A method for co-culturing Candida albicans and Staphylococcus aureus

Abstract

The present invention belongs to the technical field of microbial culture. In order to solve the problem that the co-culture of Staphylococcus aureus and Candida albicans using 96-well cell culture plates is not conducive to observing the adhesion of Candida albicans and Staphylococcus aureus, a method for co-culturing Candida albicans and Staphylococcus aureus is provided. Under 50% FBS conditions, Candida albicans and Staphylococcus aureus are co-cultured on a glass slide, and the glass slide is rotated at a low speed during the culture process. An experimental basis is provided for mixed infection and related research during the observation period in the future. A new experimental method for more clearly observing the adhesion of the two. The scheme proposed in this article can be used as a starting point for studying the interaction between Candida albicans and various other bacteria.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture, and particularly relates to a method for co-culturing Candida albicans and Staphylococcus aureus. Background Art

[0002] In real clinical environments, microbial infections are often not a single entity. The interaction of multiple microorganisms can affect the infection, virulence, and drug resistance of pathogens, leading to different outcomes of the disease. After the human body is infected with bacteria, the bacteria often do not exist alone. One or several microorganisms can form a mixed infection, which brings greater challenges to clinical anti-infection treatment.

[0003] Candida albicans (CA) and Staphylococcus aureus (SA) are both common microorganisms that colonize the human mucosa[1] and can cause a range of clinical diseases from mucosal to systemic, such as bacteremia, urinary tract infection, pneumonia, and diabetic foot[2-4].

[0004] Previous studies have shown that Candida albicans hyphae can serve as a scaffold structure due to their unique physical properties. Staphylococcus aureus can adhere to the scaffold to form a mixed biofilm, which is more likely to exist on the surface of catheters and medical devices[5].

[0005] Since Candida albicans and Staphylococcus aureus have synergistic effects in clinical practice and can make disease treatment more difficult and cause higher mortality, in recent years, more and more studies have been conducted on mixed infections of Staphylococcus aureus and Candida albicans, mainly focusing on various factors that affect the adhesion of the two. However, most of the above studies observed mixed infections on 96-well cell culture plates. Summary of the invention

[0006] The invention provides a method for co-culturing Staphylococcus aureus and Candida albicans in order to solve the problem that the co-culturing of Staphylococcus aureus and Candida albicans using a 96-well cell culture plate is not conducive to observing the adhesion of Candida albicans and Staphylococcus aureus.

[0007] The present invention is achieved by the following technical scheme: a method for co-culturing Candida albicans and Staphylococcus aureus, wherein Candida albicans and Staphylococcus aureus are co-cultured on a glass slide under 50% FBS conditions, and a three-dimensional vibration shaker is used to rotate the glass slide at a low speed of 60 revolutions per minute during the culturing process.

[0008] The specific cultivation method is:

[0009] (1) Preparation of logarithmic growth phase Staphylococcus aureus: Overnight culture of Staphylococcus aureus in TSB medium: Add about 50ul of overnight cultured Staphylococcus aureus to 24ml TSB liquid medium and adjust the concentration of bacterial suspension to OD600 = 0.02; incubate in a 37℃ water bath for about 3.5 hours to allow Staphylococcus aureus to reach logarithmic growth phase, OD600 = 1.0; aspirate 24ml of logarithmically growing bacterial suspension and centrifuge at 4500rpm and 4℃ for 25 minutes; aspirate the supernatant and leave 0.8-1.2ml of resuspended colonies; transfer the resuspended bacterial suspension to a 1.5ml Eppendorf tube and rotate at 14.5Krpm for 30 seconds; wash with 1ml HBSS + / + for ≥2 times; resuspend the colonies with 1ml HBSS+ / + (Hank's Balanced Salt Solution Ca2+ / Mg2+); place on ice for later use;

[0010] (2) Preparation of Staphylococcus aureus labeled with the cell proliferation tracer fluorescent probe CFDA SE: Take 1 ml of Staphylococcus aureus suspension and wash twice with HBSS + / +; add 1ul of 5mM CFDA SE per ml of bacterial suspension, with a final concentration of 5uM / ml; dark field: incubate in a 37℃ water bath for 15 min, wash 4 times with HBSS + / +; resuspend the cells in 1ml of HBSS+ / + solution; leave at room temperature for 30 min;

