CN104789508A - Culture medium for promoting bacterial biofilm formation and culture method - Google Patents
Culture medium for promoting bacterial biofilm formation and culture method Download PDFInfo
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Abstract
The invention discloses a culture medium for promoting bacterial biofilm formation and a culture method. Per liter of the culture medium contains 8-12g of sodium chloride, 7-13g of tryptone, 4-7g of yeast powder, 10-50g of calcium chloride and the balance of water. The culture medium for promoting the bacterial biofilm formation is taken as a bacterium culture medium for culturing the bacterium. The culture medium for promoting the bacterial biofilm formation is simple in formula, easy in preparation, low in cost and obvious in effect, has promoting effect for the Gram-positive and negative bacterium biofilm formation and is wide in application scope.
Description
Technical field:
The invention belongs to microorganism field, be specifically related to a kind of promote bacterial biof iotalm to be formed substratum and cultural method.
Background technology:
Bacterial biof iotalm (Bacterial biofilm, BF) be that one is attached to biology or non-biological material surface, wrapped up by the extracellular macromolecule of self secretion and there is the bacterial population of certain three-dimensional structure feature, biomembranous basic comprising comprises: the nutritive substance of water, bacterium, high polymer, absorption, meta-bolites and bacterial lysates etc., wherein biomacromolecule comprises protein, polysaccharide, DNA, RNA, peptidoglycan, fat and phosphatide etc.Different from the bacterium of unicellular floating state, show a series of new biological property on the whole in the bacterial biof iotalm of carrier surface propagation and there is the ability that stronger adaptation external environment coerces, as drug-fast raising, thus cause microbial film to be not easily eliminated, cause human diseases and Industrial products putrid and deteriorated etc.
The formation of bacterial biof iotalm is the process of a complexity and dynamic change, generally comprises four to double teacher: initial field planting stage, irreversible adhesion phase, structure differentiation stage, full-fledged stage and last bacterium come off and replant the stage.Research shows, said process can be subject to the impact of factors, as adhered to the material character of carrier, comprises carrier interface morphological specificity, electrostatic interaction, hydrophilic and hydrophobic and surfaceness; Cell surface physico-chemical property, comprises exocellular polysaccharide, extracellular protein and extracellular dna; Nutrition and envrionment conditions, comprise temperature, substratum, pH value, metal ion (Na
+, K
+, Ca
2+, Mg
2+, Fe
2+, Mn
2+, Zn
2+, Fe
3+and Al
3+deng) and sterilant etc.
How to promote in bacterial biof iotalm formation, more existing patents and article are announced or deliver.In " method and composition (200680023920.9) of regulating biofilm development " patent, author provides a kind of at least one of the nitrogen protoxide of significant quantity or significant quantity that utilizes and produces or discharge nitric oxide production reagent to the method promoting bacterial biof iotalm to be formed.With BMM substratum (containing following component in often liter of volume: 9.0mM Sodium Glutamate, 50mM glycerine, 0.02mM magnesium sulfate, 0.15mM SODIUM PHOSPHATE, MONOBASIC, 0.34mM dipotassium hydrogen phosphate, 145mM sodium-chlor, 20 μ l trace metal solutions and 1ml vitamin solution) induce Pseudomonas aeruginosa (Pseudomonas aeruginosa FRD1) biomembranous formation, find when adding the calcium chloride of 1.0mM and 10mM in BMM, biomembranous thickness increase about ten times of (Sarkisova can be made, S., M.Patrauchan, etal.2005.Calcium-induced virulence factors associated with the extracellular matrix of mucoidPseudomonas aeruginosa biofilms.Journal of Bacteriology 187 (13): 4327-4337).With PD2 substratum (containing following component in often liter of system: 4.0g Tryptones, 2.0g phytone, 1.0g trisodium citrate, 1.0g disodium succinate, 0.01g protohemine, 1.0g magnesium sulfate heptahydrate, 1.0g potassium primary phosphate, 1.5g dipotassium hydrogen phosphate and 2.0g bovine serum albumin component five) cultivate the shrivelled bacterium of leaf margin (Xylella fastidiosa), find when adding the ferrous sulfate of 1.0mM-2.5mM calcium chloride and 1.0-1.8mM in PD2 substratum, its biomembranous formation (Cruz can be promoted, L.F., Cobine P.A., et al.2012.Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility.Applied and Environmental Microbiology 78 (5): 1321-1331).With modified version TSB substratum (containing following component in often liter of system: 17g pancreatic digest of casein, 3g papaic digest of soybean meal, 5g sodium-chlor, 2.5g dipotassium hydrogen phosphate, 2.5g glucose and 1.5g biliary salts) cultivate sphingomonas paucimobilis (Sphingomonas paucimobilis), when adding magnesium chloride or the calcium chloride of 250 μm of concentration in substratum, the attachment of flcating germ on stainless steel substrates surface and biomembranous formation (Guvensen can be promoted, N.C., Demir S., et al.2012.Effects of magnesium and calcium cations on biofilm formation by Sphingomonas paucimobilisfrom an industrial environment.Fresenius Environmental Bulletin 21 (12): 3685-3692).With TRIS-G substratum (containing following component: 6.057g tri-(methylol) aminomethane in often liter of system, 7.88g Tris-HCl, 4.675g sodium-chlor, 1.4912g Repone K, 1.0698g ammonium chloride, 4.261g sodium phosphate, 2.033g six water cure magnesium, 0.294g Calcium dichloride dihydrate, 40mg sodium dihydrogen phosphate dihydrate, 4.8mg/L ferric ammonium citrate and 100 μ l trace substance liquid storages) cultivate pseudomonas mendocina (P.mendocina NR802), find in above-mentioned substratum, add 10mM or 20mM calcium chloride or magnesium chloride, biomembranous formation (Mangwani can be promoted, N., Shukla S.K., et al.2014.Calcium-mediatedmodulation of Pseudomonas mendocina NR802biofilm influences the phenanthrene degradation.Colloids and Surfaces B:Biointerfaces 114:301-309).With King B broth culture (containing following component in often liter of system: 20g peptone, 1.5g dipotassium hydrogen phosphate, 1.5g magnesium sulfate heptahydrate and 10ml glycerine) cultivate Pseudomonas fluorescens (P.fluorescens PCL1701), when adding the calcium chloride of finite concentration (as 15mM) in substratum, can promote that biomembranous biomass and surface coverage improve, and can not to have an impact (Safari to biomembranous thickness, A., O.Habimana, et al.2014.The significance of calcium ions on Pseudomonas fluorescens biofilms – a structural andmechanical study.Biofouling 30 (7): 859-869).With LB substratum (containing following component in often liter of system: 10g sodium-chlor, 10g Tryptones and 5g yeast powder) cultivate Aeromonas hydrophila (Aeromonas hydrophila) and enterococcus faecalis (Enterococcus faecalis) respectively, when adding the Calcium dichloride dihydrate of 1000mM in culture system simultaneously, biofilm formation (the Das of above-mentioned two strain bacterium can be promoted during the magnesium chloride of DNaseI and 5mM of 25units, T., Sehar S., et al.2014.Influenceof calcium in extracellular DNA mediated bacterial aggregation and biofilm formation.PLoS One9 (3): e91935).
The method of the promotion biofilm formation provided in above-mentioned patent or document is provided, some culture medium prescriptions are too complicated, what have is not easy to operate, composition in some formulas is expensive, therefore, a kind of simple and cheap culture medium prescription that simultaneously can have promoter action to the biofilm formation of gram negative bacterium and positive bacteria of filling a prescription of necessary exploitation.
Simultaneously, at present about the research of bacterial biof iotalm, mainly biomembranous generation development and corresponding gene regulating rule thereof, and how to reduce biomembranous generation and how to clear up microbial film, but, bacterial biof iotalm also has can be utilized namely useful one side, can prevent or reduce corrosion of metal as bacterial biof iotalm; Utilize bacterial biof iotalm can the function such as Adsorption of Heavy Metal Ions and denitrogenation, be used widely in sewage and wastewater treatment system; The also profit had is transformed into bacterial biof iotalm micro-factory (Nguyen of albumen synthesis, P., et al.2014.Programmable biofilm-based materials from engineered curlinanofibres.Nature Communications 5:4945), as can be seen here, bacterial biof iotalm will obtain advantageous application more widely in future.Utilize microbial film on a large scale if think, certain technology or method just must be used to enable bacterium form more microbial film on dirt settling surface.
