CN101718802B - Method for fixing mineral leaching rod-shaped bacteria for AFM analysis under strong acid condition - Google Patents

Abstract

The invention discloses a method for fixing mineral leaching rod-shaped bacteria for AFM analysis under a strong acid condition. In the method, a filter membrane (mixed cellulose ester millpore filter membrane) with an aperture of 0.22 micron is used as a fixing material, a domestic filter (the diameter is 25 millimeters) is used as a filter device, and a disposable medicine injection is utilized for filtration through pressurization. In a preparation process of fixing the mineral leaching bacteria, the filter condition and the filter device have small influences on the activity of the bacteria, no toxicity and convenient operation. In a solution with a pH value of 2, the prepared bacteria have low drift rate, good fixing effect and higher stability, thus an atomic force microscope (AFM) is suitable for analyzing the mineral leaching bacteria under the strong acid condition. The method can be widely applied to the field of AFM researches and has good application prospect and practical values.

Description

The fixing means that is used for the mineral leaching rod-shaped bacteria of AFM analysis under a kind of strong acid condition

Technical field

The present invention relates to a kind of microorganism fixing means, relate to the fixing means that is used for the mineral leaching rod-shaped bacteria that AFM analyzes under a kind of strong acid condition on especially.

Background technology

Atomic force microscope (AFM) has atomic resolution, can survey under vacuum, atmosphere, liquid and temperature control condition, and sample can be conductor, nonconductor, also can be flexible or even the living body biological sample.It can characterize the surface topography of sample, and the surface nature of study sample survey the dynamic process of intermolecular interaction etc. under nearly physiological condition, so AFM gets more and more people's extensive concerning.

AFM is the atomic interaction by the probe on the micro-cantilever and sample surfaces mainly.Yet in air ambient, because water vapour (perhaps other gas molecules) forms hydration shell (perhaps other adsorption films) at needle point and sample surfaces, therefore when the needle point scanning sample, needle point-sample room can form capillary force, and capillary force has very important impact to the interaction force of needle point-sample room.In order to eliminate the impact of capillary force, and the complete actual environment of analog sample, often needle point and sample are dipped in fully in the water (perhaps in other transparency liquids) and test.

The organic substances such as glutaraldehyde commonly used or poly-D-lysine fix free biological cell both at home and abroad at present, with the fresh mica sheet that dissociates, slide or through the slide of special processing as substrate, carry out at last the configuration of surface observation of cell with contact mode or the pattern of rapping.But regrettably, imaging effect is also very undesirable.This mainly is because surface of cell membrane is soft, with substrate fixing a little less than, the pressure of probe can make the contact area of probe and cell surface greatly increase, thus damaging cells or cause the drift of cell.And these method steps are loaded down with trivial details, increased the spending of operation easier and expense.Also there is the expert to fix circular biological cell with miillpore filter, but fixes bacillus with filter membrane, also do not report both at home and abroad, and ore-leaching bacteria mostly is shaft-like.

Summary of the invention

It is easy that technical matters to be solved by this invention provides a kind of method, economy, and be used for the fixing means of the mineral leaching rod-shaped bacteria that AFM analyzes under the high strong acid condition of fixed effect.

In order to solve the problems of the technologies described above, be used for the fixing means of the mineral leaching rod-shaped bacteria of AFM analysis under the strong acid condition that the present invention adopts, step is as follows:

(1), filtrator is prepared

The filtrator paper using individual packaging that each diameter is 25 millimeters, then conventional high pressure steam sterilization is dried, and is for subsequent use;

(2), filter membrane is prepared

Be that 0.22 micron filter membrane lies against in the cleaning vessel with the aperture, soak with sterilized water, make all wettingly, use again an amount of sterile water wash once before the use;

(3), preparation bacterial suspension

Adopting bacteria concentration is～10 ⁷The bacterium liquid of individual/mL is filtered fluid, and filtrate is through pre-filtering, in order to avoid filter membrane stops up;

(4), the wet filter membrane that will wash down packs in the filtrator, filtrator and filter membrane do not leak;

(5), filter

Extract liquid to be filtered with syringe, be contained on the filter inlet, push away gently a little liquid first, make filter membrane moistening, then slightly firmly advance;

(6), AFM Analysis.

Adopt technique scheme, be used for the fixing means of the mineral leaching rod-shaped bacteria that AFM analyzes under strong acid condition.Adopting bacteria concentration is～10 ⁷The bacterium liquid of individual/mL is filtered fluid, and the aperture is that 0.22 micron filter membrane (mixed cellulose ester microporous membrane) is immobilization material, and domestic filtrator (25 millimeters of diameters) is filtration unit, draws bacterium liquid by pressure filtration with the Dispoable medical syringe.

