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Pátráme po mikrobech Část XV.a Opakování v angličtině Ondřej Zahradníček K praktickým cvičením z VLLM0421c [email protected] (Z technických důvodů neobsahuje téma Mykologie a Biofilm) Pozor! Téma se vůbec neprobírá v prezenční výuce. Jeho absolvování se však kontroluje ve 14. praktiku. Upozornění • Toto je v podstatě opakovací prezentace pro zahraniční studenty (totéž mají ve své částí studijních materiálů) • Prostudování této prezentace není povinné a není ani předmětem kontrolních otázek. • Lze ho však vřele doporučit. A microbe (microogranism): what does it mean? • It should be living. and grain of dust is not and microbe, although it is microscopical • It should be microscopical. and giraffe is not and microbe, although it is living The second condition is not absolute. For example, and tapeworm can measure 10 m. But the eggs are microscopical, so it belongs to the microbiology. Main medically important microbes • Viruses (and prions) • Bacteria (e. g. and Streptococcus or an Escherichia) • Fungi (yeasts and molds) • Parasites – not all of them are microbes: – Inner parasites • Protozoa (e. g. Plasmodium malariae) • Flukes (e. g. Schistosoma haematobium) • Roundworms (e. g. Ascaris lumbricoides) • Tapeworms (e. g. Taenia saginata) – Outer parasites (lice, fleas, bugs) Survey of methods • Direct methods: We search for a microbe, its part or its product (e. g. a bacterial toxin) – Direct detection in specimen – we use the whole specimen (blood, urine, CSF etc.) – Strain identification – isolate determination • Indirect methods: We search for antibodies. An antibody is neither a part nor a product of a microbe – it is a macroorganism product, after being challenged by a microbe Survey of direct methods Method Microscopy Specimen Identification examination yes yes Cultivation yes yes Biochemical identificat. no yes Antigen detection yes yes Animal experiment yes usually not Molecular methods yes usually not* *but in molecular epidemiology – detection of simillarity of strains - yes Microscopy Microscopy • We observe microbes, in specimen also cells of host organism (epitheliae, WBCs etc.) • Wet mount – for large and/or motile microbes (parasites, fungi, motile bacteria) • Dark field wet mount (mainly spirochets) • Fixated and stained preparations – Gram staining, Giemsa staining, Ziehl Neelsen staining (use for various groups of bacterií, fungi, parasites) • Electron microscopy – in viruses; rather for research than for common virological diagnostics Microscopy of a specimen Microscopy of a strain Photo O. Zahradníček Main microscipical methods in medical microbiology Drying and Coverslip fixation Imersion system Wet mount no yes no Darkfield wet mount no yes yes yes no yes Stained preparat. Preparing a microscopical preparation • We make a smear of a swab made by a cotton swab (in stained preparations only) • Liquid specimen are dropped on a slide • If we have a strain, we make a drop of physiological saline onto the slide. We sterilize a microbiological loop in flame and after drying we take a little of bacterial mass. We mix it in a drop of saline. Wet mount – procedure An example of a wet mount C. A. T. http://www.kcom.edu/faculty/chamberlain/Website/lectures/lecture/image/clue3.jpg Simple staining The result may look like this (yeasts): http://biology.clc.uc.edu/fankhauser/ Labs/Microbiology/Yeast_Plate_Count /09_Yeast_Meth_Blue_P7201177.jP72 01179.jpg Gram stained preparation Photo: Helena Janochová and Zuzana Jurčíková Bacterial cell wall • There are bacteria, that are mechanically strong, their cell wall is thick and simple. They are called Gram-positive bacteria. • There are other bacteria, that are rather chemically strong, their cell wall is rather thin, but more complex. They are called Gram-negative bacteria. • Besides these and those, there are also so named Gram non-staining bacteria. Gram-positive cell wall Gram-negative cell-wall Gram staining – principle •Gram-positive bacteria have thick peptidoglycan layer in the cell wall. So, gentiane/crystallin violet binds more firmly to them, and after confirmation of this bound by Lugol solution even alcohol is not able to decolorize them. Gram-negative bacterie are decolorized by alcohol and thed stained red by safranin. Chemical Gram-positive Gram-negative Crystal. violet Staining violet Staining violet Lugol iodine Confirmation Less confirm. Alkohol Not decolorized Decolorized Safranin Remain violet Stain to red Mixture of gram-positive and gram-negative bacteria G+ Photo: Helena Janochová and Zuzana Jurčíková G– Culture Do matter the conditions for bacterial growth? Of course yes! Majority of bacteria need their