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1Author of the slideshow: Ondřej Zahradníček Searching for Microbes Part I. Introduction to Diagnostics Microscopy of microbese-learning versionAuthor of the slideshow: Ondřej ZahradníčekTo practicals of VLLM0421cContact to me:
2Hyperlinks to individual parts Two tales to startLaboratory safety and practical adviceClinical microbiology, definition and relationsMorphology of bacteriaDiagnostics of bacteriaMicroscopyWet mountStained preparationGram stainingMore staining methodsSome practical notes to microscopy
4Tale 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…
5Tale 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
6Tale continuesHe 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.
7And 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.
9Practical adviceBoxes 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 keyboardNext to the keys from boxes you can find and WC key. After coming back from WC, put it back to its placeIt is forbidden to students to enter the space behind the practical‘s hall!
10What to do with waste and garbage Small pieces may be placed to the small vessels directly on your tablesUsed matches have their own boxLarger 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 baskedMicroscopical preparations prepared by yourselves should be placed to the disinfection after usePlease, do not let mess on the tables (e. g. debris from rubbering, pen sharpening etc.)
11LiteratureYou 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!
12Credit 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
13More instructionsYou 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 picturesDraw 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 assistantYou will show your lab reports to the examinator at the examination and they will definitelly influence your final result at the examination!
14Here you can see, that lab reports are really important (and card of and Slovak student, partially translated into English)
15The cocci really ARE confluent in the microscope!
16ROPOT 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.
17Clinical microbiology, its definition and relations to other subjects
18Classification of our branch Plant microbiologyMolecular biology and geneticsInfectious medicineHuman medical microbiologyEpidemiology of infectious diseasesGeneral microbiologyVeterinary medical microbiologyDermato- venerologyCell biology
19Who are clinical microbiologists Basic microbiological researchMedical microbiologyOther medical branchesIndustrial microbiologyPolytechnical institutesFaculties of ScienceMedical facultiesYour teachers are blue
20A microbe (microogranism): what does it mean? It should be living. A grain of dust is not a microbe, although it is microscopicalIt should be microscopical. A giraffe is not a microbe, although it is livingThe second condition is not absolute. For example, and tapeworm can measure 10 m. But the eggs are microscopical, so it belongs to the microbiology.
21Various microbesAmong 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 springsFor us, clinical microbiologists, these microbes are not target of our interest. Nevertheless, they are very interesting.
22What these microbes can They live in depth of 10 kmThey survive temperatures around 110 °CThey stand strong radioactivityInstead 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.
24Classification of living organisms Prions – no DNA, usually not counted to be living organisms at allViruses and bacteriophagesCellullar organismsArchaea (archeobacteria)Eubacteria (eubacteria)Eucarya (eukaryotic organisms)monocellullarpolycellullar
25Medically 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!
26Medically important microbes 2 Each organism has its medically important microbes: human, each species of animal or plantEven some microbes (e. g. bacteria) have their own microbes (bacteriophagi).
28Main 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 parasitesProtozoa (e. g. Plasmodium malariae)Flukes (e. g. Schistosoma haematobium)Roundworms (e. g. Ascaris lumbricoides)Tapeworms (e. g. Taenia saginata)Outer parasites (lice, fleas, bugs)
29Bacteria are interesting! Picture is made byas. MUDr. Petr Ondrovčík, CSc.,former lecturer of the Institute, deceased young in autumn 2007Bacteria are interesting!
31Morphology of medically important microorgamisms Viruses are composed of DNA or RNA and proeins; some viruses possess an envelope „stolen“ to a host cellViruses 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.
32Morphology 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 wayCoccobacilliSpirochets – thin spiral bacteriaAmorph bacteria, e. g. mycoplasms (they do not have any wall, their shape is changing)
33Cocci in chains (electronoptic microphotograph of Enterococcus sp.)
36Fimbriae and a flagella Many bacteria are able to moveThey move mostly by a flagellaFimbriae (pilli) are used to movement, adhesion and to excange of genetical informationBacterial flagellae are different from flagellae of eukaryotic organismsFimbriae and flagellae are invisible in optical, but often in electronoptical microscopy
38Capsule and biofilmCapsule 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.
39Endospore forming (sporulation) Endospore forming is something like winter sleep, but much strongerEndospores can survive high temperatures, drying, disinfection and so onAn endospore is formed : cell is divided, but not entirelly: one part is transformed into an endospore, that comes inside the second partBacterial endospores × fungal spores!