Summary of the invention:
It is simple that first object of the present invention is to provide a kind of composition, raw material is easy to get, cheap, prepare easy, no matter be to gram positive bacterium or gram negative bacterium, its biomembranous formation can be promoted, the substratum that the promotion bacterial biof iotalm that can meet large-scale promotion needs is formed.
The substratum that promotion bacterial biof iotalm of the present invention is formed, it is characterized in that, often liter contains: sodium-chlor 8-12g, Tryptones 7-13g, yeast powder 4-7g, calcium chloride 10-50g, and surplus is water.
Second object of the present invention is to provide a kind of cultural method promoting bacterial biof iotalm to be formed, and it is characterized in that, it is that the substratum formed using above-mentioned promotion bacterial biof iotalm is cultivated as the substratum of bacterium bacterium.
Described bacterium is the bacterium etc. of Staphylococcus (Staphylococcus spp.), bacillus (Bacillus spp.), Rhodopseudomonas (Pseudomonas spp.), Citrobacter (Citrobacter spp.) or enterobacter (Enterobacter spp.).
Further preferably, described bacterium is streptococcus aureus (Staphylococcus aureus), subtilis (Bacillus subtilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Wei Shi citric acid bacillus (Citrobacterwerkmanii) or enterobacter cloacae (Enterobacter cloacae) etc.
The substratum that promotion bacterial biof iotalm of the present invention is formed, its proportioning is simple, prepare easy, cheap, successful, and has promoter action to the biofilm formation of Gram-positive and negative bacteria, and range of application is more extensive.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Testing method example
Test strain in following examples is all, with the substratum not adding calcium chloride in this embodiment, test strain is diluted to OD
600=1.0 are mixed with bacteria suspension mother liquor, then in aseptic triangular flask, add the easy of this embodiment and effectively promote the substratum that bacterial biof iotalm is formed, and then add bacteria suspension mother liquor according to the inoculum size of the volume fraction of 5%, become bacteria suspension working fluid, after bacteria suspension working fluid is mixed, draw 200 μ l bacteria suspension working fluids in 96 porocyte culture plates, do 8 repetitions, and establish blank (this embodiment easy and effectively promote not add in the substratum that bacterial biof iotalm is formed the substratum of calcium chloride), 96 orifice plates are put into 30 DEG C or 37 DEG C of constant incubator quiescent culture certain hours (24-48h), then 96 orifice plates are taken out, gently suck flcating germ, then each cultivation plate hole is cleaned respectively 3 times with aseptic deionized water, porous plate is placed at room temperature dry (now with the naked eye can see have more bacterial biof iotalm to be formed at the inwall of plate hole and bottom), and then in each plate hole, add 200 μ l 1% violet staining liquid, leave standstill room temperature dyeing 45min, suck violet staining liquid, again with aseptic washing plate hole three times to remove unnecessary Viola crystallina, after drying at room temperature 30min, the ethanol decolorization 30min of 200 μ L 95%, the long microplate reader of all-wave is utilized to survey its absorbance (OD at 595nm place
595).
Embodiment 1:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 8g, Tryptones 12g, yeast powder 7g, calcium chloride 10g, surplus is water, and after being mixed by the composition of above-mentioned substratum, sterilizing is for subsequent use.With streptococcus aureus (Staphylococcus aureus ATCC6538) as test strain, test according to the method in above-mentioned testing method example, with 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, the biofilm formation ability of bacterial strain is measured, the OD recorded by crystal violet staining assay and the long microplate reader of all-wave
595average is: 1.82; Use sodium chloride-containing 8g/L, Tryptones 12g/L, yeast powder 7g/L, surplus is that the control medium of water cultivates streptococcus aureus (Staphylococcus aureus ATCC6538), measured OD under similarity condition
595average is: 1.29 (contrasts); As can be seen here, use the easy and culture medium culturing streptococcus aureus (Staphylococcus aureus ATCC6538) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 41.09%.
