A method and apparatus for simultaneous cultivation and staining of fungi

Abstract

This invention relates to the field of microbial detection technology, specifically to a method and apparatus for simultaneous culture and staining of fungi. This invention creatively proposes a culture-integrated staining scheme, premixing a specific staining agent A at an optimal concentration in the culture medium. In use, only sample inoculation and culture are required, followed by direct in-situ microscopic observation, eliminating the need for any subsequent picking, smearing, and staining steps. This method significantly simplifies the operational process, improves detection efficiency and biosafety, and, due to its low cost and effectiveness against a variety of common pathogenic fungi, has promising prospects for clinical application and widespread adoption.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and more specifically, to a method and apparatus for simultaneous culture and staining of fungi. Background Technology

[0002] Fungi are widely distributed and diverse in nature, playing a significant role in human health, especially in clinical medicine where pathogenic fungi are major causes of infectious diseases. Despite increasing attention to fungal infections both domestically and internationally, the detection of pathogenic fungi in clinical settings still faces many limitations. Experienced laboratory personnel can significantly shorten the diagnosis and treatment cycle for clinical fungal infections, especially those caused by filamentous fungi, by performing morphological observation of stained cultured fungi under a microscope. The traditional method for staining filamentous fungi involves transferring mature cultured fungi from a petri dish to a glass slide, staining with lactophenol blue or fluorescent dye, and then observing the hyphae and spore morphology under a microscope to identify the fungal species. This traditional method has significant drawbacks: First, if the fungal staining and microscopic examination fail, continuous culture and repeated observation are impossible, inevitably prolonging the clinical reporting cycle. Second, the transfer process can easily interfere with the normal growth of fungi in the petri dish, damaging the morphology and structure of fungal colonies and hyphae, hindering microscopic observation. Furthermore, the transfer process increases the safety risks of fungal contamination for staff and the laboratory environment.

[0003] Currently, research in the differential diagnosis of pathogenic fungi, both domestically and internationally, mainly focuses on improving the accuracy of identification by integrating staining techniques with instrumental analysis to achieve precise identification of pathogens. Domestic research has made some progress; for example, gene analysis methods based on ITS sequences have shown excellent performance in the identification of specific fungi. However, the high cost of equipment and significant technical barriers limit their promotion and widespread adoption in primary healthcare institutions. Meanwhile, while traditional staining microscopy is inexpensive, the procedures are cumbersome and highly subjective, and the professional competence of the operators affects the accuracy and reproducibility of the results. In recent years, domestic researchers have attempted to develop rapid staining kits and portable instruments to meet clinical needs for fungal infection detection. For example, disposable culture chambers provide convenience for the culture and observation of small-scale pathogenic fungi, but the culture, staining, and identification steps still need to be performed in stages, making the experimental process cumbersome and time-consuming. Internationally, pathogenic fungi identification technologies are relatively mature. For instance, the disposable blood culture identification panel for fungal pathogens (BCID-FP) can rapidly and accurately identify 15 target Candida species within a specific range, demonstrating high clinical application value. However, this technology has a narrow scope of application, mainly targeting some species in the Candida genus. It is also highly dependent on supporting instruments, complex to operate, and time-consuming. In addition, it is protected by patents, making it difficult to meet the needs of clinical work in my country for efficient, inexpensive, and universally applicable fungal identification. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes an integrated method and apparatus for simultaneous fungal culture and staining. A specific staining agent A is pre-mixed uniformly in the fungal culture medium, allowing the fungi to simultaneously complete biological staining during growth. This method integrates traditional multi-step isolation procedures into a continuous process of inoculation, culture, and in-situ observation, eliminating the need for additional staining or sample processing steps.

[0005] In a first aspect, the present invention provides a method for simultaneous culture and staining of fungi, comprising the following steps: adding staining agent A to a sterilized fungal basal culture medium, maintaining the final concentration of staining agent A within the activity range of 10 mg / L to 320 mg / L, to form a pre-prepared culture medium that can be used directly; inoculating clinical specimens, after standard pretreatment, onto the surface of the pre-prepared culture medium containing staining agent A, and culturing under suitable conditions for fungal growth; during or after culture, without any picking, smearing, fixation, or additional staining operations, directly observing the fungal colonies growing on the surface of the culture medium. Because the live fungi actively absorb and utilize staining agent A during their growth and metabolism, their hyphae, spores, and cell structures will exhibit specific staining that contrasts sharply with the background color of the culture medium, thus allowing for direct morphological identification.

