CN109883796A - A kind of simple color development enhancing method of candida albicans as detection applications - Google Patents
A kind of simple color development enhancing method of candida albicans as detection applications Download PDFInfo
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- CN109883796A CN109883796A CN201910305862.8A CN201910305862A CN109883796A CN 109883796 A CN109883796 A CN 109883796A CN 201910305862 A CN201910305862 A CN 201910305862A CN 109883796 A CN109883796 A CN 109883796A
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- candida albicans
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- dyeing
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Abstract
The embodiment of the invention discloses a kind of simple color development enhancing methods of the candida albicans as detection applications, candida albicans are transferred in YPD fluid nutrient medium in the candida albicans that the weak culture medium culture in husky fort obtains, 30 DEG C of shaken cultivation 6-8h obtain candida albicans bacterium solution；The candida albicans bacterium solution is centrifuged and obtains first solution of the candida albicans body addition containing sodium bicarbonate, and the second solution mixing containing FITC is added, dyeing mixed liquor is obtained, obtains the candida albicans for showing enhancing.The embodiment of the present invention carries out dyeing colour developing to Candida albicans by the first solution containing sodium bicarbonate and the second solution containing FITC, it is simple that it dyes process color, it is good to dye color developing effect, and the candida albicans not inactivated can be dyed, has widened the purposes of dyeing candida albicans significantly.
Description
Technical field
The present embodiments relate to technical field of microbial detection, and in particular to a kind of candida albicans as detection applications
Simple color development enhancing method.
Background technique
Candida albicans is as a kind of conditionity pathogenic bacteria, and opportunistic fungal infection caused by candida albicans is in rising year by year
Gesture.The virulence factor of candida albicans, drug resistant gene and its with the correlation of host be still research hot spot.How is candida albicans
It is its most important condition for infecting host by body cell identification, is also considered as its pathogenic key.Cell swallows candida albicans
Test is to study the pattern receptors on inherent immunity cell to the important method of fungal molecule recognition mode, in the process of the experiment
In, it needs to dye candida albicans, convenient for the process that observation candida albicans is phagocytized by cells, helps further to disclose white
Candida albicans is by cell recognition and the mechanism of infection cell.
Currently, the color staining method of candida albicans routine is to use fluorescein isothiocynate (FITC), it is added to inactivation
In candida albicans suspension, 37 DEG C are protected from light incubation 1h.The color staining method of this candida albicans imitates the dyeing of candida albicans
Rate is lower, and dyeing effect is poor, as the candida albicans for detection, has be easy to cause with the test work of these dyeing candida albicans
There are errors for the experimental data that tool obtains.
Summary of the invention
For this purpose, the embodiment of the present invention provides the simple color development enhancing method of candida albicans as detection applications, it is existing to solve
Have the candida albicans dyeing effect in technology as detection poor, dyeing efficiency is low, cannot to do not inactivate thallus dyed with
And the problem of dyeing course complexity.
The purpose of the present invention is to provide a kind of easy to operate, it is easy to accomplish the side for not inactivating candida albicans fluorescent staining
Method, to overcome the problems of current colouring method.
To achieve the goals above, the embodiment of the present invention provides the following technical solutions:
A kind of simple color development enhancing method of candida albicans as detection applications trains candida albicans in the weak culture medium in husky fort
It supports the candida albicans obtained to be transferred in YPD fluid nutrient medium, 30 DEG C of shaken cultivation 6-8h obtain candida albicans bacterium solution；
The candida albicans bacterium solution is centrifuged and obtains first solution of the candida albicans body addition containing sodium bicarbonate, and is added
The second solution mixing containing FITC, obtains dyeing mixed liquor, obtains the candida albicans of display enhancing.
Preferably, which comprises 1-2h is dyed into 37 DEG C of oscillations of the dyeing mixed liquor.
Preferably, the method includes being protected from light 10-14h for described 4 DEG C of dyeing mixed liquor.
Preferably, first solution further includes the EDTA, mass fraction 0.01%Tween- that molar concentration is 1mM/L
20 and mass fraction be 0.1%NaN3；
The molar concentration of the sodium bicarbonate is 0.05M/L.
Preferably, second solution is that 50mg FITC is dissolved in 1ml DMSO to be prepared.
Preferably, it the method also includes by after thallus centrifugation, then is washed twice with PBS, and bacterial concentration is adjusted
It is 5 × 105The process of cfu/mL.
The embodiment of the present invention has the advantages that
The embodiment of the present invention reads white by the first solution containing sodium bicarbonate and the second solution containing FITC
Pearl bacterium carries out dyeing colour developing, and dyeing process color is simple, and dyeing color developing effect is good, and dyeing efficiency is high, and can be to not inactivating
Candida albicans dyed, widened significantly dyeing candida albicans purposes.
