Transcript J03_Identification_EL.pps

Searching for microbes
Part III.
Identification methods
(mostly biochemical)
Ondřej Zahradníček
To practical for VLLM0421c
Contacts to me:
[email protected]
Survey of parts of this slide show
A tale for introduction
Identification methods – global principles
Identification methods – problems & solutions
Identification methods – not based on substrates
Identification methods – practical approaches (details inside)
New approaches in identification methods
Check-up questions
Tale
• Once a sick student told to his sister: I have
borrowed some things from my school mates,
and now I have to give them back. But I am
sick. Would you be so kind and go and give the
things to them?
• Microbiological textbook belongs to Peter.
He has blue eyes and blonde hair.
• Coloured pencils belong to Ahmed. He has
brown eyes and black hair.
• The photograph of Britney Spears belongs
to John. He has brown eyes and blonde
hair.
The sister knew now:
• It is not possible to use eye color only to
identify her brother mates.
• It is also not possible to use hair color only
to identify them.
• It is necessary to use combination of both.
Peter
Ahmed
John
Eyes
blue
brown
brown
Hair
blonde
black
blonde
Remember:
• If the sister would meet a monster, halfJohn, half-Ahmed, she would not be able
to identify it.
• We require pure strains of bacteria to
be able to identify them. It is not
possible to work with mixtures of bacteria!
Identification
methods –
global
principles
Position of the method in system
• Direct methods (microbe – part – product):
–
–
–
–
Microscopy – both detection in specimen and identification
Cultivation – both detection in specimen and identification
(Biochemical) identification – identification only!
Antigen detection – both detection in specimen and
identification
– Nucleic acid detection – usually only detection in specimen
– Animal experiment – usually only detection in specimen
• Direct methods (antibodies)
Differences in metabolism of
various bacterial genera and species
• It is big difference, whether bacteria perform
fermentation or aerobic respiration
• It is a difference, whether bacteria breakdown
rather proteins and aminoacids (e. g.
genus Proteus) or rather sugars (e. g. genus
Klebsiella)
Breakdown of a certain substrate is often
a sign of adaptation to a certain
environment (well adapted
enterobacteria download lactose, that
they find in our intestine)
General principle I
• Bacteria have their specific metabolism
• Industrial microbiology exploits bacterial
metabolism (mostly fermentative catabolism)
to production various stuff, including a lot of
food
• Medical microbiology exploits differences
in metabolism between various bacteria
• Inter-species differences interest us
here. Differences between individual
strains of the same species are rather a
complication
General principle II
• We offer certain substrate to a bacterium,
and we search, whether bacteria change it into
product(s) using an enzyme. At least one of
products has to be different from substrate by
physical phase or color. If it is not different,
we use an indicator
• There are a lot of ways technical form of
this test type.
Even between mammals there are differences.
Human body is not able to produce vitamin C,
body of some mammals is.
To review…
Do you know, that you have already met
such a biochemical test? No? But yes,
yes, at cultivation! It is ENDO AGAR
There is a biochemical
test in it: it differenciates
bacteria into lactose
positive, and lactose
negative species.
Photo O. Z.
Identification
methods –
problems &
solutions
Problems
• There are also differences between strains of
the same species, not only between species
• Rarely we can see, that 100 % or 0 % strains of a
certain species produce a given enzyme
• More often it is 90 %, 10 %, 70 %, 30 %…
• How it can be in practice:
Avinella produces joanellase in 90 % cases
Saantella produces joanellase in 10 % cases
Joanase positive microbe can be:
a typical Avinella, but also
an atypical Saantella
Problems – solution
• If we search for one atribute only, is big
probability, that we will meet an atypical
strain and identification will be false
• Nevertheless, it is very small probability,
that a strain would behave atypically e.