[0006] Preferably, the staining agent A is Congo red, with the molecular formula C. 32 H 22 N6Na2O6S2, CAS No. 573-58-0, with a final concentration controlled at 80 mg / L. This concentration, verified by the examples in this application, ensures vivid and stable staining of fungi while minimizing the background color depth of the culture medium, providing optimal contrast for microscopic observation, and reducing potential impact on the normal growth rate of fungi.

[0007] Preferably, the direct observation of fungal colonies growing on the surface of the culture medium includes in-situ microscopic observation using an optical microscope; furthermore, in order to record the dynamic process of fungal growth and development, a time-lapse video recording device can also be used to visually display key morphological changes such as hyphal extension and spore formation, which is suitable for teaching and / or scientific research observation and / or auxiliary diagnosis.

[0008] Preferably, the basal culture medium for fungi is Sabouraud dextrose agar (SDA). SDA is an internationally recognized standard fungal culture medium with clearly defined components, good reproducibility, and optimal compatibility with the staining agent A system described in this invention, ensuring normal fungal growth and staining effect.

[0009] Preferably, the staining agent A has broad-spectrum staining specificity against a variety of common clinical pathogenic fungi, such as Aspergillus fumigatus, Penicillium, Penicillium wannii, Trichophyton rubrum, and Candida glabrata, which ensures the wide clinical application value of this method.

[0010] Secondly, the present invention provides a culture apparatus for carrying out the above-described method, comprising: a sealed cavity having a light-transmitting observation window, and a culture medium containing a concentration of 10-320 mg / L staining agent A pre-placed in the cavity.

[0011] Preferably, the sealed cavity is made of transparent plastic or glass and is provided with a dedicated inoculation port or top cover with a sterile diaphragm.

[0012] Thirdly, the application of staining agent A in the preparation of products for simultaneous culture and staining of fungi, including but not limited to Aspergillus fumigatus, Penicillium, Penicillium wannii, Trichophyton rubrum, or Candida glabrata.

[0013] The significant advantages of this invention compared to the prior art are as follows: Firstly, this application creatively eliminates the steps of picking, smearing, fixing, and staining in the traditional model through its core design of culture followed by staining. Only inoculation and observation are required, shortening the traditional operation cycle of several days to direct microscopic examination during / after culture, eliminating unnecessary waiting and processing time between steps.

[0014] Secondly, compared with traditional methods, the method of this invention does not require physical picking, allowing fungi to maintain their original natural growth morphology on the culture medium. Multiple, dynamic observations of the same colony can be conducted at different time points, which is particularly beneficial for observing key identification characteristics such as hyphal growth and spore formation, resulting in more accurate and reliable morphological identification results.

[0015] Thirdly, the entire detection process of the method of the present invention can be completed in a pre-sealed device, which fundamentally eliminates the generation and spread of fungal spore aerosols, helps to eliminate the risk of operator infection and cross-contamination in the laboratory, and meets higher-level laboratory biosafety standards.

[0016] Fourth, the method of this invention only requires a common optical microscope and does not require expensive fluorescence equipment or special reagents. The core component, staining agent A, is inexpensive, making this technology particularly suitable for large-scale application in resource-constrained primary hospitals, clinics, and epidemiological field investigations.

[0017] Fifth, the method of this invention has good universality for a variety of common clinical pathogenic fungi. Examples of this application demonstrate that staining agent A can effectively stain Aspergillus fumigatus, Penicillium, Paecilomyces wannii, Trichophyton rubrum, and Candida glabrata, without affecting the normal morphology of the fungi. This provides a broad application basis for their rapid screening and preliminary identification of common clinical fungal infection pathogens. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the staining agent screening experiment results in Example 1 of the present invention; wherein, A is a staining effect image of Candida glabrata cultured on Sabouraud dextrose agar (SDA) with added staining agent A; B to H are respectively images of Candida glabrata cultured on Sabouraud dextrose agar (SDA) with added staining agents B, C, D, E, F, G, and H; I is a negative control image of Candida glabrata cultured on SDA without any added staining agent; the magnification of the optical microscope field of view is 10×10.

