Download Gram staining

Searching for Microbes
Part I.
Introduction to Diagnostics
Microscopy of microbes
e-learning version
Author of the slideshow: Ondřej Zahradníček
To practicals of VLLM0421c
Contact to me:
[email protected]
Hyperlinks to individual parts
Two tales to start
Laboratory safety and practical advice
Clinical microbiology, definition and relations
Morphology of bacteria
Diagnostics of bacteria
Microscopy
Wet mount
Stained preparation
Gram staining
More staining methods
Some practical notes to microscopy
Tales for
introduction
Tale 1
• Once and wolf, and tiger and HIV virus met
together.
• The wolf said: I wanted to bite a man, but he
was armed, and so he would kill me. So I had to
run away. But you, tiger, you are stronger than I
am, maybe you could kill him.
• The tiger said: No, no, I can‘t, the man‘s arms
are stronger then I am.
• Just now something laughed – something
definitelly invisible. „But I‘ll get him, you‘ll see!“
It was HIV virus…
Tale 2 (based on real story, but still more fiction than truth )
• There was a man from Denmark, named
Christian Gram. He stained bacteria and
was angry. Sometimes he stained a
pacient‘s sample, but beside bacteria,
epitheliae were also stained. „Awful
epitheliae, they hide my bacteria!“, said he.
• So he started a research. He wanted to find
something that would stain him bacteria,
but not epitheliae…
To be continued
Tale continues
• He found, that when the sample is stained
by gentiane or crystaline violet, and then
binding of the dye to the cell wall is
supported by Lugol iodine, bacteria do
not decolorize even by alcohol. Epitheliae,
on the other hand, decolorize. „Great!“
• But soon he saw, that with epitheliae, part
of bacteria decolorize, too. „Stupid
staining“, said he, drunk remaining
decolorizing alcohol, and throught his work
into the corner of the room.
And end of the tale…
• Some twenty years later, one young researcher
found in a corner of a laboratory the dusty work
of Mr. Christian Gram.
• As he read it, he thougt – it is not bad, it only
needs a bit more to be added.
• And so, to the end of the processus, he added
counterstain of safranin (or Gabbet =
carbolfuchsin). Not only bacteria, but also
epitheliae stained red, but he said – why not? To
know, if epitheliae are present, may be quite
usefull!
• And so Gram staining of today was developped.
Laboratory
safety &
practical
advices
Practical advice
• Boxes for bags and outer coats are the
1st, the 2nd and the 4th from window. Do
not use the 3rd box, even when open!
It is and box for secondary medical school.
• Keys from boxes should be let at the
table next to the computer keyboard
• Next to the keys from boxes you can
find and WC key. After coming back from
WC, put it back to its place
• It is forbidden to students to enter
the space behind the practical‘s hall!
What to do with waste and garbage
• Small pieces may be placed to the small vessels
directly on your tables
• Used matches have their own box
• Larger pieces of garbage should be placed to the
waste basked (next to sinks)
• Broken glass should be placed to a tin close to the
waste basked
• Microscopical preparations prepared by yourselves
should be placed to the disinfection after use
• Please, do not let mess on the tables (e. g. debris
from rubbering, pen sharpening etc.)
Literature
• You need your protocols. You will find
them in Study Materials to VLLM0421c in
is.muni.cz website. This week,
exceptionally, I have printed some lab
report forms for you.
• You will need and textbook –
Greenwood‘s one, Murray‘s one or any like
these can be used. The textbook should
have virological, mycological and
parasitological parts – not all textbooks
have this!
Credit conditions
• If possible, presence in practicals should be 100 %
– Exceptionally justified absences (disease) are possible, but
even in this case you have to complete your protocol and
all you might be asked for substitution (especially for > 2
absences)
• One condition for credits is also filling in all check-up
questions in ROPOT questionaires (they should be
filled in every week)
• You will also have to show your vigillance in
practicals at the credit test that would show us that
not only your body, but your soul, too, was present
here
More instructions
• You have to draw the pictures (draw cocci, do
not write „I can see cocci“)
• Draw the pictures in colours, you have to use
couloured pencils. Fill in the pictures
• Draw what you really see (if your cocci touch
each other in the microscope, draw them so)
• If you cannot see what you think you have to see,
tell to the assistant
• You will show your lab reports to the
examinator at the examination and they will
definitelly influence your final result at the
examination!
