Table 3.
Example of the variability in protocol conditions of crystal violet assays for three different example microorganisms.
| Condition | Organism |
||
|---|---|---|---|
| Staphylococcus aureus spp. | Pseudomonas aeruginosa spp. | Candida albicans | |
| Inoculum preparation | |||
| Media | TSB, TSB wS*, LBb*, Water [8,11,18,[36], [37], [38], [39], [40], [41]] | TSB, TSB wS*, LBb*, LB*, BHI*, MHI*, T-broth*, AB* [8,[42], [43], [44], [45], [46], [47], [48], [49]] | YNB*, YPD*, RPMI-1640*, SDB* [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Inoculum incubation temperature (°C) | 35-37 [8,11,18,[36], [37], [38], [39], [40], [41]] | 25-37 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 30-37 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Incubation time (hours) | 0**-24 [8,11,18,[36], [37], [38], [39], [40], [41]] | 0**-24 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 12-24 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Inoculum shaking conditions | 0–200 rpm/min [8,11,18,[36], [37], [38], [39], [40], [41]] | 0–250 rpm/min [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 0–200 rpm/min, Roller drum [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Inoculum concentration/OD/growth phase at harvest | 103–108 CFU/mL, 0.5 McFarland, OD600nm=0.1 [8,11,18,[36], [37], [38], [39], [40], [41]] | 10–108 CFU/mL, OD600nm=0.0025, OD595=1.5 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 104–108 CFU/mL, OD600nm=1 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Biofilm growth | |||
| Media | TSB, LB*, BHI* [8,11,18,[36], [37], [38], [39], [40], [41]] | TSB, T-broth*, AB*, BHI*, MHI* [8,[42], [43], [44], [45], [46], [47], [48], [49]] | YNB*, YPD*, RPMI-1640*, ASM*, SDB*, PBS* [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Incubation temperature (°C) | 35-37 [8,11,18,[36], [37], [38], [39], [40], [41]] | 25-37 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 37 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Incubation time (hours) | 18-48 [8,11,18,[36], [37], [38], [39], [40], [41]] | 2-48 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 2-48 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Shaking conditions | 0–200 rpm/min [8,11,18,[36], [37], [38], [39], [40], [41]] | 0–180 rpm/min [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 0–120 rpm/min [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Biofilm Assessment | |||
| Washing agent | Water, Saline, PBS*, MilliQ water [8,11,18,[36], [37], [38], [39], [40], [41]] | Saline, Water, PBS* [8,[42], [43], [44], [45], [46], [47], [48], [49]] | PBS*, Water, Saline [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Washing (x times) | 1-3 [8,11,18,[36], [37], [38], [39], [40], [41]] | 1-3 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 1-3 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Crystal violet concentration | 0.01–2.3% [8,11,18,[36], [37], [38], [39], [40], [41]] | 0.1–2% [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 0.02–1% [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Staining time | 1–20 min [8,11,18,[36], [37], [38], [39], [40], [41]] | 5–30 min [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 5–45 min [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Solubilisation agent | 33% acetic acid, 95–100% ethanol [8,11,18,[36], [37], [38], [39], [40], [41]] | 30–33% acetic acid, 95–100% ethanol, DMSO* [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 30–33% acetic acid, 95% ethanol, 0.1% Triton-X [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
| Absorbance wavelength (nm) | 540-595 [8,11,18,[36], [37], [38], [39], [40], [41]] | 550-595 [8,[42], [43], [44], [45], [46], [47], [48], [49]] | 540-595 [8,[50], [51], [52], [53], [54], [55], [56], [57]] |
*wS - with Supplement (i.e. added yeast and/or glucose); TSB- Tryptic Soy Broth; LBb – Luria Bertani broth; BHI- Brain Heart Infusion; LB – Lysogeny broth; MHI – Mueller-Hinton broth; T-broth – Terrific broth; AB – minimal growth media; YNB – Yeast Nitrogen Base; YPD – Yeast Peptone Dextrose; SDB – Sabauraund Dextrose Broth; RPMI-1640 - Roswell Park Memorial Institute–1640 medium; ASM – Artificial Saliva Medium; PBS - Phosphate buffered saline; DMSO - Dimethyl sulfoxide.
**0 – Inoculum prepared directly from agar culture.