temperature, moisture, salts concetration and many other characteristics to be in a quite narrow range. lower survival lower growth limit (bactericidal) limit (inhibitory) lower growth upper survival limit (inhibitory) limit (bactericidal) Values, that enable microbial survival, are not sufficient. They should be able to multiply. Various microbes need various conditions! Medically important bacteria • Temperature usually needed around 37 °C – but bird pathogens more (42 °C), microbes coming from outside less (30 °C) • Value of pH needed around pH 7 – but gastric helicobacter by far less • NaCl concentration needed around 0.9 % (physiological saline) – but staphylococci, that have to be able to multiply on sweated skin, multiplies even at 10 % of salt! In practice part of parameters (e. g. temperature) is derived from thermostat settings, and remainder (e. g. NaCl concentrations) by composition of the culture medium. Culture thermostat Besides box thermostats, like this one, our Institute has a chamber thermostat, too. It is a whole room with 37 °C. Majority of bacteria is cultured in a thermostat overnight, so about 24 h. Photo O. Z. Relation of bacteria to oxygen • Aerobic and facultative anaerobic (eventually aerotolerant) bacteria can be grown at normal athmosphere • Strictly anaerobic bacteria need athmosphere without oxygen • bacteria with special need for oxygen require special athmosphere (microaerophile and capnophile bacteria) Why we culture bacteria • Why bacteria are cultured in the laboratory? – To keep them living and to multiply them. This is gained by cultivation in both liquid and solid media (jelly-consistence media, based on agar algae) – To obtain a strain – solid media only – To differentiate and divide them mutually – diagnostic and selective media are used, for identification Specimen and strain • Specimen is taken from a patient. Specimen contains cells macroorganism, various number of microbial species (zero to maybe twenty) and more items • A strain – an isolate – is a population of one bacteria, isolated from a specimen on a solid medium • To gain a strain, we have to grow a bacterium on a solid medium and inoculate carefully Term „colony“ www.medmicro.info • A colony is a formation on a surface of a solid media. It is developped from one cell or a small group (couple, chain, cluster) • In some cases number of colonies on an agar shows us number of microbes in the specimen – or more preciselly, number of „colony forming units“ (CFU) • Description of colonies has an important place in.bacterial diagnostics Liquid media and solid media • Liquid media are based on je meatpeptonic broth (exctract of cooked beef meat + protein hydrolysate). They are used mostly to multiplication. It is difficult to evaluate the result, in fact, only „non turbid broth – turbid broth“ (growth – no growth) • Majority of solid media are based on the same broth, but supplied by an agar alge extract. Bacteria grow slower on solid media, but the result is very variable, and it is possible to get a strain. Liquid media www.medmicro.info Classification of liquid media • Liquid media have two categories only: • multiplying media are common and universal. Example: broth for aerobic culture and VL-broth for anaerobic culture (VL = viande-levure, from french – contains meat-yeas extract) • selectively multiplying media were developped to multilply some bacteria and to supress multiplication of other. Example: selenite broth for salmonella Solid media www.medmicro.info Why an isolated colony is so important • Only so we can identify larger number of mixed pathogens • But also because only isolated colonies enable to observe typical colony characteristics. The best clown is not able to show you his art, when kept with many other clowns in a small cupboard. In case of a mixture, each bacterium forms its own colonies (at a proper dilution inoculation) 1 – inoculation of bacterial mixture (dots), 2 – result of cultivation: in first parts of inoculation a mixture, at the end – isolated colonies What to describe at colonies • • • • • Size Colour Shape (round…) Profile (convex…) Edges • • • • • Surface (smooth, rough…) Consistence (dry…) Transparency Smell Colony surroundings* *Definition is related to the medium used. For example, haemolysis is observed around some bacteria grown on media with RBCs. Solid selective media • They have to select (separate) from a bacterial mixture only one of several groups of genera • An example is blood agar with 10 % NaCl used for stafylococci • Sometimes, selectivity is reached by an antibiotic addition. Blood agar with amikacin is selective for streptococci and enterococci Diagnostic media • They do