Based on experience, one of the most challenging aspects of identifying filamentous bacteria is utilizing Gram and Neisser staining. Many incorrect identifications over the years have likely been related to over-emphasis of staining reactions in various dichotomous keys. Many of these keys create a path to follow based on filament staining reactions, and unfortunately, this concept is more complicated than originally believed.
Potential complications
Possible complications in staining reactions are likely related to factors such as chemistry reactions in the wastewater, the techniques applied during staining and the highly broad potential genetic diversity within various filamentous bacteria morphotypes (how we name them based upon how they look).
For example, in the not too distant past, we thought we may be identifying Thiothrix I and Thiothrix II under the microscope. The MiDAS field guide currently recognizes 46 separate species within the Thiothrix genus. In addition to this, there are many closely related genera that we call “Thiothrix” under the microscope, that are technically something else. The truth is that we cannot correctly assume the genetic properties of any filament based only on microscopy.
The good news, however, is that the morphology of filamentous bacteria and their associated growth conditions has and continues to be an excellent tool to utilize microscopy in process control and troubleshooting decisions. The big takeaway from this is that we are identifying filamentous bacteria morphotypes, not filamentous bacteria directly. This leads to minor grammar changes, such as not italicizing the name (which is for genus only, not morphotype), but more importantly, for the context of this article, offers another potential explanation on why many of the traditional staining reactions associated with various filament types are inconsistent in practice.
Staining remains important
For a thorough microscopic evaluation, Gram and Neisser staining remain critical. Several elements remain true, such as Actinomycetes-Mycolata (Nocardioforms) and Microthrix must stain Gram-positive; type 0092 filaments must stain Neisser positive; and type 0581 filaments (generally rarely occurring) must stain Gram-negative for identification. Even in these instances, there are potential technicalities such as Actinomycetes-Mycolata and Microthrix often losing their Gram-positive staining reactions when they are dead or non-viable. On the positive side, staining does often allow better viewing of filamentous bacteria abundance within the flocs, and it is not uncommon to miss certain filaments (such as Actinomycetes-Mycolata) completely using phase contrast alone.
Practical application
Using a dichotomous key in filamentous bacteria identification appears to be outdated and inefficient. Some common roadblocks encountered include staining reactions associated with Nostocoida limicola, Thiothrix, type 021N, Beggiatoa, type 1851 filaments and others. For filament identification, the cell shape and cell diameters appear most critical. The most practical way of learning filamentous bacteria identification involves the general morphotype descriptions, “bird-watching” techniques — as in, once you have seen it, you understand how to identify it — and taking into context surrounding microbe types that are present.
About the author: Ryan Hennessy is the principal scientist at Ryan Hennessy Wastewater Microbiology. He was trained and mentored by Dr. Michael Richard for over 10 years in wastewater microbiology, and serves as a microbiology services consultant. Hennessy is a licensed wastewater treatment and municipal waterworks operator in the state of Wisconsin and fills in as needed for operations at several facilities. He can be reached at ryan@rhwastewatermicrobiology.com. Hennessy's new book Wastewater Microbiology: Filamentous Bacteria Morphotype Identification Techniques, and Process Control Troubleshooting Strategies is now available on Amazon.
