Chapter VI - Med Biol - Course Content to Biotechnology 2023-2024 PDF

Part II Biotechnology Exercise 6 Topic: Biotechnology of food- impact on human health. The significance of microbiota for the proper functioning of the body. Parasitological and mycological diagnostics – classical methods. Functional food - food for which, apart from the nutritional effect, other be...

Part II Biotechnology Exercise 6 Topic: Biotechnology of food- impact on human health. The significance of microbiota for the proper functioning of the body. Parasitological and mycological diagnostics – classical methods. Functional food - food for which, apart from the nutritional effect, other beneficial effects on one or several functions of the organism/body have been proved , and its pro-health effect should be documented by scientific research (FUFOSE Functional Food Science in Europe research program). Metabolome - all products of the metabolism of microorganisms included in the microbiota. Metagenome - genetic material of all microorganisms included in the microbiota (genophor + plasmids). Microbiome - The collective genomes of all microorganisms symbiotic and pathogenic (with their metabolic products), inhabiting a particular environment and in the context of human health, especially the human body. Microbiota - all microorganisms, including bacteria, yeasts, and viruses, coexist in particulat environment, in the context of human health, existing in various sites of the human body (gut, skin, lung, oral cavity). Microbiota-gut-brain axis - a bi-directional communication pathway connecting the intestine and intestinal bacterial biota to the CNS (central nervous system), including neural, endocrine and immunological mechanisms. Prebiotic - selectively fermented ingredient that allows specific changes in the composition and/or activity of gastrointestinal microorganisms with beneficial effects on the health and well-being of the host; nutrients that have a beneficial effect on host health due to the modulation of the intestinal microorganism (WHO 2007). Ontocenosis - all microorganisms that inhabit a specific niche within the macroorganism. Ontohabitat - a macroorganism or a fragment that is the seat of microorganisms. Probiotic - live microorganisms that, when consumed by humans or animals, have a beneficial effect on health through quantitative and qualitative effects on the microbiota of the digestive tract and/or modification of the immune system. Axenic strain - pure (uncontaminated e.g. with bacteria) culture of the microorganism consisting of a population of cells derived from one cell, having identical genetic material and having the same biochemical characteristics. Colony - visible to the naked eye culture of the microorganism on a solid medium derived from one cell. Isolate - pure culture/culture of the fungus obtained from biological material collected from the patient. Macroculture - culture of a microorganism carried on a solid medium in a petri dish or slant in a test tube, or on liquid medium in the tube. Microbiological medium (growth/culture medium) - a solid, liquid or semi-solid product used in microbiology, containing all required nutrients to support the growth of microorganisms. Slide microculture - microorganism culture carried out on primary slides in moist chambers. The growth of bacteria/fungi is assessed microscopically. Biotechnology of food - impact on human health Biotechnology is a scientific branch that connects biology with technology; it is the application of biological techniques to product research and development. Biotechnology can be defined as an interdisciplinary field that focuses on the practical applications of biology (including microbiology, cell biology, genetics, ecology), chemistry with biochemistry, physics, mathematics and computer science, economics and medicine. Depending on the origin of organisms used in biotechnological processes, we can distinguish: - classical biotechnology - uses organisms/microorganisms (selected strains) or their products of natural origin; - molecular biotechnology - uses organisms/microorganisms or their products through genetic engineering as a result of modification of their genetic material. Contemporary biotechnology provides products and technologies to protect human and animal health, reduce anthropogenic impact on the environment, agriculture, energy industry and efficient technological processes for industry. Due to the range of use, biotechnology is divided into several areas, currently marked with colours (Table 1). Table 1. Areas of biotechnology. Colours of biotechnology Red White Grey Green Yellow Blue Brown Gold Violet Black/dark Area Product example