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VLLM0421c – Medical Microbiology I, practical sessions. Protocol to topic J02 Topic J02: 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: Characterization of media and their production Task 1a) The most important media in medical microbiology Take a look at the presented media and write down the type of medium according to the explanation given by your teacher. 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 Colour Type of medium Used for bacteria*: Petri dish/test t. (selective, …) 1. Broth 2. VL-broth 3. Selenite broth 4. Sabouraud agar 5. LöwensteinJenssen 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 (and similar MAL) 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) Preparing the blood agar Watch the video. Fill in the missing parts of the following text: If we want to prepare blood agar, we have to mix together the following components: Now, the components are heated using Arnold apparatus and sterilized. After that, we let the temperature decrease. At the temperature below 55 °C we add ________________________________. Then we pour the agar into ______________________________ or we use____________________________________________. Further notes to preparing blood agar: Name _____________________ General Medicine Date ___. ___. 2016 Page 1/4 VLLM0421c – Medical Microbiology I, practical sessions. Protocol to topic J02 Task 2: Effects of physical and chemical conditions on bacterial growth Task 2a) Effect of oxygen Five strains (J, K, L, M, N) were cultured on agar plates in four types of conditions: a) Normal atmosphere b) Elevated CO2 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. Results: Strain normal air elevated CO2 traces of no oxygen conclusion oxygen only J K L M N Task 2b) Effect 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 + NaCl (6,5 BA + NaCl (10 Slanetz Bartley (BA) %) %) agar (Na-azide) E – Enterococcus BA with amikacine SR – Streptococcus ST – Staphylococcus Task 2c) Effect of temperature The same as in the previous task but on the one type of medium and at different temperatures. Mark: GROWS – DOES NOT GROW Strain 4 °C 37 °C 42 °C PSAE PSFL Task 3: Properties of the two most common diagnostic and selectively-diagnostic media Task 3a) Blood agar – viridation and haemolysis Blood agar may be considered to be an enriched medium (with RBCs) but it is also a diagnostic medium. There can be observed the following changes on it: Total haemolysis – the activity of the bacteria completely destroys erythrocytes around them, the blood agar becomes serum-coloured and transparent. Partial hemolysis – the activity of the bacteria destroys erythrocytes only partially, the blood agar around colonies is only half-translucent and its colour is yellowish (no greenish tone). Viridation (viridis = green in Latin) – change of red blood colour to green; the agar around colony becomes greenish. No change – the majority of bacteria do not change the blood agar Describe hemolytic properties of four strains on the blood agar. Read against light. Observe the colour of the hemolysis, not the color of the bacterial colony itself. Name _____________________ General Medicine Date ___. ___. 2016 Page 2/4 VLLM0421c – Medical Microbiology I, practical sessions. Protocol to topic J02 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 impossible to fill in a cell, enter the reason (e. g. “too small“) Strain Strain Strain Size Color Shape Profile Surface Edges Translucency Consistency Odor Surroundings Name _____________________ General Medicine Date ___. ___. 2016 Page 3/4 VLLM0421c – Medical Microbiology I, practical sessions. Protocol to topic J02 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. Name _____________________ General Medicine Date ___. ___. 2016 Page 4/4