Here you can see, that lab reports are
really important (and card of and Slovak
student, partially translated into English)
The cocci really ARE confluent
in the microscope!
ROPOT questionaire
After having studied this slideshow, try to answer these questions. After
finding the answers, fill in the ROPOT questionaire.
1. In what situations wet mount is used in microbiology?
2. What are the basic shapes of bacterial cells?
3. What are possible types of arrangement of cocci?
4. What is the type of microscopy with object illuminated from side?
5. What are differences between wet mount and stained preparations?
6. How do the chemicals in Gram staining follow one after another?
7. What bacteria do not Gram stain or stain poorly?
8. What is the color of various microorganisms in Gram staining?
9. What are other staining methods commonly used in microbiology?
10. How epithelial cells and white blood cells stain in Gram staining?
Similarly you would fill in the questionaire to all other practical sessions.
Clinical
microbiology, its
definition and
relations to
other subjects
Classification of our branch
Molecular
biology and
genetics
Plant
microbiology
Infectious
medicine
Human
Epidemiology
medical
General
of infectious
microbiology microbiology diseases
Veterinary
DermatoCell
medical
venerology
biology
microbiology
Who are clinical microbiologists
Basic microbiological research
Industrial
microbiology
Medical
microbiology
Polytechnical Faculties of
institutes
Science
Other medical
branches
Medical
faculties
Your teachers are blue 
A microbe (microogranism):
what does it mean?
• It should be living. A grain of dust is not
a microbe, although it is microscopical
• It should be microscopical. A giraffe is
not a 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.
Various microbes
• Among microbes we can find
microscopical algae and
cyanobacteria, archea (formerly
archeobacteria), various organisms able
to live long time deep in the sea or in
extreme conditions of hot springs
• For us, clinical microbiologists, these
microbes are not target of our interest.
Nevertheless, they are very interesting.
What these microbes can
•
•
•
•
They live in depth of 10 km
They survive temperatures around 110 °C
They stand strong radioactivity
Instead of oxygen, they are able to „breath“
sulphur of nitrogen (or: they have another
electrone acceptor than oxygen)
• Nevertheless, many interesting thing are
performed even by medically important
microbes, as you‘ll see later.
www.meningitis.de/erreger/meningokokken.html
Neisseriae
Classification of living organisms
• Prions – no DNA, usually not counted to
be living organisms at all
• Viruses and bacteriophages
• Cellullar organisms
– Archaea (archeobacteria)
– Eubacteria (eubacteria)
– Eucarya (eukaryotic organisms)
• monocellullar
• polycellullar
Medically important microbes 1
• Medically important microbes are such,
that are important for human body (so
not for human = creator, but for human =
object)
• „Important for body“ is not at all the
same as „harmful for body“. On the
contrary, many of them are harmless,
or even helping us!
Medically important microbes 2
• Each organism has its medically
important microbes: human, each
species of animal or plant
• Even some microbes (e. g. bacteria) have
their own microbes (bacteriophagi).
Neisseria gonorrhoeae
http://medicine.plosjournals.org/archive/1549-1676/2/1/figure/10.1371_journal.pmed.0020024.g001-M.jpg
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)
Picture is made by
as. MUDr. Petr Ondrovčík, CSc.,
former lecturer of the Institute,
deceased young in autumn 2007
Bacteria are
interesting!
Morphology
of bacteria
Morphology of medically
important microorgamisms
• Viruses are composed of DNA or RNA and
proeins; some viruses possess an envelope
„stolen“ to a host cell
• Viruses have cubic or helicoidal symetry.
Several of them are able to form „pseudocrystals“
• Yeasts are egg shaped, they can form buds and
so named pseudomycelia. On the surface they
have a cell wall. They are eucaryotic.
• Filamentous fungi and parasites are very
variable in their shapes and they have various
development stages. Also they are eucaryotic.