g. in ten various tests in the same time
• Therefore the more tests, the bigger
reliability of such a testing
• More tests also enable us to detect
more different genera and species
Probability of the result
• As we have said, the more tests we use, the better
chance that we are not mistaken we have
• Nevertheless, that chance is never entire 100 %
• It is possible to say e. g., that our hypothetical strain
is
– for 99.3 % Avinella elegans
– for 0.5 % Saantella pulcherima
– for 0.2 % something different
• It is on decision of identificator, whether such a
probability ratio is enough, or other discrimination
tests are necessary
Not only percent of probability,
but also typicity index of a strain
• In reality biochemical identification result is
usually characterized by two numbers, not
just one:
– % probability: e. g. there is 90% probability,
that strain really is Avinella elegans and not
something other
– Typicity Index: ratio of identity with an „ideal
strain“ of Avinella elegans. When a strain is
ideal, Tin = 1.00; when strain e. g. does not
produce joannase, although 90 % of Avinella
strains produce it, Tin will be less than 1.00
Examples
• A strain has identification 99 %, typicity
index 0.95. Ideal situation, probably „is to that“.
• A strain has identification 99 %, but typicity
index only 0.63. It might be an atypical strain (is
good to know, what is the „test against the
identification“), but also a diagnostic mistake
• The typicity index is 1.00 for two different
strains, percent of probability is 49.5 % for
each of them (one percent of remains to
„other“). It means, that it is nearly certain that it
is one of them, but without discrimination tests we
do not find, which one is it.
Identification
methods –
practical
approaches
Practical ways of doing it
• Quick tests (seconds to minutes)
– Catalase test Catalase test
– Tests with
strips
(oxidase)
Testdiagnostic
with diagnostic
strips
• Tests with incubation (hours to days)
Simple test
tube tests
– Simple test-tube
tests
– Complex Complex
test-tube
testtests
tube tests
– Sests
test-tube
tests
Sestsofofsimple
simple/complex
test
tube tests
– Tests inTest
microtitration
plate
in microtitration
plates
– 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
• Some examples of practical use:
medic.med.uth.tmc.edu/path/oxidase.htm
Practical note
• The recommended way how to perform the catalase
test is to make a drop of hydrogen peroxide on a
slide and to mix the strain with it
• Some microbiologists make a drop directly to the agar.
In case of blood agar, this is risky – microbiologists
should be experienced to distinguish between true
catalase reaction of bacteria and week catalase
reaction of red blood cells
• In practical sessions, do not use this form also for
another reason: the H2O2 kills the bacteria and so no
living bacteria would remain for other students
Tests with diagnostic strips
•Tests with dg. strips – We touch colonies
by reaction area. If positive, the area changes
its color. The more common are:
–oxidase – reaction area becomes blue (or at
least the spots where we touched the colonies).
For examples of use of oxidase test, click on „“.
–INAC – reaction area after several minutes
becomes blue-green; the strip should be
moistened before being used
–PYR – reaction area after 5–10 minutes, addition
of a reagent and one more minute of waiting
becomes red
–betalactamase strip – testing of some resistance factors
(more in J06)
Strip test in practice
Photo: ?U archive
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. Examples of technical details –
see following slides
Simple test tube tests – I
• 1) The substrate is diluted in the liquid,
during the test a strain is mixed with the
liquid, after incubation we observe change
of color (in whole volume, or as a ring at the
surface). Example: Arabinose test for
differenciation of streptococci (liquid remains
green = test negative, Enterococcus
faecalis; liquid turns to yellow = test
positive, Enterococcus faecium)
Simple test tube tests – II
• 2) Firstly, the strain is mixed with a liquid
(physiological saline). Then a strip (similar
to oxidase strip) is added. The strip
area contains substrate. Substrate is
released from reaction area. Example:
ONPG test (positive = fluid becomes yellow,
negative = fluid remains colorless), VPT
(after addition of two different reagent in
negative case nothing changes, in positive
case a red ring at the surface is formed)
ONPG test
• ONPG: An example of a simple test-tube
test. The substrate is poured to a test-tube
and a strip is added. When the fluid turns
yellow, it means test positivity.
Example of use:
differentiation between
Citrobacter (positive)
and Salmonella
(negative)
Simple test tube tests – III
• 3) An agar is prepared
containing substrate. The
color either remains
original, or is changed.
