Pátráme po mikrobech Díl XIV. Opakování + doplňkové téma

1. Pátráme po mikrobechDíl XIV.Opakování + doplňkové téma Ondřej Zahradníček K praktickému cvičení pro VLLM0421c Kontakty na mne: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100

2. Ještě jedna pohádka • Jednou se tři policisté přeli o to, která metoda je nejlepší. • Jeden řekl: Nejdůležitější je snímání otisků prstů, to pomůže určit zločince. • Druhý zas: Ale nejdřív ho musíte chytit! A tak je důležitější pročesávání terénu. • Ale třetí, nejstarší, jim řekl: Nejdůležitější je umět používat metody dohromady, a vědět, kdy kterou použít.

3. Angličtina v odborném kontextu • Angličtina je mezinárodním jazykem vědecké komunikace, podobně jako kdysi latina. Bez ní se lékař neobejde. • Učitelé jazyků vás mohou naučit konverzaci a základní pojmy, avšak přesné kontextové použití odborných slovíček z jednolivých oborů samozřejmě nemohou znát • Existují i „zrádná slova“ – uvidíte sami • Proto vzniklo toto doplňkové téma

4. A abychom si zároveň zopakovali, co jsme probrali v tomto semestru • Začneme opakováním v angličtině • Poté si ukážeme některá zrádná slovíčka v mikrobiologické angličtině • Poté si napíšeme zápočtový test • Studenti se splněnými podmínkami zápočtu poté mohou obdržet zápočty

5. A microbe (microogranism): what does it mean? • It should be living. and grain of dust is not and microbe, although it is microscopical • It should be microscopical. and giraffe is not and 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.

6. 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)

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 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 simillarity of strains - yes

9. Microscopy

10. Microscopy • We observe microbes, in specimen also cells of host organism(epitheliae, WBCs etc.) • Wet mount – for large and/or motile microbes (parasites, fungi, motile bacteria) • Dark field wet mount (mainly spirochets) • Fixated and stained preparations – Gram staining, Giemsa staining, Ziehl Neelsen staining (use for various groups of bacterií, fungi, parasites) • Electron microscopy – in viruses; rather for research than for common virological diagnostics

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

12. Main microscipical methods in medical microbiology

13. 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.

14. Wet mount – procedure

15. An example of a wet mount C. A. T. http://www.kcom.edu/faculty/chamberlain/Website/lectures/lecture/image/clue3.jpg

16. Simple staining

17. 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

18. Gram stained preparation Cocci in chains Streptococci, genus Enterococcus

19. 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 tenčí, thin, but more complex. They are called gram-negative bacteria. • Besides these and those, there are also so named Gram non-staining bacteria.

20. Gram-positive cell wall

21. Gram-negative cell-wall

22. Gram staining – principle • Gram-positive bacteria have thick peptidoglycan layer in the cell wall. So, gentiane/crystallin violet binds more firmly to them, and after confirmation of this bound by Lugol solution even alcohol is not able to decolorize them. Gram-negative bacterie are decolorized by alcohol and thed stained red by safranin.

23. Mixture of gram-positive and gram-negative bacteria G– G+

24. Culture

25. Do matter the conditions for bacterial growth? Of course not! Majority of bacteria need their temperature, moisture, salts concetration 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!

26. Medically important bacteria • Temperature usually needed around 37 °C • but bird pathogens more (42 °C), microbes coming from outside less (30 °C) • Value of pH needed around pH 7 • but gastric helicobacter by far less • NaClconcentration needed 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.