Morphology of bacteria
• Cocci in pairs (diplococci), in chains and
clusters (do not say „streptococci“ and
„staphylococci“, it would be confusing)
• Rods straight or curved (vibria), eventually
several times curved (spirillae), short or long,
forming filaments or branched filaments; their ends may be round or
edged and also rods may be arranged in various way
• Coccobacilli
• Spirochets – thin spiral bacteria
• Amorph bacteria, e. g. mycoplasms (they
do not have any wall, their shape is
changing)
Cocci in chains (electronoptic
microphotograph of Enterococcus sp.)
http://www.morgenwelt.de/typo3temp/5ce14d39b5.jpg
Several times curved rods – Helicobacter
http://vietsciences.free.fr/nobel/medecine/images/helicobacter%2520pylori.JPG
www.primer.ru/std/gallery_std/treponema.htm
Spirochets
http://nl.wikipedia.org/wiki/Afbeelding:TreponemaPallidum.jpg
Fimbriae and a flagella
• Many bacteria are able to move
• They move mostly by a flagella
• Fimbriae (pilli) are used to movement,
adhesion and to excange of genetical
information
• Bacterial flagellae are different from
flagellae of eukaryotic organisms
• Fimbriae and flagellae are invisible in
optical, but often in electronoptical
microscopy
Bacteria with flagellae (Escherichia coli)
http://www.biotox.cz/toxikon/bacteria/bacteria/obr/escherichia_coli_1.htm
www.cbc.ca
Capsule and biofilm
• Capsule surrounds an individual bacterium or a
couple of bacteria. It is not an integral part of a
bacterial cell, rather a layer of molecules (mostly
polysaccharides) that protect the cell. Usually
negative staining is used (capsule is an unstained
place on a stained background)
• Biofilm is a complex layer, composed of bacteria,
their capsullae and other material. Biofilm is much
stronger than individual bacteria, living in so
named planctonic form.
Endospore forming (sporulation)
• Endospore forming is something like
winter sleep, but much stronger
• Endospores can survive high temperatures,
drying, disinfection and so on
• An endospore is formed : cell is divided, but
not entirelly: one part is transformed into an
endospore, that comes inside the second part
• Bacterial endospores × fungal spores!
Endopores of various species of genus
Bacillus
http://membres.lycos.fr/neb5000/BacteriologieI/Groupe
s%20Bacteriens/Batonnets%20et%20coque%20Grampositifs%20formant%20des%20endospores.htm
Endospores do not stain themselves. They
are visible as „empty place“ inside bacteria
www.cropsoil.uga.edu/~parrottlab/Bugs/index.shtml.
Diagnostics
of bacteria
Diagnostics: detection of bacteria
and their determination
• Practical medical microbiology means
that a doctor (general pactitioner,
specialist, doctor from hospital) sends a
specimen to the lab
• A laboratory of medical microbiology
has to prove eventual presence of
bacteria in such a specimen, and
eventually to determine them.
• The determination does not need to be
perfect, but it has to give enough
information for treatment
Specimen versus strain I:
specimen
Specimen is what is taken from the pacient
and comes for laboratory examination
• liquid or solid material in a test tube or
other test tube (blood, serum, urine...)
• cotton swab, usually in trasport medium.
At diagnostics, sometimes we work with the
whole specimen. In other cases we work just
with a strain or strains of pathogenic
microbes
Specimen versus strain II: strain
Strain is pure culture („cultivate“) of one
species of a microbe
Strain can be gained only by
cultivation of a microbe on a solid
medium.
Koch‘s discovery, that bacteria can be
cultured like that, was essential for
modern microbiology.
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 (usually)
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
Microbiological laboratory
Foto O. Z.
Microscopy
What can we see in a microscope
• When we work with a strain, we can see
one type of microbial cells
• When we work with a specimen, we can
see
– microbes – sometimes no microbes, sometimes
more than ten various species of organisms
– cells of host organism – usually epitheliae,
WBCs, sometimes RBCs and other cells
– other structures, e. g. fibrin fibers, cellullar
detritus etc.
Types of microscopy
• Electron microscopy – in viruses, rather
research than routine diagnostics
• Optical microscopy
– Wet mount – large and/or motile organisms
(parasites, fungi, motile bacteria)
– Wet mount – dark field (mostly spirochets)
– Fixated and stained preparations, e. g.
• Gram staining – most important bacteriological stain
• Ziehl-Neelsen staining – e. g. for TB bacilli
• Giemsa staining – to some protozoa
• Gomori trichrom staining – for some other protozoa
• Fluorescent staining – for better visualisation
Comparison of size: yeast of genus
Candida and bacterium Staphylococcus
Photo: archive of the institute, from www.medmicro.info
Microscopy
of a
specimen
Microscopy of
a strain
Photo O. Zahradníček
Microscopy of a strain
(grampositive rods)
Foto O. Z.