Example: Simmons
citrate. When the medium
remains green, it is
negative, when it becomes
blue, it is positive.
medic.med.uth.tmc.edu
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
www.arches.uga.edu
MIU could be also done as three
individual tests: motility…
…indol and urea
medic.med.uth.tmc.edu
Hajna medium
(Kligler Iron
Agar – KIA in Hajna modification)
•
•
•
•
•
•
Color of the bottom part of the
medium unchanged: bacterium does
not ferment glucose (differenciation of so
named G- non-fermenters ×
enterobacteria)
Bottom part turns black – formation
of H2S
Broken medium, with bubbles – gas
formed from glucose
Bottom part yellow, upper part red
– bacterium is a glucose fermenter,
lactose non fermenter
The whole medium is yellow –
lactose
fermented, too
www2.ac-lyon.fr/
Example of conclusion
of Hajna + MIU
Test
Reaction
Pseudomonas
aeruginosa
Escherichia coli
Proteus mirabilis
Salmonella
enterica
Citrobacter freundi
Hajna
MIU
Glc Lac H2S Mot Ind Ure
-
-
-
-
-
+
+
+
+
-
+
+
+
+
-
+
+
-
+
+
-
-
+
+
(+)
+
-
-
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 dried,
bacteria are mixed with saline of
suspension medium and then it is mixed
with the dried substrate
Tests by Erba Lachema
(most common in Czechia)
• The factory produces plates with dried substrates,
placed in the bottom of the wells
• We prepare a bacterial suspension in saline or
suspension medium
• To each well we add one or two suspension drops
• The remainder of suspension is often used as test
tube test with a diagnostic strip (ONPG, VPT)
• Both the plate and the test tube is incubated in a
thermostat
NEFERMtest 24
Erba Lachema: one
frame enables testing of
four triple-strips (four
tests, determination of
four various strains)
Photo: O. Z.
Other producers
www.ilexmedical.com/products_engl/api.htm.
(the same principle, small
differences in practical form)
Foto: O. Z.
http://www.oxoid.com/blue
Press/uk/en/images/PR020
505.jpg
www.ilexmedical.com/products_engl/api.htm.
www.ucd.ie/kyr/Images/jpgs/Photo16.htm
Evaluation of plate tests
• Such a test gives us a row of results –
usually in form of „+“ (test is positive,
substrate changed) or „-“ (test negative,
substrate unchanged, original color).
• An example of a: + - + + + - - - - - --++++
• There are several ways, how to convert
such a row into a „legible result“
Ways of evaluation
• Comparison with a table is possible for
simple tests and clear results only.
• Conversion into octal codes plus
searching result in the code list. Common.
• Result input directly into a computer,
which gives us the result. Not always
practical
Computer evaluation is often used when the
reading itself is automatic, e. g. on a
spectrophotometer.
Octal codes – what is it, and why
• Mathematically it is conversion of binary
system (+ + – – + + – – –, or 110011000)
into octal system (written 630)
• For practical reasons, reading inside triplets is
usually reverted – normally 1 1 0 converted
into octal code should be six and 0 1 1 three;
in our „microbiological re-counting“ is uses to
be the opposite
Octal codes – II
• In practice, each triplet of result is
converted into a number 0 to 7.
• Of course, when a test has e. g. 17
reactions, there is a pair instead of triplet
at the end (so the final number can be
only 0, 1, 2, 3).
• When we have e. g. 16 (19, 22…)
reactions, the final number should be zero
or one only.
Practical example
• We mark positive and negative reaction
results
• Under each triplet we write 1 – 2 – 4
• For each triplet we read only „+“
numbers, not „ –“ (these go out)
Test
Result
Code
JAN
LEN
MAG
TOM
PET
KAR
FRA
HAN
+
1
–
2
5
+
4
+
1
+
2
3
–
4
–
1
–
2
0
Re-counting the triplets
–––
+––
–+–
++–
––+
+–+
–++
+++
124
124
124
124
124
124
124
124
1
2
1
4
1
2
1
+2
+4
+4
+2+4
0
1
2
3
4
5
6
7
For example, in
ENTEROtest16 (17 tests)
ONPG
1
2
H
3
G
4
F
5
E
6
D
7
C
8
B
9
A
10 11 12 13 14 15 16 17
H G F E D C B A
First row of the plate 2nd row of the plate
+S llllllllllllllll
– S llllllllllllllll
? S llllllllllllllll
? +
–
+ + +
– – – – – – – –
+ + + +
1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2
5
3
0
0
6
3
Identification
methods –
not based on
substrates
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
Plasmacoagulase and hyaluronidase
(both tests are used in
staphylococci)
www.hardydiagnostics.com
Photo O. Z.
