Searching for microbes Part III. Culture of bacteria & yeasts Ondřej Zahradníček to practical of VLLM0421c contacts to me: 777 031 969 zahradnicek@fnusa.cz ICQ 242-234-100 Survey of parts of this slideshow Tale • It was once a bacterium, and it was very small, and so without the microscope nobody has seen it, and in the microscope it was very difficult to see its shape. It was very unhappy, because it emphasized very much its beauty. • Once, Mr. Koch came. He put the bacterium onto a solid medium and hide it into a thermostat. Bacterium was very happy and quickly started to multiply… And Day After… • Pan Koch came, opened the thermostat, took out the Petri dish with medium, and he saw: his liked, tiny bacterium was visible by a naked eye! Of course, not as one bacterium, but as a strain of totally identical cells, that raised from that bacterium in form of a colony • The bacterium was very happy, and it showed its beauty to mr. Koch. It showed him its shape, size, profile of its colony, pigments and many other things. Multiplication of bacteria, growth curve Multiplication of bacteria • Each bacteria has its generation period • During one gereration period one makes two, in ten times of the period one makes 1024 bacteria (theoretically) and so on,. • Ideal multiplication would exist only if we would add all the time nutrients and eventually oxygen and we would remove waste products. Real growth curve • Phase of latence – we let bacteria grow, but they still do not multiply • Exponencial phase – growth accelerates • Stational phase – they grow with the same speed all the time • Slowing and stopping of growth – lack of nutrients, to many waste, or bacteria regulate themselves by „quorum sensing“ Conditions needed for bacterial (fun- gal) culture Do the conditions for bacterial growth matter? 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 • NaCl concentration needed around 0,9 % (physiological saline) – but staphylococci, that have to be able to multiply on sweated skin, multiplies even at 10 % of salt! Culture thermostat Example of use of relation to temparature 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 molds grow at lower temperature. Elevated temperature is suitable e. g. for Campylopacter Influence of NaCl concentrations to the growth of some bacterial genera Besides various NaCl concentrations • Adding of sodium azide enables growth of enterococci, but neither staphylococci, nor streptococci are able to grow • Amikacin enables growth of streptococci and enterococci. Staphylococci, sensitive to amikacin, are eliminated. A little about catabolism of bacteria • We have three types of catabolism: – Fermentation – nutrients breakdown without need for oxygen. Little energetically effective, but does not need oxygen. Forduct is e. g. lactate, ethanol etc. – Aerobic respiration – little nutrients gives a lot of energy, but oxygen is needed. Forduct is CO[2] and H[2]O – Anaerobic respiration – another electron acceptor • Type of catabolism is closely connected with relation to oxygen. Fermentating bacteria are usually facultatively or scrictly anaerobic. On the other hand, aerobically respirating bacteria use to be scrictly aerobic. Relation to oxygen • Strict aerobes grow only in presence of oxygen • Strict anaerobes they grow only in environment without oxygen • Facultative anaerobes and aerotolerant bacteria (it is not possible to differentiate them) grow at all conditions • Microaerophile bacteria grow only in conditions with traces of oxygen [• ]Capnophile bacteria need more CO[2] Growing anaerobic bacteria Bacterial culture – introduction 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; invented by Robert Koch – To differentiate and divide them mutually –diagnostic and selective media are used, for identification 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 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 Solid media Is it good, or bad, that various bacteria need different conditions? • It is bad, because it complicates deffinition of conditions, that would be suitable for majority of bacteria • It is good, because this enables to use it in diagnostics (e. g. growth ability on medium with 10 % NaCl differenciates well stafylococci) Media globally versus media in medical microbiology • In industrial microbiology or in some other applications we use mostly chemically defined media. We know their composition, and it is possible to observe, how much of something increased or decreased. • In clinical microbiology we have no need to know a detailed composition. Often some parts of media are not definable (red blood cells, yeast extract). 