Standard Operating Procedures (SOP) for Sterilization and Aseptic Methods in AnimalCell Culture Lab.

Prepared by: Oumalkaire Yacin Abdi

ID Number: B12090014

Department of Biosciences

Malaysia University of Science and Technology

Kelena Square, Bloc C, Kelena Jaya, Petaling Jaya,

Selangor, Malaysia

Table of Contents Pages

Introduction............................................................1

i. Overview.........................................................1

ii. Purpose...........................................................1

iii. Benefits...........................................................2

Aseptic Techniques.................................................3

i. Procedures for aseptic techniques.................3

ii. Laminar flow biosafety hoods.........................5

Sterilization Methods.............................................7

i. Wet Heat (Autoclave).....................................7

ii. Dry Heat ( Flaming, Baking)............................8

iii. Filtration.........................................................9

iv. Disinfectants(Solvents)...................................9

v. Radiation........................................................10

Appendix................................................................11

Introduction

i. Overview:

Cell Culture has become an eminent tool used in the life sciences today. In any tissue and cell culture laboratory, there is always a constant threat of contamination. This SOP is designed to serve as a guide in sterilizing and keeping laboratory materials sterile prior any experimental work.

ii. Purpose : This SOP details some basic laboratory procedures and it is appropriate for lab

workers and those who deal with cell lines to apply. For accurate and precise lab workresults , it is highly advised to follow these procedures.

iii. Benefits :

The use of this SOP will help lab workers or staff to deal with a sterilized environment. Therefore risk of contamination are minimized. Thus, growth, inoculations or any other experiment is likely to be successful.

Aseptic Techniques

i. Procedures for aseptic techniques in cell culture lab:

Aseptic techniques ensure that all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi, mycoplasma and cross

contamination with other cell lines. All work is done under a tissue culture hood. A movable glass panel or sash covers the face area of the tissue culture hood and acts as a physical barrier that helps to maintain a particulate-free environment and laminar air flow.Before you start your work:

be sure the UV light is off; let the air flow for 2-3 minutes to filter and establish air flow;

spray interior surfaces with 70% ethanoland wipe surfaces dry;

avoid rapid movements that will disrupt laminar air flow;

all equipment that will be taken into the cabinet such as media bottles, pipette tip boxes, and your gloves should be sprayed with 70% ethanol.

avoid talking while performing works with the laminar air flow hood, germs from the body could contaminate the culture or the cell lines.

Among the most common procedures performed at the clean bench are liquid

transfers using a pipette or micropipette.When using a pipette: 1. Begin by loosening the cap of the conical tube inside the hood before you open the pipette cover. Open the cover from the bottom of the pipette.

2. Make sure the tip of the pipette does not touch areas that are not sterile.

3. Put the pipetter on the pipette. Aspirate the appropriate amount of liquid.

4. Again, be very careful that the tip of your pipette does not touch the outside of the tube or any other area that is not sterile.

5. Release the media. Do not reuse the pipette unless you are making multipleplates that are identical.

6. Use a fresh pipette if you use different cells or different media.When using the micropipette: 1. spray it with 70% ethanol and wipe it dry. Inside the hood all

work is done with sterile pipette tips.

2. Once again, loosen the cap of the conical tube and your flask before you start.

3. Insert the tip on the micro-pipette, taking good care that the tip does not touch any surface.

4. Aspirate the appropriate amount and loosely set the cap back on theconical tube.

5. Again, ensure that the tip does not touch any other surface, open the flask and release the liquid. Discard the tip.

When your work is done:

dispose of all waste spray the work surface inside the hood with 70%

ethanol dispose of tissue by autoclaving at night, turn the germicidal UV lamp back on

ii. Laminar flow biosafety hoods :

Principle of laminar flow cabinet:sterilize air though filter and blow it across work surface as a particle-free laminar air stream

Typical laminar air flow velocity: 0.3 - 0.5m/s

Purpose of a laminar flow cabinet: Product protection from room contaminants(does not protect operator)

Advantage of Vertical Laminar Flow Cabinet:- No blocking caused by large object- Not blowing air straight to operator’sface 8hrs/day

Advantage of Horizontal Laminar Flow Cabinet:- Easier to put sensitive object near HEPA filter- Not blowing dust on straight to sensitive object

Sterilization Methods

Wet Heat (Autoclave):

The method of choice for sterilization in most labs is autoclaving; using pressurizedsteam to heat the material to be sterilized. This is a very effective methodthat kills all microbes, spores and viruses, although for some specific bugs, especially high temperatures or incubation times are required.

Autoclaving kills microbes by hydrolysis and coagulation of cellular proteins, whichis efficiently achieved by intense heat in the presence of water.

The intense heat comes from the steam. Pressurized steam has a high latent heat; at 100degC it  holds 7 times more heat than

water at the same temperature. This heat isliberated upon contact with the cooler surface of the material to be sterilized, allowing rapid delivery of heat and good penetration of dense materials.At these temperatures, water does a great job of hydrolyzing proteins… so those bugs don't stand a chance.

Dry Heat (Flaming, Baking):

Dry heating has one crucial difference fromautoclaving. You've guessed it – there's nowater, so protein hydrolysis can't take place.

Instead, dry heat tends to kill microbes byoxidation of cellular components. This requires more energy than protein hydrolysis so higher temperatures are required for efficient sterilization by dryheat.

For example sterilization can normally be achieved in 15 minutes by autoclaving at 121deg C, whereas dry heating would generally need a temperature of 160deg C tosterilize in a similar amount of time.

FILTRATION:

Filtration is a great way of quickly sterilizing solutions without heating. Filters, of course, work by passing the solution through a filter with a pore diameter that is too small for microbes to pass through.

Filters can be scintered glass funnels madefrom heat-fused glass particles or, more commonly these days, membrane filters made from cellulose esters. For removal of bacteria, filters with an average pore diameter of 0.2um is normally used.

But remember, viruses and phage can pass through these filters so filtration is not a good option if these are a concern.

Disinfectants(Solvents):

Ethanol is commonly used as a disinfectant,although since isopropanol is a better solvent for fat it is probably a better option.

Both work by denaturing proteins through a process that requires water, so they must be diluted to 60-90% in water to be effective.

Again, a it's important to remember that although ethanol and IPA are good at killing microbial cells, they have no effect on spores.

Radiation:

UV, x-rays and gamma rays are all types of electromagnetic radiation that have profoundly damaging effects on DNA, so makeexcellent tools for sterilization.

The main difference between them, in terms of their effectiveness, is their penetration.

UV has limited penetration in air so sterilization only occurs in a fairly smallarea around the lamp. However, it is relatively safe and is quite useful for sterilising small areas, like laminar flow hoods.

X-rays and gamma rays are far more penetrating, which makes them more dangerous but very effective for large scale cold sterilization of plastic items (e.g. syringes) during manufacturing.

Appendix:

AUTOCLAVE:

HORIZONTAL LAMINAR AIR FLOW HOOD:

VERTICAL LAMINAR AIR FLOW HOOD: