PREPARATION OF 10-100 CELLS CULTURE SUSPENSION

• PROCEDURE FOR PREPARATION OF 10-100 CELLS CULTURE SUSPENSION:

• Cell culture is a technique for growing cells from an organism in a controlled environment outside of their natural bodies.

• Preparation of Initial Stock Suspension:

• Select the type of organisms to which 10-100 cells suspensions need to be prepared.
• Take 10 mL of sterile saline solution in test tube and label as initial stock suspension.
• Take a loop full of culture from the working culture (selected organism) and make the initial stock suspension and swirl the test tube for proper mixing.
• Preparation of serial dilution:
• Prepare a series of 9 test tubes with each of 9 mL of saline solution and sterilize them as per validated cycle.
• Label the tubes serially by numbering from 1 to 8 and mark the remaining one (9th) as negative control (blank) for saline.
• Now pipette out 1.0 mL from initial stock suspension using sterile glass pipette/micro pipette using sterile tips and transfer in the sterile saline solution test tube containing 9 mL numbered as 1, mark the dilution as 10 -1.
• Mix the suspension tubes carefully by swirling motions without spilling them and transfer 1.0 mL of the content from 10 -1 dilution using sterile glass pipette/micro pipette with sterile tips.
• Aseptically inoculate them in 9 mL of sterile saline solution labelled as 2, again mix the suspension well and mark the dilution as 10 -2.
• Likewise serially transfer 1.0 mL and perform the serial dilution method as per Format-II for all the remaining tubes up to 10 -8 dilutions.
• Transfer the suspensions aseptically and mix the suspensions by swirling after each transfer, to avoid any error and label the dilutions correctly.
• After completion of serial dilution, transfer 0.1mL from all the dilution (10 -1 to10 -8) into sterile petri plates under Bio safety cabinet.
• Then pour the sterilized Soyabean casein digest agar media into the petri plates containing bacterial cultures and use the Sabouraud dextrose agar for fungal cultures.
• Perform one diluent control and media negative control.Preserve all the dilution tubes in the refrigerator or Cooling incubator below 8°C.
• Allow the plates to get solidified under Bio safety cabinet and incubate the bacterial cultures in inverted position at 30 – 35°C for 48 hours and fungal cultures at 20 – 25°C for 48-72 hours and wrap the Aspergillus plates with Parafilm/Aluminum foil under incubation.
• After completion of incubation period, observe the plates and count the number of colonies obtained in each plate and record the suspension preparation in Format-I.

NOTE: – Preserve the 10 -100 cells containing dilutions at 2-8°C in Cooling Incubator.

• ANNEXURES: 

• ABBREVIATIONS:

Annexure-I
STOCK SUSPENSION PREPARATION RECORD

Annexure-II
SERIAL DILUTION METHOD