Searching for MicrobesPart XIV.

Review to the spring term

Ondřej ZahradníčekTo practicals of VLLM0421c

zahradnicek@fnusa.cz

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!• The second: But firstly you have to

catch him! So, terrain searching is the primary

• But 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 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 methodsMethod Specimen

examination

Identification

Microscopy 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 fixation

Coverslip Imersion system

Wet mount

no yes noDarkfieldwet mount

no yes yes

Stained preparat.

yes no yes

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.jP7201179.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+

G–Photo: Helena Janochová and Zuzana Jurčíková

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 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“

• 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

www.medmicro.info

Liquid media and solid media

• Liquid 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 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

• 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

www.oxoid.com

Selective diagnostic media • 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.

www.medmicro.info

Selective, diagnostic and selective diagnostic media – review

Selective medium

Strain A does not grow

Strain B grows

Diagnostic medium

Strain C grows, colonies

Strain D grows, colonies

Selective diagnostic medium

Strain E does not grow

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

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

Special use media www.medmicro.info

www.medmicro.info

Survey of media – part oneName Class Colour Type For

broth liquid media

yellowish

multiplying

aerobes

VL-broth

darker anaerobes

selenite broth

pinkish selective multiplying

Salmonella

Sabouraud agar

solid media in a test tube

white selective* fungi

Löwentein-Jensen

green enriched TBC

Blood agar solid media in.dish

red enriched diagnostic

majority of bacteria

Endo agar pink selective diagnostic

mostly enterobacteria

*only with antibiotics

Survey of media – part twoName Class Colour Type For

MH solid media on Petri dish

nearly white

special atb suseptibility

NaCl brown selective

staphylococci

VL-agar red like BA anaerobes

XLD orange selective diagnostic

Salmonella

chocolat agar

brown enriched haemophilli, neisseriae

Levinthal agar

yellowish

enriched haemophilli

Slanetz-Bartley

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.edu/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 triple-strips (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.

Primarilly, we are interested in survival limit (not growth limit, important for microbial cultivation).

lower survival limit (bactericidal)

upper survival limit (bactericidal)

lower growth limit (inhibitory)

upper growth limit (inhibitory)

Above the line: we act by a certain temperature, 24 h

Below: 4 h only, then back to optimal temperature

Met

hodo

logi

cal

differ

ence

dies

grows

no growth

growth limit

survival limit

Microbes and outer influences II

Sometimes the action of factor combines

The factor allways important is the timeA resistant, spore forming bacterium

160 °C 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 synteticIn recent period, the last two groups are often

put into one group called „antibiotics“• Locally acting agents: antiseptics

Results of the diffusion disc test

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.

REZISTENTNÍ

CITLIVÝ

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 – resultWe 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 methodAntibody 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• 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)

1 first pacient‘s visit

1

2 after 2 – 3 weeks

2

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 (haemolysin)

RBChaemolysis

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

PCR

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-problems-solving, as some other people suppose.

Survey of interpretationProper reaction

Internal control

Interpretation

negative positive negative

negative negative inhibition of reaction

positive positive positive

positive negative (highly) positive

An expample of a gel

Patients 1 and 4 – positive, patient 2 – negative, patient 3 – inhibition of reaction. 5 – positive control, 6 – negative control, 7 - ladder

www.medmicro.info

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

AM – amniotic sac, YS – yolk sac, AL – allantois

CH – chorioallantoic membrane (CAM)

SH – shell

AB - albumen

http://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

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

Foto: Ondřej Zahradníček

Here the swab is broken to fit into the test tube

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 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 drop

Pictures taken from CD-ROM „Parasite-Tutor“ – Department of Laboratory Medicine, University of Washington, Seatle, WA

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 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 laboratory

In some filarioses the sampling is recomended to perform during night only, or during day only.

Nice summer!

Trichomonas vaginalis, photo O. Z.