Method for detecting growth condition of aspergillus fumigatus in cement before and after flood soaking

Abstract

The invention relates to the field of building material science, environmental microbiology and structural engineering, in particular to a method for detecting the growth condition of aspergillus fumigatus in cement before and after flood soaking. The problems that a traditional chemical spraying disinfection method is limited in prevention and control effect and prone to causing secondary pollution are solved. According to the technical scheme, the method comprises the steps that S1, aspergillus fumigatus samples are cultured; S2, cement blocks are poured and maintained, and experiment grouping is conducted; s3, the young aspergillus fumigatus blocks are transplanted to the surfaces of the cement blocks; s4, dyeing bacterial plaques on the surfaces of the cement blocks by using a lactic acid phenol cotton blue dyeing solution; s5, carrying out qualitative analysis; s6, quantitative analysis is carried out; and S7, detecting the change of the breaking strength and the compressive strength of the cement block through a compression testing machine. The method has the beneficial effects that the detection process is extremely convenient; the detection cost is low, expensive analytical instruments are not needed, the steps are simple, and the operation threshold is low.

Description

TECHNICAL FIELD

[0001] The application relates to the fields of building material science, environmental microbiology and structural engineering, and particularly relates to a detection method for growth of Aspergillus fumigatus in cement before and after flood soaking. BACKGROUND

[0002] Reference paper: Wu Y, Wen B, Gasevic D, et al. Climate Change, Floods, and Human Health. [J]. The New England journal of medicine, 2024, 391(20): 1949-1958. Floods, as a natural disaster, have a significant impact on human life and property, and can directly deprive life, and also produce secondary health threats, including: not only significantly increasing the risk of infectious diseases (including water-borne, mosquito-borne and rodent-borne diseases), but also causing high incidence of skin and respiratory tract infections; and may induce acute onset of cardiovascular diseases, exacerbate the condition of chronic respiratory diseases and the like.

[0003] As a frequently occurring natural disaster, floods have direct hazards (such as damage to infrastructure, acute water-borne diseases caused by water pollution, etc.), which have been widely concerned and researched, but the hidden and temporary health risks at the level of building materials after floods have not been fully recognized, and there is a significant gap in the related research.

[0004] Cement-based materials have become the core building materials for modern urban construction and infrastructure (such as residential walls, public building structures, municipal pipelines, etc.) due to their excellent durability, mechanical strength, and economy. In normal conditions, cement-based materials can effectively inhibit the adhesion and reproduction of microorganisms (including fungi, bacteria, etc.) due to their high alkalinity (pH usually 12-13) and low porosity. Cement has natural resistance to fungi that can produce allergenic and pathogenic spores, ensuring the biological safety of indoor environments. However, flood disasters can significantly disrupt the surface stability of cement-based materials: floodwater carries silt and pollutants that adhere to the cement surface, and water penetration can cause temporary moisture absorption in the cement surface layer, leading to a temporary decrease in pH (due to the neutralization of CO2 and organic acids in the water body) and a local increase in porosity. This series of temporary changes creates an ideal microenvironment for fungal colonization and reproduction. Compared to the normal state of cement-based materials, the moisture-absorbed cement surface layer not only provides the necessary water conditions for fungal growth, but also provides organic components in pollutants and silt as nutrient sources, significantly promoting fungal growth. More importantly, these fungi are mostly species that can release airborne spores (such as Aspergillus and Penicillium), which can spread through indoor air currents and be inhaled by the human body, causing or exacerbating respiratory diseases (such as asthma, rhinitis, and pneumonia), posing a potential and ongoing public health threat.

[0005] The changes in cement-based materials caused by floods are temporary and local, and as the cement-based materials naturally dry, the alkalinity and porosity of the surface layer gradually recover, and the conditions for fungal growth disappear. However, before this happens, fungal spores have already been released and spread. Due to the temporary and local nature of these changes, conventional material performance testing methods (such as mechanical property testing and overall pH testing) are difficult to effectively identify and evaluate them, leading to long-term "neglect" of this health risk, and there are obvious gaps in related research in the fields of materials science and public health.

[0006] Epidemiological data further confirm the real harm of this risk: the existing article Zhengyu Y, Wenzhong H, E JM, et al. Mortality risks associated with floods in 761 communities worldwide: time series study. [J]. BMJ (Clinical research ed.), 2023, 383 e075081-e075081. shows that after the occurrence of flood disasters, the mortality risk of the population increases significantly within 60 days, and the risk rises rapidly within 14-47 days; It is particularly worth noting that about 25 days after the flood, the incidence of respiratory diseases reaches a peak, but the potential mechanism for this incidence peak (especially the role of building material-related fungal spores) has not been clearly identified. The article Kundzewicz W Z, Hirabayashi Y, Kanae S. River Floods in the Changing Climate-Observations and Projections [J]. Water resources management, 2010, 24(11): 2633-2646. further analysis shows that, affected by climate change, the frequency and intensity of global floods are on the rise, and the increase in flood frequency is significantly positively correlated with the incidence of allergic respiratory diseases such as asthma and rhinitis, and the core correlation path is believed to be that floods promote the spread of fungal spores in the building environment. The existing article J M M, G A M, Kerry C, et al. Respiratory and allergic health effects of dampness, mold, and dampness-related agents: a review of the epidemiologic evidence. [J]. Environmental health perspectives, 2011, 119(6): 748-56. has confirmed that exposure to certain molds (such as Aspergillus) can directly induce respiratory infections and exacerbate symptoms in patients with existing respiratory diseases (such as acute asthma attacks), and the dampness problem that is common in the building environment after floods and the growth of mold are the most common but not fully recognized indirect health impact factors in flood disasters.

[0007] Among the common fungal species in the post-flood building environment, Aspergillus, Cladosporium, Penicillium, Trichoderma and Paecilomyces dominate, among which Aspergillus fumigatus is one of the most concerned species due to its wide distribution and strong pathogenicity. Aspergillus fumigatus is ubiquitous in the natural environment, and its colony is fluffy or flocculent, initially white, then gradually changes to dark green or smoke green, and the color of the old colony is further deepened. The spores of Aspergillus fumigatus have extremely strong air transmission ability, and can be detected in indoor and outdoor air. Normal people can inhale hundreds of such spores per day. For the post-flood building environment, the residual situation of Aspergillus fumigatus spores can directly reflect the removal effect of fungal pollution during building repair, and is an important indicator for evaluating the biological safety of building indoor environment. If Aspergillus fumigatus spores can still be detected after repair, it indicates that there is still a potential risk of fungal pollution in the building, therefore, Aspergillus fumigatus has become the core model species for studying the fungal pollution and health risk of building materials after flood.

[0008] In summary, the current research and prevention and control technology for fungal pollution and respiratory system health risk of cement-based material surface layer after flood still has the following key deficiencies:

[0009] (1) Lack of risk awareness: existing research focuses more on the direct consequences of floods, and pays insufficient attention to the risk of fungal growth caused by temporary changes in the surface layer of cement-based materials, resulting in a long-term research gap in this hidden risk, and an effective risk assessment system has not been established.

[0010] (2) Due to the local and transient nature of the changes in the surface layer of cement-based materials, conventional material detection methods cannot accurately identify them, and there is a lack of rapid and efficient detection technology for fungal spores (especially Aspergillus fumigatus spores) in post-flood building environments, making it difficult to achieve early warning and intervention of risks.

[0011] (3) Lack of in-depth mechanism research: Although epidemiological data confirm that the incidence of respiratory diseases increases after floods, the mechanism research on the correlation chain of "changes in the surface layer of cement-based materials-fungal growth-spore diffusion-respiratory diseases" is still insufficient, especially the lack of basic data such as the colonization pattern of key fungi such as Aspergillus fumigatus in the surface layer of cement-based materials and spore release kinetics, which cannot provide scientific basis for risk prevention and control.

[0012] (4) Current prevention and control measures for fungal pollution of building materials after floods rely mainly on conventional methods such as ventilation and drying, chemical disinfection, and lack of targeted prevention and control technology based on the characteristics of cement-based materials and the growth pattern of fungi, resulting in limited prevention and control effect and secondary pollution (such as chemical disinfectant residues).

[0013] How to solve the above problems is the subject of the present application. SUMMARY

[0014] The present application aims to study the fungal colonization potential of cement material surface and its correlation with respiratory diseases by simulating the conditions of flood erosion of building environment, and to evaluate the effects of flood soaking on fungal growth and mechanical properties of cement materials, to provide technical support for the dual evaluation of building structure safety and indoor biological safety after flood. The present application uses solution soaking of cement test blocks to simulate the action of flood, and then carries out Aspergillus fumigatus inoculation to reproduce the process of mold invasion; on the one hand, the micro-imaging technology is used to dynamically monitor the colony growth dynamics, spore diffusion characteristics and biofilm formation process, to obtain direct experimental evidence of flood-induced fungal colonization on material surface, on the other hand, through the bending and compressive strength test, the change rule of mechanical properties of cement-based materials in the process of flood erosion of living environment is found out, and the correlation analysis system of "environmental action-biological colonization-material performance" is constructed. In order to achieve the above application purposes, the technical scheme of the present application is specifically as follows:

[0015] A method for detecting the growth of Aspergillus fumigatus in cement before and after flood soaking, comprising the following steps:

[0016] Step S1: culture of Aspergillus fumigatus sample: Aspergillus fumigatus is cultured using potato glucose agar medium, which provides necessary nutrients and environmental conditions for Aspergillus fumigatus;

[0017] Step S11: under sterile environment, uniformly coat the glycerol mixture containing Aspergillus fumigatus spores on the surface of the high-temperature sterilized potato glucose agar medium;

[0018] Step S12: place the culture medium in a constant temperature incubator and culture at a constant temperature for a period of time to obtain a young Aspergillus fumigatus strain with high activity and strong reproductive ability, which is convenient for subsequent experiments, until the surface of the culture medium is completely covered with Aspergillus fumigatus mycelium;

[0019] Step S2: cement block pouring, curing and experimental grouping;

[0020] Step S21: configure Portland cement with a specified water-cement ratio of 0.5; pour the prepared cement paste into the mold;

[0021] Step S22: according to the "Technical Code for Curing of Cast-in-place Concrete" JC / T 60018-2023, take out the cement from the mold after curing for a certain period of time;

[0022] Step S23: divide the cement block into two parts, and perform different strain transfer operations on each part;

[0023] Step S24: divide each part into three different groups.

[0024] The first part is directly inoculated with the strain after being soaked in water for a period of time; the second part is first recovered and then inoculated with the strain after being soaked in water for a period of time;

[0025] Step S241: The first part of the cement is divided into three different groups according to the soaking time;

[0026] The cement blocks in the first group are not soaked in water;

[0027] The cement blocks in the second group are soaked in water for a short time;

[0028] The cement blocks in the third group are soaked in water for a long time;

[0029] Step S242: The cement blocks in each group are divided into an observation group, a control group, and a test group;

[0030] The observation group is the cement block inoculated with the Aspergillus fumigatus block with potato glucose agar medium;

[0031] The control group is the cement block inoculated with the Aspergillus fumigatus block with the same area as the Aspergillus fumigatus block without medium;

[0032] The test group is the cement block for compressive and flexural strength test;

[0033] Step S243: The second part is divided into three different groups according to the soaking time;

[0034] The cement blocks in the first group are not soaked in water, and are recovered for a period of time;

[0035] The cement blocks in the second group are soaked in water for a short time, and are recovered for a period of time;

[0036] The cement blocks in the third group are soaked in water for a long time, and are recovered for a period of time;

[0037] Step S244: The cement blocks in each group are divided into an observation group, a control group, and a test group;

[0038] The observation group is the cement block inoculated with the Aspergillus fumigatus block with potato glucose agar medium;

[0039] The control group is the cement block inoculated with the Aspergillus fumigatus block with the same area as the Aspergillus fumigatus block without medium;

[0040] The test group is the cement block for compressive and flexural strength test;

[0041] Step S3: The young Aspergillus fumigatus block is inoculated on the surface of the cement block;

[0042] Step S31: using high-temperature sterilized professional inoculation shovel and inoculation ring, cutting uniform size of Aspergillus fumigatus block from the potato glucose agar medium, inoculating on the surface of the cement block of different observation groups and control groups in step S242;

[0043] Step S32: using high-temperature sterilized professional inoculation shovel and inoculation ring, cutting uniform size of Aspergillus fumigatus block from the potato glucose agar medium, inoculating on the surface of the cement block of different observation groups and control groups in step S244;

[0044] Step S33: placing all the cement blocks in a constant-temperature bacteria maintenance box for constant-temperature culture, and continuing to culture for a period of time;

[0045] Step S4: staining the surface of the cement block with lactic acid phenol cotton blue staining solution; (lactic acid phenol cotton blue staining solution is a common staining agent used in microbiology laboratories, which can stain Aspergillus fumigatus without staining the cement, facilitating the observation of the morphological structure of Aspergillus fumigatus such as hyphae and spores under a microscope.)

[0046] Step S41: taking out the cultured cement block;

[0047] Step S42: high-temperature inactivation; the cement block is subjected to high-temperature inactivation by a high-pressure steam sterilization pot to ensure the safety of the experimental personnel during the microscopic observation experiment;

[0048] Step S43: applying lactic acid phenol cotton blue staining solution to the surface of the high-temperature sterilized observation group and control group for staining;

[0049] Step S5: observing under a microscope whether Aspergillus fumigatus hyphae are produced at the stained site and observing the length of the Aspergillus fumigatus hyphae for qualitative analysis;

[0050] Place the cement block under a microscope (a microscope with a reflected light source) for observation and take photos to save the growth of Aspergillus fumigatus on the cement block at different soaking times for qualitative analysis and comparison.

[0051] Step S6: quantifying the spore density of Aspergillus fumigatus by gradient dilution method and conducting quantitative analysis combined with image processing technology;

[0052] Step S61: using high-temperature sterilization and weighing the initial mass of the rubber bulb pipette in advance, randomly and uniformly (the purpose of random and uniform suction position is to ensure the representativeness and accuracy of the experimental results, avoid deviation, reduce human influence, and make the experimental results more objective) sucking several sample points on the inoculation point of the cement block surface (ensuring sufficient sample quantity), and weighing;

[0053] Step S62: Gradient dilution of the extracted Aspergillus fumigatus spores using sterile water to achieve a certain dilution multiple; the present application adopts 10 9 times dilution

[0054] Step S63: After the diluted solution is added dropwise on the glass slide, staining is performed using lactic acid phenol cotton blue staining solution, and then observation is performed under a microscope; the present application extracts 1 milliliter of the diluent.

[0055] Step S64: Randomly and uniformly select several observation points for shooting. Through analysis of these images, the colony growth on the surface of the cement block is quantitatively evaluated according to the spore density. (Ensure sufficient sample quantity)

[0056] Step S7: Detect the change of the flexural strength and the compressive strength of the cement block through a pressure testing machine

[0057] Step S71: Test the flexural strength of the test group and record it;

[0058] Step S72: Collect and clean the cement block that is broken by the pressure testing machine after the flexural strength test.

[0059] Step S73: Test the compressive strength of the test group and record it.

[0060] The size of the test group cement block is: a cuboid cement block with a size of 40*40*160mm (a compressive strength test piece according to the CBT50081-concrete mechanics and physical test method standard), and three pieces are used for the flexural and compressive strength test of the cement.

[0061] The size of the observation group and the control group is: a cylindrical cement block with a diameter of 60mm and a thickness of 12mm (used for inoculating Aspergillus fumigatus on a smooth surface, facilitating the observation of the growth of Aspergillus fumigatus spores under a microscope)

[0062] All operations are performed under sterile and direct contact-free conditions to ensure the accuracy of the experimental results, avoid the infection of miscellaneous bacteria and the entry of mold into the human body, and affect the health of the human body.

[0063] Compared with the prior art, the present application has the following beneficial effects:

[0064] (1) The detection process is extremely convenient. The growth of Aspergillus fumigatus colonies on the surface of the dyed cement test piece can be directly observed under a microscope. The colony morphology, distribution density and coverage area after staining can be directly interpreted, which greatly improves the detection efficiency and the visualization degree of the results, and is suitable for rapid screening and long-term dynamic monitoring of a large number of samples.

[0065] (2) The detection cost is low, and expensive analytical instruments (such as a scanning electron microscope, an X-ray diffractometer, and other expensive large instruments) or a large amount of consumable chemical reagents are not required, and the detection process is green and environmentally friendly. Microscopes are basic optical equipment and are widely used in most laboratories, and are simple to operate and maintain; common and economical dyeing agents. The entire detection process is pollution-free, produces no harmful products, does not stain the test piece, and meets the green and environmentally friendly concept;

[0066] (3) The method steps are simple, and the operation threshold is low, so even non-professionals can master it after short-term training. From sample preparation, dyeing to microscopic observation, the process is highly standardized and has good repeatability, which is conducive to comparison and data integration between different researchers, and has good promotion applicability;

[0067] (4) The present application can not only effectively evaluate the growth of fungi on the surface of cement, but also be used for systematic study on the chemical changes and microstructure evolution of the surface of cement materials after flood soaking, such as the increase of porosity, the decrease of alkalinity and the transformation of phase composition, so as to reveal the dynamic coupling relationship between the performance degradation of building materials and the colonization of microorganisms under the influence of flood. This method fills the gap of detection means in the field of micro-mechanism and process research.

[0068] (5) By integrating the cross perspectives of material science, microbiology and environmental health science, the detection strategy established by the present application helps to establish a systematic cognitive chain of “flood disaster-cement porosity increase-microbial proliferation-health risk”, and provides key technical support for building environment health assessment and disease control under the background of disasters, which has important scientific significance and engineering application value.

[0069] The accompanying drawings are used to provide a further understanding of the present application, and constitute a part of the specification, together with embodiments of the present application, to explain the present application, and do not constitute a limitation of the present application.

[0070] Figure 1 It is a flowchart of the method of the present application.

[0071] Figure 2 It is a grouping schematic diagram of the present application.

[0072] Figure 3 It is a schematic diagram of the qualitative and quantitative detection system of Aspergillus fumigatus growth of the present application.

[0073] Figure 4 It is a colony growth qualitative and quantitative analysis diagram of the embodiment of the present application; wherein: 4(a) is a spore diagram after the microscope is enlarged 100 times, and 4(b) is a black and white binary processing spore diagram.

[0074] Figure 5The figure of the flexural and compressive strength of the cement test piece of the present application; wherein: 5(a) is the flexural strength column chart of the cement block, and 5(b) is the compressive strength column chart of the cement block. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical scheme and advantages of the present application more clear and explicit, the present application will be further described in detail below in combination with the drawings and examples. Of course, the specific examples described herein are only used to explain the present application and not to limit the present application.

[0076] Example 1

[0077] As Figure 1 shown, a method for detecting the growth of Aspergillus fumigatus in cement before and after flood soaking, comprising the following steps:

[0078] Step S1: culture of Aspergillus fumigatus sample: using potato glucose agar medium to culture Aspergillus fumigatus, which provides necessary nutrients and environmental conditions for Aspergillus fumigatus;

[0079] Step S11: under sterile environment, uniformly coat the glycerol mixture containing Aspergillus fumigatus spores on the surface of the high-temperature sterilized potato glucose agar medium;

[0080] Step S12: place the culture medium in a constant temperature bacteria culture box and culture at a constant temperature of 26℃ for a period of time (3-5 days), 3-5 days of culture at a constant temperature of 26℃ can obtain young Aspergillus fumigatus strains with high activity and strong reproductive ability, which is convenient for subsequent experiments, until the surface of the culture medium is completely covered with Aspergillus fumigatus mycelium;

[0081] Step S2: cement block pouring, curing and grouping for experiment;

[0082] Step S21: configure Portland cement with a specified water-cement ratio of 0.5; pour the prepared cement paste into the mold;

[0083] Step S22: according to the "cast-in-place concrete curing technical specification" JC / T 60018-2023, take out the cement from the mold after curing for a certain period of time (28 days);

[0084] As Figure 2 described, step S23: divide the cement block into two parts (group A and group B), and perform different strain transfer operations on each part;

[0085] Step S24: divide each part into three different groups.

[0086] The first part is directly subjected to strain transfer after being soaked in water for a period of time; the second part is first recovered and then subjected to strain transfer after being soaked in water for a period of time;

[0087] Step S241: Divide the cement from the first part into three different groups according to the soaking time of the blocks;

[0088] The first group of cement blocks was not soaked in water;

[0089] The second group of cement blocks were soaked in water for a short time;

[0090] The third group of cement blocks were soaked in water for a long time;

[0091] Step S242: Each group of cement blocks is divided into an observation group, a control group, and a test group;

[0092] The observation group consisted of cement blocks inoculated with Aspergillus fumigatus var. mongolica culture medium; specifically, A11 was a cement block that had been cured for 28 days, not soaked in the solution, and inoculated with Aspergillus fumigatus var. mongolica culture medium; A21 was a cement block that had been cured for 28 days, soaked in the solution for 1 day, and inoculated with Aspergillus fumigatus var. mongolica culture medium; and A31 was a cement block that had been cured for 28 days, soaked in the solution for 7 days, and inoculated with Aspergillus fumigatus var. mongolica culture medium.

[0093] The control group consisted of cement blocks inoculated with Aspergillus fumigatus var. flavomarginata (ASF) spores, but without culture medium. Specifically, A12 was a cement block inoculated with ASF spores, cured for 28 days, not soaked in solution, and without culture medium; A22 was a cement block inoculated with ASF spores, cured for 28 days, soaked in solution for 7 days, and without culture medium; A32 was a cement block inoculated with ASF spores, cured for 28 days, soaked in solution for 7 days, and without culture medium.

[0094] The test set consists of cement blocks used for testing compressive and flexural strength.

[0095] Step S243: Divide the second part into three different groups according to the soaking time;

[0096] The first group of cement blocks were not soaked in water and were allowed to recover for a period of time.

[0097] The second group of cement blocks were soaked in water for a short time, and then allowed to recover for a period of time.

[0098] The third group of cement blocks were soaked in water for a long time and then allowed to recover for a period of time.

[0099] Step S244: Each group of cement blocks is divided into an observation group, a control group, and a test group;

[0100] The observation group consisted of cement blocks inoculated with Aspergillus fumigatus mold blocks coated with potato dextrose agar. Specifically, B11 consisted of cement blocks that were cured for 28 days, not soaked in the solution, then recovered for 63 days, and inoculated with Aspergillus fumigatus mold blocks coated with potato dextrose agar. B21 consisted of cement blocks that were cured for 28 days, soaked in the solution for 1 day, then recovered for 63 days, and inoculated with Aspergillus fumigatus mold blocks coated with potato dextrose agar. B31 consisted of cement blocks that were cured for 28 days, soaked in the solution for 7 days, then recovered for 63 days, and inoculated with Aspergillus fumigatus mold blocks coated with potato dextrose agar.

[0101] The control group consisted of cement blocks inoculated with Aspergillus fumigatus var. flavomarginata (ASF) ...

[0102] The test set consists of cement blocks used for testing compressive and flexural strength.

[0103] Step S3: Transfer the young Aspergillus fumigatus mold blocks to the surface of the cement block;

[0104] Step S31: Using a professional inoculation shovel and inoculation loop that have been sterilized at high temperature, cut out blocks of Aspergillus fumigatus of uniform size from potato dextrose agar medium and inoculate them on the surface of cement blocks of different observation groups and control groups in step S242. The present invention uses four preset points, which are the four vertices of a square with a side length of 40mm.

[0105] Step S32: Using a professional inoculation shovel and inoculation loop that have been sterilized at high temperature, cut out blocks of Aspergillus fumigatus of uniform size from potato dextrose agar medium and inoculate them on the surface of cement blocks of different observation groups and control groups in step S244. The present invention uses four preset points, which are the four vertices of a square with a side length of 40mm.

[0106] Step S33: Place all cement blocks in a constant temperature microbial incubator and maintain a constant temperature (26℃) for incubation for a period of time (21 days);

[0107] Step S4: Stain the bacterial spots on the cement block surface with lactophenol indigo dye; (Lactophenol indigo dye is a commonly used staining agent in microbiology laboratories. It can stain Aspergillus fumigatus without staining the cement, making it easy to observe the morphology and structure of Aspergillus fumigatus, such as hyphae and spores, under a microscope.)

[0108] Step S41: Remove the cultured cement block;

[0109] Step S42: Perform high-temperature inactivation; This invention uses a high-pressure steam sterilizer to perform high-temperature inactivation on cement blocks to ensure the safety of experimental personnel during microscope observation experiments;

[0110] Step S43: Apply lactophenol cotton blue dye solution to the surfaces of the observation group and control group, which have been sterilized at high temperature, for staining;

[0111] Step S5: Observe under a microscope whether Aspergillus fumigatus hyphae are produced on the stained area and observe the length of the Aspergillus fumigatus hyphae for qualitative analysis;

[0112] The cement blocks were observed under a microscope (with a reflective light source), and photographs were taken to perform qualitative analysis and comparison of the growth of Aspergillus fumigatus on cement blocks soaked for different times.

[0113] Step S6: Quantify the density of Aspergillus fumigatus spores using a gradient dilution method, and perform quantitative analysis using image processing techniques;

[0114] Step S61: Using a dropper that has been sterilized at high temperature and weighed beforehand, randomly and evenly (the purpose of random and even sampling is to ensure the representativeness and accuracy of the experimental results, avoid deviation, reduce human influence, and make the experimental results more objective) take a number of (8) sample points on the inoculation point on the surface of the cement block (to ensure sufficient sample quantity) and weigh them.

[0115] Step S62: The extracted Aspergillus fumigatus spores are serially diluted with sterile water to achieve a certain dilution factor; this invention uses 10 9 Dilution

[0116] Step S63: Add the diluted solution to a glass slide, stain it with lactophenol cotton blue dye, and observe it under a microscope; in this invention, 1 ml of the diluted solution is taken.

[0117] Step S64: Randomly and evenly select several (20) observation points for image capture. Analyze these images to quantitatively assess colony growth on the cement block surface based on spore density. (Ensure sufficient sample quantity.)

[0118] Step S7: Detect the changes in flexural and compressive strength of the cement block using a pressure testing machine.

[0119] Step S71: Perform flexural strength tests on the test group and record the results;

[0120] Step S72: Collect and clean the cement blocks that were broken by the compression testing machine after the flexural strength test;

[0121] Step S73: Perform compressive strength tests on the test group and record the results.

[0122] The test group of cement blocks consisted of rectangular cement blocks measuring 40*40*160mm (compressive strength specimens as specified in CBT50081-Standard for Test Methods of Concrete Mechanics and Physics), with 3 blocks per group (28-day curing group, 28-day curing and 7-day soaking group, and 28-day curing and 7-day soaking group followed by 63-day recovery group) used for testing the flexural and compressive strength of cement.

[0123] The observation group and control group consisted of cylindrical cement blocks A11, A12, A21, A22, A31, A32, B11, B12, B21, B22, B31, and B32, each with a diameter of 60 mm and a thickness of 12 mm. These blocks were used to inoculate Aspergillus fumigatus on smooth surfaces, facilitating microscopic observation of Aspergillus fumigatus spore growth.

[0124] All operations are performed under sterile conditions without direct contact, ensuring the accuracy of experimental results while preventing contamination by other bacteria and mold from entering the human body and affecting human health.

[0125] Example 2

[0126] Based on implementation 1

[0127] like Figure 3 As shown, Figure 3 This is a schematic diagram of a qualitative and quantitative detection system for Aspergillus fumigatus growth, focusing on the entire process of Aspergillus fumigatus colony staining, sample processing, dilution, and microscopic observation on cement block surfaces. The experiment revolves around five key operations: staining, sampling, dilution, slide preparation, and observation, as detailed below:

[0128] (1) Staining treatment: Take the inactivated cement block, use a sterile dropper to draw lactic acid cotton blue dye solution, and apply it evenly to the inoculation point and the surrounding 20mm area (covering the possible hyphal growth area).

[0129] (2) Microscopic observation: Place the stained cement block under a microscope, magnify it 100 times, and observe whether Aspergillus fumigatus hyphae are produced on the stained part and observe the length of Aspergillus fumigatus hyphae for qualitative analysis.

[0130] (3) Sample collection and weighing: Using a dropper that has been sterilized at high temperature and pre-weighed to its initial mass (referred to as m0), 8 sample points are randomly selected on the surface of the stained cement block. The stained bacterial plaques and a small amount of surface matrix attached to the surface are collected. After collection, the sample is weighed again (referred to as m1). The mass of a single sampling is calculated by the difference (m1-m0) to ensure the accuracy and representativeness of the sample quantity and avoid subsequent quantitative deviations due to differences in the sampling quantity.

[0131] (4) Serial dilution: Transfer the collected bacterial sample to a sterile centrifuge tube, add sterile physiological saline, and gradually dilute using a 10-fold serial dilution method until a final concentration of 10 is reached. 9 Diluted multiple times.

[0132] (5) Slide preparation and counterstaining: Take 10 9 Add 1 ml of the diluted solution to the center of a clean glass slide, gently spread it into a thin, even layer with a sterile toothpick, add a small amount of lactic acid cotton blue dye for counterstaining, and then cover with a coverslip (avoiding air bubbles to prevent affecting the microscopic field of view) to prepare a standardized microscopic observation slide.

[0133] (6) Microscopic observation: Place the prepared slide on the stage of an optical microscope with a reflective light source, adjust the magnification to 100x, and randomly select several observation points for observation and photography. Visually identify the stained Aspergillus fumigatus spores (which appear as clear blue granules) through the microscope, distinguish impurities from the target spores, and provide raw observation data for the subsequent step S64 (image analysis to quantify spore density).

[0134] This process achieves two core objectives: first, through staining and microscopic observation, to preliminarily determine the growth status of Aspergillus fumigatus on the surface of cement blocks (whether there are identifiable spores); second, through standardized sampling, dilution, and slide preparation operations, to provide standardized samples and observation conditions for subsequent quantification of spore density using image processing techniques (such as ImageJ software), ultimately achieving precise quantitative analysis of Aspergillus fumigatus growth and supporting the core research conclusion of "the impact of flood immersion on fungal colonization of cement materials".

[0135] like Figure 4 (a) As described in step S64: 20 observation points are randomly and uniformly selected for image capture. The colony growth on the cement block surface is quantitatively assessed based on spore density through analysis of these images (ensuring sufficient sample quantity). Then, ImageJ software is used to analyze the images. Figure 4 (a) After performing black-and-white binarization, the result is... Figure 4 (b), in Figure 4 In (b), the screening was conducted using the criterion that the roundness of Aspergillus fumigatus spores was greater than or equal to 0.8 (roundness of a perfectly round spore is 1). Substances with a roundness greater than or equal to 0.8 were identified as Aspergillus fumigatus spores, while those not meeting the criterion were considered impurities. The circled portion in the diagram represents spores, while the others are impurities.

[0136] like Figure 5 As stated in step S7: the changes in flexural strength and compressive strength of the cement block are detected using a pressure testing machine.

[0137] Step S71: Perform flexural strength tests on the test group and record the results. Figure 5 (a);

[0138] Step S72: Collect and clean the cement blocks that were broken by the compression testing machine after the flexural strength test;

[0139] Step S73: Perform compressive strength tests on the test group and record the results. Figure 5 (b);

[0140] Figure 5 Depend on Figure 5 (a) "Column chart of flexural strength of cement block" and Figure 5 (b) The “Cement Block Compressive Strength Bar Chart” consists of two charts, both of which use “Grouping of Cement Blocks with Different Treatment Methods” as the horizontal axis and “Mechanical Strength Value” as the vertical axis. The height of the bars visually presents the differences in mechanical properties of each group of cement blocks. The core purpose is to quantitatively analyze the impact of flood immersion and the recovery process after immersion on the flexural and compressive strength of cement materials.

[0141] Figure 5 (a) and Figure 5 (b) The x-axis groupings are completely consistent, all based on the variable setting of "flood soaking time + recovery time", and are divided into 3 groups. Each group uses "40mm*40mm*160mm rectangular cement blocks" as test specimens (compliant with the "Standard for Test Methods of Concrete Mechanics and Physical Methods" CBT50081). Three cement blocks are set in each group, and the final strength value is the average of the three cement blocks to ensure data reliability. The specific groupings are as follows:

[0142] (1) Unsoaked group: After 28 days of standard curing, the cement blocks were not soaked in water and were directly used for mechanical performance testing as a blank control group to reflect the benchmark mechanical strength of cement materials.

[0143] (2) 7-day soaking group: After 28 days of standard curing, the cement blocks were soaked in water for 7 consecutive days (simulating the long-term soaking environment of flood). After soaking, they were taken out directly, the surface moisture was cleaned and mechanical property tests were carried out to evaluate the impact of short-term flood soaking on cement strength.

[0144] (3) 7-day soaking and 63-day recovery group: After 28 days of standard curing and 7 days of water soaking, the cement blocks were transferred to a standard curing environment for 63 days of recovery curing (simulating the natural recovery process of building materials after the flood recedes). After the recovery was completed, mechanical performance tests were conducted to evaluate the repair effect of the recovery period on the strength of cement after soaking.

[0145] Figure 5 (a): Bar graph of flexural strength of cement block

[0146] Unsoaked group: The height of the column corresponds to approximately 8 MPa on the vertical axis, which is the highest value among the three groups. It represents the benchmark flexural strength of cement material after standard curing and reflects its resistance to bending under normal use conditions.

[0147] The 7-day soaking group: The height of the column corresponds to about 4 MPa on the ordinate, which is significantly lower than that of the unsoaked group, with a strength loss of about 50%. This indicates that 7 days of flood soaking will greatly weaken the flexural strength of cement materials, possibly due to the generation of internal microcracks caused by water penetration or the loosening of the structure of cementitious material hydration products.

[0148] The 7-day soaking recovery group (63-day recovery): The bar height corresponds to approximately 6 MPa on the ordinate, which is between the unsoaked group and the 7-day soaking group. Compared to the 7-day soaking group, the strength is increased by about 50%, but it is still about 25% lower than the unsoaked group. This indicates that the 63-day recovery curing after soaking can partially repair the flexural strength of the cement, but it cannot fully restore it to the baseline level. This may be because the irreversible microscopic damage (such as increased porosity) generated during the soaking process has not been completely eliminated.

[0149] ​ (b): Bar chart of compressive strength of cement block

[0150] Unsoaked group: The column height corresponds to approximately 65 MPa on the ordinate, which is the highest value among the three groups. It represents the benchmark compressive strength of cement materials, reflects their core ability to withstand axial pressure, and is within the normal range of the 28-day standard compressive strength of silicate cement.

[0151] The 7-day soaking group: The height of the bar corresponds to approximately 40 MPa on the ordinate, which is about 38.5% lower than that of the unsoaked group. The strength loss is significant but lower than the loss of flexural strength. This indicates that the compressive strength of cement materials is less sensitive to flood soaking than the flexural strength. This may be because the compressive strength is more dependent on the density of the material, and short-term soaking has less impact on the overall density than on the interfacial bonding (which affects flexural strength).

[0152] The group that recovered from 7 days of soaking to 63 days of recovery: The height of the bar corresponds to approximately 55 MPa on the ordinate, which is about 37.5% higher than the group that was soaked for 7 days. However, it is still 15.4% lower than the group that was not soaked. This indicates that the recovery period has a better effect on the repair of the compressive strength of cement than on the flexural strength. This may be because the repair of compressive strength is easier to achieve through subsequent hydration reactions to supplement the cementitious materials. However, it is still impossible to fully recover to the baseline level, which confirms that flood soaking has an impact on the mechanical properties of cement materials.

