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Searching for Microbes Part XIV. Review to the spring term Ondřej Zahradníček To practicals of VLLM0421c zahradnicek @ fnusa . cz

Searching for Microbes Part XIV. Review to the spring term

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Searching for Microbes Part XIV. Review to the spring term. Ondřej Zahradníček To practicals of VLLM0421c [email protected] One more tale…. Once three policemen discussed what method is the most important. One said: Dactyloscopy is the best! It helps to identify any criminal! - PowerPoint PPT Presentation

Text of Searching for Microbes Part XIV. Review to the spring term

  • Searching for MicrobesPart XIV.Review to the spring termOndej ZahradnekTo practicals of [email protected]

  • One more taleOnce three policemen discussed what method is the most important.One said: Dactyloscopy is the best! It helps to identify any criminal!The second: But firstly you have to catch him! So, terrain searching is the primaryBut the third, the eldest, said: The most important is to use all our methods together, and to know, when to use each of them.

  • A microbe (microogranism): what does it mean?It should be living. and grain of dust is not and microbe, although it is microscopicalIt should be microscopical. and giraffe is not and microbe, although it is livingThe 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 microbesViruses (and prions)Bacteria (e. g. and Streptococcus or an Escherichia)Fungi (yeasts and molds)Parasites not all of them are microbes:Inner parasitesProtozoa (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 methodsDirect 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 determinationIndirect 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*but in molecular epidemiology detection of simillarity of strains - yes

    MethodSpecimen examinationIdentificationMicroscopyyesyesCultivationyesyesBiochemical identificat.noyesAntigen detectionyesyesAnimal experimentyesusually notMolecular methodsyesusually not*

  • Microscopy

  • MicroscopyWe 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 bacteri, fungi, parasites)Electron microscopy in viruses; rather for research than for common virological diagnostics

  • Microscopy of a specimenMicroscopy of a strainPhoto O. Zahradnek

  • Main microscipical methods in medical microbiology

    Drying and fixationCoverslipImersion systemWet mountnoyesnoDarkfieldwet mountnoyesyesStained preparat.yesnoyes

  • Preparing a microscopical preparationWe make a smear of a swab made by a cotton swab (in stained preparations only)Liquid specimen are dropped on a slideIf 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.jP7201179.jpg

  • Gram stained preparationPhoto: Helena Janochov and Zuzana Jurkov

  • Bacterial cell wallThere 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.

    ChemicalGram-positiveGram-negativeCrystal. violetStaining violetStaining violetLugol iodineConfirmationLess confirm.AlkoholNot decolorizedDecolorizedSafraninRemain violetStain to red

  • Mixture of gram-positive and gram-negative bacteriaG+GPhoto: Helena Janochov and Zuzana Jurkov

  • 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.Various microbes need various conditions!Values, that enable microbial survival, are not sufficient. They should be able to multiply.lower survival limit (bactericidal)upper survival limit (bactericidal)lower growth limit (inhibitory)lower growth limit (inhibitory)

  • Medically important bacteriaTemperature usually needed around 37 Cbut bird pathogens more (42 C), microbes coming from outside less (30 C)Value of pH needed around pH 7but gastric helicobacter by far lessNaCl 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 thermostatBesides 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 oxygenAerobic and facultative anaerobic (eventually aerotolerant) bacteria can be grown at normal athmosphereStrictly anaerobic bacteria need athmosphere without oxygenbacteria with special need for oxygen require special athmosphere (microaerophile and capnophile bacteria)

  • Why we culture bacteriaWhy 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 onlyTo differentiate and divide them mutually diagnostic and selective media are used, for identification

  • Specimen and strainSpecimen is taken from a patient. Specimen contains cells macroorganism, various number of microbial species (zero to maybe twenty) and more itemsA strain an isolate is a population of one bacteria, isolated from a specimen on a solid mediumTo gain a strain, we have to grow a bacterium on a solid medium and inoculate carefully

  • Term colonyA 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 diagnosticswww.medmicro.info

  • Liquid media and solid mediaLiquid media are based on je meat-peptonic 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 mediawww.medmicro.info

  • Classification of liquid mediaLiquid 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 mediawww.medmicro.info

  • Why an isolated colony is so importantOnly so we can identify larger number of mixed pathogensBut 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 coloniesSizeColourShape (round)Profile (convex)EdgesSurface (smooth, rough)Consistence (dry)TransparencySmellColony surroundings**Definition is related to the medium used. For example, haemolysis is observed around some bacteria grown on media with RBCs.