[0011] (3) Co-culture of Candida albicans and Staphylococcus aureus: Cultivate Candida albicans overnight in YPD liquid medium, aspirate 1 ml of bacterial suspension, wash twice with HBSS + / +; resuspend the cells with 1 ml of HBSS + / +; aspirate 100 µl of the cell suspension into 900 ul of HBSS + / + solution to dilute the cells; gently drop 300 ul of the diluted cell suspension onto a glass slide and culture at 37°C;

[0012] Tilt the slide and gently rinse the slide with 1 ml HBSS + / + to wash off the non-adherent cells; pipette 200 ul of 50% FBS onto the slide and incubate at room temperature for 30 min; add 300 ul of the labeled Staphylococcus aureus suspension in step (2) onto the slide; gently place the slide on a horizontal rotator and incubate at 37°C for 1 hour, and observe the adhesion under a fluorescence microscope.

[0013] The rotating speed of the rotator is 60 rpm.

[0014] Compared with the prior art, the present invention has discovered a new carrier and culture conditions for the co-culture of Candida albicans and Staphylococcus aureus, providing an experimental basis for future observation of mixed infection and related research. A new experimental method for more clearly observing the adhesion of the two. The scheme proposed in this article can serve as a starting point for studying the interaction between Candida albicans and various other bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The fluorescence value changes of Candida albicans and Staphylococcus aureus in 96-well plates before and after washing with HBSS+ / + and TWEEN20;

[0016] Figure 2 The adhesion of Candida albicans and Staphylococcus aureus on a 96-well plate; in the figure: A: Candida albicans + Staphylococcus aureus + 50% FBS; B: Candida albicans + Staphylococcus aureus; the pictures are all after washing twice with HBSS+ / +, and under a fluorescence microscope at 200 times, it can be seen that Candida albicans and Staphylococcus aureus are mixed together in clusters, and the adhesion structure is unclear;

[0017] Figure 3 The adhesion of Candida albicans and Staphylococcus aureus on the slide; in the figure: A: Candida albicans + Staphylococcus aureus + 50% FBS, it can be seen that Staphylococcus aureus is clearly adhered to the hyphae of Candida albicans; B: Candida albicans + Staphylococcus aureus, it can be seen that Candida albicans and Staphylococcus aureus grow evenly and mixedly, without obvious adhesion (fluorescence microscope 200 times). DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0020] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0022] 1. Experimental materials: Candida albicans: SC5314 WT, obtained from the Center for Fungal and Fungal Diseases Research, Peking University Health Science Center; Staphylococcus aureus UAMS1 WT was donated by the Institute of Microbiology, Chinese Academy of Sciences.

[0023] 2. Instruments and reagents: UV analyzer (WD-9403F, Liuyi Instrument Factory, Beijing); constant temperature oscillator (THZ-C, National Laboratory Equipment Institute, Changzhou); pressure steam sterilizer (SYQ-DSX-280B, Shanghai Shen'an Medical Equipment Co., Ltd.); fluorescence microscope (L3201, Guangzhou); constant temperature biochemical incubator (SPX-250B-Z, Boxun Industrial Co., Ltd. Medical Equipment Factory); horizontal rotator (RPR-100B, Nanjing); fluorescence plate reader (SpectraMax Gemini EM) COM1, costar clearplate 96 wells, bottom read, well scan, cross-pattern, Density 3, Excite 485, Emit: 535, Cutoff: 530); 96-well cell culture plate (Costar 3595); FBS (Shanghai Shenggong Co., Ltd.); HBSS+ / + (Gibco cat#: 14025); CFDA SE (Shanghai Bioengineering Co., Ltd.); TWEEN20 (Shanghai Bioengineering Co., Ltd.); YPD medium (Shanghai Bioengineering Co., Ltd.); TSB medium (Shanghai Bioengineering Co., Ltd.).

[0024] The culture medium formula used in the present invention is as follows:

[0025] YPD medium (yeast medium): Formula: 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose). If making solid culture medium, add 2% agar powder.