Mikroscopy of a specimen (WBCs, G– cocci)
Foto O. Z.
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.
Wet mount × stained preparations
• In wet mount there is a layer between the
slide and the coverslip. It is difficult to focus
the objects in that layer. Therefore it is
recommended to start with 4× magnifying
objective, and to continue with 10×, (20×)
and 40× magnifying ones.
• In stained preparations all objects lay in
one plane (surface of the slide). It is quite
easy to focus it. Use 100× immersion
objectiv only. Never use non-imersion
objectives, even not before droping the oil!
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
Wet mount (native
preparation)
• In case of a wet mount a drop, in which there is
a specimen or mixed strain, we do not dry. We
only cover the preparation by a coverslip and we
observe by objectives, magnifying e. g. 4×, 10×,
20× or 40×.
• We use no immersion oil
• Wet mount can be also done from the specimen,
e. g. C..A..T. medium with pacient‘s swab. Then
we observe microbes, but also epithelial cells,
eventually leucocytes.
Wet mount – procedure (with a strain)
An example of a wet mount C. A. T.
http://www.kcom.edu/faculty/chamberlain/
Website/lectures/lecture/image/clue3.jpg
Stained
preparation
Preparing a stained preparation
• We start again by a drop of specimen or of
a strain mixed in saline. In this case, the
smaller the drop is, the better.
• A drop is let to dry. It is allowed to help
drying by placing near to the burner.
• After drying, the preparation is fixated by
drawing the slide throught the flame of
the burner. It is necessary to check the
temperature by your hand.
Simple staining
• May be used if necessary directly by
a general practicioner, that is far
from the nearest microbiological
laboratory and has methylene blue and
a microscope.
• Not commonly used in practice
• Shows us size, shape and
arrangement of microbes
Simple staining – how to do it
• Fixated preparation place on a grid in
sink and pour methylene blue on it
• Let is be some two minutes
• Then wash by tap water, and dry by
filtration paper
• Add a small drop of imersion oil and
observe by an objective magnifying 100 ×
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 staining
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 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
www.arches.uga.edu
Gram-negative cell-wall
www.arches.uga.edu
Gram staining – principle 1
•Gram -positive bacteria have a thick
peptidoglycan layer in the cell wall.
– So, gentiane/crystallin violet binds more firmly
to them, and…
– …after confirmation of this bound by Lugol
iodine solution…
– …even alcohol is not able to decolorize them.
•Gram-negative bacteria are decolorized
by alcohol and then stained pink by
safranin.
Gram staining – principle 2
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
Other bacteria than typical G+ and G– bacteria:
Mycobacteria are acid fast. Their cell wall is hydrophobic. They stain neither
violet, nor red; they do not stain at all. Sometimes we describe them as
„Gram-non-staining bacteria“
Mycoplasmas have no cell wall at all, but their cytoplasm stains weakly red.
Nevertheless, they are poorly visible (also becasuse they are small), Gram
staining is usually not used for them
Spirochetes have gram-negative cell wall type, but they are very thin, so they
are poorly visible and Gram staining is usually not used for them
Other structures than
bacteria: how do they stain?
• Yeasts mostly stain violet like G+ bacteria.
They have a cell wall of their own type, but in
Gram staining it works similarly like that of
Gram positive bacteria
• Human cells stain mostly red, although nuclei
may be partially blue
Do not forget, that preparations of specimens
might contain various fibers, cell detritus etc.,
and all preparations may contain staining
artifacts, too. Sometimes they are very
confusing and may be mistaken for bacteria by
a non-experienced observer!
Gram stained preparation
http://textbookofbacteriology.net/Enterococcus.jpeg
Streptococci, genus
Enterococcus
Gram stain – procedure
Part One: fixated preparation
1.We make a small
saline drop
2.We sterilize your loop
and wait until it stops
to be too hot
3.We take some mass of
microbes by your loop
(stains A to E)
4.We mix in the drop
5.We sterilize your loop
again and place back
6.We let the drop dry, or
dry AROUND your
burner
7.We fixate the slide by
passing it THOUGH the
flame of the burner
8.We move the slide to
the sink for staining
Part Two: proper Gram staining
•
•
•
•
•
•
•
•
•
•
Gentian/crystaline violet (20 –) 30 sec.