  • Solid selective mediaThey have to select (separate) from a bacterial mixture only one of several groups of generaAn 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 mediaThey do not supress growth of any microbeOn the other hand, their composition enable them to differenciate microbes according to some propertiesAn example is blood agar to observe haemolytical properties, and VL blood agar (simillar, but to anaerobes)Special case are chromogenic and fluorogenicmediaPhoto: O. Z.Photo: O. Z.

  • Chromogenic and fluorogenic media Chromogenic media contain a dye with bound specific substrate it loses it colour, it is no more a dye, but a chromogenbacteria able to breakdown the specific substrate change the chromogen againt to the original dyeThe medium may contain more chromogens (for more species)Fluorogenic media: similar, with a fluorescent dyewww.oxoid.com

  • Selective diagnostic media Combine selective and diagnostic propertiesExample Endo agar:Only some G bacteria can grow on it (selectivity)The growing bacteria can be differentiated into lactose fermentative and lactose non fermentativeA simillar is McConkey medium, more common in world (but not used in OUR laboratory)Selective diagnostic are also XLD, CIN media etc.www.medmicro.info

  • Selective, diagnostic and selective diagnostic media review

    Selective mediumStrain A does not growStrain B growsDiagnostic mediumStrain C grows, colonies Strain D grows, colonies Selective diagnostic mediumStrain E does not growStrain F grows, colonies Strain G grows, colonies

  • Enriched and selective enriched media For bacteria with specific need for nutrientsThey are enriched by different chemicalsEven 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 agarwww.medmicro.info

  • In vitro antibiotic susceptibility testing: Mller-Hinton agar; also to pigments production observationRigth, a non-pigmented Staphylococcus strain, left down a pigmented Pseudomonas strainSpecial use mediawww.medmicro.infowww.medmicro.info

  • Survey of media part one*only with antibiotics

    NameClassColourTypeForbrothliquid mediayellowishmultiplyingaerobesVL-brothdarkeranaerobesselenite brothpinkishselective multiplyingSalmonellaSabouraud agarsolid media in a test tubewhiteselective*fungiLwentein-JensengreenenrichedTBCBlood agarsolid media in.dishredenriched diagnosticmajority of bacteriaEndo agarpinkselective diagnosticmostly enterobacteria

  • Survey of media part two

    NameClassColourTypeForMHsolid media on Petri dishnearly whitespecialatb suseptibilityNaClbrownselectivestaphylococciVL-agarredlike BAanaerobesXLDorangeselective diagnosticSalmonellachocolat agarbrownenrichedhaemophilli, neisseriaeLevinthal agaryellowishenrichedhaemophilliSlanetz-Bartley pinkselective diagnosticenterococci

  • Biochemical identification

  • PrincipleEven 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 indicatorThere are a lot of ways technical form of this test type.

  • Practical ways of doing itQuick tests (seconds to minutes)Catalase testTests with diagnostic strips (oxidase)Tests with incubation (hours to days)Simple test-tube testsComplex test-tube testsSests of simple test-tube testsTests in microtitration plate (miniaturisation)Other tests (e. g. vejcar's plate)

  • Catalase testCatalase 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 stripsTests with dg. strips We touch colonies by reaction area. If positive, the area changes its colour. The more common are:oxidase strip becomes blueINAC strip after minutes becomes blue-greenPYR strip after minutes , addition of a reagent and one more minute of waiting becomes redbetalactamase strip testing of some resistance factors (see in two weeks)

  • Oxidase testmedic.med.uth.tmc.edu/path/oxidase.htm

  • Simple test-tube testsThey 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 testsIn one test-tube we have more reactionsFor 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-tubesComplex 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 testsIt 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 platesMiniaturisation 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 fiftyTechnical 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 triple-strips (four tests, determination of four various strains)Photo: O. Z.

  • Other identification testsBesides 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 plasmaTest of ability to agglutinate rabbit plasmaTest of ability to decapsulate an encapsulated strain (hyaluronidase test)Motility testing we have had it already

  • Outer influences, decontamination

  • Microbes and outer influences IAt decontaminationch methods, it is absolutelly necessary to reach such value of the acting physical or chemical factor, to kill the microbe.Primarilly, we are interested in survival limit (not growth limit, important for microbial cultivation).