Embodiment 2:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, calcium chloride 20g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With streptococcus aureus (Staphylococcus aureus ATCC6538) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.17; With often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, surplus is that the control medium of water cultivates streptococcus aureus (Staphylococcus aureus ATCC6538), measured OD under similarity condition
595average is: 1.32 (contrasts); As can be seen here, use the easy and culture medium culturing streptococcus aureus (Staphylococcus aureus ATCC6538) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 64.39%.
Embodiment 3:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 12g, Tryptones 7g, yeast powder 6g, calcium chloride 30g, surplus is water, is mixed by mentioned component, and sterilizing is for subsequent use; With streptococcus aureus (Staphylococcus aureus ATCC6538) as test strain, test according to the method for above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.32; Use sodium chloride-containing 12g, Tryptones 8g, yeast powder 6g, surplus is that the control medium of water cultivates streptococcus aureus (Staphylococcusaureus ATCC6538), measured OD under similarity condition
595average is: 1.35 (contrasts); As can be seen here, use the easy and culture medium culturing streptococcus aureus (Staphylococcus aureusATCC6538) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 71.85%.
Embodiment 4:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 10g, Tryptones 10g, yeast powder 5g, calcium chloride 40g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With streptococcus aureus (Staphylococcus aureus ATCC6538) as test strain, test according to the method for above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.05; With often liter of sodium chloride-containing 10g, Tryptones 10g, yeast powder 5g, surplus is that the control medium of water cultivates streptococcus aureus (Staphylococcus aureus ATCC6538), measured OD under similarity condition
595average is: 1.38 (contrasts); As can be seen here, use the easy and culture medium culturing streptococcus aureus (Staphylococcus aureus ATCC6538) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 1.21 times.
Embodiment 5:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 11g, Tryptones 9g, yeast powder 4g, calcium chloride 50g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With streptococcus aureus (Staphylococcus aureus ATCC6538) as test strain, test according to the method for above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.77; With often liter of sodium chloride-containing 11g, Tryptones 9g, yeast powder 4g, surplus is that the control medium of water cultivates streptococcus aureus (Staphylococcus aureus ATCC6538), measured OD under similarity condition
595average is: 1.39 (contrasts); As can be seen here, use the easy and culture medium culturing streptococcus aureus (Staphylococcus aureus ATCC6538) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 27.34%.
Embodiment 6:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter of sodium-chlor 8g, Tryptones 12g, yeast powder 7g, calcium chloride 10g, and surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With subtilis (Bacillus subtilis ATCC6051) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.36; With often liter of sodium chloride-containing 8g, Tryptones 12g, yeast powder 7g, surplus is that the control medium of water cultivates subtilis (Bacillus subtilisATCC6051), measured OD under similarity condition
595average is: 1.01 (contrasts); As can be seen here, use the easy and culture medium culturing subtilis (Bacillus subtilis ATCC6051) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 34.65%.
Embodiment 7:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, calcium chloride 20g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With subtilis (Bacillus subtilis ATCC6051) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.49; With often liter of sodium chloride-containing 9g, Tryptones 13g, yeast powder 6g, surplus is that the control medium of water cultivates subtilis (Bacillussubtilis ATCC6051), measured OD under similarity condition
595average is: 0.98 (contrast); As can be seen here, use the easy and culture medium culturing subtilis (Bacillus subtilis ATCC6051) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 52.04%.
Embodiment 8:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 12g, Tryptones 8g, yeast powder 6g, calcium chloride 30g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With subtilis (Bacillus subtilis ATCC6051) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.30; With often liter of sodium chloride-containing 12g, Tryptones 8g, yeast powder 6g, surplus is that the control medium of water cultivates subtilis (Bacillus subtilisATCC6051), measured OD under similarity condition
595average is: 1.09 (contrasts); As can be seen here, use the easy and culture medium culturing subtilis (Bacillus subtilis ATCC6051) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 19.27%.