medical and pharmaceutical biotechnology related to health care, molecular and serological diagnostics, drug searching, vaccine and antibiotic production, searching for new therapies industrial biotechnology focuses on the production of artificial biodegradable plastics, processing of natural resources, production of biofuels, production of enzymes, development of new production processes environmental biotechnology focuses on maintaining biodiversity as well as removing environmental contaminants using biological processes human insulin (microorganisms); blood coagulation factor (transgenic sheep) "smart" polymers; biofuels agricultural biotechnology concerns agricultural production, food production, creation of transgenic plants (GMOs), selective cultures, in vitro cultures feed and food biotechnology deals with the production and improvement of food (fermentation processes, etc.), the creation of functional food biotechnology of seas and oceans which concerns the exploitation of marine environments for industrial purposes, searching for new materials biotechnology of dry and desert areas deals with management in arid regions, creation of crop plants resistant to lack of water, water treatment bioinformatics and computer technology deals with the creation of computer systems, creation of bioinformatic databases, sequencing and nanobiotechnology biotechnology law concerns patents, ethical principles in biotechnology, problems of philosophical biotechnology bioterrorism biotechnology deals with the topic of biological weapons and protection against bioterrorism sewage treatment plants - filters from activated sludge; bioremediation using microorganisms "golden rice" - producing the precursor of vitamin A wine, beer, cheese; hypoallergenic rice; probiotics hydrocolloids used in medicine and food technology hydroponic crops (use of salt water) online databases; starter search engine The Act on Genetically Modified Organisms Cartagena Protocol on Biological Safety Some biotechnology activities include the creation of transgenic organisms or stem cell therapies which encounter opposition from the public due to the religious and legal-ethical aspects. Therefore, individual countries are introducing numerous laws regarding the possibilities of using genetic engineering. The significance of microbiota for the proper functioning of the body Microbiota can be defined as a group of microorganisms that typically inhabit a particular environment, such as the soil, a body of water, or a site on/ or in an macroorganism. In the context of human health, this term is used to describe all microorganisms, including bacteria, yeasts, and viruses, coexist in various sites of the human body (gut, skin, lung, oral cavity). The human body is believed to be occupied by around 4×1013 microorganism cells, which is ten times the number of human body cells. Depending on the location, between 104 and 1011 cells are found per 1ml of content of the digestive tract, about 107 are found per 1 cm2 on the skin. The microorganisms inhabiting the human body may include bacteria (the most numerous group: 10-100 billion cells), Archeobacteria, viruses - including bacteriophages and giant viruses and fungi which constitute 0.1% and play an essential role in maintaining microbial balance. For this reason, the human body is treated as a unique ecosystem inhabited by numerous microorganisms. All microorganisms and their genetic material and metabolism products are referred to as the microbiome. The genomes of all microorganisms (composed of a genofore and plasmids) in the human body consist of ~ 3.3 million genes, with up to 80-90% differing in individuals, while the human genome contains only ~ 22,000 genes (in 99.9% identical to all people). Natural microbiota maintains many functions in the human body:  competition for pathogens;  stimulation of the immune system:  invasiveness;  impediness;  stimulation of intestinal epidermal cells proliferation;  digestion of carbohydrates (e.g. lactose, dietary fibre), proteins (e.g. casein), fatty acids (e.g. cholesterol);  vitamin synthesis (e.g. group B, K);  absorption of minerals;  regulation of gut motility;  breaking down of toxins and mutagens;  pH regulation. The human microbiota is highly dynamic and changes throughout life. Mature microbiota (especially the gut microbiota) is formed around the age of two years. During this period, it resembles the composition of the microbiota of adults. At every stage of individual life, the structure and size of microbiota are influenced by environmental factors such as lifestyle, diet, feeding in infancy, nutrients in children, malnutrition, use of antibiotics in the treatment