  • Above the line: we act by a certain temperature, 24 hBelow: 4 h only, then back to optimal temperatureMethodological differencediesgrowsno growthgrowth limitsurvival limit

  • Microbes and outer influences II Sometimes the action of factor combinesThe factor allways important is the time

    A resistant, spore forming bacterium160 C170 C180 C20 minsurvivessurvivesdies30 minsurvivesdiesdies60 mindiesdiesdies

  • Checing up, whether sterilisation was done, and its quality assessmentOrientation checking up e. g. by typical smellingAssessment of real concentration of disinfectants (chemically)Chemical check up of sterilization uses indicators that change colour at a certain temperatureBiological way uses resistant strains of Bacillus genus. These absolve the whole cycle and then their survival is assessed.

  • Antibiotics

  • Methods of fight with microbesImmunisation exploits natural mechanisms of a macroorganismDecontamination 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 agentsAgents acting to the whole body:Antiparasital agents against parasitesAntimycotics against yeasts and moldsAntivirotics against virusesAntituberculotics against mycobacteriaAntibiotics against bacteria (natural origin)Antibacterial chemoterapeutics also against bacteria, but synteticIn recent period, the last two groups are often put into one group called antibioticsLocally acting agents: antiseptics

  • Results of the diffusion disc test1 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.REZISTENTNCITLIV

  • Diffusion disc test in practice: zones are measured and compared with reference zoneswww.medmicro.info

  • Microdillution testAtb-s are in a row of wells in a plastic microtitration plate, concetration decreasesThe lowest concetration, that inhibits the growth, is the MIC valueFor interpretation, we need breakpoint values for each antibiotic. MIC < breakpoint => the strain is susceptible. MIC > breakpoint => resistanceOne plate is usually used for one strain,e. g. 12 antibiotics, each in 8 concentrations

  • Microdilution test example Photo: O. Z.

  • E-testsPrincipially simillar to diffusion disc testInstead of a disc, a strip is usedThe 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-shapedThe test is quantitativeThe strip has a scale sipmle reeding(see image on the next screen)

  • E-tests resultWe can read the MIC value directly on the strip in place, where the margins cross the stripwww.uniklinik-ulm.de

  • Assessment of resistance factorsSometimes, instead of susceptibility testing, we should rather assess the presence of individual resistance factors by special methods, e. g. betalactamasesSome 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 onePhoto 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 responseAntibody = 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 + pacients sample or microbial strain.Direct methodAntibody detection: laboratory antigen (microbial) + pacients serum (or saliva).Indirect method

  • InterpretationAntigen detection: it is a direct method. Positive result means presence of the microbe in the pacients bodyAntibody 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/IgGAvidity of antibodies

  • TitreAfter serum dilution, we add the antigenIn 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 resultsThe highest dilution, where a positive reaction is still visible, is called titre.

  • Dynamics of titreAbsolute 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)1 first pacients visit12 after 2 3 weeks2

  • Precipitation and aglutination common characteristicsPrecipitation and aglutination are the two most simple serological reactions, we work here really with antigen and antibody only without other componenesEither we decect antigen using animal antibody, or antibody using laboratory antigenOnly in the second example, we count titers!

  • Precipitation, agglutination, agglutination on carriersPrecipitation: 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 reactionFor CFT, we use animal (guinea-pig) complement. The patients complement is inactivated before the reactionComplement is not able to get bound to isolated antigenComplement is not able to get bound to isolated antibodyComplement is able to get bound to COMPLEX antigen antibody

  • CFT principle

  • Problems existing in CFTToo 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 principleThere are many ways, how antibodies do work. One of them is direct neutralising effectThis effect is rarely present in whole bacteria. On the other hand, it may be observed in whole viruses, and in bacterial toxinsNevertheless, sometimes antibodies neutralise some characteristic of the whole bacteria, e. g. motility of Treponema in Nelsons test

  • Examples of neutralisation reactions

    TaskNeutralisedObjectReaction1Bacterial toxin (haemolysin)RBChaemolysisASO2VirusRBC agglutinationHIT3VirusCell metabolic efectVNT

  • Reactions with labelled componentsIndividual 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 boundWhen all components bind respectivelly, a not-interrupted chain is formedAt the end there is a labelling agent

  • Washing out and its senseWhen 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 agentFluorescent dye is labelling agent in immunofluorescenceRadioisotope is labelling agent in RIAEnzyme is labelling agent in ELISAWestern blotting is a special type of an ELISA, where individual antigens are divided electroforeticallyWhen an enzyme is used as a labelling agent, the very last component should be the substrate so one more component.

