Simple culture method of pseudohyphal form of candida meraukensis

Abstract

The application discloses a simple pseudohyphal phase of M. capitata culture method, and belongs to the technical field of yeast culture methods. Specifically, a strain is taken from a -80 DEG C refrigerator, activated at room temperature of 20 DEG C, inoculated on YPD culture medium on an ultraclean table, and placed in a 28 DEG C constant temperature incubator for 48 h to recover the strain and form a colony; a single colony is taken and inoculated on YPD culture medium again, and placed in a 28 DEG C constant temperature incubator for 48 h to obtain a second-generation strain; the second-generation strain of the single colony is picked and inoculated on a modified V8 juice culture medium, and incubated at 25 DEG C for 10 days to obtain the simple pseudohyphal phase of M. capitata. The application can stably culture the simple pseudohyphal phase of M. capitata, provides a scientific basis for enriching the life history of M. capitata, provides technical guidance for reasonably using drugs, provides a new idea for preventing the occurrence of M. capitata disease, and has important significance for promoting the healthy development of the crustacean industry.

Description

Technical Field

[0001] This invention belongs to the technical field of yeast culture methods, and in particular relates to a simple pseudohyphae-phase *Dacronis mitochondritis* culture method. Background Technology

[0002] *Megasteria micrantha*, belonging to the Ascomycota and the *Megasteria* genus, is an opportunistic pathogen widely found in both freshwater and seawater. It can infect various economically important aquatic species, including the Chinese mitten crab, giant freshwater prawn, swimming crab, Chinese long-armed shrimp, and brine shrimp. In recent years, its incidence in aquaculture has been gradually increasing, and the lack of effective treatment has made it difficult to manage, resulting in significant economic losses and seriously hindering the healthy development of the aquaculture industry. Only by adhering to the scientific principle of combining prevention and treatment can the spread of this disease be effectively controlled. Therefore, exploring the life cycle of *Megasteria micrantha* is crucial.

[0003] The cell morphology of *Saccharomyces cerevisiae* is mainly divided into four categories: budding cells, simple pseudohyphae, coupled cells, and ascospore cells. However, in the culture studies of *Saccharomyces cerevisiae*, most observations have focused on the budding cell morphology, and there are currently no records of stable culture and observation of its simple pseudohyphae phase cells, making it impossible to construct a complete life cycle.

[0004] Hyphae formation was observed in *Saccharomyces cerevisiae* after incubation at 37°C for 6 hours on Spider solid medium. While solid media such as maltose agar, corn broth, Sabouraud dextrose broth, V8 juice broth, and YPD broth could produce a small amount of simple pseudohyphae, the resulting cells were rich in yeast phase cells and lacked purity. There is a lack of cultivation methods for determining the growth conditions of simple pseudohyphae cells in *Saccharomyces cerevisiae*. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a simple method for culturing pseudomycelial *A. mitochondritis* by improving and optimizing the composition of the culture medium.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention discloses a method for culturing a simple pseudomycelial phase of *Saccharomyces cerevisiae*. The method involves taking a strain from a -80°C freezer and activating it at room temperature (20°C). The strain is then streaked onto YPD medium in a clean bench and incubated at 28°C for 48 hours to allow for revival and colony formation. A single colony is then re-inoculated onto YPD medium and incubated at 28°C for 48 hours to obtain a second-generation strain. A single colony of the second-generation strain is picked and streaked onto a modified V8 juice medium and incubated at 25°C for 10 days to obtain *Saccharomyces cerevisiae* with a simple pseudomycelial phase.

[0008] Furthermore, the modified V8 juice culture medium is based on the V8 juice culture medium with the addition of mannitol and dipotassium hydrogen phosphate. The medium consists of 1 part V8 juice culture medium, 0.5 parts nutrient broth, 1 part mannitol, and 0.1 parts dipotassium hydrogen phosphate. The nutrient broth is composed of 10g peptone, 3g beef powder, and 5g sodium chloride.

[0009] The beneficial effects of this invention are as follows:

[0010] 1. This invention optimizes the culture conditions of *Saccharomyces cerevisiae*, prepares an improved culture medium, and cultivates *Saccharomyces cerevisiae* with a high purity of simple pseudomycelial phase.

[0011] 2. This invention can stably culture a simple pseudohyphae of *Saccharomyces cerevisiae*, providing a scientific basis for enriching the life history of *Saccharomyces cerevisiae*, providing technical guidance for clarifying how to use drugs rationally, and offering a new approach to preventing *Saccharomyces cerevisiae* diseases. It is of great significance to promoting the healthy development of the crustacean industry.

[0012] 3. The reason this invention can obtain *Saccharomyces cerevisiae* with a simple pseudohyphae phase is that, based on the cultivation of a small amount of *Saccharomyces cerevisiae* with simple pseudohyphae in V8 juice medium, the key is to explore how to adjust and improve the medium formula to induce a larger quantity and higher proportion of simple pseudohyphae. This invention, based on V8 juice medium, uses a controlled variable method, comparing mycelial growth states by employing different proportions of mannitol and dipotassium hydrogen phosphate, optimizing the formula ratio and cultivation time, and ultimately obtaining the optimal cultivation method. Attached Figure Description

[0013] Figure 1 Simple pseudohyphae cells of *Saccharomyces cerevisiae* were cultured under conditions of twice the amount of mannitol.

[0014] Figure 2 Simple pseudohyphae cells of *Saccharomyces cerevisiae* were cultured under conditions of twice the dipotassium hydrogen phosphate.

[0015] Figure 3 Simple pseudohyphae cells of *Saccharomyces cerevisiae* were cultured in V8 juice medium.