of infectious diseases or physiological state of the body. The early establishment of microbiota is affected by several factors such as delivery mode (caesarean delivery vs vaginal delivery), breast milk vs formula feeding, antibiotic usage, and timing of the introduction of solid foods and cessation of milk feeding. The microbiome of the digestive tract Gastrointestinal microbiota accounting for the majority of the biomass of microorganisms is the best explored. The number of microbial cells varies in individual sections of the gastrointestinal tract. There are 10 1 cells/g in the stomach, including Helicobacter pylori, Firmicutes (Lactobacillus, Streptococcus), Proteobacteria (Escherichia) and fungi (Candida). The small intestine contains 103-107 cells/g, mainly Lactobacillus, Streptococcus, Enterococcus and Enterobacteriaceae. In contrast, the large intestine is colonized by 1012-1014 cells/g, including as many as 50 clusters of bacteria, with Firmicutes (Lactobacillus, Clostridium, Streptococcus) and Bacteroidetes (Bacteroides thetaiotaomicron) as those predominating ones. The microbiome of the intestine can be divided into two sets of microorganisms: - a stable assembly constituting 30% of the cells - the primary set that occurs in most people; - a variable assembly of 70% of all cells - depends on nutrition, age, health, environmental conditions, consumption of medicines, and diseases. The composition and distribution of bacteria in the following sections of the gastrointestinal tract are determined by several factors, including: - host-dependent factors (pH, immunoglobulins, bile salts, enzymes, intestinal motility, mucins, tissue secretions); - microbiological factors (adhesion, mobility, ability to absorb carbon from various compounds, spores, enzymes, antibacterial compounds, generation time); - interactions between microorganisms - protectionist/antagonistic interactions (cooperation in the use of various carbon sources, competition), growth factors and vitamin synthesis, short-chain fatty acids, amines, changes in electrochemical potential Eh, pH, oxygen concentration, antibacterial compounds, siderophores (carriers of iron ions), nutritional requirements; - diet (composition, non-digestible cellulose fibres, medicines, etc.). Taking into account the diversity of microorganisms, three enterotypes of the intestinal microbiota are distinguished in the human population: - enterotype 1 - with a predominance of Bacteroides, - enterotype 2 - with a predominance of Prevotella, - enterotype 3 - with the predominance of Ruminococcus - the most frequently recorded one. Research conducted within the framework of the Human Microbiome Project (an international research program aimed at understanding the relationship between humans and microbiota) revealed a difference in the composition of the intestinal microbiome in different parts of the world: in Europe, dominate Firmicutes, in Africa - Bacteroidetes. Disturbances of natural microbiota Dysbiosis is a condition in which an imbalance in the microbiota occurs that promotes changes in the normal physiological activities of the human organism, possibly resulting in health problems. Any imbalance in the number and composition of the microbiota, referred to as dysbiosis, causes changes in the normal functioning of the human body. Dysbiosis most often concerns intestinal microbiota, which is particularly exposed to the numerous toxic components consumed by man. The most important in the regulation of homeostasis is the ontocenosis of the intestine. Disturbances in the quantitative and qualitative composition of intestinal microbiota may contribute to the development of numerous metabolic, organ, oncologic, immune, nervous and infectious diseases (Table 2). Table 2. Changes in microbiota structure in different diseases. Disease obesity diabetes type 1 (children) diabetes type 2 colorectal cancer Changes in microbiota structure Bacteroides ↓ Firmicutes ↑ - Lactobacillus sp., Enterococcus sp., Peptostreptococcus sp. - polisaccharides fermentation, increased assimilation of sugars and fatty acids Proteobacteria ↑ Lactobacillus sp. ↓ Bifidobacterium sp. ↓ Prevotella sp. ↓ Clostridium sp. ↑ Bacteroides sp. ↑ Clostridium sp. ↓ Firmicutes ↓ β-Proteobacteria ↑ Prevotella sp. ↓ Ruminococcus spp. ↓ Pseudobutyrivibrio ruminis ↓ Phascolarctobacterium sp. ↑ Citrobacter farmer ↑ Escherichia coli – some strains produce colicin (inhibit proliferation of tumor cell) The following factors can have a negative impact on the microbiota status: - antibiotics, - laxatives, - dysfunction of the gallbladder, - sugar and fats, - industrial preservatives and toxins, - a diet that causes