[0020] Figure 2 This is a schematic diagram of the results of the screening experiment for the working concentration of staining agent A in Example 2 of the present invention; wherein, A is the negative control cultured on Sabouraud dextrose agar (SDA) without the addition of staining agent A; B to E are the culture effects of adding staining agent A to Sabouraud dextrose agar (SDA) medium at final concentrations of 80 mg / L, 160 mg / L, 240 mg / L, and 320 mg / L, respectively; F is the staining effect of Candida glabrata under high magnification (10×40) when the final concentration of staining agent A is 80 mg / L, and the magnification of the other microscope fields is 10×10.

[0021] Figure 3 This is a comparative diagram showing the effect of staining agent A on the growth of *Candida glabrata* in Example 3 of the present invention; wherein, A is the state of the control group without staining agent A on day 1 of culture; B is the state of the experimental group containing 80 mg / L staining agent A on day 1 of culture; C is the state of the control group on day 2 of culture; D is the state of the experimental group on day 2 of culture; E is the state of the control group on day 2 of culture under high magnification; F is the state of the experimental group on day 2 of culture under high magnification. The magnification of the optical microscope field of view is 10×40; the magnification of the other microscope fields of view is 10×10.

[0022] Figure 4 This is a comparative diagram showing the effects of staining agent A on the staining and growth of various fungi in Example 4 of the present invention; where A represents the control group and experimental group of Trichophyton rubrum (containing 80... Comparison images of day 1 and day 2 of culture using mg / L staining agent A. The first row of photos was taken on the first day after fungal inoculation, and the second row on the second day. The first column of photos shows the blank control group, and the second column shows the experimental group with added staining agent A. Comparison images of day 1 and day 2 of culture using Penicillium control and experimental groups. The first row of photos was taken on the first day after fungal inoculation, and the second row on the second day. The first column of photos shows the blank control group, and the second column shows the experimental group with added staining agent A. Comparison images of day 1 and day 2 of culture using Penicillium wanyi control and experimental groups. The first row of photos was taken on the first day after fungal inoculation, and the second row on the second day. The first column of photos shows the blank control group, and the second column shows the experimental group with added staining agent A. Comparison images of day 1 and day 2 of culture using Aspergillus fumigatus control and experimental groups. The first row of photos was taken on the first day after fungal inoculation, and the second row on the second day. The first column of photos shows the blank control group, and the second column shows the experimental group with added staining agent A. The magnification of the optical microscope field of view is 10×10.

[0023] Figure 5 This is a comparative diagram showing the effect of staining agent A on the staining and growth of Trichophyton mentagrophytes in Example 4 of the present invention; wherein, the first row is the image of the Trichophyton mentagrophytes control group (without staining agent A), and the second row is the image of the Trichophyton mentagrophytes experimental group (containing 80 mg / L staining agent A); the magnification of the first column of the optical microscope field of view is 4×10, and the magnification of the second column of the optical microscope field of view is 10×10.

[0024] Figure 6 This is a comparative diagram showing the effects of staining agent A on the staining and growth of Fusarium solani in Example 4 of the present invention; wherein, the first row is the image of the control group of Fusarium solani (without staining agent A), and the second row is the image of the experimental group of Fusarium solani (containing 80 mg / L staining agent A); the magnification of the first column of the optical microscope field of view is 4×10, and the magnification of the second column of the optical microscope field of view is 10×10. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] The term "embodiment" as used herein means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in multiple embodiments of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0027] Unless otherwise specified, specific techniques or conditions in the embodiments described herein shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all conventional commercially available products.

[0028] I. Reagents, Consumables and Equipment 1.1 Consumables The fungal strains used in this application, such as Aspergillus fumigatus, Penicillium expansum, Candida parapsilosis, and Fusarium solani, were all purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd. in China. Detailed information is shown in Table 1. Information on the raw materials and consumables used in the preparation of Sabouraud Dextrose Agar (SDA) medium is shown in Table 2.

[0029] Table 1. Information on fungal strains used in the experiment.

[0030] Table 2 SDA Raw Materials and Consumables Information

[0031] 1.2 Reagents Information on the various dyes and other chemical reagents used in this application is shown in Table 3.