[0016] Figure 4 For cultured *Saccharomyces cerevisiae* mycelial phase cells;

[0017] Figure 5 To culture simple pseudohyphae phase cells of *Saccharomyces cerevisiae* in a modified V8 juice culture medium;

[0018] Table 1 shows the formulations for different culture media;

[0019] Table 2 shows the formula for the modified V8 juice culture medium. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0021] Example:

[0022] 1.1 Research Equipment and Materials

[0023] Constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.)

[0024] Digital electron microscope (Shenyang Hejun Biotechnology Co., Ltd.)

[0025] High-pressure steam sterilizer (Zhiwei Instruments Co., Ltd.)

[0026] 90mm sterile petri dish (Shenyang Guyue Trading Co., Ltd.)

[0027] V8 Fruit Juice Medium (2x Solution) (MM7051, Coolaber)

[0028] Maltose agar medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0029] Sabouraud dextrose medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0030] YPD culture medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0031] Corn agar medium (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0032] Mannitol (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.)

[0033] Dipotassium hydrogen phosphate (Tianjin Ruijinte Chemicals Co., Ltd.)

[0034] 1.2 Experimental Procedure

[0035] 1.2.1 Strain resuscitation

[0036] Experimental strain: Wild strain isolated from diseased crabs

[0037] The bacterial strain was taken from a -80°C freezer and activated at room temperature. It was then streaked onto YPD medium in a clean bench and incubated at 28°C for 48 hours to allow the strain to recover and form colonies. A single colony was then inoculated again into YPD medium and incubated at 28°C for 48 hours to obtain the second-generation strain.

[0038] 1.2.2 Culture of Simple Pseudohyphae of *Saccharomyces cerevisiae*

[0039] Colonies approximately 2 mm in diameter were picked and incubated in modified V8 juice medium at 25°C for 10 days to obtain *Saccharomyces cerevisiae* with simple pseudomycelial phase cells. Figure 5 As shown.

[0040] The modified V8 juice culture medium described in this example includes: 1 part V8 juice culture medium, 0.5 parts nutrient broth, 1 part mannitol, and 0.1 parts dipotassium hydrogen phosphate.

[0041] During the cultivation process, colonies with a diameter of approximately 2 mm were picked and incubated on various solid media (as shown in Table 1), including maltose agar, corn agar, Sabouraud dextrose agar, V8 juice medium, and YPD medium, at three temperatures: 20°C, 25°C, and 28°C. The results showed that only yeast-phase *Saccharomyces cerevisiae* was cultured on maltose agar, corn agar, Sabouraud dextrose agar, and YPD medium at different temperatures. However, after 30 days of incubation on V8 juice medium at 25°C, a certain amount of simple pseudohyphae cells (e.g., *Saccharomyces cerevisiae*) were obtained. Figure 3 As shown), individual colonies of the strain and a second generation of *Saccharomyces cerevisiae* were picked and incubated in Spider liquid medium (3g nutrient broth, 3g mannitol, 0.6g dipotassium hydrogen phosphate, 300ml pure water) at 37°C for 6 hours. A large number of pseudohyphae (as shown) were obtained in both cases. Figure 4 As shown in Table 2, since V8 juice medium can obtain a certain amount of simple pseudohyphae cells and Spider liquid medium can induce them to grow into longer pseudohyphae, a modified version of V8 juice medium was prepared using the components in Spider liquid medium that induce pseudohyphae, in order to shorten the culture time and obtain simple pseudohyphae cells with higher purity. However, the components that produce the inducing effect in Spider liquid medium are still unclear, so their dominant role was analyzed. Under the same nutrient conditions of 0.5g nutrient broth and 30ml V8 juice, two versions of medium were prepared. The first version contained 0.6g mannitol and 0.24g dipotassium hydrogen phosphate; the second version contained 1.2g mannitol and 0.12g dipotassium hydrogen phosphate. The concentrations of mannitol and dipotassium hydrogen phosphate were increased by two times for comparison (e.g., ...). Figure 1 , Figure 2 As shown in the image, the formation of simple pseudohyphae was observed by inoculating second-generation *Saccharomyces cerevisiae*. The nutrient broth consisted of 10g peptone, 3g beef powder, and 5g sodium chloride.

[0042] Simple pseudohyphae cells of *Saccharomyces cerevisiae* can be obtained by incubating colonies on modified V8 juice medium at 25°C for 10 days. Figure 5 As shown.

[0043] Table 1: Formulations of different culture media

[0044]

[0045] Table 2: Formula for Modified V8 Fruit Juice Culture Medium

[0046]

[0047] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; as long as the use needs are met, they are all within the protection scope of the present invention.

Claims

1. A method for culturing a simple pseudohyphae-phase *Saccharomyces cerevisiae*, characterized in that: The strain was taken from a -80°C freezer and activated at room temperature (20°C). It was then streaked onto YPD medium in a clean bench and incubated at 28°C for 48 hours to allow the strain to recover and form colonies. A single colony was taken and inoculated again into YPD medium and incubated at 28°C for 48 hours to obtain the second-generation strain. A single colony of the second-generation strain was picked and streaked onto a modified V8 juice medium and incubated at 25°C for 10 days to obtain the simple pseudomycelial cells of *Saccharomyces cerevisiae*. The improved V8 juice culture medium is based on the V8 juice culture medium with the addition of mannitol and dipotassium hydrogen phosphate. The medium consists of 1 part V8 juice culture medium, 0.5 parts nutrient broth, 1 part mannitol, and 0.1 parts dipotassium hydrogen phosphate. The nutrient broth is composed of 10g peptone, 3g beef powder, and 5g sodium chloride.

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