Table 3.
Advantages ( +) and disadvantages (–) of CFW and WGA staining for the detection of chytrids
| CFW | WGA | |||
|---|---|---|---|---|
| Binding specificity | ± | non-specific fluorochrome that binds to beta-(1,4) and (1.3)-glucans, including chitin | + | chitin-binding lectin, which specifically binds to N-acetyl-D-glucosamine (in chitin) and N-acetyl-D-neuraminic (sialic) acid residues |
| Cross-reactivity | – | cross-reactivity with cellulose, chitosan, keratin, collagen, and elastin | – | cross-reactivity with peptidoglycan layer of Gram-positive bacteria |
| – | binding affinity to cellulosic cell walls of phytoplankton can hamper a successful distinction between phytoplankton cells and associated sporangia | |||
| – | oomycetes with cellulosic cell walls can be misidentified as chytrids | |||
| – | binding to cell walls of choanoflagellates expected | – | binding to cell walls of choanoflagellates observed | |
| Staining efficiency* | + | Sporangia: 88–100% | – | Sporangia: 0–100% |
| – | Rhizoids: 0–100% | + | Rhizoids: 100% | |
| + | Resting spores: 100% | – | Resting spores: 8–100% | |
| + | Stalks: 80–100% | – | Stalks: 0–100% | |
| Fluorescence | – | fixed wavelength (Exmax/Emmax 355/433 nm) | + | flexible wavelengths: WGA lectins are commercially available with different conjugated fluorophores (e.g., Alexa-Fluor 350, 488, 555, 647, etc., Fluorescein) |
| Co-staining | + | DAPI co-staining is not possible (but co-staining with SYTOX green has been applied) | + | DAPI co-staining possible, combined applications with CARD-FISH have been published |
| Sample preparation | + | Utermöhl chambers: good applicability | + | Utermöhl chambers: good applicability |
| – | PC filters: limited applicability (high | + | PC filters: good applicability (moderate | |
| background already at 1 µg CFW mL−1) | background even at 25 µg WGA mL−1) | |||
| ± | Flow cytometry: not tested herein | + | Flow cytometry: good applicability | |
| Sample fixation | + | live, non-fixed cells: successful staining of sporangia | – | live, non-fixed: partly unsuccessful staining of mature sporangia (limited accessibility to WGA binding sites) |
| + | Lugol and PFA-preserved cells: successful staining of sporangia | + | Lugol and PFA-preserved cells: successful staining of sporangia | |
| + | acidic Lugol did not induce formation/visualization of unspecific CFW-stained flocs | – | acidic Lugol induced formation/visualization of unspecific WGA-stained flocs (neutral/alkaline Lugol preferable) | |
| Storage | + | room temperature | – | -20 °C, repeated freeze/thawing cycles should be avoided |
| + | long-term storage (> 1 year) | + | stable for at least one year (manufacturer instructions) | |
| Costs | + | inexpensive (0.10 USD per 1 mL sample) | + | inexpensive (0.40 USD per 1 mL sample) |
*Based on entirely/partly stained features as shown in Fig. 2
± indicates a neutral evaluation.