43Diagnostics: detection of bacteria and their determination Practical medical microbiology means that a doctor (general pactitioner, specialist, doctor from hospital) sends a specimen to the labA 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
44Specimen versus strain I: specimen Specimen is what is taken from the pacient and comes for laboratory examinationliquid 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
45Specimen versus strain II: strain Strain is pure culture („cultivate“) of one species of a microbeStrain 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.
46Survey of methodsDirect 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 determinationIndirect 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
47Survey of direct methods Specimen examinationIdentificationMicroscopyyesCultivationBiochemical identificat.noAntigen detectionAnimal experimentusually notMolecular methodsusually not**but in molecular epidemiology – detection of simillarity of strains - yes
50What can we see in a microscope When we work with a strain, we can see one type of microbial cellsWhen we work with a specimen, we can seemicrobes – sometimes no microbes, sometimes more than ten various species of organismscells of host organism – usually epitheliae, WBCs, sometimes RBCs and other cellsother structures, e. g. fibrin fibers, cellullar detritus etc.
51Types of microscopyElectron microscopy – in viruses, rather research than routine diagnosticsOptical microscopyWet 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 stainZiehl-Neelsen staining – e. g. for TB bacilliGiemsa staining – to some protozoaGomori trichrom staining – for some other protozoaFluorescent staining – for better visualisation
52Comparison of size: yeast of genus Candida and bacterium Staphylococcus Photo: archive of the institute, from
53Microscopy of a specimen Microscopy of a strainPhoto O. Zahradníček
54Microscopy of a strain (grampositive rods) Foto O. Z.
55Mikroscopy of a specimen (WBCs, G– cocci) Foto O. Z.
56Main microscipical methods in medical microbiology Drying and fixationCoverslipImersion systemWet mountnoyesDarkfieldwet mountStained preparat.
57Wet 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!
58Preparing 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 slideIf 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.
60Wet 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 oilWet 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.
64Preparing 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.
65Simple stainingMay 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 practiceShows us size, shape and arrangement of microbes
66Simple staining – how to do it Fixated preparation place on a grid in sink and pour methylene blue on itLet is be some two minutesThen wash by tap water, and dry by filtration paperAdd a small drop of imersion oil and observe by an objective magnifying 100 ×
70Bacterial cell wallThere 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.
73Gram 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.
74Gram staining – principle 2 ChemicalGram-positiveGram-negativeCrystal. violetStaining violetLugol iodineConfirmationLess confirm.AlkoholNot decolorizedDecolorizedSafraninRemain violetStain 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
75Other 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 bacteriaHuman cells stain mostly red, although nuclei may be partially blueDo 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!
76Gram stained preparation Cocci in chainsStreptococci, genus Enterococcus
77Gram stain – procedure Part One: fixated preparation We make a small saline dropWe sterilize your loop and wait until it stops to be too hotWe take some mass of microbes by your loop (stains A to E)We mix in the dropWe sterilize your loop again and place backWe let the drop dry, or dry AROUND your burnerWe fixate the slide by passing it THOUGH the flame of the burnerWe move the slide to the sink for staining
78Part 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 waterdry by filtration papermicroscopy as in Task One
79Mixture of gram-positive and gram-negative bacteria G–G+
81Burri capsular staining pathmicro.med.sc.eduIn 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.
82Giemsa stainingGiemsa 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 malariaVaginal smearGiemsa staining
83Ziehl-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 actinomycetesMycobakteriaZiehl-Neelsen stain
84Gomori trichrom staining Gomori trichrom staining is used for intestinal protozoa (Giardia intestinalis, Entamoeba histolytica etc.)It is very complex with many stepsIt enable us differentiation between pathogenic and non-pathogenic species of intestinal amoebaeAs alternative, it is also possible to use staining using Heidenheim‘s haematoxylin
86Practical 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
87After having finished the microscopy, it is necessary: When using imersion, clean the imersion objective by gase and petrolEventually clean like this also other objectives, if they are dirtyThe table of the microscope clean by a square of paper, eventually also with petrolSwitch off and cover your microscope
89Lugol 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 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.Back
90Prof. Christian Gram Back Hans Christian Joachim Gram (September 13, 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 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.Back
91Lipopolysacharide 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)Back