Searching for Microbes Part XIV. Review to the spring term

1. Searching for MicrobesPart XIV.Review to the spring term Ondřej Zahradníček To practicals of VLLM0421c zahradnicek@fnusa.cz

2. One more tale… • Once three policemen discussed what method is the most important. • One said: Dactyloscopy is the best! It helps to identify any criminal! • The second: But firstly you have to catch him! So, terrain searching is the primary • But the third, the eldest, said: The most important is to use all our methods together, and to know, when to use each of them.

3. 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.

4. 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)

5. 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

6. Survey of direct methods *but in molecular epidemiology – detection of simillarity of strains - yes

7. Microscopy

8. 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

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

10. Main microscipical methods in medical microbiology

11. 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.

12. Wet mount – procedure

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

14. Simple staining

15. 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

16. Gram stained preparation Photo: Helena Janochová and Zuzana Jurčíková

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

18. Gram-positive cell wall

19. Gram-negative cell-wall

20. 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.

21. Mixture of gram-positive and gram-negative bacteria Photo: Helena Janochová and Zuzana Jurčíková G– G+

22. Culture

23. Do matter the conditions for bacterial growth? Of course yes! 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!

24. 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.

25. 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. Photo O. Z.

26. 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)

27. 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 a strain – solid media only • To differentiate and divide them mutually –diagnostic and selective media are used, for identification

28. 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

29. 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 www.medmicro.info

30. 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.

31. Liquid media www.medmicro.info

32. 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 developped to multilply some bacteria and to supress multiplication of other. Example: selenite broth for salmonella

33. Solid media www.medmicro.info

34. 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.

35. 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

36. 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.

37. 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 stafylococci • Sometimes, selectivity is reached by an antibiotic addition. Blood agar with amikacin is selective for streptococci and enterococci

38. 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 Photo: O. Z. Photo: O. Z.

39. Chromogenic and fluorogenic media • Chromogenic 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) • Fluorogenic media: similar, with a fluorescent dye www.oxoid.com

40. Selective diagnostic media www.medmicro.info • 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.

41. Selective, diagnostic and selective diagnostic media – review

42. 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)

43. www.medmicro.info Chocolate agar

44. Special use media www.medmicro.info 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 www.medmicro.info

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

46. Survey of media – part two

47. Biochemical identification

48. Principle • Even between mammals there are differences. Human body is not able to produce vitamin C, body of some mammals is. • We offer certain substrate to a bacterium, and we search, whether bacteria change it into a product using an enzyme. A product has to be different from substrate by physical phase or colour. If it is not different, we use an indicator • There are a lot of ways technical form of this test type.

49. Practical ways of doing it • Quick tests (seconds to minutes) • Catalase test • Tests with diagnostic strips (oxidase) • Tests with incubation (hours to days) • Simple test-tube tests • Complex test-tube tests • Sests of simple test-tube tests • Tests in microtitration plate (miniaturisation) • Other tests (e. g. Švejcar's plate)

50. Catalase test • Catalase test: very simple: we mix bacteria with substrate (H2O2 solution). Bubbles = positivity. Principle: 2 H2O2 2 H2O + O2 medic.med.uth.tmc.edu/path/oxidase.htm