Embodiment 9:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 10g, Tryptones 10g, yeast powder 5g, calcium chloride 40g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With subtilis (Bacillus subtilis ATCC6051) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.27; With often liter of sodium chloride-containing 10g, Tryptones 10g, the control medium of yeast powder 5g cultivates subtilis (Bacillus subtilisATCC6051), measured OD under similarity condition
595average is: 1.03 (contrasts); As can be seen here, use the easy and culture medium culturing subtilis (Bacillus subtilis ATCC6051) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 23.30%.
Embodiment 10:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 11g, Tryptones 9g, yeast powder 4g, calcium chloride 50g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With subtilis (Bacillus subtilis ATCC6051) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 37 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 1.23; With often liter of sodium chloride-containing 11g, Tryptones 9g, yeast powder 4g, surplus is that the control medium of water cultivates subtilis (Bacillus subtilisATCC6051), measured OD under similarity condition
595average is: 1.02 (contrasts); As can be seen here, use the easy and culture medium culturing subtilis (Bacillus subtilis ATCC6051) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 20.59%.
Embodiment 11:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 8g, Tryptones 12g, yeast powder 7g, calcium chloride 10g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.34; With often liter of sodium chloride-containing 8g, Tryptones 12g, the control medium of yeast powder 7g cultivates Pseudomonas aeruginosa (Pseudomonasaeruginosa ATCC9027), measured OD under similarity condition
595average is: 1.82 (contrasts); As can be seen here, use the easy and culture medium culturing Pseudomonas aeruginosa (Pseudomonas aeruginosaATCC9027) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 28.57%.
Embodiment 12:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, calcium chloride 20g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.62; With often liter of sodium chloride-containing 9g, Tryptones 13g, yeast powder 6g, surplus is that the control medium of water cultivates Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027), measured OD under similarity condition
595average is: 1.79 (contrasts); As can be seen here, use the easy and culture medium culturing Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 1.02 times.
Embodiment 13:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 12g, Tryptones 8g, yeast powder 6g, calcium chloride 30g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.68; With often liter of sodium chloride-containing 12g, Tryptones 8g, yeast powder 6g, surplus is that the control medium of water cultivates Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027), measured OD under similarity condition
595average is: 1.85 (contrasts); As can be seen here, use culture medium culturing Pseudomonas aeruginosa provided by the present invention (Pseudomonas aeruginosa ATCC9027), compared with the control, its biofilm formation ability improves 44.86%.
Embodiment 14:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 10g, Tryptones 10g, yeast powder 5g, calcium chloride 40g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.55; With often liter of sodium chloride-containing 10g, Tryptones 10g, yeast powder 5g, surplus is that the control medium of water cultivates Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027), measured OD under similarity condition
595average is: 1.88 (contrasts); As can be seen here, use the easy and culture medium culturing Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 35.64%.
Embodiment 15:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 11g, Tryptones 9g, yeast powder 4g, calcium chloride 50g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.26; With often liter of sodium chloride-containing 11g, Tryptones 9g, yeast powder 4g, surplus is that the substratum of water cultivates Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027), measured OD under similarity condition
595average is: 1.86 (contrasts); As can be seen here, use the easy and culture medium culturing Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC9027) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 21.51%.
Embodiment 16:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 8g, Tryptones 12g, yeast powder 7g, calcium chloride 10g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635) as test strain, test according to the method for above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.41; With often liter of sodium chloride-containing 8g, Tryptones 12g, yeast powder 7g, surplus is that the control medium of water cultivates Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635), measured OD under similarity condition
595average is: 2.03 (contrasts); As can be seen here, use the easy and culture medium culturing Wei Shi citric acid bacillus (Citrobacterwerkmanii ATCC51635) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 67.98%.
Embodiment 17:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, calcium chloride 20g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.75; With often liter of sodium chloride-containing 9g, Tryptones 13g, yeast powder 6g, surplus is that the control medium of water cultivates Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635), measured OD under similarity condition
595average is: 2.02 (contrasts); As can be seen here, use the easy and culture medium culturing Wei Shi citric acid bacillus (Citrobacterwerkmanii ATCC51635) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 85.64%.