[0026] TSB medium (Tryptone Soy Broth Medium): 1.5% (g / 100ml) tryptone, 0.5% (g / 100ml) soy peptone, 0.5% (g / 100ml) sodium chloride, prepared with distilled water, and the pH was adjusted to 7.2±0.2.

[0027] The reagents required for HBSS+ / + are listed in Table 1.

[0028] Table 1

[0029]

[0030] 3. Experimental methods:

[0031] 1. Co-culture of Candida albicans and Staphylococcus aureus in 96-well cell culture plates:

[0032] A: Cultivation of Candida albicans hyphae in 96-well cell culture plates: Cultivate SC5314 overnight in YPD medium. Take 1 ml of overnight culture suspension and wash twice with HBSS + / +. Take 100 µl of washed suspension and add it to 900 ul HBSS + / + solution, or adjust the concentration of suspension to 5×10 8 . Pipette 10 µl of the diluted bacterial suspension into 1 ml of HBSS + / + solution and shake well. Add 100 ul of bacterial suspension to a 96-well cell culture plate and incubate in a 37°C incubator for 3 hours. Wash twice with HBSS + / + to remove non-adherent cells. Add 100 ul of 50% FBS or HBSS + / + and incubate at room temperature for 30 minutes. Wash carefully twice with HBSS + / +.

[0033] B. Preparation of Staphylococcus aureus labeled with the cell proliferation tracer fluorescent probe CFDA SE: Culture Staphylococcus aureus overnight in TSB liquid medium. Pipette 1 ml of bacterial suspension and wash twice with HBSS + / +. Add 1ul 5mM CFDA SE to each 1ml of washed cell suspension. Dark field operation: Incubate in a 37℃ water bath for 15 minutes. Wash 4 times with HBSS + / +. Resuspend in 1ml HBSS + / +. Let stand at room temperature for 30 minutes. Dark field operation: Dilute 50µl of cells per 5ml HBSS + / +.

[0034] C. Co-culture of Candida albicans and labeled Staphylococcus aureus: Remove the wash solution from the wells and add 100ul diluted Staphylococcus aureus cells to the Candida albicans hyphae wells and control wells. Centrifuge in parallel at 1500 rpm for 5 minutes. Incubate at 37°C for 60 minutes. Gently wash 1-2 times with HBSS + / + or 1-2 times with TWEEN20. Measure GFP fluorescence on a fluorescent plate reader.

[0035] 2. Cultivate Candida albicans hyphae on a glass slide:

[0036] A. Preparation of S. aureus in logarithmic growth phase: Culture S. aureus in TSB medium overnight. Add about 50ul of overnight cultured S. aureus to 24ml TSB liquid medium and adjust the concentration of bacterial suspension to OD600= 0.02. Incubate in a 37°C water bath for about 3.5 hours to allow S. aureus to reach logarithmic growth phase (S. aureus has the strongest growth activity during this period) OD600=1.0. Pipette 24ml of logarithmic growth bacterial suspension into a 50ml conical tube. Centrifuge for 25 minutes at 4°C, 4500rpm. Pipette the supernatant and leave about 1ml of resuspended colonies. Transfer the resuspended bacterial suspension to a 1.5ml centrifuge tube and spin in a tabletop centrifuge for 30 seconds at 14.5Krpm. Wash with 1ml HBSS + / + for more than 2 times. Resuspend the colonies with 1ml HBSS+ / +. Place on ice for later use.

[0037] B. Preparation of CFDA SE-labeled S. aureus: Take 1 ml of S. aureus suspension and wash twice with HBSS + / +. Add 1ul of 5mM CFDA SE per ml of suspension to a final concentration of 5uM / ml. Dark field: Incubate in a 37°C water bath for 15 min and wash 4 times with HBSS + / +. Resuspend the cells in 1ml of HBSS + / + solution. Leave at room temperature for 30 min.