27. Culture thermostat 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.

28. Relation of bacteria to oxygen • Aerobic and facultative anaerobic (eventually aerotolerant)bacteria can be grown at normal athmosphere • Strictly anaerobic bacteria need athmosphere without oxygen • bacteria with special need for oxygen require special athmosphere (microaerophile and capnophile bacteria)

29. Why we culture bacteria • Why bacteria are cultured in the laboratory? • To keep them living and to multiply them. This is gained by cultivation in both liquid and solid media (jelly-consistence media, based on agar algae) • To obtain strain – solid media only • To differentiate and divide them mutually –diagnostic and selective media are used, for identification

30. Specimen and strain • Specimen is taken from a patient. Specimen contains cells macroorganism, various number of microbial species (zero to maybe twenty) and more items • A strain – an isolate – is a population of one bacteria, isolated from a specimen on a solid medium • To gain a strain, we have to grow a bacterium on a solid medium and inoculate carefully

31. Term „colony“ • A colony is a formation on a surface of a solid media. It is developped 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 preciselly, number of „colony forming units“ (CFU) • Description of colonies has an important place in.bacterial diagnostics

32. Liquid media and solid media • Liquid media are based on je meat-peptonic broth (exctract of cooked beef meat + protein hydrolysate). They are used mostly to multiplication. It is difficult to evaluate the result, in fact, only „non turbid broth – turbid broth“ (growth – no growth) • Majority of solid media are based on the same broth, but supplied by an agar alge extract. Bacteria grow slower on solid media, but the result is very variable, and it is possible to get a strain.

33. Liquid media

34. Classification of liquid media • Liquid media have two categories only: • multiplyingmedia are common and universal. Example: broth for aerobic culture and VL-broth for anaerobic culture (VL = viande-levure, from french – contains meat-yeas extract) • selectively multiplyingmedia were developped to multilply some bacteria and to supress multiplication of other. Example: selenite broth for salmonella

35. Solid media

36. Why an isolated colony is so important • Only ao 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.

37. 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

38. 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.

39. Solid selective media • They have to select (separate) from a bacterial mixture only one of several groups of genera • An example is blood agar with 10 % NaCl used for stafylococci • Sometimes, selectivity is reached by an antibiotic addition. Blood agar with amikacin is selective for streptococci and enterococci Illustration photo: NaCl crystals

40. Diagnostic media • They do not supress growth of any microbe • On the other hand, their composition enable them to differenciate microbes according to some properties • An example is blood agar to observe haemolytical properties, and VL blood agar (simillar, but to anaerobes) • Special case are chromogenic and fluorogenicmedia

41. Chromogenic and fluorogenic media • Chromogeninc media contain a dye with bound specific substrate  it loses it colour, it is no more a dye, but a chromogen • bacteria able to breakdown the specific substrate change the chromogen againt to the original dye • The medium may contain more chromogens (for more species) • Fluorogenoc media: similar, with a fluorescent dye

42. Selective diagnostic media • Combine selective and diagnostic properties • Example – Endo agar: • Only some G– bacteria can grow on it (selectivity) • The growing bacteria can be differentiated into lactose fermentative and lactose non fermentative • A simillar is McConkey medium, more common in world (but not used in OUR laboratory) • Selective diagnostic are also XLD, CIN media etc.

43. Selective, diagnostic and selective diagnostic media – review

44. Enriched and selective enriched media • For bacteria with specific need for nutrients • They are enriched by different chemicals • Even blood agar is an enriched medium, although shown as a diagnostic medium (it may be considered a member of both groups). • An expample of „pure enriched medium“ is chocolat and Levinthal agar for pathogenous Neisseriae and hemophili (that do not grow even on blood agar) • Media may be selective enriched (e. g. GC agar, – chocolat agar with anibiotics for culture of Neisseria gonorrhoeae)

45. Chocolate agar

46. Special use media – 1 • Observation of virulence factors • Picture shows a medium with Congo red for staphylococcal biofilm • or e. g. yolk agar for histotoxic clostridia Dry, black colonies show biofilm production Red, wet colonies mean no production of biofilm

47. Special use media – 2 In vitro antibiotic susceptibility testing: Müller-Hinton agar; also to pigments production observation Rigth, a non-pigmented Staphylococcus strain, left down a pigmented Pseudomonas strain

48. *only with antibiotics Survey of media – part one

49. Survey of media – part two

50. Biochemical identification