Embodiment 18:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 12g, Tryptones 8g, yeast powder 6g, calcium chloride 30g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.81; With often liter of sodium chloride-containing 12g, Tryptones 8g, yeast powder 6g, surplus is that the control medium of water cultivates Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635), measured OD under similarity condition
595average is: 1.98 (contrasts); As can be seen here, use the easy and culture medium culturing Wei Shi citric acid bacillus (Citrobacterwerkmanii ATCC51635) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 92.42%.
Embodiment 19:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 10g, Tryptones 10g, yeast powder 5g, calcium chloride 40g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.87; With often liter of sodium chloride-containing 10g, Tryptones 10g, yeast powder 5g, surplus is that the control medium of water cultivates Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635), measured OD under similarity condition
595average is: 1.92 (contrasts); As can be seen here, use the easy and culture medium culturing Wei Shi citric acid bacillus (Citrobacterwerkmanii ATCC51635) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 1.02 times.
Embodiment 20:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 11g, Tryptones 9g, yeast powder 4g, calcium chloride 50g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With Wei Shi citric acid bacillus (Citrobacter werkmanii ATCC51635) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.53; With often liter of sodium chloride-containing 11g, Tryptones 9g, the control medium of yeast powder 4g cultivates Wei Shi citric acid bacillus (Citrobacterwerkmanii ATCC51635), measured OD under similarity condition
595average is: 2.01 (contrasts); As can be seen here, use the easy and culture medium culturing Wei Shi citric acid bacillus (Citrobacter werkmaniiATCC51635) effectively promoting bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 75.62%.
Embodiment 21:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 8g, Tryptones 12g, yeast powder 7g, calcium chloride 10g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With enterobacter cloacae (Enterobacter cloacae ATCC39978) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.28; With often liter of sodium chloride-containing 8g, Tryptones 12g, yeast powder 7g, surplus is that the control medium of water cultivates enterobacter cloacae (Enterobactercloacae ATCC39978), measured OD under similarity condition
595average is: 1.62 (contrasts); As can be seen here, use the easy and culture medium culturing enterobacter cloacae (Enterobacter cloacaeATCC39978) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 40.74%.
Embodiment 22:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 9g, Tryptones 13g, yeast powder 6g, calcium chloride 20g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With enterobacter cloacae (Enterobacter cloacae ATCC39978) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.89; With often liter of sodium chloride-containing 9g, Tryptones 13g, yeast powder 6g, surplus is that the substratum of water cultivates enterobacter cloacae (Enterobactercloacae ATCC39978), measured OD under similarity condition
595average is: 1.59 (contrasts); As can be seen here, use the easy and culture medium culturing enterobacter cloacae (Enterobacter cloacaeATCC39978) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 81.76%.
Embodiment 23:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 12g, Tryptones 8g, yeast powder 6g, calcium chloride 30g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With enterobacter cloacae (Enterobacter cloacae ATCC39978) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 3.08; With often liter of sodium chloride-containing 12g, Tryptones 8g, yeast powder 6g, surplus is that the control medium of water cultivates enterobacter cloacae (Enterobactercloacae ATCC39978), measured OD under similarity condition
595average is: 1.61 (contrasts); As can be seen here, use the easy and culture medium culturing enterobacter cloacae (Enterobacter cloacaeATCC39978) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 91.30%.
Embodiment 24:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 10g, Tryptones 10g, yeast powder 5g, calcium chloride 40g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With enterobacter cloacae (Enterobacter cloacae ATCC39978) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.15; With often liter of sodium chloride-containing 10g, Tryptones 10g, yeast powder 5g, surplus is that the substratum of water cultivates enterobacter cloacae (Enterobactercloacae ATCC39978), measured OD under similarity condition
595average is: 1.59 (contrasts); As can be seen here, use the easy and culture medium culturing enterobacter cloacae (Enterobacter cloacaeATCC39978) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 35.22%.