(rinse by tap water – not necessary)
Lugol (20 –) 30 sec.
(rinse by tap water –not ncessary)
Alkohol 15 (– 20) sec.
rinse by tap water!!! imporant!
Safranin 60 – 120 sec.
rinse by tap water
dry by filtration paper
microscopy as in Task One
Mixture of gram-positive and
gram-negative bacteria
www.arches.uga.edu
G+
G–
More staining
methods
Burri capsular staining
In Burri staining,
bacteria were stained
red and the
bacground by black
ink. Capsule is the
unstained place
between the red
bacterium and the
black ink.
pathmicro.med.sc.edu
Giemsa staining
• Giemsa staining is a staining method
with methylalcohol fixation (not flame
fixation) and after that staining with blue
stain according to Giemsa-Romanowski.
We use it for staining of vaginal
secretions and smears for malaria
Vaginal smear
Giemsa staining
http://en.microdigitalworld.ru
Ziehl-Neelsen staining
• Ziehl-Neelsen staining is used for so
called acidoresistant (acid fast) bacteria,
that have hydrophobic cell wall. Such
bacteria are e. g. mycobacteria, partially
also nocardiae and actinomycetes
Mycobakteria
Ziehl-Neelsen stain
www.primer.ru
Gomori trichrom staining
• Gomori trichrom staining is used for
intestinal protozoa (Giardia intestinalis,
Entamoeba histolytica etc.)
• It is very complex with many steps
• It enable us differentiation between
pathogenic and non-pathogenic species of
intestinal amoebae
• As alternative, it is also possible to use
staining using Heidenheim‘s haematoxylin
Practical
notes to
microscopy
Practical advice:
• Objectives should be cleaned by soft
tissue only. Once used tissue is
thrown out and not used once more.
• The microscopical table is cleaned by
paper square. Also this is thrown out
after use
After having finished the microscopy, it is
necessary:
• When using imersion, clean the
imersion objective by gase and petrol
• Eventually clean like this also other
objectives, if they are dirty
• The table of the microscope clean by a
square of paper, eventually also with
petrol
• Switch off and cover your microscope
The End
Lugol iodine = mixed I2 + KI
Jean Guillaume Auguste Lugol (18 August 1786 – 16
September 1851) was a French physician. He was born
in Montauban. He studied medicine in Paris and
graduated MD in 1812. In 1819 he was appointed
acting physician at the Hôpital Saint-Louis a post he
held until he retired. Lugol was interested in
tuberculosis and presented a paper to the Royal
Academy of Science in Paris in which he advocated the
use of fresh air, exercise, cold bathing and drugs. He
also published four books on scrofulous diseases and
their treatment (1829, 1830, 1831, 1834). He
suggested that his iodine solution could be used to
treat tuberculosis. This assertion attracted much
attention at the time. Although not efficacious in
treating tuberculosis, Lugol's iodine was successfully
used to treat thyrotoxicosis by Plummer.
http://en.wikipedia.org/wiki/Jean_Guillaume_Auguste_Lugol
www.jergym.hiedu.cz
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Prof. Christian Gram
Hans Christian Joachim Gram
(September 13, 1853 - November 14,
1938) was a Danish bacteriologist. Gram
studied botany at the University of
Copenhagen and and was an assistant in
botany to the zoologist Japetus
Steenstrup. He entered medical school
in 1878 and graduated in 1883. In
Berlin, in 1884, he developed a method
for distinguishing between two major
classes of bacteria. In 1891, Gram
became a lecturer in pharmacology, and
later that year was appointed professor
at the University of Copenhagen. In
1900 he his Chair in Pharmacology to
become Professor of Medicine.
en.wikipedia.org/wiki/Hans_Christian_Gram.
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Lipopolysacharide of G– cell wall
• It contains lipid A. This lipid is also called
endotoxin. It is released when the cell is
broken. It is an important factor of virulence.
• In also contains the polysaccharide part.
It is a virulence factor; it also contains body
antigens (called also O-antigens). These
antigens are often important in diagnostics
(especially in enterobacteria like Escherichia
coli or Salmonella)
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