Ambe 101 @ prctl-2

Ambe 101 @ prctl-2

• 1.
  PRACTICAL CLASS-2 TYPES OF MEDIA& MEDIA PREPARATION
• 2.
  • A growthmedium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells • It is either an organic or a synthetic substance that provides both the biophysical and the biochemical factors necessary for the growth of bacteria
• 3.
  • Microorganisms neednutrients, a source of energy and certain environmental conditions in order to grow and reproduce. • In the environment, microbes have adapted to the habitats most suitable for their needs, whereas in laboratory, these requirements must be met by a culture medium. • This is basically an aqueous solution to which all the necessary nutrients have been added
• 4.
  Based on chemical composition •Synthetic media • Non synthetic media Based on consistency • Solid medium • Liquid medium • Semi solid medium Based on utility or purpose • Basal medium • Enriched medium • Differential medium • Selective medium • Assay medium • Maintenance medium • Fermentation medium
• 5.
  The percent elementalcomposition of all the chemicals in the media is clearly known Also known as Chemically defined media Both autotropic and heterotropic organisms can grow Useful in studying the nutritional requirements of bacteria and also for microbiological assay of vitamins, amino acids Ex: Peptone water: 1 % peptone + 0.5 % NaCl, Czapek dox medium
• 6.
  Complex type andcontain various ingredients of unknown percent elemental composition such as animal plant and microbial extracts. Also known as un-defined media Used for cultivation of wide range of microorganisms Ex: yeast extract, beef extract, potato dextrose agar
• 7.
  Agar powder isadded to the nutrient broth it becomes nutrient agar- solid media Used for making nutrient agar plates, slants and stabs ADVANTAGES:  COLONY MORPHOLOGY CAN BE STUDIED  PURE CULTURES CAN BE OBTAINED  ENUMERATION CAN BE DONE DISADVANTAGES: HIGH POPULATIONS CAN NOT BE OBTAINED
• 9.
  No agar isadded or used while preparing the media ADVANTAGES:  HIGH POPULATION SCAN BE OBTAINED  ENUMERATION BY TURBIDOMETER DISADVANTAGES:  COLONY MORPHOLOGY CANNOT BE STUDIED  PURE CULTURES CANNOT BE OBTAINED
• 10.
  Contains agar at0.5% Soft curd like consistency Studying bacterial motility Cultivation of microaerophilic bacteria Ex: Nitrogen free malic acid BTB agar
• 14.
   Differential orindicator media distinguish one microorganism type from another growing on the same medium.  This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism.  These media are used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.
• 15.
  For example, ifa mixture of bacteria is inoculated into a blood containing agar medium (blood agar), some of the bacteria may hemolyze (destroy) the red blood cells, others do not thus one can distinguish between hemolytic and non-hemolytic bacteria on the same medium. e.g., Congored yeast extract mannitol agar
• 18.
  Transport media shouldfulfill these criteria:  Temporary storage of specimens being transported to the laboratory for cultivation  Maintain the viability of all organisms in the specimen without altering their concentration  Contain only buffers and salt  Lack of carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication  Transport media used in the isolation of anaerobes must be free of molecular oxygen.
• 19.
  Examples of transportmedia include:  Thioglycolate broth is for strict anaerobes.  Stuart transport medium is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralize.  Certain bacterial inhibitors are used for gonococci, and buffered glycerol saline for enteric bacilli.  Venkataraman Ramakrishna (VR) medium is used for V. cholerae
• 20.
   Enriched mediacontain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones.  They are commonly used to harvest as many different types of microbes as are present in the specimen.  Addition of extra nutrients in the form of blood, serum, egg yolk, etc, to basal medium makes enriched media.  Enriched media are used to grow nutritionally exacting (fastidious) bacteria.
• 21.
   Blood agaris an enriched medium in which nutritionally rich whole blood supplements the basic nutrients.  Chocolate agar is enriched with heat-treated blood (40– 45 °C), which turns brown and gives the medium the color for which it is named
• 22.