constipation, - fungal and bacterial infections of the intestines, - contraceptives, - stomach acid deficiency, - high level of stress. The influence of diet on the composition and number of microbiota was also demonstrated. In people eating a high-carbohydrate diet, a large proportion of Prevotella, Methanobrevibacter, and fungi of the genera Candida and Saccharomyces were found. A high proportion of Candida was observed in high-protein and high-fat diets. In contrast, people having a low-carbohydrate diet had microbiota with a greater proportion of Firmicutes (Lactobacillus). A balanced diet is essential for maintaining the correct/beneficial state of microbiota. Interactions of natural microbiota with the central nervous system The microbiota-gut–brain axis is the biochemical signalling pathway between the intestinal microbiota, the gastrointestinal tract (GI tract) and the central nervous system (CNS). Gut microorganisms produce neurotransmitters which send signals to CNS via the vagus nerve. Immune cells, stimulated by gut microbiota, produce cytokines, which are transported via the circulatory system throughout the whole body into CNS. Metabolites of gut microorganisms (especially short fatty acids) stimulate epithelial cells of the intestine to produce neurotransmitters, activate the vagus nerve or are directly transported with blood to CNS. On the other hand, neuronal and biochemical signals from the CNS may result in a change in the structure and abundance of the intestinal microbiota. The relationship between microbiota and the secretion of neurotransmitters, such as serotonin and γ-aminobutyric acid, is extremely important. Serotonin improves mood, concentration and learning ability, and regulating appetite, sleep and muscle tension; 80-90% of serotonin is produced by enterochromaffin cells (EC) in intestine. EC is stimulated by 20 species of gut spore-forming bacteria. γ-aminobutyric acid (GABA) regulates and reduces pain in CNS and inhibits the inflammatory response. GABA can be produced by Bifidobacterium dentium, while Lactobacillus sp. and Bifidobacterium sp. increase GABA activity. Mechanisms of interaction of microbiota upon the CNS:  prevention of increased permeability of the intestinal barrier to bacterial LPS by microbiota; and inhibition of the activation of inflammation;  reduction of levels of inflammatory cytokines;  increasing the tryptophan content - the precursor of serotonin;  reduction of cortisol content;  production of numerous neurotransmitters and neurometabolites;  change the amount of GABA receptors in the CNS. Numerous quantitative and qualitative disturbances concerning natural microbiota were found in the course of diseases of the nervous system (Table 3). Table 3. Changes in microbiota structure in CNS diseases. Disease Autism/Asperger’s paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS) Toxoplasma gondii infection Symptoms difficulties with social interaction and communication, restricted and repetitive behavior aggression fears personality changes changes in behaviour increased courage suicidal risk schizophrenia Change in microbiota Lactobacillus ↑↑ Streptococcus ↓ yeast (Candida) Bacteroidetes (mostly Bacteroides, Odoribacter i Oscillospira) ↑ In animal model: Enterobacteria ↑ Prevotella ↑ Clostridium ↓ Firmicutes ↓ Modifications of microbiota Currently, various methods of microbiota modification are used in the treatment of human diseases, among others faecal microbiota transplantation (FMT). The FMT procedure involves transferring a healthy donor's faeces to a recipient's digestive tract, mainly in oral capsules. However, the faecal content can also be administered to the duodenum using the endoscope. FMT is widely used to treat Clostridium difficile (C. difficile) infections with a global cure rate for C. difficile of over 95% with a single or double FMT infusion versus a 20% cure with vancomycin. FMT has been also used to treat patients with ulcerative colitis (UC) unresponsive to the standard therapy and has proved to be successful in helping up to 80% of the most difficult cases of irritable bowel syndrome (IBS). Faecal transplants originated in ancient Chinese medicine more than 1,700 years ago. In the past, this procedure involved drinking a liquid suspension of another person's faeces — a highly risky technique. Today's faecal transplants are sterile and safe, and there is an increasing number of studies to support their use. Probiotic, prebiotic and functional food Probiotics are defined as live microorganisms consumed by humans or animals and have a beneficial effect on health through quantitative and qualitative effects on the intestinal microflora and/or immune