Embodiment 25:
Easy and effectively promote the substratum that bacterial biof iotalm is formed, the formula of this substratum is as follows: often liter containing sodium-chlor 11g, Tryptones 9g, yeast powder 4g, calcium chloride 50g, surplus is water, and after being mixed by mentioned component, sterilizing is for subsequent use; With enterobacter cloacae (Enterobacter cloacae ATCC39978) as test strain, test according to the method in above-mentioned testing method example.With 96 porocyte culture plates at 30 DEG C of quiescent culture bacterial strain 24h, measure the biofilm formation ability of bacterial strain by crystal violet staining assay and the long microplate reader of all-wave, the OD recorded
595average is: 2.03; With often liter of sodium chloride-containing 11g, Tryptones 9g, yeast powder 4g, surplus is that the control medium of water cultivates enterobacter cloacae (Enterobactercloacae ATCC39978), measured OD under similarity condition
595average is: 1.68 (contrasts); As can be seen here, use the easy and culture medium culturing enterobacter cloacae (Enterobacter cloacaeATCC39978) that effectively promotes bacterial biof iotalm to be formed provided by the present invention, compared with the control, its biofilm formation ability improves 20.83%.
Claims (4)
1. promote to it is characterized in that the substratum that bacterial biof iotalm is formed, often liter contains: sodium-chlor 8-12g, Tryptones 7-13g, yeast powder 4-7g, calcium chloride 10-50g, and surplus is water.
2. promote to it is characterized in that the cultural method that bacterial biof iotalm is formed, it is that the substratum formed using promotion bacterial biof iotalm according to claim 1 is cultivated as the substratum of bacterium bacterium.
3. cultural method according to claim 2, it is characterized in that, described bacterium is the bacterium of Staphylococcus (Staphylococcusspp.), bacillus (Bacillus spp.), Rhodopseudomonas (Pseudomonas spp.), Citrobacter (Citrobacter spp.) or enterobacter (Enterobacter spp.).
4. cultural method according to claim 2, it is characterized in that, described bacterium is streptococcus aureus (Staphylococcus aureus), subtilis (Bacillus subtilis), Pseudomonas aeruginosa (Pseudomonasaeruginosa), Wei Shi citric acid bacillus (Citrobacter werkmanii) or enterobacter cloacae (Enterobactercloacae).
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| CN105543325A (en) * | 2016-01-29 | 2016-05-04 | 珠海市现代农业发展中心 | Bacillus biofilm forming capability evaluation method and used culture media |
| CN109475170A (en) * | 2016-05-29 | 2019-03-15 | 以色列国家农业和农村发展农业研究组织沃尔坎尼中心 | The method for generating bacteria composition |
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| CN107022505A (en) * | 2017-03-30 | 2017-08-08 | 东北师范大学 | A kind of citric acid bacillus and its application for heavy metal copper ion remaval |
| CN111254100A (en) * | 2020-03-27 | 2020-06-09 | 山东中医药高等专科学校 | Pseudomonas aeruginosa biofilm and preparation method and application thereof |
| CN111607607A (en) * | 2020-04-27 | 2020-09-01 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for improving formation of Citrobacter williamsii biofilm |
| CN111607607B (en) * | 2020-04-27 | 2021-05-28 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for improving formation of Citrobacter williamsii biofilm |
| CN113149326A (en) * | 2020-12-14 | 2021-07-23 | 呼伦贝尔东北阜丰生物科技有限公司 | Comprehensive utilization process of threonine mother liquor |
| CN113583931A (en) * | 2021-09-28 | 2021-11-02 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Citrobacter williamsii ansB gene knockout mutant strain and application thereof |
| US11555193B1 (en) | 2021-09-28 | 2023-01-17 | Institute Of Microbiology, Guangdong Academy Of Sciences (Guangdong Detection Center Of Micr | Gene ANSB knockout mutant of citrobacter werkmanii and application thereof |
| CN115193350A (en) * | 2022-07-18 | 2022-10-18 | 齐鲁工业大学 | Method for microencapsulating lactobacillus in low-pH fruit juice |
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