cells per volume of sample (i.e. mL, g, bead). These values have been converted from gene copies of the target organism to cells based upon the average number of gene copies typically found within that organism.

For CENSUS-Expression (RNA), results are reported as gene copies per volume of sample, since gene copy number can change depending on the activity of the organism.

How do these numbers compare to typical culture based methods (i.e. plate counts, MPNs, etc)?

Results obtained from either CENSUS or CENSUS-Express provide a direct assessement on the abudance of a specific microbe or microbial group and are not subject to the limitations associated with more traditional based approaches.

Limitations of traditional culture based approaches are:

  • not very representative of in-situ community.
  • highly selective based upon conditions used to grow the bacteria (media, temp, etc.)
  • it is well documented that less than 10% of bacteria can be cultured.

How many Bio-Trap Samplers do I need to analyze for both CENSUS and PLFA?

One Bio-Trap sampler per location can be used to analyze for a variety of analyses, including PLFA and CENSUS.  However, if you analyzing for PLFA and mRNA, then you would need a larger Bio-Trap sampler for that location.  Bio-Trap Samplers were designed specifically for improving the data produced by MBTs, so the ability to have a sufficient amount of beads for multiple analyses is critical to their application.

How many samples should I collect to characterize a site?

For most sites we recommend at a minimum collecting samples across a gradient (e.g. upgradient, source, mid-plume, downgradient, etc.) Sample locations can also be selected based upon changes in contaminant concentrations. For example, select sampling locations in which the primary contaminant varies by one order of magnitude. Unfortunately, collecting just one sample will not provide a lot of information about what is going on at a given site.

How much sample do I need to collect?

Almost any type of sample matrix can be analyzed using qPCR. The majority of samples received for CENSUS are collected either by our bio-trap samplers or by filtering groundwater using our bio-flo sampling approach. The following table provides typical volumes required for this analysis.

Sampling Matrix

CENSUS (DNA)

CENSUS -Expression (RNA)

Groundwater (Bio-Flo)
Filter ~1-2L
Filter ~1-2L
Groundwater (Bailer)
1L Poly
NA
Soil/Sediment/Solid
10-20g
NA
Culture/Isolate/Biofilm
10mL
10mL
 Bio-Trap Sampler
 1
 1

Should a monitoring well be purged prior to Bio-Trap deployment?

If the monitoring well has not been purged recently, it may be necessary prior to deployment.  MI recommends that three well volumes be removed to ensure contact with formation water and reduce well bore effects.

Should a well be purged prior to sample collection?

MI recommends that the same procedure be used for the microbial sampling as for the geochemical parameters.  As these results are often compared, it is important that samples be collected under similar monitoring well conditions.

Since Dehalococcoides (DHC) is present reductive dechlorination must be occurring, right?

No not necessarily, there are several situations where complete dechlorination may not be observed when DHC has been detected at low concentrations. These sites may be limited in the amount of electron donor or have compounds which would inhibit dechlorination.

What are the hold times?

The hold times for each analysis are listed below:

 

PLFA 24-48 hours
CENSUS 24-48 hours
CENSUS-Expression (RNA) 24 hours
Heterotrophic Plate Counts 24-48 hours
ORm 7-10 days
Acidity Testing 7-10 days
DGGE 24-48 hours
Most Probable Numbers (MPNs) 24-48 hours

What are the QA/QC procedures used during this analysis?

MI uses a variety of QA/QC procedures including, positive controls, negative controls, internal recovery standard, etc. to ensure that our clients can depend upon the data they have obtained.

What do I mark on the chain of custody to request bvcA and tceA functional genes?

Please mark qDHC and DHC functional genes under the CENSUS heading on the chain of custody form.

What does a positive or negative results mean?

Positive results for samples analyzed using the CENSUS based approach show that organisms with genetic potential are present in that particular sample. So if the environmental conditions at this sampling point are appropriate, then degradation of the specific compound should occur. Positive results for samples analyzed by CENSUS-Expression (RNA) confirm that this target group is actively expressing a desired function (e.g. Dehalococcoides is actively producing genes which encode for VC degradation).

Negative results for CENSUS and CENSUS-Expression (RNA) mean that the organism of interest was not detected within a given sample or is present at levels below our limit of detection (LOD).

What does the concentration of Dehalococcoides mean?

The abundance of Dehalococcoides spp. within a given sample is often used as an indicator of the potential for reductive dechlorination to occur. The following are benchmarks that can be used to understand whether the biomass levels are low, moderate or high as compared with other sites.

Low: <10e2 cells/mL

Moderate: 10e3 to 10e4 cells/mL

High: >10e5 cells/mL

What does the target EBAC quantify?

This target is used to provide an index of the total amount of eubacteria within a given sample by targeting a conserved region found in all eubacteria. Results from this target can be used to correlate changes in the ratio of total biomass to a specific group of bacteria (i.e. Dehalococcoides spp.).

What if the Bio-Flo filter clogs during collection prior to passing 1 liter through the filter?

If the filter clogs prior to 1 liter, you can submit an additional filter for that sample.  A maximum of two Bio-Flo filters can be used for the CENSUS analysis.  If you are still concerned with the low volume passed through both filters, you can submit a 1 liter bottle of the sample to MI for lab filtration.

What is the difference between Dehalococcoides (DHC), BAV1 R-Dase, and TCE R-Dase?

DHC targets Dehalococcoides specific sequences of the 16S rRNA gene which is present in all bacteria. The functional gene BAV1 R-Dase (bvcA) is present in a strain of Dehalococcoides (Bav1) that is known to catalyze the direct dechlorination of VC.  The functional gene TCE R-Dase (tceA) is found in strains 195 and FL2 and catalyzes the dechlorination of TCE to VC.

What is the interaction between Dehalococcoides (DHC), Methanogens and Methanotrophs?

The choice of these targets is dependent upon site specific conditions such as redox state.  Please refer to the section on reductive dechlorination and cometabolism of chlorinated solvents for more information.

What is the limit of detection for Dehalococcoides?

The limit of quantification for Dehaloccoides spp. is 500 cells/sample.  Therefore, the larger volume used, the greater the chance of detection.  For instance, if 1000 mls of groundwater is submitted for analysis the limit of quantification is <1 cell/ml.

What is the turn around time for analysis?

The turn around time for each analysis is listed below.

 

PLFA (groundwater, Bio-Trap) 21-30 days
PLFA (soil) 30-45 days
CENSUS 7-10 days
CENSUS-Expression (RNA) 10-14 days
Heterotrophic Plate Counts 10-14 days
Most Probable Numbers (MPNs) 30-40 days
Oil Retention (ORm) 14-20 days
Acidity Testing 14-20 days
DGGE 21-30 days

What levels of reports are available from MI?

Microbial Insights has a variety of reporting levels available for each analysis. For information on additional levels of QAQC reports, please contact us.  There are also report styles available for help with data assessment.