Aseptic Techniques and sterile handling in atc lab

ASEPTIC TECHNIQUES AND STERILE HANDLING IN ANIMAL BIOTECHNOLOGY

PRESENTED BY:AKSHDEEP SHARMAREG NO: 12BTB003

B TECH BIOTECH6TH SEM

CELL CULTURE IS THE COMPLEX PROCESS BY WHICH CELLS ARE GROWN UNDER CONTROLLED CONDITIONS. IN PRACTICE, THE TERM "CELL CULTURE" HAS COME TO REFER TO THE CULTURING OF CELLS DERIVED FROM MULTICELLULAR EUKARYOTES, ESPECIALLY ANIMAL CELLS .

Animal Cell/Tissue Culture:

Aseptic techniques :

In culturing animal cells, it is essential that all procedures are carried out using aseptic or sterile techniques. Laminar flow facilities or sterile rooms provide a suitable environment, but even then aseptic techniques should be employed.

Basic aseptic technique is to ensure that the work area is clear, swabbed down regularly with 70% ethanol and that all the equipment used has been sterilized.

A: Performing all procedures in a laminar flow hood.

B: Using flames to fix microorganisms on container necks .

C: Holding a bottlecap with the little finger.

D: Avoid touching tops of open vessels while transferringtheir content.

Aseptic techniques :

Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger.

Work either left to right or vice versa, so that all material goes to one side.

Clean up spills immediately & always leave the work place neat & tidy.

Aseptic techniques :

Switch on UV light at least 20 minutes before and after use.

Avoid placing pipettes upside down.

Use gloves, masks and lab coats while working.

Sterilization :

Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or kills all forms of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media.

Sterilization Technique:

The various media constituents and other reagents used in cell cultures must be carefully sterilized either by autoclaving or by filtration.

Heat stable constituents like water, salts , supplements like peptone or tryptose, etc. are autoclaved at 121°C for 20 minutes.

Sterilization Technique: Heat labile constituents like

serum, trypsin, proteins, growth factors etc. must be sterilized by filtration through a 0.2 micron porosity membrane filter.

Autoclaving is preferred to filtration:

Autoclaving is preferred to filtration since it is cheaper.

Autoclaving needs less labour. Autoclaving is uniformly

effective.

Most of the media, now available commercially are usually presterlized.

Safety aspect in cell culture:

Possibly keep cultures free of antibiotics in order to be able to recognize the contamination.

Never use the same media bottle for different cell lines.

Necks of glass bottles prefer heat at least for 60 secs at a temperature of 200°C.

Safety aspect in cell culture: Switch on the laminar flow cabinet 20 minutes prior to start working.

Cell cultures which are frequently used should be subcultered & stored as duplicate strains.

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