[0038] C. Co-culture of Candida albicans and Staphylococcus aureus on slides: C. albicans was cultured overnight in YPD liquid medium. Pipette 1 ml of bacterial suspension and wash twice with HBSS + / +. Resuspend the cells with 1 ml HBSS + / +. Pipette 100 µl of cell suspension into 900 ul HBSS + / + solution to dilute the cells. Gently drop 300 ul of the diluted cell suspension onto a glass slide and incubate at 37°C. Tilt the slide and gently rinse the slide with 1 ml HBSS + / + to wash off the non-adherent cells. Pipette 200 ul of 50% fetal bovine serum (FBS) onto each slide and incubate at room temperature for 30 minin. Half of the slides were used as controls without 50% FBS. Add 300 ul of the labeled Staphylococcus aureus suspension to each slide. Gently place on a horizontal rotator and incubate at 37°C for 1 hour. The adhesion was observed under a fluorescence microscope.

[0039] 4. Statistical analysis: SPSS12.0 statistical software was used to perform statistical analysis using analysis of variance and factorial design, and P < 0.05 was considered statistically significant.

[0040] V. Experimental results:

[0041] 1. Adhesion of Candida albicans and Staphylococcus aureus in 96-well plates

[0042] Adhesion of Candida albicans and Staphylococcus aureus to 96-well plates Figure 2 As shown in Figure 2, the fluorescence value changes of Candida albicans and Staphylococcus aureus before and after washing with HBSS+ / + and TWEEN20 in a 96-well plate are shown in Figure 2. Figure 1 After washing twice with HBSS+ / +, under a fluorescence microscope at 200 times, Candida albicans and Staphylococcus aureus were seen mixed together in clusters, and the adhesion structure was unclear.

[0043] The results of variance analysis showed that +CA+FBS+LABEL SA and +CA-FBS+LABEL SA were statistically significant among different elution times (P<0.05).

[0044] The fluorescence values ​​of Candida albicans and Staphylococcus aureus before and after washing with HBSS+ / + and TWEEN20 in 96-well plates are shown in Table 1. The results of pairwise comparison showed that compared with the unwashed group, the groups washed once, washed twice, washed once with TWEEN 20, and washed twice with TWEEN 20 were statistically significant (P<0.05). No statistical differences were found between the other groups in pairwise comparisons (see Table 2).

[0045] Table 2: Fluorescence values ​​of Candida albicans and Staphylococcus aureus in 96-well plates before and after washing with HBSS+ / + and TWEEN20

[0046]

[0047] Note: * indicates P < 0.05 compared with the unwashed group.

[0048] The results of the effects of elution times and FBS on adhesion are shown in Table 3. Through factorial design analysis, it can be seen that in the 96-well plate, the number of washes and whether FBS was added had a significant effect on the fluorescence value of adhesion, P < 0.05, which was statistically significant and had nothing to do with the type of washing solution.

[0049] Table 3 Effects of elution times and FBS on adhesion

[0050]

[0051] 2. Adhesion of Candida albicans and Staphylococcus aureus on the slide: Figure 3 As shown, Candida albicans and Staphylococcus aureus can be seen growing evenly and mixedly without obvious adhesion.

[0052] VI. Conclusion:

[0053] When cultured on a 96-well cell culture plate, the adhesion status can be observed by the fluorescence value, but the value varies greatly. After washing, most of the non-adherent cells fall off and the fluorescence value drops significantly.

[0054] As can be seen from Tables 1 and 2, after mixed culture, the fluorescence value of the 96-well plate without washing and the fluorescence value of the 96-well plate without FBS addition and without washing were not significantly different (P>0.05), but the fluorescence value decreased significantly after washing once, whether it was washed with HBSS+ / + or TWEEN20, which was statistically significant compared with before washing (P<0.01). After washing twice, the fluorescence value further decreased, which was statistically different from before washing (P<0.01) and statistically different from washing once (P<0.01). The results of different control wells were consistent. There was no statistical difference in the fluorescence value of the 96-well plate without FBS addition before washing (P>0.05), but whether it was washed with HBSS+ / + or TWEEN20, after washing once and twice, the fluorescence value of the well without FBS addition was significantly higher than that of the well with FBS addition, which was statistically different (P<0.05). It can also be seen through fluorescence microscopy that, whether before or after cleaning, Staphylococcus aureus grows in clusters in the wells of the 96-well plate, mixed with Candida albicans, and the adhesion structure is unclear. The type of cleaning solution has no obvious effect on the change of fluorescence value.

