Conditioned medium samples harvested from the MRC–5 cultures displaying deterioration were filtered (0.45 μm pore size), and
the filtrate inoculated onto fresh cultures of MRC–5 cells. When the secondary MRC–5 cultures were observed for 14 days, the
authors did not see a reappearance of the floating clumps or the progressive cell deterioration. This procedure was repeated,
with similar results being obtained. In contrast, passage of the same conditioned medium samples (albeit without filtration)
onto fresh MRC-5 cultures resulted in the reappearance of the floating clumps and progressive cell deterioration within a
The filtration results described above indicated a microbial, not a viral, origin for the effects noted. The authors subsequently
verified this idea through transmission electron microscopic examination of the affected MRC–5 cells, which revealed organisms
both within (see Figure 1) and outside of the cells (see Figure 2). These organisms were rod-shaped, with diameters ranging
from 0.34 to 0.39 μm and overall lengths of up to 1.1 μm. The organisms were not apparent on visual observation of the MRC–5
cell monolayers, using light microscopy. They were, however, readily observed when the focal plane was raised to that of the
medium–air interface. At this level, which is not typically viewed when scoring cell cultures for cytopathic effect, the authors
could easily visualize both the floating clumps noted at the monolayer and mats of dark, branching organisms (see Figure 3).
Figure 1: Transmission electron micrograph of an MRC–5 cell from an infected culture, showing intracellular organisms (B),
36700 x magnification. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
A Sabouraud dextrose agar (SAB) plate streaked with conditioned medium samples from the affected MRC–5 plates and incubated
aerobically at 20–25 °C led to white- to cream-colored colonies by day 7. The organism also was isolated using fluid thioglycollate
medium incubated aerobically at 30–35 °C. In the latter case, growth was observed by day 7 as particulates that were found
in a band about two thirds of the distance to the top of the liquid, and in a band floating at the liquid/air interface. Isolation
of the organisms on tryptic soy agar (TSA) plates resulted in white- to cream-colored colonies by day 7 after streaking of
conditioned medium samples from the affected MRC–5 plates. These colonies were present both on TSA plates incubated aerobically
at 20–25 °C and in plates incubated at 30–35 °C. Gram staining of the organisms revealed a weak, inconsistent positive reaction.
The organisms gave a positive reaction, staining as dark red and pink rods, when evaluated using Kinyoun's acid-fast staining
procedure. Colonies taken from the TSA plates were submitted to a subcontractor for identification. Using the 16s rRNA gene
sequencing method, the isolate was found to be a member of the Mycobacterium fortuitum complex. Differentiation of members of this nontuberculosis complex (M. chelonae, M. abcessus, and M. immunogenum) can be problematic. An assignment of M. chelonae or M. abscessus was obtained, and definitive identification to the species level was not pursued.
Figure 2: Transmission electron micrograph of an MRC–5 cell from an infected culture, showing extracellular organisms (B),
23600 x magnification.
The authors subsequently evaluated additional retained bioreactor harvest samples that had been collected at various intervals
to establish whether the microbe was present in the bioreactor before termination of the run. The earliest bioreactor harvests
did not cause MRC–5 cell deterioration, and organisms were not detected in the inoculated detector cell cultures. In contrast,
all samples from a point approximately midway through the run onward elicited these effects. The latency of onset of the observed
effects in the detector cell cultures correlated generally with bioreactor harvest day, such that the most rapid effects were
observed in the samples harvested just before termination of the bioreactor run. Isolation of organisms from the affected
studies led, in each case, to identification of the M. chelonae or M. abscessus.
Figure 3: Light micrograph of an infected MRC-5 culture, showing mats of organisms and floating clumps at the plane of the
medium/air interface (50 x magnification).