system modification (WHO 2004). Probiotic microorganisms are i. e. Lactobacillus sp., Bifidobacterium sp., Saccharomyces boulardii. The pro-health effects of probiotics on the human body include: - a beneficial modification of the intestinal microbiota, - improving the digestion of lactose (reducing the symptoms of lactose intolerance), - reducing the risk of obesity, - lowering cholesterol, - activation of specific and nonspecific immune responses (Figure 1), - preventing constipation, - shortening the duration of rotavirus diarrhoea as well as diarrhoea of other aetiology, - increasing the absorption of nutrients and minerals, - decreasing the activity of faecal enzymes, - protecting against colon cancer, - carcinogens detoxification, - inhibiting the growth and translocation of pathogens, reducing the risk of intestinal infection. ↑ cytokines Th1, IFNγ, IL6, TNF ↑Number and function of T regulatory lymphocytes, IL10, TGFβ ↑IgG, ↓IgE ↑membrane IgA probiotic ↑Barrier epithelial, protein expression, ↓ bacterial translocation ↓cytokines Th2, IL4, IL5, IL1 ↑Inflammatory factors, SCFA, ↓ inflammatory infiltrates ↑Pathogen removal, bacteriocin production, ↓adhesion to mucous membrane and epithelium. Figure 1. Immunomodulation function of probiotics. Prebiotics are selectively fermented ingredients that allow for specific changes in the composition and / or activity of micro-organisms in the gastrointestinal tract with beneficial effects on the health and well-being of the host (2004); food substances that are undigested by humans but that stimulate beneficial microbial activities in the gut (WHO 2007). Probiotics are inulin, cellulose, oligofructose, etc. Numerous studies are underway to find new substances that could be used as prebiotics. They should meet certain criteria, i.e. they may not be digested by endogenous host enzymes, need to be a selective substrate for one or a limited number of commensal colonies living in the colon, and the products of their breakdown by intestinal bacteria should lower the pH of the digestive content. Prebiotics should simultaneously stimulate the growth and activity of healthpromoting gastrointestinal microbiota and induce host-beneficial local effects in the lumen of the digestive tract or systemic effects. The basic feature of prebiotics must be known and documented in chemical structure and the ease of obtaining them on an industrial scale. Synbiotic is a nutritional supplement composed of prebiotic and probiotic ingredients, with potential immunomodulating and gastrointestinal (GI) biota-restoring activity. The prebiotics and probiotics work synergistically in the GI tract, thereby modulating the GI microbiota ecosystem and may improve the functions of the intestinal barrier. In addition, synbiotics may have a beneficial effect on the immune system. It has been proven that using synbiotics increases the activity of probiotic bacteria and more effectively regulates the composition of natural intestinal microbiota. The prebiotic component positively regulates the activity of the intestinal microbiota and a given probiotic strain, while the probiotic strain stimulates the commensal microorganisms. Functional foods can be defined as dietary items that, apart from providing nutrients and energy, beneficially modulate one or more targeted functions in the body by enhancing a certain physiological response and/or by reducing the risk of disease. The types of functional foods due to product types are presented in Table 4. Table 4. Types of functional food. Functional food/product type fortified enriched altered non-altered enhanced Definition Example a food fortified with additional nutrients a food with added new nutrients or components not normally found in a particular food a food from which a deleterious component has been removed, reduced or replaced with another beneficial substance foods naturally containing increased content of nutrients or components a food in which one of the component has been naturally enhanced through special growing conditions, new feed composition, genetic manipulation etc. fruit juices fortified with vitamin C margarine with plant sterol ester, probiotics or prebiotics fibres as fat releasers in meat or ice cream products natural foods eggs with increased omega-3 content obtained by altered chicken feed The most common products of this type include fermented dairy products or containing probiotic bacteria, spreads containing esters of phytosterols and phytostanols, beverages enriched with vitamins A, C and E or calcium and magnesium, beef enriched with conjugated linoleic acid (CLA), eggs enriched with polyunsaturated fatty acids from the n3 family. In