Detailed description of the invention
It, below will be to embodiment party in order to illustrate more clearly of embodiments of the present invention or technical solution in the prior art
Formula or attached drawing needed to be used in the description of the prior art are briefly described.It should be evident that the accompanying drawings in the following description is only
It is merely exemplary, it for those of ordinary skill in the art, without creative efforts, can also basis
The attached drawing of offer, which is extended, obtains other implementation attached drawings.
Fig. 1 is the candida albicans observation figure dyed under fluorescence microscope provided in an embodiment of the present invention, and amplification factor is
20X；
Fig. 2 is the candida albicans observation figure dyed under optical microscopy provided in an embodiment of the present invention, and amplification factor is
20X；
Fig. 3 is the candida albicans observation figure dyed under fluorescence microscope provided in an embodiment of the present invention, and amplification factor is
40X；
Fig. 4 is the candida albicans observation figure dyed under optical microscopy provided in an embodiment of the present invention, and amplification factor is
40X；
Fig. 5 is the candida albicans of improvement colouring method dyeing under ordinary optical microscope provided in an embodiment of the present invention, is put
Big multiple is 20X；
Fig. 6 is that the candida albicans that conventional coloring method dyes under fluorescence microscope observes figure, amplification factor 10X；
Fig. 7 is that the candida albicans that conventional coloring method dyes under ordinary optical microscope observes figure, amplification factor 10X.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily, it is clear that described embodiment is the present invention one
Section Example, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not doing
Every other embodiment obtained under the premise of creative work out, shall fall within the protection scope of the present invention.
The simple color development enhancing method of candida albicans provided in an embodiment of the present invention as detection applications, the colour developing enhancing side
Method be especially for not inactivating candida albicans fluorescent staining color development enhancing method, specifically includes the following steps:
One, the configuration of staining solution
1, the first solution is prepared: various composition is as follows: the NaHCO of 0.05M3, 1mM/L EDTA, mass fraction 0.01%
Tween-20, mass fraction 0.1%NaN3；
2, the second solution is prepared: being weighed 50mg FITC and is dissolved in 1ml DMSO, 4 DEG C are kept in dark place.
3, the preparation of the weak culture medium in husky fort: 1% peptone, 2% agar, 4% maltose.
4, the preparation of YPD fluid nutrient medium: 2% glucose, 1% tryptone, 0.5% yeast extract.
Two, candida albicans thallus culture
Candida albicans reference culture ATCC5314 plants collection skin by Chinese Academy of Medical Sciences pathogenic microorganism bacterium (poison)
Grind institute (medical mycology preservation) branch center preservation, 30 DEG C of culture 48h of the weak culture medium of Yu Shabao, with the white beads of aseptic inoculation ring picking
Bacterium bacterium colony transferred species is in YPD fluid nutrient medium, 30 DEG C of shaken cultivation 6-8h.Take above-mentioned cultured candida albicans, 10000rmp from
Heart 5min, PBS are cleaned 2 times, are counted under microscope, adjustment bacterial concentration to 5 × 105cfu/mL。
Three, candida albicans fluorescent staining
After centrifugation candida albicans liquid outwells supernatant, 1ml NaHCO is added3Solution, 10 μ l fluorescein isothiocynate FITC are molten
Liquid label, after mixing, 37 DEG C are protected from light 1.5h or 4 DEG C of shaking dyeing and are protected from light a night, and PBS is rinsed 2 times, obtains the white thought of dyeing
Pearl bacterium, it is spare.
Four, the candida albicans simple microscope and fluorescence microscope of fluorescent staining
Comparative example 1
Taking concentration is 5 × 105After discarding supernatant, it is molten that 200 μ l FITC are added in the candida albicans bacterium solution of cfu/mL, centrifugation
Liquid at 37 DEG C, is protected from light shaking dyeing 1.5h, and PBS is rinsed 2 times, obtains the candida albicans of dyeing, spare.Wherein, FITC solution
Preparation method is that 1.25mmol/l FITC is dissolved in the 0.1mmol/L NaHCO containing 0.5%DMSO3In solution, the pH of solution is
9.0.And by the candida albicans of dyeing in simple microscope and fluorescence microscopy microscopic observation.