[0055] Cultivated on a glass slide, due to the special culture carrier, the fluorescence value cannot be measured and needs to be observed under a fluorescence microscope. Through the fluorescence microscope, it can be seen that the Staphylococcus aureus on the glass slide with FBS added is densely adhered to the hyphae of Candida albicans, and it is still tightly adhered after washing and is not easy to wash off. On the glass slide without FBS added, Staphylococcus aureus and Candida albicans grow mixedly, and a small amount of Staphylococcus aureus adheres to the hyphae of Candida albicans. This shows that FBS is conducive to the adhesion and growth of Candida albicans and Staphylococcus aureus, and also shows that the participation of serum can better simulate the environment of the human body cavity tube or various cannulas, and the data is more real and reliable than that without serum.

[0056] Gentle rotation on a horizontal rotator on a glass slide can better allow Staphylococcus aureus to adhere to the hyphae of Candida albicans, with uniform distribution and clear structure. By observing the adhesion of the two, a laboratory basis is provided for further research on the adhesion of Candida albicans and Staphylococcus aureus and the formation of biofilms.

[0057] The fungus Candida albicans and the bacterium Staphylococcus aureus can coexist in a polymicrobial biofilm [6]. Staphylococcus aureus can tightly bind to the hyphae of Candida albicans, but not to the spores. Candida growing in hyphae has obvious toxicity, and mixed infection with a large number of Staphylococcus aureus has stronger toxicity, aggravating the clinical condition and bringing great difficulties to treatment.

[0058] Domestic and foreign scholars have conducted a large number of studies on the mechanism of biofilm formation in mixed infection of Candida albicans and Staphylococcus aureus, and there are many studies on the diagnosis and inhibition of biofilm formation, mainly focusing on adhesion factors [7,8]. Adhesins are a large family. In the adhesion process of Candida albicans and Staphylococcus aureus, Candida albicans adhesion lectin (Als3p) plays an important role in the adhesion process. It was found that the adhesion of the two was significantly inhibited in the ALS3 gene knockout strain. Gao et al. studied the effects of amphotericin B and berberine on the in vitro antibacterial activity of Candida albicans and Staphylococcus aureus biofilms [9], and confirmed that amphotericin B and berberine can affect the in vitro antibacterial activity of the two after adhesion, providing new ideas for the treatment of mixed infection of the two. At the same time, Zhang Yongchen et al. studied the role of dihydroxyanthraquinone in reducing mixed biofilms of Staphylococcus aureus and Candida albicans

[10] .

[0059] However, current studies on mixed infection of Candida albicans and Staphylococcus aureus in vitro are basically carried out in 96-well plates or 24-well plates. Professor Toni M Förster reviewed the interaction between Candida albicans and Gram-positive bacteria, and studied the synergistic or antagonistic effects between different microorganisms

[11] , so that we can better understand clinical mixed infections, but these studies were all conducted on PVC cell culture plates[12,13]. The adhesion of Staphylococcus aureus to Candida albicans is mostly observed by fluorescence microscopy, but the staining methods vary. Harriott et al.

[14] used SYTO 9 green fluorescent nucleic acid dye to dye live Staphylococcus aureus green, and fluorescent brightener to dye the fungal cell wall blue, and observed the adhesion of the two by fluorescence microscopy. Subsequently, Ovchinnikova et al.

[15] also used this staining method to evaluate the difference in adhesion of Staphylococcus aureus to different regions of Candida albicans hyphae. In addition, some studies used crystal violet staining to stain mixed biofilms and evaluated the growth and reproduction of bacteria by spectrophotometer

[16] . The differences in these staining methods are helpful for observing the adhesion of the two, but they are all performed in 96-well plates or 24-well plates.

[0060] The present invention observed that cells in the 96-well plate easily adhered to form a mass, and under a microscope or even an electron microscope, they were all mixed and distributed in a mass. Without washing, there may be some Staphylococcus aureus that did not really adhere to the hyphae of Candida albicans. In the past, mixed infection in vitro culture was washed with PBS multiple times to remove non-adherent Staphylococcus aureus, and generally 1×PBS was used for washing 3 times.

