Maintenance of Sterility in Animal Tissue Culture

As introduced in the Animal Biotechnology - Tissue Culture and Cell Culture post, we are continuing the study by understanding the method to maintain sterility in this techniques. 
In Animal Tissue Culture (ATC) Laboratories, the animal tissues are surgically separated from the source (examples- patients or laboratory animals) and this tissue is immediately transferred to an adequate volume of sterile medium or to a balanced salt solution. To avoid microbial contaminants, antibiotics are also added to this medium.

These are the methods through which Aseptic conditions or Sterility is maintained in an ATC lab.

1.    First thing to remember and obey is- to clean hands till elbows with a disinfectant soap before and after working with any cell culture in any tissue culture lab. Each student or laboratory worker should strictly follow this step.

2.    All the surfaces of reagent bottles and other laboratory tools should be cleaned with disinfectants or they should be wiped with 70% ethanol or isopropanol before they are kept for use in the working laboratory.

3.    Dissection instruments like blades, scalpels, scissors, forceps, needles etc. should be sterilized by flame.

These instruments can also be sterilized by using dry heat at 180°C for 2 hours.

4.    Glassware should be sterilized by autoclaving. Sometimes instead of autoclaving, 10% bleach solution can also be used for soaking the glass bottles and tubes and then rinsed with distilled water.

5.    Use of mouth for pipetting liquid medium and cell suspension should be avoided as it causes microbial contamination in the culture. Instead, bulb type pro-pipettes, pi-pumps etc. are generally used to transfer the medium or culture from one vessel to other.

6.    The work room or the vertical Laminar air flow (LAF) hoods must be properly sterilized before the culturing the cells or tissues.

The working area of laminar air flow hood is first cleaned by 70% ethanol by inside to outward sweeping. Then after closing the glass windows of the LAF, the Ultra Violet lamp is left on for 15-20 minutes (do not expose human skin and eyes to the UV lamp as it is carcinogenic). After UV sterilization, allow the High Efficiency Particulate Air (HEPA) filters are present before the air blower in a LAF, these filters provide sterile and contamination free air to the working area. The air flow is kept on for 5 minutes to remove contaminated air out of the LAF cabinet.

Sterilization of substrate and medium and other reagents should be done by autoclaving at 121°C at a pressure 15 psi for 20 minutes.

Heat labile constituents like serum, trypsin, proteins, growth factors etc. should not be autoclaved but can be sterilized by filtration through 0.2 µm porosity membrane filter.

also do not forget to read : 
Animal Biotechnology - Tissue Culture and Cell Culture
Prevention of Cross-contamination in Animal cell cultures 
 & 

Use of Antibiotics in Animal Tissue Culture for maintaining sterility