As a result with analysis
As shown in Figure 1 and Figure 5, to the candida albicans of above-mentioned dyeing fluorescence microscopy under the microscope, amplification factor 20X,
And as shown in figure 3, the candida albicans dyed under fluorescence microscope, amplification factor 40X, in comparative example, such as Fig. 6 institute
Show, the candida albicans that conventional coloring method dyes under fluorescence microscope, amplification factor 10X, under green exciting light, in Fig. 6,
It can be seen that only a small amount of candida albicans is dyed to green, fluorescence intensity is weak.What colouring method through the embodiment of the present invention obtained
The fluorescence microscopy of candida albicans under the microscope the candida albicans of figure and conventional method dyeing fluorescence microscope figure it is found that
Under green exciting light, in the candida albicans that the colouring method dyeing of the embodiment of the present invention obtains, it is seen that candida albicans is dyed to
Green, fluorescence intensity is higher, and form is more typical, it is easier to observe.It can be seen from the figure that by with fluorescent staining result ratio
It is right, it is seen that improve the dyeing efficiency of colouring method 98% or more.
As shown in Fig. 2, the candida albicans dyed under optical microscopy, amplification factor 20X, and it is illustrated in figure 5 this
The candida albicans of improvement colouring method dyeing, amplification factor 20X under the ordinary optical microscope that inventive embodiments provide.With biography
The candida albicans of the colouring method dyeing of system, as shown in fig. 7, the white beads that conventional coloring method dyes under ordinary optical microscope
Bacterium, amplification factor 10X, the white beads that the colouring method of the embodiment of the present invention obtains candida albicans and conventional coloring method obtains
Bacterium, compared with the fluorescent staining result that conventional coloring method obtains, conventional coloring method is only to the dyeing efficiency of candida albicans
In 3-5% or so, it is unable to satisfy the requirement of follow-up test.In addition, the colouring method and biography of the candida albicans of the embodiment of the present invention
The colouring method of the candida albicans of system is compared, and the colouring method of the embodiment of the present invention has efficient characteristic.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (6)
1. a kind of simple color development enhancing method of candida albicans as detection applications, which is characterized in that
Candida albicans is transferred in YPD fluid nutrient medium in the candida albicans that the weak culture medium culture in husky fort obtains, 30 DEG C of oscillations
6-8h is cultivated, candida albicans bacterium solution is obtained；
The candida albicans bacterium solution is centrifuged and obtains first solution of the candida albicans body addition containing sodium bicarbonate, and is added and contains
The second solution of FITC mixes, and obtains dyeing mixed liquor, obtains the candida albicans of display enhancing.
2. the simple color development enhancing method of candida albicans as detection applications as described in claim 1, which is characterized in that
The described method includes: 1-2h is dyed in 37 DEG C of oscillations of the dyeing mixed liquor.
3. the simple color development enhancing method of candida albicans as detection applications as described in claim 1, which is characterized in that
The method includes being protected from light 10-14h for described 4 DEG C of dyeing mixed liquor.
4. the simple color development enhancing method of candida albicans as detection applications as described in claim 1, which is characterized in that
First solution further includes the EDTA that molar concentration is 1mM/L, and mass fraction is 0.01%Tween-20 and quality
Score is 0.1%NaN3；
The molar concentration of the sodium bicarbonate is 0.05M/L.
5. the simple color development enhancing method of candida albicans as detection applications as described in claim 1, which is characterized in that
Second solution is that 50mg FITC is dissolved in 1ml DMSO to be prepared.
6. the simple color development enhancing method of candida albicans as detection applications as described in claim 1, which is characterized in that
It is washed twice after being centrifuged the thallus, then with PBS, and bacterial concentration is adjusted to 5 × 105cfu/
The process of mL.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014185151A1 (en) * | 2013-05-13 | 2014-11-20 | コニカミノルタ株式会社 | Cell-staining method and specimen collection tube for use in method |
| CN104165872A (en) * | 2014-07-11 | 2014-11-26 | 广西医科大学 | Detection method for aspergillus fumigates biomembrane exopolysaccharide |
| CN105463056A (en) * | 2016-01-21 | 2016-04-06 | 中国人民解放军第四军医大学 | Culture medium for rapid culture and developing of candida albicans and detection method |
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2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014185151A1 (en) * | 2013-05-13 | 2014-11-20 | コニカミノルタ株式会社 | Cell-staining method and specimen collection tube for use in method |
| CN104165872A (en) * | 2014-07-11 | 2014-11-26 | 广西医科大学 | Detection method for aspergillus fumigates biomembrane exopolysaccharide |
| CN105463056A (en) * | 2016-01-21 | 2016-04-06 | 中国人民解放军第四军医大学 | Culture medium for rapid culture and developing of candida albicans and detection method |
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| 侯红漫: "《食品安全学》", 31 October 2014, 中国轻工业出版社 * |
| 王锐 等: "异硫氰酸荧光素标记鳗弧菌条件优化及应用条件对荧光信号的影响", 《水产学报》 * |
| 赵俊英 等: "《真菌病诊断与治疗》", 31 March 2007, 人民卫生出版社 * |
| 邢庆昌 等: "RNAⅢ抑制肽抑制葡萄球菌对Hela细胞的黏附", 《中国组织工程研究》 * |
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