Beyond Cell Culturing

Microbiological activity is a related factor for many pipe corrosion problems found today.  Given that it is present in most circulating systems to some degree, and even in drinking water, the possible role of Microbiologically influenced corrosion (MIC) should always be considered in any corrosion investigation.  Proper identification and quantification of an MIC condition is critical since different forms will demand very different methods for treatment and correction.

Evidence that an MIC condition might exist includes:

• • • High steel corrosion rates exceeding 20 mils per year (MPY)
    • Extreme pitting activity
    • Extremely premature pipe failure
    • Tubercular deposits
    • High microbiological plate counts
    • Foul smelling or hydrogen sulfide smelling deposits
    • Pinhole leaks
    • Growths in the cooling tower basin, or pans
    • Thick black muddy deposits
    • Microbiological growths or slimes on the tower fill, condenser tubes, corrosion coupons, or any interior piping surface

Though most often found at carbon steel pipe and in open condenser water systems, MIC has also been shown to cause severe deterioration to copper and galvanized steel domestic water pipe.

• First Indications

The first suggestion of an MIC condition is often visual.  Heavily fouled pipe surfaces, tower pans, heat exchangers, water boxes, and other areas indicate microbiological activity.  The potential for MIC is always suggested by high colony counts at a standard field dip slide test, even though the microbes responsible for the MIC may not be present.  Metallurgical testing, however, usually offers the first solid evidence of an MIC problem by exposing the distinctive profile of the corroded pit area caused by specific microbiological organisms.  A follow-up chemical analysis of the pit area for the metabolic by-products of such microorganisms will provide further evidence that MIC exists.  The presence of sulfuric acid or sulfur based compounds, for example, would strongly suggest an MIC condition since it is a common by-product of MIC related sulfur reducing bacteria.

Providing absolute proof of a biological condition, however, often requires additional steps in follow-up to metallurgical testing.  Two additional methods for positively identifying the presence of MIC related microorganisms are:

• Identification Through Cell Cultures

The first method involves incubating, in a qualified lab, samples of the suspected microorganisms on culture plates having the necessary energy source, nutrients, pH, and temperature.  If present, and if in viable form, the microorganisms will grow and reproduce to yield visible colonies having very specific, shape, color, and texture – and which are reactive to specific indicating tests and identification procedures.  Mixed colonies of microorganisms generally require further culturing in order to make an identification.

Microbiological plate cultures are incubated for as long as 1-2 weeks, after which a report of colony forming units (CFU), or an estimate of cell quantity, can be established.  This culture method of cell identification provides a low cost means to not only identify specific microorganisms, but to estimate their quantity, or level of infection, within the piping system.

Providing representative and viable samples for testing is as important as the laboratory testing method itself.  Anaerobic microorganisms, those which cannot live in the presence of air or oxygen, and which are common to many MIC conditions, are especially difficult to properly sample and save.  All suspected microbiological samples should be taken immediately prior to pick-up by the lab facility or shipped via next day service.  Samples should also remain wet at all times, and should be collected into a sterile container whenever possible.

• Identification Through DNA

Plate culture identification may not always provide the information necessary to identify a suspected MIC problem.  This may be due to an insufficient sample, due to old, dried, or otherwise dead and non-viable organic material, or to the inability to conclusively identify suspected organisms.  In such cases, or where a greater and more finite degree of information is desired, advanced DNA identification is available.

Under this method, microbial DNA is directly isolated from the sample – which gives a more accurate representation of the microbial population of the sample without the bias of slower growing microorganisms not being represented.  In addition, injured cells, which may not be able to grow on standard cell culture media, will also be detected and named.

Such calculated colony forming units based upon DNA will be higher than at cultured colony forming units because of the inclusion of recently killed or dead cells, as well as cells that may not grow due to injury or failure of the media to provide favorable conditions for growth.  Unlike cell culturing, DNA testing does not require live viable microbiological

Varying levels of DNA analysis exist:

Preliminary Analysis
This “quick look” provides a breakdown of microbial DNA present for the three basic groups of bacteria, fungi, and algae.  Bacterial DNA is then further isolated according to whether it is gram negative or gram positive.

• Full DNA Analysis

This investigation reports the percentage of bacterial DNA found in the sample sediment and the percentage of each bacterial genus identified from a library of 21 bacterial genus-specific probes.  In addition, colony forming units are calculated for each bacterial genus detected.

The calculated CFU value is based on the amount of 1.6 fentagrams/bacterial cell.  Such calculated CFU’s will be higher than cultured CFU’s because of inclusion of recently killed or dead cells as well as cells that may not grow due to injury or failure of the media to provide favorable conditions for growth.

• Specific Genus

There are times when only information concerning a particular bacterial type is required without doing a full DNA analysis.  In such cases, a calculated colony forming unit (CFU) of the genus detected is given.

Again, the calculated CFU value is based on the amount of 1.6 fentagrams/bacterial cell.  Such calculated CFU’s will be higher than cultured CFU’s because of inclusion of recently killed or dead cells as well as cells that may not grow due to injury or failure of the media to provide favorable conditions for growth.

General categories of microorganisms tested for include: Slime formers, iron bacteria, sulfate reducers, and sulfuric acid producers.

• Final Confirmation

Often the second or third step in the evaluation of a pipe corrosion problem, DNA and cell culture testing offers the final proof most property managers require before committing to a long and likely expensive remedial effort.  Thorough knowledge of the problem is also critical to the water treatment contractor in order that they can evaluate and choose the best chemical cleaning option.

MIC is commonly suspected in most fire piping failures although in reality it is far less common.  This belief to an MIC problem, often “verified” by field MIC tests or less accurate lab analysis showing false positives for other common but non-threatening microbacterial forms, will be positively confirmed or refuted by DNA testing.

DNA testing is especially helpful where live cell samples cannot be provided, or where the interest to test for MIC is an after thought based upon a dried and stale pipe or deposit sample.  DNA offers one additional option toward ensuring that the steps taken in any pipe cleaning or remedial effort are the rights ones, and that they will be effective.

© Copyright 2024 – William P. Duncan, CorrView International, LLC