The present invention adopts simple filtration unit just can fix ore-leaching bacteria, it is simple in structure, easy and simple to handle, impact on bacterial activity is little, avirulence, and the bacterium that makes is that the bacterium drift rate is low in 2 the solution at pH, good fixing effect, stability is higher, the reduction that these are a large amount of workload and expense, more simple and with strong points than other method.

Description of drawings

Fig. 1 is filter apparatus configuration schematic diagram of the present invention;

Fig. 2 is the torus that the filter membrane after filtering shows under common binocular microscope light;

Fig. 3 is the filter membrane (Fig. 2 representative) of seeing under common binocular microscope.

Embodiment

The invention will be further described below in conjunction with drawings and Examples.

Embodiment 1: fixing ore-leaching bacteria Thiobacillus ferrooxidans (Acidithiobacillus ferrooxidans)

(1) filtrator is prepared

Check whether the filtrator 2 of 25 millimeters of diameters and edge and the plane of filter plate have burr, or the irregular place cushion rubber of the inside, make it level and smooth.Each filtrator 2 paper using individual packaging, then conventional high pressure steam sterilization is dried, and is for subsequent use.

(2) filter membrane is prepared

Filter membrane is isotropy, regardless of pros and cons.With 0.22 micron mixed cellulose ester microporous membrane 3 (25 millimeters the filter membrane that available and filtrator is supporting, also can oneself be cut into and be fit to size) lie against in the cleaning vessel, soak with sterilized water, make all wettingly, use again an amount of sterile water wash once before the use.

(3) bacterial suspension preparation

Original strain is with aseptic 9K medium culture; PH2.0; 120 ℃ of sterilization 20min.With Fe ²⁺During for the cultivation energy, add the FeSO of 44.7g/L ₄7H ₂O; During take S as the cultivation energy, add 10g/L sulphur powder.At 30 ℃, enrichment culture some generations under the 170r/min condition.

Bacterial growth filters with common qualitative filter paper first during to exponential phase.Then, get 2mL filtrate, centrifugal 5min under 1000r/min removes the bottom solids, stays supernatant stand-by.Get an amount of stand-by liquid, with directly count bacterial population in the bacterium liquid of binocular microscope and blood cell counting plate.Bacteria concentration is higher than 10 ⁷Diluting that individual/order of magnitude of mL is above is rear stand-by.

(4) filter front the preparation

To pack in the filtrator 2 of 25 millimeters of diameters by the 0.22 micron wet mixed cellulose ester microporous membrane 3 that step (2) wash down, check whether filtrator 2 has leakage.Take out several milliliters of air with the syringe of new unlatching, again be contained in the filter inlet place, inject air, will feel that at this moment air can not pass through filtrator on the contrary, slightly loose one's grip, plunger will rebound, and this just illustrates that filtrator and filter membrane do not leak.

(5) filter

Referring to Fig. 1, because ore-leaching bacteria is difficult for polluting, the method can be done in common table top drilling.Extract 1mL bacterium liquid to be filtered with syringe 1, be contained in filtrator 2 imports, push away gently a little liquid first, make mixed cellulose ester microporous membrane 3 moistening, then slightly firmly advance.After having filtered, waited 3 to 4 minutes, mixed cellulose ester microporous membrane 3 is taken out, use an amount of sterile water wash once again, on filter paper unnecessary water is sopped up, filter membrane is stand-by.

(6) analyze

Referring to Fig. 2 and Fig. 3, the back side (going out the water surface) of filter membrane is stained with double faced adhesive tape, stick on 2 * 2 centimetres the cover glass, again cover glass is bonded on the operation round platform of atomic force microscope, add suitable sterilization 9K, carry out surface analysis.

Claims (1)

1. be used for the fixing means of the mineral leaching rod-shaped bacteria that AFM analyzes under the strong acid condition, it is characterized in that: step is as follows:

(1), filtrator is prepared

The filtrator paper using individual packaging that diameter is 25 millimeters, then conventional autoclaving is dried, and is for subsequent use;

(2), filter membrane is prepared

Be that 0.22 micron filter membrane lies against in the cleaning vessel with the aperture, soak with sterilized water, make all wettingly, use again an amount of sterile water wash once before the use;

(3), preparation bacterial suspension

Adopting bacteria concentration is 10 ⁷The bacterium liquid of individual/mL is filtered fluid, and filtrate is through pre-filtering, in order to avoid filter membrane stops up;

(4), the wet filter membrane that will wash down packs in the filtrator, filtrator and filter membrane do not leak;

(5), filter

Extract liquid to be filtered with syringe, be contained on the filter inlet, push away gently a little liquid first, make filter membrane moistening, then slightly firmly advance;

(6), AFM Analysis.

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