Searching for MicrobesPart I.

Introduction to Diagnostics

Microscopy of microbese-learning version

Author of the slideshow: Ondřej Zahradníček

To practicals of VLLM0421cContact to me:

zahradnicek@fnusa.cz

Laboratory safety and practical advice

Clinical microbiology, definition and relations

Morphology of bacteria

Diagnostics of bacteria

Microscopy

Wet mount

Stained preparation

Hyperlinks to individual parts

Gram staining

Some practical notes to microscopy

More staining methods

Two tales to start

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 questionaireAfter 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

Human medical microbiologyVeterinary

medical microbiology

General microbiology

Molecular biology and genetics

Cell biology

Infectious medicine

Dermato- venerology

Epidemiology of infectious diseases

Plant microbiology

Who are clinical microbiologists

Medical microbiology

Industrial microbiology

Other medical branches

Polytechnical institutes

Faculties of Science

Medical faculties

Basic microbiological research

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.

Neisseriae

www.meningitis.de/erreger/meningokokken.html

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)

Bacteria are interesting!

Picture is made by

as. MUDr. Petr Ondrovčík, CSc.,

former lecturer of the Institute, deceased young in autumn 2007

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

Spirochets

http://nl.wikipedia.org/wiki/Afbeelding:TreponemaPallidum.jpg

www.primer.ru/std/gallery_std/treponema.htm

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

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.

www.cbc.ca

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/Groupes%20Bacteriens/Batonnets%20et%20coque%20Gram-positifs%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 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

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 fixation

Coverslip Imersion system

Wet mount

no yes noDarkfieldwet mount

no yes yes

Stained preparat.

yes no yes

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.jP7201179.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 2Chemical Gram-positive Gram-

negative

Crystal. violet Staining violet Staining violet

Lugol iodine Confirmation Less confirm.

Alkohol Not decolorized

Decolorized

Safranin Remain violet Stain to redOther 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 themSpirochetes 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

Streptococci, genus Enterococcus

Cocci in chains

http://textbookofbacteriology.net/Enterococcus.jpeg

Gram stain – procedure Part One: fixated preparation1.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

G+

G–

www.arches.uga.edu

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 smearGiemsa 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

MycobakteriaZiehl-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 tomicroscopy

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 + KIJean 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 GramHans 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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