Download Wet mount

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.