[0032] Table 3. Information on reagents used in the experiment

[0033] Example 1: Screening of staining agents for simultaneous fungal culture and staining According to the reference formula, weigh out 5g of peptone, 20g of glucose, and 10g of agar, and dissolve them in deionized water by heating. Adjust the pH of the culture medium to between 5.8 and 6.2, add deionized water to 500ml, mix well, dispense into Erlenmeyer flasks, autoclave at 115.6℃ for 15min, remove, and after passing the sterility test, store at 4℃ for later use.

[0034] Equal volumes of different candidate staining agents (AH) were added to Sabouraud dextrose agar (SDA) medium to a final concentration of 100 mg / L. After rapid mixing, approximately 2 mL of the medium was injected into each well of a 6-well cell culture plate and allowed to solidify. Using a disposable inoculation loop, the standard strain of *Candida parapsilosis* (BNCC378088) was inoculated using a zigzag streak. The 6-well plates were incubated upside down at 20–35°C in the dark. The growth and staining of the fungi at the streak inflection points were observed daily using an optical microscope (10 × 10 magnification).

[0035] The results are as follows Figure 1 As shown, *Candida glabrata* exhibits clear and stable staining (1A) only on Sabouraud dextrose agar (SDA) medium supplemented with staining agent A, with distinct cell outlines that are easily identifiable. However, in the medium supplemented with staining agent BH (…),… Figure 1 In BH (Bacterial Hematology) samples, the fungi were either unstained or severely affected by background interference, making clear observation impossible. The control group (without any added staining agent) Figure 1 The fungi in (I) were also not stained. Therefore, staining agent A was selected as the staining agent for the method of the present invention.

[0036] Example 2: Determination of the optimal working concentration of dye A A concentrated stock solution of staining agent A (2.5 g / L) was prepared and sterilized by filtration through a 0.22 μm microporous membrane. The stock solution was added to warm (approximately 50°C) SDA medium at specific ratios and mixed thoroughly to achieve final concentrations of 80 mg / L, 160 mg / L, 240 mg / L, and 320 mg / L. SDA without staining agent served as a negative control. Following the method described in Example 1, the staining medium at each concentration was injected into 6-well plates, inoculated with *Candida glabrata*, and cultured for observation.

[0037] The results are as follows Figure 2 As shown, with the increase of staining agent A concentration, the background color of the culture medium itself gradually deepens. Figure 2 BE). When the concentration is 80 mg / L ( Figure 2 B), Candida glabrata showed good staining and had the lightest background on the culture medium, resulting in the best contrast for observation. Figure 2F represents a 10×40x magnification field of view at a concentration of 80 mg / L. While higher concentrations (160-320 mg / L) can also stain fungi, excessively dark background colors severely interfere with microscopic observation. Furthermore, considering cost savings and minimizing the potential impact of the dye on microorganisms, 80 mg / L was determined to be the optimal working concentration of staining agent A in the method of this invention.

[0038] Example 3: Evaluation of the effect of staining agent A on fungal growth Two parallel experiments were set up: the experimental group used SDA medium containing 80 mg / L dye A; the control group used ordinary SDA medium without dye. Candida glabrata was inoculated according to the method in Example 1 and cultured at 20-35℃ in the dark. For two consecutive days, the growth rate, colony morphology, and details under high magnification of the two groups of fungi were observed daily under an optical microscope.

[0039] The results are as follows Figure 3 As shown: Day 1 of cultivation ( Figure 3 A vs 3B) and the next day ( Figure 3 In both C vs 3D magnification, there were no significant differences in the growth status of *Candida glabrata* between the experimental and control groups. At high magnification (10×40), the experimental group ( Figure 3 F) and control group ( Figure 3 The yeast cells in E) showed completely consistent morphology and budding, with no abnormalities observed. This indicates that at the concentration of 80 mg / L used in this invention, staining agent A has no adverse effects on the normal growth and reproduction of Candida glabrata and will not interfere with accurate morphology-based identification.

[0040] Example 4: Verification of the universality of the method of the present invention against a variety of pathogenic fungi. To verify the broad applicability of the method of the present invention, standard strains of *Trichophyton rubrum*, *Penicillium*, *Penicillium wannii*, *Aspergillus fumigatus*, *Fusarium solanum*, and *Trichophyton mentagrophytes* were selected as test subjects. Each group of strains included an experimental group (SDA containing 80 mg / L staining agent A) and a control group (ordinary SDA). They were inoculated and cultured according to the method in Example 1, and observed for two consecutive days.