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. Various specimens – various cultivation • How the specimen type influences culture? – Specimens, where microbes use to be rare are inoculated into liquid media only. Microbes multiply quickly. Example: conjunctival swab – Specimens, where the amout of microbes may vary, but even small amounts are important are cultured on both liquid and solid media. Example: wound swab – Specimens, where usually we have many microbes, eventually even common microflora, are cultured on solid media only. Example: throat swab Liquid media Liquid media Classification of liquid media • Liquid media have two categories only: • multiplying media 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 multiplying media were developped to multilply some bacteria and to supress multiplication of other. Example: selenite broth for salmonella Solid media: inoculation of specimen and strain Solid media Solid (agar) media • To have all advantages, given by solid media, we have both the specimen musíme specimen (cultivation specimen à 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 halfth 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. 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. In case of a mixture, each bacterium forms its own colonies (at a proper dilution inoculation) How to inoculate of a specimen to a medium How to reinoculate of an agar culture What to describe at colonies • Size • Colour • Shape (round…) • Forfile (convex…) • Edges • Surface (smooth, rough…) • Consistence (dry…) • Transparency • Smell • Colony surroundings* Difference between shape and profile Solid media: classification and examples 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 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 Changes on blood agar • All media with RBCs (blood agar, VL bloodní agar, agar with washed RBCc etc. –but not blood agar with 10 % NaCl, where RBCs are lyzed) enable us to see: Chromogenic and fluorogenic media • Chromogenic media contain a dye with bound specific substrate à 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 Chromogenic media for yeasts Properties of Endo agar • Endo agar enable growth of G- bacteria only, and and even not all of them (selectivity) • The growing bacteria can be differenciated into lactose positive (red) and negative (pale). XLD and MAL media for Salmonella Selective, diagnostic and selective diagnostic media – review 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) Chocolate agar Dry, black colonies show biofilm production Special use media – 2 In vitro antibiotic susceptibility testing: Müller-Hinton agar; also to pigments production observation Note In bacteria requiring growth factors even antibiotic testing should be perfromed on enriched media Modern trends in culture • Despite development of genetic methods, cultivation still keeps its key role in mostly bacterial diagnostics • Because of standardization, laboratories have to switch from „home made“ media to comercial products • Chromogenic and fluorogenic media start to be used more and more, despite the price Survey of the most important media 1. Broth 2. VL-broth 3. Selenite broth 4. Sabouraud 5. Löwenstein-Jenssen 6. Blood agar (BA) 7. Endo agar 8. MH 9. BA + 10 % NaCl 10. VLA (VL BA) 11. XLD (and MAL) 12. CHA 13. Levinthal 14. Slanetz-Bartley Survey of media – part one Survey of media – part two The End Robert Koch Once more Robert Koch Robert Koch in Egypt expedition during a cholera epidemics Solid media in a test tube? Why? Among given media, two of theme are in test tubes, although they are solid. The reason is that they are used for slowly growing organisms. Both mycobacteria (Löwenstein-Jensen) and some molds (Sabouraud) grow slowly and the medium would be dry before the organism would grow on a Petri dish In case of Hajna medium (see J04) the reason is different: the medium is used for biochemical testing and the difference between the lower part (no access to oxygen) and upper part (surface of medium) is important for its function Löwenstein Jensen medium is also interesting as is it solid although agar is absent; it is solid because of coagulated eggs. Blood agars • It is possible to use blood agar with red blood cells of various organisms (horses, chicken, cattle, and even humans). Nevertheless, the sheep RBCs are far most used ones • It is possible to add blood cells to various bases. For example, if you add blood to VL broth (simplified), you get VL agar (VL blood agar) • For haemolytical interaction testing (e. g. CAMP test, see P02) it is recommended to use agar with washed red blood cells. Endo agar and its principle • Endo agar contains lactose as a substrate • It also containts basic fuchsin • This fuchsin acts as factor of selectivity • The same fuchsin (together with Na[2]SO[3]) also acts as indicator (Schiff reagent). Bacteria forming lactaldehyde from lactose are visualised by purple colour Endo agar should be kept away from light, otherwise it becomes purple without bacteria.