Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.