addition, functional foods include numerous vegetable products. Selected products included in functional food are presented in Table 5. Table 5. Examples of functional food and their beneficial effect to human health. Product whole-grain products oatmeal grape juice green or black tea fatty fish soy garlic spinach, greens nuts kale, Key components fiber, lignans, phytoestrogens β-glucan, soluble fiber phenols tannins, catechins omega-3 fatty acids phytoestrogens, protein collard organic sulphur compounds lutein, zeaxanthin Potential benefits reduction of the risk of cancer and heart diseases reduction of cholesterol level improvement of cardiovascular system activity reduction of the risk of cancer reduction of the risk of heart diseases reduction of the risk of cancer and heart diseases and symptoms of menopause reduction of the risk of cancer and heart diseases reduction the risk of age-related blindness monosaturated fatty acids reduction the risk of heart diseases Parasitological and mycological diagnostics – classical methods Diagnostic algorithm Methods used in the diagnosis of fungal infections and parasitic invasions can be divided into classical (techniques based on microscopic diagnostics and cultures) as well as serological and molecular ones. In routine diagnostics, results of several simultaneously applied methods should be taken into account (Figure 2). The first, preliminary stage for the identification of the microorganism is proper collection of biological material from the patient. Biological material Microscopic slide Direct preparation (direct microscopic slide) Culture (liquid ad solid media) Serological tests Molecular methods Macroculture Microculture Biochemical tests Molecular methods Identification Drug susceptibility Therapy Figure 2. Diagram of a diagnostic algorithm in medical mycology. I. Collection of biological material The diagnostic material should be taken from the affected site or a spot around it, at the right time and in the appropriate quantity/volume. The material must be taken aseptically (sterile). All collected diagnostic materials must be immediately delivered to the microbiological laboratory. Adequate conditions for storage and transport of a diagnostic material should be provided (proper temperature, transport media). The types of biological materials collected are included in Table 6. Table 6. Types of biological materials collected for mycological diagnostic. Infection site/location Systems and Skin organs Genitourinary system Digestive tract Central nervous system Respiratory tract General testing for fungal infections Hearing organ Eyeball Hematopoietic system - bone marrow Systemic mycoses Organ mycoses Biological material type skin scales nail scraps skin scrapings affected hair urine the urethra swabs catheter swabs in catheterized patients glans penis swabs vagina swabs stool gastrointestinal content bile cerebrospinal fluid Diseases of the lungs and bronchi: sputum nose, throat and larynx swabs bronchoalveolar lavage Laryngological diseases: secretions from the paranasal sinuses lavage from the mouth tongue, gums, palate swabs external auditory canal swabs and scrapings conjunctival sac and cornea swabs bone marrow samples collected by biopsy blood Histopathological examination: obtained by biopsy punctuates, fragments of tissue, fluids from the chest, abdomen, joint, ulcers tissue sections Serological tests: blood serum urine cerebrospinal fluid II. Analysis of the presence of microorganisms - Classical methods 1. Direct preparations/microscopic slides In mycology, non-stained or stained preparations/microscopic slides are often prepared directly from material taken from the patient. They allow to identify the presence of fungal cells in the body, determine the type of fungi [yeast (Y) or mould (M)], in the case of yeast they allow to determine the development phase (Y or M), determine the extent of hyphye or blastospores adherence to epithelial cells and assess the ratio of fungi to bacteria. At the same time, they facilitate the proper selection of the growing medium, shorten the diagnosis time and allow to detect the aetiological factor of fungal infections. In the case of evaluation of microscopic slides made from fungal cultures (mainly mould and dermatophytes), it is also possible to identify the etiological factor. The characteristics of fungi used in microscopic identification include: shape and structure of hyphae/pseudocysts, morphology of spore-forming structures and type of sporulation, as well as types of produced asexual and sex spores. Microscopic preparations/slides are performed by several techniques (Figure 3). unstained nonsustaind stained sterile