   Media thatcontains an ingredient that encourages the growth of some bacteria, but discourages the growth of other bacteria  Ex: Eosin methylene blue ANTIBIOTICS DYES CHEMICALS ALTERING pH
• 24.
  • These mediaare used for the assay of vitamins, amino acids, and antibiotics. • Ex: Antibiotic assay media are used for determining antibiotic potency by the microbiological assay technique.
• 25.
   Media usedfor storing the bacteria for a long period of time.  Examples: Egg saline medium, chalk cooked meat broth
• 26.
   Basal mediaare those that may be used for growth (culture) of bacteria that do not need enrichment of the media.  Examples: Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media.
• 27.
  The media whichis used for fermentation purpose
• 28.
  NUTRIENT BROTH NUTRIENTAGAR BEEF EXTRACT – 3 GMS PEPTONE – 5 GMS GLUCOSE - 5 GMS SODIUM CHLORIDE – 5 GMS DISTILLED WATER – 1000 ML BEEF EXTRACT – 3 GMS PEPTONE – 5 GMS GLUCOSE - 5 GMS SODIUM CHLORIDE – 5 GMS AGAR – 20 GMS DISTILLED WATER – 1000 ML
• 31.
  To prepare nutrientagar media, nutrient broth, nutrient agar slants & stabs Providing the required nutrients in an appropriate container for the growth of bacteria
• 37.
  • Weigh requiredquantities of media components. ( beef extract 0.45 grams, peptone 0.75 grams, glucose 0.75 grams, sodium chloride 0.75 grams for 150 ml of distilled water) • Dissolve the chemicals one by one • Check the pH of the medium and adjust the to 7.0 if necessary using diluted alkali or acid • Transfer 10 ml of nutrient broth into 4 test tubes, cover them with cotton plug or cap and label the nutrient broth tubes
• 38.
  • Transfer 100ml of nutrient broth into 150 ml of conical flask • Weigh 2 % agar and add it into nutrient broth ( nutrient agar media) • Cover the conical flask with cotton plug wrap with newspaper and tie it up with rubberbands • Label the conical flask • Boil the nutrient broth with agar in boiling water bath till agar melts
• 39.
  • Transfer about5 ml molten agar medium into each screw cap tubes and screw them loosely • Again recap the flask cover with cotton plug and wrap paper and secure them properly with rubber band • Keep the material for autoclaving at 121֯C at 15 lbs pressure for 15 minutes
• 40.
  • After sterilizationcool sterile nutrient agar medium to about 45 ֯C • Pour sterile nutrient agar medium into sterile petriplates under asceptic conditions • Leave the plates till the medium is solidified • Keep the screw cap tubes vertically to prepare a nutrient agar stab and in slanting position for preparing a nutrient agar slant till the medium is solidified
• 41.
  • Verify thelabel for the materials and leave them in an incubator at 30 ֯C for overnight incubation to allow the growth, if any contamination has occured • Observe the nutrient agar plates, nutrient broth, slants and stabs for any growth i.e., turbidity in broth, colonies in nutrient agar plates, slants and stabs • Materials without any growth only can be used for experiment and can be preserved in a refrigerator till their use • Discard the contaminated media only after autoclaving
• 43.
  Sterile nutrient agar plates,broth, slants and stabs i.e., No growth of microorganisms is observed Contaminated plates, broth, slant and stabs i.e., growth of microorganisms is observed
• 46.
  No growth of microorganismswas observed so I followed asceptic conditions Growth of microorganisms was observed so I did not followed asceptic conditions
• 47.
  What is thechemical nature of the agar agar? What is the melting temperature of agar and gelatin? What does it indicates, if any turbidity is seen in the sterilized nutrient broth tubes? How do you test that nutrient agar plates are contaminated? Mention the uses of solid medium and liquid medium? What is the temperature at which molten agar medium solidifies?
• 48.
  Pseudomonas Azospirillum Azatobacter Rhizobium Escherichia coli Fungi Yeast Actinomycetes FIND OUTTHE MEDIA USED FOR ISOLATING THE FOLLOWING MICROORGANISMS