Pátráme po mikrobech Díl XV. Opakování v angličtině + anglická terminologie v mikrobiologii

1. Pátráme po mikrobechDíl XV.Opakování v angličtině +anglická terminologie v mikrobiologii Ondřej Zahradníček K praktickým cvičením z VLLM0421c zahradnicek@fnusa.cz Pozor! Téma se vůbec neprobírá v prezenční výuce. Jeho absolvování se však kontroluje ve 14. praktiku.

2. Přehled témat Opakování jarního semestru v angličtině Zrádná slovíčka v angličtině Upozornění: Z technických důvodů tato prezentace může obsahovat některé pasáže, které již byly v příslušných tématech aktualizovány, avšak v této prezentaci (zaměřené spíše na anglická slovíčka) aktualizovány nejsou.

3. Opakování jarního semestru v angličtině

4. Diagnostics: detection of bacteria and their determination • Practical medical microbiology means that a doctor (general practitioner, 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

5. Specimen versus strain I: specimen Specimen is what is taken from the patient and comes for laboratory examination • liquid or solid material in a test tube or other test tube (blood, serum, urine...) • cotton swab, usually in transport medium. At diagnostics, sometimes we work with the whole specimen. In other cases we work just with a strain or strains of pathogenic microbes

6. 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.

7. 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

8. Survey of direct methods *but in molecular epidemiology – detection of similarity of strains – yes

9. Power switch Olympus microscope Eyepiece with ocular lens Objectives (with lenses) Stage with stage clips Diaphragm Stage control Lamp (with another diaphragm) Light regulator Fine focus and coarse focus

10. Immersion oil use • NEVER USE IMMERSION OIL WITH NON IMMERSION OBJECTIVE LENSES!!! • When using immersion oil with immersion objective, it is necessary to clean the objective after use

11. Cleaning the microscope • After use of microscope, non-immersion objectives should be only cleaned when dirty. Immersion objectives should be always cleaned by benzine („BENZÍN“) and soft gauze. Do not use filtration paper squares for cleaning objectives! It should be only used for cleaning of stage (= the flat part of the microscope, where your preparation is placed)

12. Discarding/cleaning the preparations • Preparations made by yourselves should be discarded after use to the disinfectant solution • Preparations already prepared at the start of the practical should be cleaned (see further) and placed back to the original Petri dish

13. How to clean a preparation without destroying it • Clean the preparation using benzine and filtration paper square from the underneath (bottom) side thoroughly • From upper side, do the same ONLY in areas where you see no visible preparation • Never try to clean the preparation part of your slide. It is only possible to blot out excess of immersion oil using filtration paper (without moving the paper against the slide!)

14. Main microscopic methods in medical microbiology You will learn the drying and fixation methods and all other techniques concerning preparing your preparation in first to practical sessions.

15. 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)

16. 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 capsules and other material. Biofilm is much stronger than individual bacteria, living in so named planctonic form.

17. 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 entirely: one part is transformed into an endospore, that comes inside the second part • Bacterial endospores × fungal spores!

18. 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 epithelial cells, WBCs, sometimes RBCs and other cells • other structures, e. g. fibrin fibers, cellular detritus etc.

19. 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

20. Comparison of size: yeast of genus Candida and bacterium Staphylococcus Photo: archive of the institute, from www.medmicro.info

21. Microscopy of a specimen Microscopy of a strain Photo O. Zahradníček

22. 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 patient's swab. Then we observe microbes, but also epithelial cells, eventually leucocytes.

23. 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 through the flame of the burner. It is necessary to check the temperature by your hand.

24. 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.

25. 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.

26. Gram staining – principle 2 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“ Mycoplasmashave no cell wall at all, but their cytoplasm stains weakly red. Nevertheless, they are poorly visible (also because they are small), Gram staining is usually not used for them Spirocheteshave gram-negative cell wall type, but they are very thin, so they are poorly visible and Gram staining is usually not used for them

27. 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 fibres, cell detritus etc., and all preparations may contain staining artefacts, too. Sometimes they are very confusing and may be mistaken for bacteria by a non-experienced observer!

28. Proper Gram staining • Gentian/crystaline violet (20 –) 30 sec. • (rinse by tap water – not necessary) • Lugol (20 –) 30 sec. • (rinse by tap water –not necessary) • Alkohol 15 (– 20) sec. • rinse by tap water!!! important! • Safranin 60 – 120 sec. • rinse by tap water • dry by filtration paper • microscopy as in Task One

29. Burri capsular staining pathmicro.med.sc.edu In Burri staining, bacteria were stained red and the background by black ink. Capsule is the unstained place between the red bacterium and the black ink.