[0041] The results are as follows Figure 4 As shown, staining agent A can successfully smear Trichophyton rubrum (… Figure 4 A) Penicillium ( Figure 4 B), Penicillium wansii ( Figure 4 C) and Aspergillus fumigatus ( Figure 4 D) Staining during cultivation. Compared to their respective control groups, these fungi maintained normal growth morphology, hyphal structure, and spore characteristics in the experimental groups, making them easily identifiable by examiners. However, staining agent A failed to stain Trichophyton mentagrophytes (appendage...) Figure 5 ) and staining of Fusarium solani (attached) Figure 6), and interfered with the growth of Fusarium solani (see appendix). Figure 6 This result demonstrates that the method for simultaneous culture and staining of fungi described in this invention is effective and safe for most clinically common test fungi, and possesses good clinical application potential and universality.

[0042] Example 5: Integrated culture and detection device and reagent kit Based on the above method, a device for simultaneous fungal culture and staining was designed and prepared. The main body of the device can be a flat cavity made of transparent plastic or glass, with a highly transparent sealing film or coverslip covering the top as an observation window. The bottom of the cavity is pre-filled with SDA culture medium containing 80 to 320 mg / L staining agent A, and one side of the cavity has an inoculation port with a sealed rubber septum. In use, after clinical specimens are pretreated using standard methods in the field, fungi are collected using a disposable inoculation loop or platinum wire inoculation loop and streaked on the solid culture medium in the chamber. After sealing, the entire device is inverted in an incubator for light-protected culture. Laboratory personnel can place the device directly on the microscope stage at any time and observe and identify the naturally stained fungal colonies growing within it through the observation window on top.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneous culture and staining of fungi, characterized in that, S1. Preparation of culture medium containing staining agent: Add sterile staining agent A to the sterilized fungal basal culture medium, mix well, and make the final concentration of staining agent A from 10 mg / L to 320 mg / L. S2. Inoculation and culture: The sample to be tested is inoculated onto the surface of the culture medium containing staining agent A prepared in step S1 and cultured under suitable conditions for fungal growth. S3. In situ microscopic observation: During or after the culture process, fungal colonies growing on the culture medium are observed directly without smearing or staining. The fungus exhibits a color distinct from the background of the culture medium due to the absorption of the staining agent A, and is identified by observing the morphology of its stained hyphae, spores, or cells.

2. The method for simultaneous culture and staining of fungi according to claim 1, characterized in that, In step S1, the final concentration of the staining agent A in the culture medium is 80 mg / L.

3. The method for simultaneous culture and staining of fungi according to claim 1 or 2, characterized in that, In step S1, the culture medium is Sabouraud dextrose agar medium.

4. The method for simultaneous culture and staining of fungi according to claim 1, characterized in that, In step S2, the culture conditions are a temperature of 20-35℃ and culture in the dark.

5. The method according to claim 1, characterized in that, The staining agent A has staining specificity for a variety of fungi, including but not limited to Aspergillus fumigatus, Penicillium, Penicillium wannii, Trichophyton rubrum, or Candida glabrata.

6. An apparatus for carrying out the method according to any one of claims 1-5, characterized in that, include: A sealed cavity with a light-transmitting observation window, and a culture medium containing staining agent A at a concentration of 10 mg / L to 320 mg / L pre-placed in the cavity.

7. The apparatus according to claim 6, characterized in that, The concentration of staining agent A in the culture medium is 80 mg / L; and / or, the culture medium is Sabouraud dextrose agar medium.

8. The apparatus according to claim 6 or 7, characterized in that, The sealed cavity is made of transparent plastic or glass; and / or, the sealed cavity is provided with a septate sterile inoculation port or top cover for inoculating samples.

9. Application of staining agent A in the preparation of products for simultaneous culture and staining of fungi.

10. The application according to claim 9, characterized in that, The fungi include, but are not limited to, Aspergillus fumigatus, Penicillium, Penicillium wannii, Trichophyton rubrum, or Candida parapsilosis.

Images