  • Importance of the conjugateConjugate 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 classUse of conjugate is the principle of selective diagnostic of individual immunoglobulin classes

  • PCR

  • Basic scheme of PCR reactionIn first phase we have to get isolated DNA. It is a complex processIn 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 bygel electroforesis of byELISA method ( serologic ELISA!!!)

  • Use of DNA (RNA) detection in medical microbiologyThe methods are used mostly in situations, where microscopic and culture diagnostic is difficult or impossibleIt is not very useful for common, ubiquitous pathogens. Because of its sensitivity they would detect accidental molecules comming from environmentThe methods are neither useless, as some people think, neither all-problems-solving, as some other people suppose.

  • Survey of interpretation

    Proper reactionInternal controlInterpretationnegativepositivenegativenegativenegativeinhibition of reactionpositivepositivepositivepositivenegative(highly) positive

  • An expample of a gelPatients 1 and 4 positive, patient 2 negative, patient 3 inhibition of reaction. 5 positive control, 6 negative control, 7 - ladderwww.medmicro.info

  • Virology

  • Virological diagnosticsCulture 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 possibleAnimal experiment here equal to izolationDetection of DNA in viruses > bacteriaDetection of Ag in specimen very commonIndirect diagnostics usually basis of the entire diagnostics

  • Viral isolationAnimal now less commonly. Typical animal is a suckling baby mouse. Fertilized egg is a classical method:Amniotic sacAlantoic sacYolk sacChorioallantoic 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 partsAM amniotic sac, YS yolk sac, AL allantoisCH chorioallantoic membrane (CAM)SH shellAB - albumenhttp://www.scielo.cl/fbpe/img/bres/v38n4/fig02.gif

  • www.herpesdiagnosis.com/diagnose.htmlhttp://cmir.mgh.harvard.edu/cellbio/cellculture.php?menuID_=122(HSV is Herpes Simplex virus HSV 1 causing mostly herpes labialis, HSV 2 herpes genitalis)Cytopathic effect of a virus

  • Parasitology

  • SamplingFor 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. swabFor Acantamoeba used contact lenses are sent in their own fluid, eventually corneal scraping might be performedFor tissue parasites serum is sent usuallyIn other pararasites we sample according to situation (urine, content of a cyst)

  • Sampling for intestinal parasitesTo 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 temperatureSpecimen 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 TrichomonasFoto: Ondej ZahradnekHere the swab is broken to fit into the test tube

  • Parasites: diagnostic methodsMicroscopy 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 recentlyIndirect detection is used in tissue parasitoses, mostly toxoplasmosis, larval toxocarosis etc.

  • Intestinal parasites diagnosticsAs a basis, we use methods based on modified wet mount:In Kato method counterstain with malachite green is used, to make parasites better visibleFaust 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 methodIn the second halft, Kato is already preparedPrinciple: 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 protozoaHelmint 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 hereWet mount, just stool mixed with a drop of saline, eventually a drop of Lugol solution is added after first observation to see better some structuresTrichrom 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, Milek staining (Mr. Milek was a laboratory assistant in parasitology in esk Budjovice)

  • Graham method in pinworm diagnosticsThe 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 dropIn 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 non-clotted 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.

  • Thin smearThick dropPictures taken from CD-ROM Parasite-Tutor Department of Laboratory Medicine, University of Washington, Seatle, WA

  • Trichomonas diagnosticsTrichomonads are recently diagnosed mostly using culture-microscopical:A C. A. T. swab is performedThe medium is cultured overnightA drop of medium is microscopied as a wet mount. The preparations cannot be preservedTherefore 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 diseasesIn ectoparasites majority of diagnostics is non-microbiological (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 laboratoryIn some filarioses the sampling is recomended to perform during night only, or during day only.

  • Nice summer!Trichomonas vaginalis, photo O. Z.