[0061] And the present invention adopts glass slide, simple and easy to operate, and through gentle flushing, can wash away the cells that do not adhere, leave the microorganism that really adheres together, experimental cost is reduced, simple to operate, easy to observe.Meanwhile, by giving or not giving fetal bovine serum experiment, it is found that giving FBS can make Staphylococcus aureus and Candida albicans adhere better, this may make to add FBS after can better simulate the internal environment, suitable for the adhesion of the two, while mixed culture gives slow gentle rotation, then wash away free cells, under fluorescence microscope, Staphylococcus aureus can be more clearly observed to adhere to the hyphae of Candida albicans, structure is clear, for further studying Candida albicans and Staphylococcus aureus, even Candida and bacterial mixed infection provide laboratory basis.By slide method, Candida albicans and Staphylococcus aureus mixed infection observation and by the size of fluorescence value detection two adhesion situations in the past document has not yet mentioned, in this study is first application.

[0062] References:

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[10] Zhang Yongchen, Yu Ling, Huang Jing, Chen Fei. Study on the effect of 1,2-dihydroxyanthraquinone on reducing mixed biofilm of Staphylococcus aureus and Candida albicans[J]. Chinese Journal of Infection and Chemotherapy. 2021, 21(1): 73-77.

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[0078]

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[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for co-culturing Candida albicans and Staphylococcus aureus, characterized in that: Candida albicans and Staphylococcus aureus were co-cultured on slides under 50% FBS conditions, and the slides were rotated at a low speed during the culture process; The specific cultivation method is: (1) Preparation of logarithmic growth phase Staphylococcus aureus: Overnight culture of Staphylococcus aureus in TSB medium: Add 50 µl of overnight cultured Staphylococcus aureus to 24 ml TSB liquid medium and adjust the concentration of the bacterial suspension to OD 600 = 0.02; incubate in a 37°C water bath for 3.5 hours to allow Staphylococcus aureus to reach the logarithmic growth phase, OD 600 =1.0; aspirate 24 ml of the logarithmically growing bacterial suspension and centrifuge at 4500 rpm and 4°C for 25 minutes; remove the supernatant and leave 0.8-1.2 ml of the resuspended bacterial colonies; transfer the resuspended bacterial suspension into a 1.5 ml Eppendorf tube and spin at 14.5K rpm for 30 seconds; use 1 ml of Hank's balanced salt solution Ca 2+ / Mg 2+ Wash with HBSS + / + for ≥2 times; resuspend the colony with 1 ml HBSS+ / +; keep on ice for later use; (2) Preparation of Staphylococcus aureus labeled with the cell proliferation tracer fluorescent probe CFDA SE: Take 1 ml of Staphylococcus aureus suspension and wash twice with HBSS + / +; add 1µl of 5mM CFDA SE per ml of bacterial suspension, with a final concentration of 5µM / ml; dark field: incubate in a 37°C water bath for 15 min, wash 4 times with HBSS + / +; resuspend the cells in 1 ml of HBSS+ / + solution; and leave at room temperature for 30 min; (3) Co-culture of Candida albicans and Staphylococcus aureus: Cultivate Candida albicans in YPD liquid medium overnight, aspirate 1 ml of bacterial suspension, wash twice with HBSS + / +; resuspend the cells in 1 ml of HBSS + / +; aspirate 100 µl of the cell suspension into 900 ul of HBSS + / + solution to dilute the cells; 300 µl of the diluted cell suspension was gently dropped onto a glass slide and incubated at 37°C; Tilt the slide and gently rinse it with 1 ml HBSS + / + to wash off the non-adherent cells; pipette 200 µl 50% FBS onto the slide and incubate at room temperature for 30 min; add 300 µl of the labeled Staphylococcus aureus suspension from step (2) onto the slide; gently place it on a horizontal rotator and incubate at 37°C for 1 hour, and observe the adhesion under a fluorescence microscope.

2. The method for co-culturing Candida albicans and Staphylococcus aureus according to claim 1, characterized in that: The rotating speed of the rotator is 60 rpm.

Images