Photo O. Z.
Diagnostic use of antibiotics
• One possibility is also in vitro
susceptibility testing to a certain
antibiotic in situation, that we know,
that strain X is in 100 % susceptible
and strain Y is in 100 % resistant. In
practice, it is, of course, never 100 %.
• An example is optochin test
• Practically it the same as normal
antibiotic susceptibility tests, see
the practical two weeks later
Optochin test negative and positive
http://www.mc.maricopa.edu
New
approaches in
identification
methods
New identification methods
• One of trends of recent period is
automatisation of biochemic identification
at use of an expert system. Sometimes it is
directly bound to (automatic) cultivation.
• Problem at using these methods is that usually
the clinical origin of the strain is not taken into
account sufficiently, and so the preciseness of
identification, choice of probable pathoten ×
accidentally contamining strain etc. is not
always suitable.
• Another approach give us new methods on new
principles, e. g. mass spectrometry type
MALDI-TOF, see further
MALDI TOF
• Principle: mass spectrometry
– already long time used in chemistry, but classic ways only
enabled detection of small molecules
– Based on division of ionised particles according to
their molecular mass
– now ionisation by laser enables to detect also large
molecules, charakteristic for individual species of
bacteria/yeasts
– uses ionisation by laser at presence of a matrix
(MALDI, matrix assisted laser desorption/ionization) in
combination with time of flight detector (TOF, time-of-flight)
• Recently very expensive (6 000 000 CZK)
• In Czechia in several places now (temporarily at our
institute – only I–III/2011)
MALDI TOF – details
• Speed of the particle (connected with its mass)
may be counted from detected time of flight
(TOF)
• Mixture of matrix (4-hydroxy-cinamic acid) and
specimen on the metalic plate is beaten by a
nanosecond laser pulse
• matrix absorbs the energy of the pulse and
molecules of the specimen are ionised by its
jejím fragmentation
• Method „finds“ specific proteins of the strains
and compares them with the databese, today
already pretty large, but still enlarged
MALDI-TOF
Link to
producer‘s websites
Work with MALDI-TOF
Preparing strain for MALDI-TOF
The End
http://www.telmeds.org
Examples of catalase test use
• The most common use it in diagnostics of
G+ cocci. Among medically important
genera, staphylococci are catalase +,
while streptococci and enterococci are
catalase –
• Nevertheless, there exist some more
examples, too, e. g. in G+ rods: Listeria
is catalase +, Erysipelothrix
(microscopically similar) catalase –
Examples of oxidase test use
• Oxidase can be used in various
situations:
• To confirm diagnostics of Neisseria,
Moraxella and Pseudomonas (oxidase
positive)
• To differentiate between Vibrionaceae
(oxidase +) and Enterobacteriaceae
(oxidase – except genus Plesiomonas)
Check-up questions
Try to find the answers, then answer to the questions in
ROPOT. The formulations of questions might be slightly
different.
1. What does abbreviation "MIU" mean?
2. Why sometimes even for differentiation of two specimens one test is not
sufficient?
3. How do we use the oxidase strip?
4. What is the visible difference between products and substrate in the
catalase test?
5. Percent of probability of identification and typicity index: is it the same?
6. There is a certain medium. When we culture bacteria there, we get the
information whether they ferment lactose. What is the name of this medium?
7. When we do not need an indicator at biochemical identification reaction?
8. When a saccharide is exploited by a bacterium, does it always mean it
fermentation?
9. Is it possible that one organism is able to split certain substrate, and
another is not?
10. And also some more surprises 