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Institute of Laboratory Medicine
Hyphae in focus: comparison of fungal
growth on agar with and without
SpinAX sample preparation
Nadine Hunn, Yannick Born, Eva Hitz, Michael Oberle
Institute of Laboratory Medicine, Cantonal Hospital Aarau AG, Aarau, Switzerland
Background
In clinical tissue samples, molds typically appear in hyphal form,
while spore formation (macro- and microconidia) is largely
restricted to superficial growth. For culture-based diagnostics,
mycologists fear that homogenization (bead-beating) of tissue
could likely damage hyphae1; therefore, it is recommended to
chop tissue into small portions. The SpinAX system (AxonLab)
offers automated, standardized, bead-free homogenization
using a grinder, but its effect on hyphal integrity and fungal Fig. 1: Study design: An agar punch containing mold was embedded in SAB agar
at 42 °C, a temperature chosen to preserve fungal structures. After 1–2 days of
viability remains unclear. incubation, the agar with grown-in hyphae was removed from the tube and a
section was cut: one piece was homogenized using the SpinAX device, while the
Aim other served as a positive control and was manually ground before being re-
inoculated onto SAB agar.
To assess whether processing of tissue with the SpinAX system
affects hyphal structure or growth of molds on agar.
No. of Tab. 1: One Candida and
Methods Species
Isolates 13 mold species of
Candida sp. 1 different origins were used
Mold isolates were embedded in tubes containing liquid Aspergillus fumigatus 2 for this study. Six Isolates
Sabouraud (SAB) agar (42 °C) to inhibit sporulation. After Aspegillus niger 1 were tested in duplicate
solidification, tubes were incubated at 28 °C for 1–2 days until Aspergillus brasiliensis 1 (indicated with (2)).
Aspergillus vitis (2)
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d at 28 °C for 1–2 days until Aspergillus brasiliensis 1 (indicated with (2)).
Aspergillus vitis (2) 1
fungal growth became visible (agar turbidity). The agar block Microsporum gypseum 1
was removed from the tube: one part was homogenized using Penicillium sp. 1
SpinAX, the other manually ground. Both preparations were Fusarium solanie complex (2) 1
microscopically examined and plated onto SAB agar. After Trichophyton mentagrophytes complex (2) 2
Mucor cicinelloides (2) 1
incubation at 28 °C for 1–2 days, fungal growth and colony Rhizomucor pusillus 1
morphology were qualitatively assessed (Fig. 1). Rhizops arrhizus (2) 1
total 14
Results
A total of 24 culture assays, including 3 negative controls and
14 Isolates (Tab. 1) were included in the study. All isolates
showed growth on SAB agar after SpinAX homogenization. Six
isolates were tested in duplicates with congruent results (Tab. 1,
isolates indicated with (2)). Growth was comparable between
conventional and SpinAX-prepared samples. Homogenization
with the SpinAX showed a uniform distribution of colonies on
the agar and a well separation of individual colonies in the
fractions (Fig. 2). Fig. 2: Growth of mold on SAB after SpinAX homogenization, single colonies are
Microscopic examination showed intact hyphal structures after detectable
SpinAX homogenization (Fig. 3).
Discussion
SpinAX offers a standardized alternative, potentially valuable for
homogenization of spore-deficient clinical samples. Here, we
established a reproducible protocol for induction of hyphal
growth to simulate the processing of tissue containing fungal
structures. We could show that hyphae were not damaged after
SpinAX homogenization and that growth was comparable to
manually processed 'tissue'. In this artificial setting, we certainly
deal with a high density of hyphae, which may not perfectly
represent patient samples. Therefore, it remains to be seen Fig. 3: Microscopic comparison of three mold species following manual and SpinAX
homogenization. The images demonstrate that hyphae remain int
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Fig. 3: Microscopic comparison of three mold species following manual and SpinAX
homogenization. The images demonstrate that hyphae remain intact after both
whether the method yields comparable results in real patient procedures, indicating that the SpinAX treatment does not cause structural
samples. damage.
1Med Mycol. 2022 Dec 7;60(12):myac081. doi: 10.1093/mmy/myac081. https://pubmed.ncbi.nlm.nih.gov/36477206/