Topic J03: Cultivation of bacteria and yeasts Materials for study (from textbooks, internet etc.): Bacterial culture. Use also your notes from Chemistry and Biochemistry (e. g. Schiff reagent etc.) Task 1: Characterisation of media and their production Task 1a: Most important media in medical microbiology Look at given media and write here the type of medium according to explanation given by your teacher. Do not forgot to write, whether the medium is solid or liquid and whether it is in a Petri dish or in a test tube. Name of medium Liquid/solid Petri dish/test t. Colour Type of medium (selective, …) Used for bacteria*: 1. Broth 2. VL-broth 3. Selenite broth 4. Sabouraud agar 5. Löwenstein-Jenssen medium 6. Blood agar (BA) 7. Endo agar (EA) 8. Mueller Hinton (MH medium) 9. BA with 10 % NaCl 10. VL agar (VLA) 11. XLD 12. Chocolate agar 13. Levinthal agar 14. Slanetz-Bartley medium *not necessary to fill everywhere, only in media used for diagnostics of certain bacteria Task 1b: Manufacturing of blood agar. Look at the video. Fill in missing parts of following text: If we want to manufacture blood agar, we have to mix together following components: Now, the components are heated using Arnold apparatus, and sterilised. Now, we let the temperature to decrease. At temperature beneath 55 °C we add ________________________________. Then we pour the agar into ______________________________ or we use _______________________________________________. Eventual more notes to blood agar manufacturing: Task 2: Influence of physical and chemical conditions on the bacterial growth Task 2a: Influence to oxygen Five strains (J, K, L, M, N) were cultured on agar plates in four types of conditions: a) normal athmosphere b) elevated CO[2] concentration c) decreased oxygen concentration d) no oxygen at all (oxygen replaced by a mixture of other gases) Write „G” (growing) or “N”(not growing); assess, which strain is strictly aerobic, facultatively anaerobic, strictly anaerobic, microaerophilic, capnophilic Result: Strain normal air elevated CO[2] traces of oxygen only no oxygen conclusion J K L M N Task 2b: Influence of salts and antibiotics You can see three strains on various types of media. Describe the presence/absence of growth Mark: GROWS – DOES NOT GROW Strain Blood agar (BA) BA + NaCl (6,5 %) BA + NaCl (10 %) Slanetz Bartley agar (Na-azide) BA with amicacine E – Enterococcus SR – Streptococcus ST – Staphylococcus Task 2c: Influence of temperature Like the previous task, only with the same medium, but different temperature. Mark: GROWS – DOES NOT GROW Strain 4 °C 37 °C 42 °C PSAE PSFL Task 3: Properties of the two most common diagnostic and selective-diagnostic media Task 3a: Blood agar – viridation and hemolysis Blood agar may be considered to be an enriched medium (with RBCs) but it is also a diagnostic medium. Following changes may be observed on it: Total haemolysis – bacterie with their activity destroy the erythrocytes around them tolally, blood agar becomes serum-colloured, it is transparent Partial haemolysis – bacterie using their activity destroy erythrocytes only partially, blood agar around colonies is only half-translucent and its colour is yellowish (no greenish tone) Viridation – change of red blood colour to a green one; agar around colony becomes greenish No change – majority of bacteria do not change the agar Describe haemolytical properties of four strains on blood agar. Read against light. Observe the colour of the haemolysis, not the colour of the bacterial colony itself. Streptococcus pyogenes (SRPY) Streptococcus agalactiae (SRAG) Streptococcus pneumoniae (SRPN) Enterococcus faecalis (ECFS) Task 3b: Endo agar – presence/absence of growth, lactose fermentation Describe growth/no growth, and changes of the medium surrounding the colonies. Staphylococcus epidermidis (STEP) Escherichia coli (ESCO) Salmonella Enteritidis (SAEN) Task 4: Description of morphologic characteristics of colonies Describe three strains of bacteria. If it is impossilbe to fill a cell, enter a reason (e. g. „too small“) Strain Strain Strain Size Colour Shape Profile Surface Edges Translucency Consistency Odour Surroundings Task 5: Inoculation of samples and strains on solid media Task 5a: Inoculation of a swab Inoculate a swab on the medium. Draw your result. Task 5b: Inoculation of a strain Inoculate a strain on the medium. Draw your result. Check-up questions: 1. Why VL broth is covered by parafin oil? 2. Why red blood cells are used only after the agar gets cold? 3. Why gelatine is usually not used at making solid media? 4. Microaerophilic and capnophilic conditions: is it the same? 5. Is the ability of staphylococci to grow at high NaCl concentrations related with its adaptation related to the macroorganism? 6. What characteristics cannot be seen by one’s eye? And what characteristic requires touching the colony? 7. Why it is so important to obtain isolated colonies at cultivation? 8. Blood agar is made of “basis for blood agar” (in fact it is nutrient agar) and defibrinated sheep blood. Is it possible to add blood to other bases?