7.2. Preparation of microbiological culture media Chapter 7. STRAIN CULTURE AND CULTIVATION-BASED TECHNIQUES
7.2. Preparation of microbiological culture media The survival and growth of microorganisms depend on available and a favourable growth environment. Culture media are nutrient solutions used in laboratories to grow microorganisms. For the successful cultivation of a given microorganism, it is necessary to understand its nutritional requirements and then supply the essential nutrients in the proper form and proportion in a culture medium. The general composition of a medium is as follows: H-donors and acceptors (approximately 1-15 g/L) C-source (approximately 1-20 g/L) N-source (approximately 0.2-2 g/L) Other inorganic nutrients e.g. S, P (50 mg/L) Trace elements (0.1-1 µg/L) Growth factors (amino acids, purines, pyrimidines, occasionally 50 mg/L, vitamins occasionally 0.1-1 mg/L) Solidifying agent (e.g. agar 10-20 g/L) Solvent (usually distilled water) Buffer chemicals Microbiological culture media could be classified according to: 1. Consistency, which could be adjusted by changing the concentration of solidifying or gelling agents, e.g. agar, gelatine (liquid media do not contain such materials) Cultures in liquid media (or broth) are usually handled in tubes or flasks and incubated under static or shaken conditions. This way, homogenous conditions are generated for growth and metabolism studies,(e.g. with the control of optical density and allowing sampling for the analysis of metabolic products). Semisolid media are usually used in fermentation and cell mobility studies, and are also suitable for promoting anaerobic growth. Solid media are prepared in test tubes or in Petri dishes, in the latter case, the solid medium is called agar plate. In the case of tubes, medium is solidified in a slanted position, which is called agar slant, or in an upright position, which is called agar deep tube. Solid media are used to determine colony morphology, isolate cultures, enumerate and isolate bacteria (e.g. using dilutions from a mixed bacterial population in combination with spreading), and for the detection of specific biochemical reactions (e.g. metabolic activities connected with diffusing extracellular enzymes that act with insoluble substrates of the agar medium). 2. Composition Chemically-defined (or synthetic) media are composed only of pure chemicals with defined quantity and quality. Complex (or non-synthetic) media are composed of complex materials, e.g. yeast extract, beef extract and peptone (partially digested protein), therefore their chemical composition is poorly defined. On the other hand, these materials are rich in nutrients and vitamins. 3. Function All-purpose media do not contain any special additives and they aim to support the growth of most bacteria. Selective media enhance the growth of certain organisms while inhibit others due to the inclusion of particular substrate(s). Differential media allow identification of microorganisms usually through their unique (and visible) physiological reactions. In the detection of common pathogens, most practical media are both selective and differential. Enrichment media contain specific growth factors that allow the growth of metabolically fastidious microorganisms. An enrichment culture is obtained with selected media and incubation conditions to isolate the microorganisms of interest. EXERCISE 22: PREPARATION OF AGAR SLANTS Object of study: preparation of agar slants Materials and equipment: distilled water measuring cylinder flask bacteriological chemicals laboratory scales chemical spoons 1N NaOH solution 1N HCl solution pH indicator paper or pH meter cotton gloves dispenser test tubes test tube caps test tube basket slanting stage autoclave incubator Practise: 1. Measure the components of the medium (e.g. TSA or nutrient, see Appendix) into a flask containing 9/10 volume of the solvent. Use a clean chemical spoon for every measurement. Dissolve the solid components and fill with the remaining solvent up to final volume. If the medium contains heat sensitive components (like sugars), they must be separately sterilised in solution (e.g. by filter sterilisation), and then mixed with the already sterilised and cooled agar medium. 2. Close the flask with cotton plug and cover with aluminium foil, put into the autoclave and start a sterilisation cycle (see EXERCISE 1). This cycle could be intermitted when the internal temperature has reached 121°C, at that temperature every component (e.g. agar-agar) will be dissolved correctly. 3. Check the pH of the medium with an indicator paper or with a pH meter and adjust to the proper value with NaOH or HCl solution. 4. Pour the 60-70°C medium into the dispenser. Add 5-6 mL medium to each test tube, close them with caps and place them into a test tube basket. 5. Place the tubes into the autoclave and complete a whole sterilisation cycle for 20 min at 121°C (see EXERCISE 1). 6. Put the test tubes onto a slanting stage to let the medium solidify in the test tubes. 7. Label the slants according to the type of the medium and perform a sterility test: incubate the test tubes at 28°C for 24 hours, and check for sterility. 8. The prepared media can be stored for 1-2 weeks at 12-15°C, or longer in a refrigerator. (Do not store medium containing agar-agar under 4-5°C as it destroys its structure!) EXERCISE 23: PREPARATION OF AGAR PLATES Object of study: preparation of agar plates Materials and equipment: distilled water measuring cylinder flask bacteriological chemicals laboratory scales chemical spoons 1N NaOH solution 1N HCl solution pH indicator paper or pH meter cotton gloves sterile, empty Petri dishes Bunsen burner autoclave incubator Practise: 1. Prepare a medium as in EXERCISE 22. 2. Cool the sterilised medium to 55°C. 3. Take out the cotton plug and flame the mouth of the flask over a Bunsen burner, and then pour the medium into sterile, empty Petri dishes (15-20 mL into each Petri dish). 4. Keep the Petri dishes horizontally until the medium completely solidifies. Turn dishes upside-down and stack them up for storage. 5. Label the plates according to the type of the medium and perform a sterility test as in EXERCISE 22 6. In case of longer storage, Petri plates must be placed into plastic bags or boxes to avoid drying out. EXERCISE 24: PREPARATION OF AGAR PLATES WITH AUTOMATIC EQUIPMENT Automatic and semi-automatic equipment, like surface spreading or medium dispenser equipment make laboratory work quicker. Inside the equipment used during the practical course, Petri dishes are moved and opened automatically and a peristaltic pump fills the medium. The dispenser pipe is opened only for the period the medium is portioned in order to avoid any drops between the Petri plates. There is also a UV lamp inside the equipment to avoid contamination (Fig. 18).
Fig. 18. Semi-automatic equipment for the preparation of agar plates (Plate pourer). Stacked closed Petri dishes (1) fall opened on a conveyor belt inside the equipment, where they move together with the lid automatically to a dispenser pipe. A peristaltic pump (2) through the dispenser pipe (3) fills a preset volume of the medium (4) into the Petri dish. The conveyor belt moves further the Petri dish, the cover falls atop the filled bottom, and finally the ready to use plates (5) come out from the equipment. Object of study: preparation of agar plates Materials and equipment: distilled water measuring cylinder flask bacteriological chemicals laboratory scales chemical spoons 1N NaOH solution 1N HCl solution pH indicator paper or pH meter cotton gloves sterile, empty Petri dishes automatic equipment for medium preparation autoclave incubator Practise: 1. Prepare a medium as in EXERCISE 22 using the special flask of the plate-pourer apparatus. 2. Cool the sterilised medium to 55°C. 3. Fix one end of the sterilised silicone pipe to the top of the flask and the filler end to the automatic dispenser equipment. Stack empty, sterile Petri dishes to the dish container. 4. Switch on the UV lamp, adjust the thickness of the plate and start dispensing. 5. Keep the Petri dishes on the cooling platform until the medium completely solidifies. 6. Label the plates and perform a sterility test as in EXERCISE 22 Prev Chapter 7. STRAIN CULTURE AND CULTIVATION-BASED TECHNIQUES
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