water  sól methylene blue staining Lugol staining Microcopic slides overexposed DMSO KOH  l fixed, stained india ink staining Gram staining Giemsa staining histopathological slides In the diagnosis of parasitic invasions, it is extremely important to directly detect the developmental form of the parasite in the body. In direct preparations of semen, vaginal content or concentrated urine Trichomonas vaginalis can be detected. Developmental forms of gastrointestinal parasites are found in direct slides from faeces, stained with Lugol liquid, overexposed with KOH or stained with malachite green. In the case of protozoa, the diagnosis and identification is based on the presence of the parasite (trofozoite, cyst, oocyst), while in the case of helminths (flukes, tapeworms, roundworms) it is possible to find the presence of eggs in the patient's stool (typical of the genus / species), and additionally in the case of tapeworms - proglodites. Concentration methods are commonly used such as flotation and sedimentation. Blood parasites are detected in peripheral blood smears most frequently stained by the Giemsa method. Some tissue/organ parasites can also be found in histological preparations. Figure 3. Types of microscopic preparations/slides. A B C D E F Photo 1. Microscopic slides used in mycological and parasitological diagnostics: A. Gram staining - visible blastospores and pseudo-hyphae of Candida parapsilosis (magnification: 1000x, photo: K. Góralska); B. india ink staining Cryptococcus neoformans cells visible in the smear of cerebrospinal fluid, surrounded by a polysaccharide capsule (magnification: 400x, photo by K. Góralska); C. aniline blue in lactofenol staining - visible hyphae of mould fungi (magnification: 400x, photo K. Góralska); D. hematoxylin and eosin staining - visible furcocercariae Schistosoma mansonii (magnification: 100x, photo K. Góralska); E. trichrome staining - visible Sarcoptes scabiei in skin section (magnification: 400x, photo: M. Dzikowiec); F. overexpressed with lactophenol - visible Demodex folliculorum on eyelashes (over 100x, photo by M. Dzikowiec). 2. Cultures Cultures are still the basis of mycological diagnostics in most laboratories. A culture of fungi indicates their presence in the patient's body and allows to obtain the right amount of material to carry out the identification by microscopic or biochemical methods. Due to their functions media used in medical mycology can be divided into: - multiplying: Sabouraud agar - most commonly used for the detection and multiplying of yeast (Candida, Saccharomyces), often with the addition of gentamicin and chloramphenicol which inhibit bacterial growth; - selective and multiplying: Czapek-Dox agar - used in the detection of mould fungi; - selectively-differentiating: Chrom-agar Candida (chromogenic medium) - used for the differentiation of species of the genus Candida (particular species grown in different colours); - special: Nickerson's medium (identification) - used for yeast microorganisms, fungi are identified on the basis of their characteristic growth and type of spores (chlamydospores) e.g. Candida albicans Cultures are rarely used in suspected parasitic invasions. The most commonly used substrates for the propagation of protozoa are among others Pavlov or Simica medium used to detect Entamoeba gingivalis, Trichmonas tenax and Trichomonas vaginalis (gold standard). 3. Biochemical methods Biochemical methods are mainly used in mycological diagnostics. For biochemical diagnostics it is necessary to first isolate the axenic strain of fungi from the biological material on the mycological medium. Identification of the microorganism based on biochemical properties uses differences in metabolism based on: - oxygen requirements, - fermentation properties, - the ability to assimilate carbon compounds, nitrogen etc., - hydrogen sulphide production, - the production of various enzymes, e.g. catalase, urease, - osmotic requirements, - absorption of vitamins, - distribution of fats, nucleic acids, - production of primary and secondary metabolites (toxins, pigments, amino acids). Currently, numerous commercial biochemical diagnostic tests are available; they are used in most laboratories (Photo 2), e.g. - API 20 C AUX - for yeast (determination of yeast isolate to species / strain), - Candida API - for fungi of the genus Candida, - ID32 - for yeast fungi, - Zym API - for yeast, evaluation of the ability to produce enzymes (virulence evaluation). A B Photo 2. Biochemical test: A. API 20 C AUX - identification, B. API-Zym - enzymatic activity (photo K. Góralska). References: 1 Campbell, J.B. Reece: Biology. Pearson, Benjamin Cummings, Seventh Edition 2005