not supress growth of any microbe • On the other hand, their composition enable them to differenciate microbes according to some properties • An example is blood agar to observe haemolytical properties, and VL blood agar (simillar, but to anaerobes) • Special case are chromogenic and fluorogenicmedia Photo: O. Z. Photo: O. Z. Chromogenic and fluorogenic media www.oxoid.com • Chromogenic media contain a dye with bound specific substrate it loses it colour, it is no more a dye, but a chromogen • bacteria able to breakdown the specific substrate change the chromogen againt to the original dye • The medium may contain more chromogens (for more species) • Fluorogenic media: similar, with a fluorescent dye Selective diagnostic media www.medmicro.info • Combine selective and diagnostic properties • Example – Endo agar: – Only some G– bacteria can grow on it (selectivity) – The growing bacteria can be differentiated into lactose fermentative and lactose non fermentative • A simillar is McConkey medium, more common in world (but not used in OUR laboratory) • Selective diagnostic are also XLD, CIN media etc. Selective, diagnostic and selective diagnostic media – review Selective medium Strain A does not grow Diagnostic Strain C medium grows, colonies Selective Strain E diagnostic does not medium grow Strain B grows Strain D grows, colonies Strain F grows, colonies Strain G grows, colonies Enriched and selective enriched media • For bacteria with specific need for nutrients • They are enriched by different chemicals • Even blood agar is an enriched medium, although shown as a diagnostic medium (it may be considered a member of both groups). • An expample of „pure enriched medium“ is chocolat and Levinthal agar for pathogenous Neisseriae and hemophili (that do not grow even on blood agar) • Media may be selective enriched (e. g. GC agar, – chocolat agar with anibiotics for culture of Neisseria gonorrhoeae) Chocolate agar www.medmicro.info Special use media www.medmicro.info In vitro antibiotic susceptibility testing: Müller-Hinton agar; also to pigments production observation Rigth, a non-pigmented Staphylococcus strain, left down a pigmented Pseudomonas strain www.medmicro.info Survey of media – part one Type *only with antibiotics Name Class Colour For broth VL-broth liquid media yellowish multiplying aerobes anaerobes darker selenite pinkish broth Sabouraud solid white agar media in a Löwentein- test tube green Jensen selective Salmonella multiplying selective* fungi enriched TBC Blood agar red enriched diagnostic majority of bacteria pink selective diagnostic mostly Endo agar solid media in.dish enterobacteria Survey of media – part two Name Class MH nearly solid media on white Petri dish brown red NaCl VL-agar Colour Type For special atb suseptibility selective staphylococci like BA anaerobes XLD orange selective diagnostic Salmonella chocolat agar brown enriched haemophilli, neisseriae Levinthal agar yellowish enriched haemophilli SlanetzBartley pink selective diagnostic enterococci Biochemical identification Principle • Even between mammals there are differences. Human body is not able to produce vitamin C, body of some mammals is. • We offer certain substrate to a bacterium, and we search, whether bacteria change it into a product using an enzyme. A product has to be different from substrate by physical phase or colour. If it is not different, we use an indicator • There are a lot of ways technical form of this test type. Practical ways of doing it • Quick tests (seconds to minutes) – Catalase test – Tests with diagnostic strips (oxidase) • Tests with incubation (hours to days) – Simple test-tube tests – Complex test-tube tests – Sests of simple test-tube tests – Tests in microtitration plate (miniaturisation) – Other tests (e. g. Švejcar's plate) Catalase test • Catalase test: very simple: we mix bacteria with substrate (H2O2 solution). Bubbles = positivity. Principle: 2 H2O2 2 H2O + O2 medic.med.uth.tmc.edu/path/oxidase.htm Tests with diagnostic strips • Tests with dg. strips – We touch colonies by reaction area. If positive, the area changes its colour. The more common are: – oxidase – strip becomes blue – INAC – strip after minutes becomes blue-green – PYR – strip after minutes , addition of a reagent and one more minute of waiting becomes red – betalactamase strip – testing of some resistance factors (see in two weeks) Oxidase test medic.med.uth.tmc.e du/path/oxidase.htm Simple test-tube tests • They may be in liquid phase, or in agar. • In both cases, substrate is in a test tube, eventually together with an indicator. Substrate may be also added in form of a strip with reaction area with it (ONPG-test). • Test positivity = colour change (in whole volume, or as a ring at the surface) Complex test-tube tests • In one test-tube we have more reactions • For example MIU test. – M = motility (turbidity is spread through a half-liquid agar, not only in site of inoculation) – I = indol (positivity = red ring) – U = urea (breakdown of urea is indicated by the whole medium turning pink) • Or Hajna medium, detecting glucose breakdown, formation of gas from glucose, laktose breakdown and sulphan formation Sets of test-tubes • Complex test-tube tests have some problems. Often positivity of one test disables to see another one. It is difficult to authomatize them and they require experienced personel. • More simple, although sometimes more expensive solution, is a set of several simple test-tube tests • It is, of course, also possible to combine both simple and complex tests (e. g. Hajna + MIU + Simmons citrate + ornithin dekarboxylase – in our laboratory) Miniaturisation: tests in microtitration plates • Miniaturisation of a simple test-tube tests set tests in microtitration plate wells. Each test-tube is replaced by a well. • Number of tests in sets is variable from seven (Neisseria Test) to more than fifty • Technical detail are various. Nevertheless, always the substrate is lyofilized, bacteria are mixed with saline of suspensium medium and then it is mixed with the lyofilized substrate NEFERMtest 24 Pliva Lachema: one frame enables testing of four triplestrips (four tests, determination of four various strains) Photo: O. Z. Other identification tests • Besides tests based on substrate breakdown, we have also other similar tests, that find presence of some bacterial enzymes or virulence factors. For example: – Test of ability to coagulate rabbit plasma – Test of ability to agglutinate rabbit plasma – Test of ability to decapsulate an encapsulated strain (hyaluronidase test) – Motility testing – we have had it already Outer influences, decontamination Microbes and outer influences I At decontaminationch methods, it is absolutelly necessary to reach such value of the acting physical or chemical factor, to kill the microbe. lower survival lower growth limit (bactericidal) limit (inhibitory) upper growth upper survival limit (inhibitory) limit (bactericidal) Primarilly, we are interested in survival limit (not growth limit, important for microbial cultivation). Above the line: we act by a certain temperature, 24 h Below: 4 h only, then back to optimal temperature dies survival limit no growth growth limit grows Microbes and outer influences II Sometimes the action of factor combines The factor allways important is the time A resistant, spore 160 °C forming bacterium 170 °C 180 °C 20 min survives survives dies 30 min survives dies dies 60 min dies dies dies Checing up, whether sterilisation was done, and its quality assessment • Orientation checking up – e. g. by typical smelling • Assessment of real concentration of disinfectants (chemically) • Chemical check up of sterilization uses indicators that change colour at a certain temperature • Biological way uses resistant strains of Bacillus genus. These absolve the whole cycle and then their survival is assessed. Antibiotics Methods of „fight“ with microbes • Immunisation – exploits natural mechanisms of a macroorganism • Decontamination methods – crude physical and chemical influences, action outside the organism (see last practical) • Antimicrobial agents – fine, targeted action inside the organism with aim of maximal effect of the microbe and minimal influence on the host macroorganism Types of antimicrobial agents • Agents acting to the whole body: – Antiparasital agents against parasites – Antimycotics against yeasts and molds – Antivirotics against viruses – Antituberculotics against mycobacteria – Antibiotics against bacteria (natural origin) – Antibacterial chemoterapeutics also against bacteria, but syntetic In recent period, the last two groups are often put into one group called „antibiotics“ • Locally acting agents: antiseptics Results of the diffusion disc test CITLIVÝ REZISTENTNÍ 1 Bacteria are affraid of antibiotics. Large zone (sometimes so large that it is impossible to measure it) 2 Bacteria are not affraid of antibiotics, they are resistant. A small zone around the disc, or no zone. Diffusion disc test in practice: zones are measured and compared with reference zones www.medmicro.info Microdillution test • Atb-s are in a row of wells in a plastic microtitration plate, concetration decreases • The lowest concetration, that inhibits the growth, is the MIC value • For interpretation, we need breakpoint values for each antibiotic. MIC < breakpoint => the strain is susceptible. MIC > breakpoint => resistance • One plate is usually used for one strain,e. g. 12 antibiotics, each in 8 concentrations Microdilution test – example Photo: O. Z. E-tests • Principially simillar to diffusion disc test • Instead of a disc, a strip is used • The strip has raising atb concentration from one end to another ( grace to a special technology – that is why they are expensive) • The zone is not round, but egg-shaped • The test is quantitative • The strip has a scale – sipmle reeding(see image on the next screen) E-tests – result We can read the MIC value directly on the strip – in place, where the margins cross the strip www.uniklinik-ulm.de Assessment of resistance factors • Sometimes, instead of susceptibility testing, we should rather assess the presence of individual resistance factors by special methods, e. g. betalactamases • Some of theme are diagnostic strips (chemical detection of a given enzyme) or tests on a different principle. • It is mostly used in situations, where susceptibility tests are not sure enough (for many reasons, e. g. a metabolite is active,l and not the antibiotic itself, etc.) One of tests for ESBL (extended sprectrum beta-lactamase) The area labelled blue is the important one Photo O. Z. Serology Antigen and antibody Antigen = a macromolecule coming from an alien organism: plant, microbe, animal. In microbiology, we are interested in microbial antigens – parts of microbial body, that challenge host body to an antibody response Antibody = an immunoglobuline, formed by the host body as a response to antigen challenge (of course not only by humans, but also by various animals) Two ways how to use it: Antigen detection: laboratory (animal origin) antibodies + pacient‘s sample or microbial strain. Direct method Antibody detection: laboratory antigen (microbial) + pacient‘s serum (or saliva). Indirect method Interpretation • Antigen detection: it is a direct method. Positive result means presence of the microbe in the pacient‘s body • Antibody detection: it is an indirect method. Nevertheless, there are some ways how to get the information – when the microbe met the body: – Amount of antibodies (relative – titre) – Class of antibodies: IgM/IgG – Avidity of antibodies Titre • After serum dilution, we add the antigen • In relation with the reaction type, either we can se the reaction result directly (aglutinate, precipitate), or we have to visualize it adding other components (complement, RBCs, etc.) • Anyway, we have to be able to discriminate positive and negative reaction results • The highest dilution, where a positive reaction is still visible, is called titre. Dynamics of titre 1 first pacient‘s visit 2 after 2 – 3 weeks • Absolute amount of antibodies is not the most sure information: some patients are poor antibody-producers, etc. • Dynamics of titre: better, means how the response gets changed during the time (usually during two or three weeks) 2 1 Precipitation and aglutination – common characteristics • Precipitation and aglutination are the two most simple serological reactions, we work here really with antigen and antibody only without other componenes • Either we decect antigen using animal antibody, or antibody using laboratory antigen • Only in the second example, we count titers! Precipitation, agglutination, agglutination on carriers • Precipitation: Antigens act alone, as macromolecules (coloid antigen) • Agglutination: Antigen act being part of its microbial cell (we work with whole microbes, corpuscular antigen) • Agglutination on carriers: Formerly isolated antibodies are bound to an alien particle – latex or RBC Precipitation Aglutination Aglutination on carriers Complement-fixing test (CFT) • Complement = one component of immunity reaction • For CFT, we use animal (guinea-pig) complement. The patient‘s complement is inactivated before the reaction • Complement is not able to get bound to isolated antigen • Complement is not able to get bound to isolated antibody • Complement is able to get bound to COMPLEX antigen – antibody CFT – principle Problems existing in CFT • Too much complement: false negative results. What to do? Titrate the complement (according to Task 2) • Something in serum binding the complement itself (anticomplementarity component): false positive results. What to do? Perform anticomplementarity test without antigen (A situation like a homeless man sweeping the plant globules from the bench) Anticomplementarity test Neutralisation reaction: general principle • There are many ways, how antibodies do work. One of them is direct neutralising effect • This effect is rarely present in whole bacteria. On the other hand, it may be observed in whole viruses, and in bacterial toxins