30. 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

31. 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

32. 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

33. Do the conditions for bacterial growth matter? Of course they do! Majority of bacteria need their temperature, moisture, salts concentration and many other characteristics to be in a quite narrow range. lower survival limit (bactericidal) lower growth limit (inhibitory) lower growth limit (inhibitory) upper survival limit (bactericidal) Values, that enable microbial survival, are not sufficient. They should be able to multiply. Various microbes need various conditions!

34. Medically important bacteria • Temperature usually needed around 37 °C • but bird pathogens more (42 °C), microbes coming from outer environment less (30 °C) • Value of pH needed around pH 7 • but gastric helicobacter by far less • Water amount should be high, especially for G– • Osmolarity usually described as NaCl concentration, that is usually 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.

35. Culture thermostat (incubator) 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.

36. Example of use of relation to temperature in bacterial diagnostics • Pseudomonas aeruginosa grows at 37 °C and 42 °C. • On the other hand, Pseudomonas fluorescens grows at 4 °C and 37 °C. Besides Pseudomonas, Listeria, too, and yeasts and moulds grow at lower temperature. Elevated temperature is suitable e. g. for Campylobacter

37. 10 % 6,5 % Influence of NaCl concentrations to the growth of some bacterial genera Majority of Enterococci Staphylococci bacteria

38. Relation to oxygen often together described as just simply „aerobes“ • Strict aerobes grow only in the presence of oxygen • Facultative anaerobes and aerotolerant bacteria (it is not possible to differentiate them) grow at all atmosphere conditions • Strict anaerobes they grow only in environment without oxygen • Microaerophile bacteria grow only in conditions with traces of oxygen • Capnophile bacteria need more CO2

39. Term „colony“ • A colony is a formation on a surface of a solid media. It is developed 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 precisely, number of „colony forming units“ (CFU) • Description of colonies has an important place inbacterial diagnostics www.medmicro.info

40. Liquid media www.medmicro.info

41. Classification of liquid media • Liquid media have two categories only: • multiplyingmediaare common and universal. Example: broth for aerobic culture and VL-broth for anaerobic culture (VL = viande-levure, from French – contains meat-yeas extract) • selectively multiplyingmediawere developed to multiply some bacteria and to suppress multiplication of other. Example: selenite broth for salmonella

42. Solid media www.medmicro.info

43. Solid (agar) media • To have all advantages, given by solid media, we have both the specimen specimen (cultivation specimen  strain), but also strain (cultivation strain  strain) dilute properly at inoculation. Classical way of dilution inoculation is so named cross inoculation. In practice, usually e. g. one half of a plate is inoculated by a swab, and then diluted by a loop. Sometimes some discs and culture lines are added – not being a topic for today.

44. 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.

45. 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

46. How to inoculate of a specimen to a medium Using the swab, inoculate the on a part of the agar plate (to about one third of Petri dish diameter) Sterilize your loop Dilute from part with specimen, making the second part of inoculation Sterilize your loop Dilute from lines inoculated in the second phase (not touching the part inoculated by the swab) Sterilize your loop Inoculate the „serpent“ on the remaining part of the plate

47. How to reinoculate of an agar culture Sterilize your loop Take the strain Inoculate first phase Sterilize your loop Do not take the strain again Inoculate second phase Sterilize your loop Do not take the strain again Inoculate third phase Sterilize your loop Do not take the strain again Inoculate the „serpent“

48. Size Colour Shape (round…) Profile (convex…) Edges Surface (smooth, rough…) Consistence (dry…) Transparency Smell Colony surroundings* What to describe at colonies *Definition is related to the medium used. For example, haemolysis is observed around some bacteria grown on media with RBCs.

49. Solid selective media • They have to select (separate) from a bacterial mixtureonly one of several groups of genera • An example is blood agar with 10 % NaCl used for staphylococci • Sometimes, selectivity is reached by an antibiotic addition. Blood agar with amicacin is selective for streptococci and enterococci

50. Photo O. Z. Diagnostic media • They do not suppress growth of any microbe • On the other hand, their composition enable them to differentiate microbes according to some properties • An example is blood agar to observe haemolytic properties, and VL blood agar (similar, but to anaerobes). WCHA agar is a variant of VL blood agar. • Special case are chromogenic and fluorogenicmedia Photo O. Z.