[0153] 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 detecting the growth of Aspergillus fumigatus in cement before and after flood immersion, characterized in that, Includes the following steps: Step S1: Culture of Aspergillus fumigatus samples: Aspergillus fumigatus was cultured using potato dextrose agar medium; Step S2: Cement blocks are poured, cured, and experimental groups are formed; Step S3: Transfer the young Aspergillus fumigatus mold blocks to the surface of the cement block; Step S4: Stain the bacterial spots on the surface of the cement block with lactic acid cotton blue dye solution; Step S5: Observe under a microscope whether Aspergillus fumigatus hyphae are produced on the stained area and observe the length of the Aspergillus fumigatus hyphae for qualitative analysis; Step S6: Quantify the density of Aspergillus fumigatus spores using a gradient dilution method, and perform quantitative analysis using image processing techniques; Step S7: Detect the changes in flexural strength and compressive strength of the cement block using a pressure testing machine.

2. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 1, characterized in that, Step S1 includes the following steps: Step S11: Under aseptic conditions, the glycerol mixture containing Aspergillus fumigatus spores is evenly spread on the surface of potato dextrose agar medium that has been sterilized at high temperature. Step S12: Place the culture medium in a constant temperature microbial incubator and incubate it at a constant temperature for a period of time.

3. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 1, characterized in that, Step S2 includes the following steps: Step S21: Prepare silicate cement that meets the specifications; pour the prepared cement slurry into the mold; Step S22: Remove the cement from the mold after it has been cured for a certain period of time; Step S23: Divide the cement block into two parts, and perform a different bacterial transfer operation on each part; Step S24: Divide each part into three different groups.

4. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 3, characterized in that, The specific steps of dividing each part into three different groups are as follows: Step S241: Divide the cement from the first part into three different groups according to the soaking time of the blocks; The first group of cement blocks was not soaked in water; The second group of cement blocks were soaked in water for a short time; The third group of cement blocks were soaked in water for a long time; Step S242: Each group of cement blocks is divided into an observation group, a control group, and a test group; The observation group consisted of cement blocks inoculated with Aspergillus fumigatus var. mongolica culture medium. The control group consisted of cement blocks inoculated on the surface of Aspergillus fumigatus blocks that had the same area as the Aspergillus fumigatus blocks but did not contain culture medium. The test set consists of cement blocks used for testing compressive and flexural strength. Step S243: Divide the second part into three different groups according to the soaking time; The first group of cement blocks were not soaked in water and were allowed to recover for a period of time. The second group of cement blocks were soaked in water for a short time, and then allowed to recover for a period of time. The third group of cement blocks were soaked in water for a long time and then allowed to recover for a period of time. Step S244: Each group of cement blocks is divided into an observation group, a control group, and a test group; The observation group consisted of cement blocks inoculated with Aspergillus fumigatus var. mongolica culture medium. The control group consisted of cement blocks inoculated on the surface of Aspergillus fumigatus blocks that had the same area as the Aspergillus fumigatus blocks but did not contain culture medium. The test set consists of cement blocks used for testing compressive and flexural strength.

5. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 4, characterized in that, Step S3 includes the following steps: Step S31: Using a professional inoculation spatula and inoculation loop that have been sterilized at high temperature, cut out pieces of Aspergillus fumigatus of uniform size from potato dextrose agar medium and inoculate them on the surface of cement blocks in different observation and control groups in step S242. Step S32: Using a professional inoculation shovel and inoculation loop that have been sterilized at high temperature, cut out pieces of Aspergillus fumigatus of uniform size from potato dextrose agar medium and inoculate them on the surface of cement blocks in different observation and control groups in step S244. Step S33: Place all cement blocks in a constant temperature microbial incubator and maintain a constant temperature for incubation for a period of time.

6. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 1, characterized in that, Step S4 includes the following steps: Step S41: Remove the cultured cement block; Step S42: Perform high-temperature inactivation; Step S43: Apply lactophenol cotton blue dye solution to the surfaces of the observation group and control group, which have been sterilized at high temperature, for staining.

7. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 1, characterized in that, Step S6 includes the following steps: Step S61: Using a dropper that has been sterilized at high temperature and pre-weighed to its initial mass, randomly and evenly collect several sample points at the inoculation sites on the surface of the cement block, and weigh them. Step S62: Use sterile water to perform serial dilution of the extracted Aspergillus fumigatus spores to achieve a certain dilution factor; Step S63: Take the diluted solution and add it to a glass slide, stain it with lactophenol cotton blue dye solution, and then observe it under a microscope; Step S64: Randomly and uniformly select several observation points for shooting; analyze these images to quantitatively assess the colony growth on the cement block surface based on spore density.

8. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 1, characterized in that, Step S7 includes the following steps: Step S71: Perform flexural strength tests on the test group and record the results; Step S72: Collect and clean the cement blocks that were broken by the compression testing machine after the flexural strength test; Step S73: Perform compressive strength tests on the test group and record the results.

9. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 3, characterized in that, The specific steps of dividing the cement block into two parts are as follows: The first part involves soaking the bacteria in water for a period of time before directly transferring the inoculum; the second part involves soaking the bacteria in water for a period of time before allowing it to recover and then transferring the inoculum.

10. The method for detecting the growth of Aspergillus fumigatus in cement before and after flood immersion according to claim 8, characterized in that: The test group of cement blocks consisted of 3 rectangular cement blocks, each measuring 40*40*160mm. The size of the observation group and the control group was a cylindrical cement block with a diameter of 60 mm and a thickness of 12 mm.

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