Nevertheless, sometimes antibodies neutralise some characteristic of the whole bacteria, e. g. motility of Treponema in Nelson‘s test Examples of neutralisation reactions Task Neutralised Object Reaction 1 Bacterial toxin RBC (haemolysin) haemolysis ASO 2 Virus RBC agglutination HIT 3 Virus Cell metabolic efect VNT Reactions with labelled components • Individual components are bound on the previous components, the first of them to the surface. • Instead of one component a specimen from pacient is used. The specimen is suspicious to contain the given component. • If it is true, the component is bound • When all components bind respectivelly, a not-interrupted chain is formed • At the end there is a labelling agent Washing out and its sense • When also the components that are not bound to the surface would remain, we would not be able to differenciate a positive reaction and a negative one. • That is why after each step washing follows. After such a washing, only bound components remain present. • When the chain is broken, the part after the missing component is washed out. Types of labelling agent • Fluorescent dye is labelling agent in immunofluorescence • Radioisotope is labelling agent in RIA • Enzyme is labelling agent in ELISA – Western blotting is a special type of an ELISA, where individual antigens are divided electroforetically When an enzyme is used as a labelling agent, the very last component should be the substrate – so one more component. Importance of the conjugate • Conjugate is used mostly in indirect reactions (detection of antibodies) • It is an antibody that has human antibody (e. g. IgM, IgA or IgG) for an antigen • It can be selective against a certain antibody class • Use of conjugate is the principle of selective diagnostic of individual immunoglobulin classes Basic scheme of PCR reaction • In first phase we have to get isolated DNA. It is a complex process • In second phase proper amplification runs (only if the specimen contains a part of DNA corresponding to a primer) • In third phase amplification product should be detected by – gel electroforesis of by – ELISA method (≠ serologic ELISA!!!) Use of DNA (RNA) detection in medical microbiology • The methods are used mostly in situations, where microscopic and culture diagnostic is difficult or impossible • It is not very useful for common, ubiquitous pathogens. Because of its sensitivity they would detect accidental molecules comming from environment • The methods are neither useless, as some people think, neither all-problemssolving, as some other people suppose. Survey of interpretation Proper reaction Internal control negative positive Interpretation negative negative positive positive inhibition of reaction positive positive negative negative (highly) positive An expample of a gel www.medmicro.info Patients 1 and 4 – positive, patient 2 – negative, patient 3 – inhibition of reaction. 5 – positive control, 6 – negative control, 7 - ladder Virology Virological diagnostics • Culture isolation Requires living cells. • Microscopy: electronoptical, optical only to examination of somenting, that viruses do in vivo / in vitro (inclusions, cytopatic effect) • Biochemical identification is not possible • Animal experiment here equal to izolation • Detection of DNA – in viruses > bacteria • Detection of Ag in specimen – very common • Indirect diagnostics – usually basis of the entire diagnostics Viral isolation • Animal now less commonly. Typical animal is a suckling baby mouse. • Fertilized egg is a classical method: – Amniotic sac – Alantoic sac – Yolk sac – Chorioallantoic membrane (only here sometimes a visible result – so called pocks) • Tissue cultures: LEP, HeLa, monkey kindney and various other. Some viruses perform a cytopathic effect (CPE) on tissue cultures, but some viruses do not. Fertilized egg and its parts SH – shell AB - albumen http://www.scielo.cl/fbpe/i mg/bres/v38n4/fig02.gif AM – amniotic sac, YS – yolk sac, AL – allantois CH – chorioallantoic membrane (CAM) http://cmir.mgh.harvard.edu/cellbio/cellculture.php? menuID_=122 www.herpesdiagnosis.com/diagnose.html Cytopathic effect of a virus (HSV is Herpes Simplex virus – HSV 1 causing mostly herpes labialis, HSV 2 herpes genitalis) Parasitology Sampling • For intestinal parasites rectal swab is not sufficient, a bit of stool is needed (see more ) • For Trichomonas either a slide for Giemsa staining is sent (alone or in pair with another one for Gram staining), or a C. A. T. swab • For Acantamoeba used contact lenses are sent in their own fluid, eventually corneal scraping might be performed • For tissue parasites serum is sent usually • In other pararasites we sample according to situation (urine, content of a cyst) Sampling for intestinal parasites • To send stool for parasitological sampling (usually using Kato and Faust methods), we need sample of stool sized like a hazel nut. A vessel for sampling need not be sterile. Unlike virological examination the sample does not need low transport temperature • Specimen sized like a coconut (as sometimes some student say) is not recomended C. A. T. swab for urethral and vaginal sampling for Candida (yeast) and Trichomonas Here the swab is broken to fit into the test tube Foto: Ondřej Zahradníček Parasites: diagnostic methods • Microscopy is important, either wet mount, or staining (trichrom, Giemsa stain, Ziehl Neelsen for intestinal coccidia) • Culture is rarelly used, in practice only in Trichomonas and Acantamoeba. • Among other direct methods PCR is used recently • Indirect detection is used in tissue parasitoses, mostly toxoplasmosis, larval toxocarosis etc. Intestinal parasites diagnostics • As a basis, we use methods based on modified wet mount: – In Kato method counterstain with malachite green is used, to make parasites better visible – Faust method is a concentration one (see later) • Graham method is used in pinworms only (see later) • Wet mount „sensu stricto“ and stained preparations (e. g. trichrom) are used in increased suspicion for intestinal protozoa (either primarilly, or after seeing Faust and Kato) Faust method In the second halft, Kato is already prepared • Principle: stool is repeatedly mixed with ZnSO4 solution, centrifugated and supernatant taken for the next step. Finally, the solution is filled up to the top of the test-tube and covered by a coverslip. The parasites adhere to the coverslip from below. Then coverslip is removed onto the slide with allready prepared Kato method. Methods for diagnostics of intestinal protozoa • Helmint eggs are found directly in Faust and Kato methods. When something resembling cysts (of trophozoites) of protozoa is found, more methods are used. We use here – Wet mount, just stool mixed with a drop of saline, eventually a drop of Lugol solution is added after first observation to see better some structures – Trichrom staining. Fixation using alcohol-sublimate and further 70% alkohol, proper trichrom, 96% alcohol and carbolxylene. Or haematoxylin stain. – for cryptosporidia eventually Ziehl Neelsen, or , in Czechia, Miláček staining (Mr. Miláček was a laboratory assistant in parasitology in České Budějovice) Graham method in pinworm diagnostics • The patient bends forward, stretches his/her buttocks, and now a special transparent sticky tape is sticked on his/her anus and mostly perianal rugae. Then the tape is removed again and sticked to a slide. • Transparency of the tape is crucial, otherwise it is not possible to microscopy. (Nevertheless, some „experts“ send a non-translucent tape, or cover all the tape by a label with patient name) • It is easier and more effective than stool examination. It is still used rather in children – adults use to have to hairy anus, so the method woudl be too painflul and difficult. Diagnostics of blood parasites: thin smear and thick drop • In diagnostics of blood parasites it is important to perform a smear using special methods of thin smear and thick drop. • For both methods, fresh blood is used, of nonclotted blood, if the smear is not performed immediatelly. The thin smear is fixated, the thick drop is not. Both of them are Giemsa stained. • Look at following pictures and short videoclips from a CD-ROM „Parazite Tutor“. Pictures taken from CD-ROM „Parasite-Tutor“ – Department of Laboratory Medicine, University of Washington, Seatle, WA Thin smear Thick drop Trichomonas diagnostics • Trichomonads are recently diagnosed mostly using culture-microscopical: – A C. A. T. swab is performed – The medium is cultured overnight – A drop of medium is microscopied as a wet mount. • The preparations cannot be preserved • Therefore in our practical we have the second possible way of diagnostics – Giemsa stained smear on a slide. When it is a part of „Microscopical appearance of vaginal microflora“ (MAVM), it is described as MAVM V. • Other ways are used rarelly Diagnostics of other parasital diseases • In ectoparasites majority of diagnostics is nonmicrobiological (everything can be observed by a laik, eventually a dermatologist in case of Sacroptes scabiei) • In tissue parasites serum for indirect diagnostics is sent usually (CFT, ELISA) • In some cases, mostly tropical parasitioses, it is better to consult sampling technique with a laboratory In some filarioses the sampling is recomended to perform during night only, or during day only. Nice summer! Trichomonas vaginalis, photo O. Z.