NEED:

This assignment is basically done due to the shortage of BARC culture. BARC culture contains the consortium bacteria ,this bacteria helps for the formation of nitrate which is helpful for the growth of plants. Due to the limited amount of BARC available in the laboratory, mass multiplication of BARC is needed. It also helps to learn the mass multiplication protocol and aquire laboratory skills.

AIM:

Mass multipliaction of BARC culture.

REQUIREMENTS:

For Plating of BARC culture:

  1. Available BARC culture
  2. PDA and NA media
  3. Petri plates
  4. Spreader
  5. Microtips and micropipette
  6. Autoclave
  7. Incubator
  8. LAF
  9. Beaker

PROTOCOL:

For Plating of BARC culture:

  1. Prepared PDA and NA media .
  2. Autoclaved media,petri plates,distilled water,test tubes,spreader,microtips at 121C temp and 15 lbp pressure for 20 min.
  3. For Maintaining asceptic condition : At the same time, clean LAF(laminar air flow) using aceton / 80% ethanol and ON UV light for 15-20 min.after 20 min OFF UV light and and ON blower and day light.
Image 1: Autoclave
  1. Then all remaining procedure carried out under asceptic conditions.
  2. After sterilization, media poured into petri plates (approximately 30 ml/plate)
Image 2: Pouring
  1. After solidification of media, add 0.2 ml of aliquot of BARC from test tube on petri plate. And spread equally on whole petri plate.
  2. Follow same protocol for all the petri plates with same concentration of BARC culture and spread equally by using spreader.
  3. Packed petri plates with parafilm. And kept it in incubator at 37 C temp for 4/5 days.
Image 3: Kept petri plates in incubator for incubation.

REQUIREMENTS:

For mass multiplication:

  1. NB broth
  2. Beaker
  3. Wire loop
  4. LAF
  5. Orbital shaker.

PROTOCOL:

For mass multiplication:

  1. After incubation of 5 days. Growth of MOS has been completed.
  • Image 4
  • Image 5
Image 4: Growth og microorganism on NA media.
Image 5: Growth of microorganism on PDA media.
  1. We can store this incubated culture in refrigerator.
  2. For Maintaining asceptic condition : Clean LAF (laminar air flow) using aceton / 80% ethanol and ON UV light for 15-20 min.after 20 min OFF UV light and and ON blower and day light.
  3. NB broth sterilized by using autoclave that is 121 C temp and 15 lbps pressure for 20 min.
  4. After the complete cooling of NB broth, microbial colonies from incubated plates are suspended into the broth with the help of wire loop.
Image 6: Suspended bacterial colonies into NB broth.
  1. Generally for 400 ml of broth requires two plates of incubated MOS.
  2. After suspension this broth is kept on orbital shaker for multiplication of bacteria at 125 rpm for 5 days.
Image 7 : Kept NB broth on orbital shaker for multiplication of microorganisms.

RESULT AND CONCLUSION:

Colony count:

After 5 days, growth of MOS has been observed by plating of multiplied BARC culture. And then incubated for 5 days. After 5 days incubation, colony count has beent calculated which was 205. Which conclude that multiplication of MOS has been done.