In our lab we have seen that for good staining of biofilms the best cv concentration is 0.5-1% in pure ethanol, while in original work from O'Toole 2011 a 0.1% solution is proposed.

Hello

For biofilm the concentration of crystal violet 0.5% is better , but also you can use 1%,because its effect on O.D.

1% Aqueous Gram Crystal Violet is a reagent recommended for use in qualitative procedures to differentiate gram-negative from gram- positive organisms

MERRITT et al, 2015: Growing and Analyzing Static Biofilms

(Curr Protoc Microbiol. Author manuscript; available in PMC 2015 Sep 14. Published in final edited form as: Curr Protoc Microbiol. 2005 Jul; 0 1: Unit–1B.1. doi:  [10.1002/9780471729259.mc01b01s00]

PM CID: PMC4568995 PMID: 18770545 )

Growing and Analyzing Static Biofilms

...0.1% (w/v) crystal violet in water

HANEY et al, 2018:

Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides (Biomolecules. 2018 Jun; 8(2): 29. Published online 2018 May 21. doi:  [10.3390/biom8020029] PMCID: PMC6022921 PMID: 29883434)

".....'One mL of a 0.1% CV solution'.....(NB: i.e. hydrous solution .....)... CV is a dye that is familiar to all microbiologists .... but also: .." assaying residual bound bacteria using crystal violet (CV) has issues, since CV stains biomass rather than living bacteria, and thus dead bound bacteria will still be stained. ... and ...."CV has certain drawbacks, including non-specific binding to anionic proteins and other negatively charged molecules, like capsules, lipolysaccharides, and DNA/nucleic acids, leading to an inability to distinguish between live and dead bacterial populations. These issues contribute to a large variability between samples that may complicate the interpretation of biofilm screening results."

I know you haven't asked for that, but - just to mention - there are also other staining methods available, like:

LATIMER et al, 2016: "A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis", as publ. in: Anal Bioanal Chem. 2016; 408: 999–1008.

Quote from their Methods section: "The stain contained 100 mL of 0.1× concentration phosphate-buffered saline (PBS) at pH 7.4 with 0.1 g of erythrosine B (Thermo Fisher Scientific, Waltham, MA), 0.2 g KeyAcid Rhodamine (Keystone Analine Corporation, Chicago, IL), and 0.3 g Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific) mixed in. ....."

(cf. also ResearchGate: Article A method for rapid quantitative assessment of biofilms with ...

find the original OA-article at (prior to pasting the following URL into your browser DELETE the underscore character " _ " in between >>https>://.> dito: https_://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709385/pdf/216_2015_Article_9195.pdf and the Supplementary Material:

ESM 1: (PDF 903 kb) 216_2015_9195_MOESM1_ESM.pdf (904K) = >>dito deleting "_ " before pasting into your browser:

https_://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709385/bin/216_2015_9195_MOESM1_ESM.pdf

Thanks for the answers. Any further info on bacterial gram stain? Some people saying its always 1%? Anyone use anything other than that?

Dominik: you haven't mentioned, in which "system" [e.g. smears, bacteria culture, tissue sections, etc.] and why CV solely you want to use.

It might be of benefit for others if they know more detailed about the conditions of our staing....

It might be that Gram staining by 'crystal violet' only might be a bit out-fashioned in terms of 'modern analysis' (but see below for smears, as well as other staining techniques) because there have been reported 'some issues' - regarding specifity and interpretation - "which contribute to a large variability between samples that may complicate the interpretation of biofilm screening results "....(see also my previous answer quoting from " HANEY et al, 2018 " .

But for sure you can find a lot of articles where use of CV with varying concentrations and solutes (water or alcohol) is described. So it may - a usual - depend on the task you have to manage....

e.g., cf: Article Use of the Gram stain in microbiology

( TJ Beveridge, May 2001: "Use of the Gram stain in microbiology" published in: Biotechnic and Histochemistry 76(3):111-118 - DOI: 10.1080/714028139; Unfortunately PPV (Pay Per View)

or

>> crystal violet. Experiment Findings >>File available Jan 2018 by Odysseas Kopsidas [ Odysseas Kopsidas ] Part of the project: 'Experimental Findings' (Experiment Findings crystal violet

Experiment Findings crystal violet several adsorbent πριονιδι spruce 15-7-2011

For finding other publications copy and paste into your browser (still already when on ResearchGate: for publications:

https://www.researchgate.net/search.Search.html?type=publication&query=Gram%20staining%20crystal%20violet ;

for Questions, Queries and answers:

https://www.researchgate.net/search.Search.html?type=question&query=Gram%20staining%20crystal%20violet.

You might also consult 'CONN's Biological Stains - A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine' (HOROBIN RW and KIERNAN JA, Eds, eg. 10th Ed. 2002, Chapter 14: Amino di- and triarylmethane dyes, stating synonyms of the dye (e.g. gentian violet ) properties CV's solubility in water: 0.2-1.7%,3-14% in ethanol, also in acetone (0.4%) and chloroform (5.1 %), insoluble in xylene.

BANCROFT JD and GAMBLE M (Eds)Theory & Practice of Histological Techniques, 6th Ed, Repr.2008 (Churchill-Livingstone/Elsevier), p. 312 / 313, presenting a

"Gram method for bacteria in smears" (flame-dried, "stain for 15 seconds in 1% [NB: meant: hydrous solution] crystal violet OR methyl violet.. counterstain for 20 seconds with carbol fuchsin....." ) and a

"Modified BROWN-BRENN method for Gram-positive and Gram-negative bacteria in paraffin sections (Churukian & Schenk, 1982)" which indicates use of commercially available Crystal violet solution [ composition given as:

• Crystal violet, 10% alcoholic: 2 ml
• Distilled water: 18 ml

Ammonium oxalate, 1%: 80 ml __________________________________________ (that means End volume 100 ml and an end concentration for CV of 0.1% ). The protocol of this particular modified BROWN-BRENN-Gram +/- stain fills two columns / 1 page (and therefore a bit more sophisticated, though, than plain CV hydrous or alcoholic ).

In the description for "The GRAM STAIN": (quote:) "In spite of more than a cenury having passed since GRAM described his technique in 1884, its chemical rationale is still obscure......

The following procedure ["Gram method for bacteria in smears"] is only suitable for the demonstration of bacteria in smears of pus and sputum." (end of quote....)

For me the perhaps serious question arises whether one can rely only on the technique of Gram when trying to quantify [to analyse quantitatively - quantising = counting?] biofilm(s).

Good luck, best wishes, W.H.M.

For bacteria staining commonly 1% aqueous solution of crystal violet is used

e.g. https://assets.thermofisher.com/TFS-Assets/MBD/Instructions/IFU40063.pdf

Regards

Vit

What can be used instead of ammonium oxalate for Crystal violet dye preparation for gram staining?