Standard Operating Procedure for Cleaning & Sterilization of Lab Equipment's
Glassware
New Glassware :- Glassware that has never been used may be slightly alkaline. In order to neutralize it use 1-2% solution of hydrochloric acid. (Add 60 ml concentrated HCL in 3 liters of water.) Immerse new glassware and leave for 24 hr. Rinse twice with ordinary water and once with Distilled water. Dry the glassware.
Dirty Glassware:- Preliminary rinsing - Rinse twice in cold or running water. Soak for 2-3 hrs. in a container of water mixed with soap oil. Brush the inside of glassware with a test tube brush. Remove articles one by one and rinse under running tap water, then soak them all in ordinary water for 30 minutes. Place containers (Petri dishes, test tubes, beakers, flasks, measuring cylinders) on a draining rack. Test tubes are placed upside down in a wire basket for draining. For drying, place the baskets in hot air oven at 60°C.
Microscopic Slides
New Slides:- Place slides overnight in a bowl of water mixed with soap oil. Rinse each slide under tap water for 15 minutes. Wipe the slides, one at a time with a soft cloth. Place on blotting paper or filter paper and allow drying. Pack the slides in stacks of 10 or 20 groups in paper.
Dirty slides:- Oil is removed by rubbing vigorously with newspaper. Cover slips are removed using a needle or forceps. Now soak in detergent solution for 24 hrs.
Cover slips:- Used cover slips can be cleaned and re used. Make the following solution in a large beaker:-Water 200 ml,Detergent 3 ml,Bleach 15 ml Put the cover slips in this one by one and soak for 2-3 hours, shaking gently from time to time. Rinse the beaker with cover slips with tap water 4 times. Finally rinse with distilled water. Dry in hot air oven at 60° C and keep in a clean petri dish.
STERILIZATION:-Sterilization is the process of freeing an article from all living organisms, including bacteria and their spores.Heat is the most commonly used and most reliable method of sterilization.It should be used wherever possible.Heat can be done in two ways,
1) Dry heat – Hot air Oven:- Dry heat is suitable for glassware, instruments, articles not spoiled by very high temperatures and for water impermeable substances like oils, waxes and powders. An oven is heated by electricity with heating elements in the wall of the chamber. It is fitted with a fan to assist the circulation of air and to ensure rapid and uniform heating of the load. For sterilization hot air oven is maintained at 160°C for 1 hour or at 180°C for 20 minutes. While preparing the load articles are either plugged with cotton wool or wrapped in aluminium foil or kraft paper to protect them from airborne contamination during storage after sterilization.
Precautions:- Glassware should be perfectly dry before being placed in the sterilizing oven because wet glassware will crack. The oven should not be overloaded and space must be left for circulation of air through the load. The holding time should be calculated only when the temperature indicator reaches the specified temperature (160°C or 180°C). The door of the oven should not be opened in between heating as the temperature is not maintained and improper sterilization occurs. After the completion of sterilization, oven should be allowed to cool to 40°C before the door is opened to remove any articles otherwise cracking of glassware may occur on contact with cold air. Wear Heat Resistant Gloves while unloading from Hot – Air Oven.
2) Moist heat – Autoclave:- Steam kills microorganism by the irreversible denaturation or heat coagulation of cellular protein. Microorganisms and their spores are killed at lower temperature by moist heat than by dry heat Used for sterilization of culture media and other liquid to retain their water content.Preparation of material for autoclaving - Media containing flasks and tubes, petri dishes etc., should be wrapped in autoclave polythene bags and tied with string. If autoclave polythene bags are not available then use Kraft paper, cotton or gauze for wrapping the tubes/ petri dishes etc.
Sterilization procedure Fill the bottom of the autoclave until the temperature electrode immense with but the water should not touch the basket. Put the basket containing the material to be sterilized in the autoclave along with sterilization indicator papers. Close the lid, making sure the rubber washer is in its groove. Screw down the lid clamps evenly and firmly but not too tightly. Open the air outlet valve and begin heating the autoclave. When a jet of steam appears at the air outlet wait for 3-4 minutes till the jet of steam becomes uniform and continuous (shows that all air has been removed) close the air outlet valve, tighten the lid clamps and reduce the heat slightly. Removing load from autoclave: When pressure gauge shows that the autoclave is at atmospheric pressure, open the discharge tap very slowly to allow air to enter as cooling of steam proceeds. Unscrew the lid clamps and take off the lid and remove the baskets of sterile equipment. During cooling process, sterilized items should never be placed on cold metal surfaces as moisture will condense onto the items and contaminant them. The sterile load should be kept in wire mesh racks or baskets till it gets cooled. Precautions while using an autoclave. In the autoclave, all parts of the load to be sterilized must be permeated by steam.. Steam should be hot, saturated and dry (free from particles of liquid water).. All the air must be removed from the autoclave chamber and articles of the load, so that the load is exposed to pure steam during sterilization.. For sterilization, exposure of microbes to 121°C for 20 minutes at 15 pounds pressure is required.. Avoid autoclaving small and large volumes in the same load because with the same holding period (eg. 20 min) small amounts (eg. 10 ml) may be damaged by overheating and large amounts (eg 2000 ml) may not be sterilized.. Do not fill the bottle, tubes, flasks etc to more than 75-80% of their capacity as the contents overflow on expansion during heating.. Tubes, flasks and bottles should be closed with cotton wool stoppers, loose slip on metal caps or loosely applied screw caps.8. There is 3-5% loss of water from media due to evaporation on cooling. So prepare media by adding 5% extra distilled water.. Never touch the drainage tap, outlet valve or safety valve while the autoclave is under pressure. Never leave the autoclave unattended while the pressure is rising. Never open the lid before the pressure has dropped to normal.. During sterilization make sure that the lid is secured and no steam escapes. Never leave the autoclave to cool for too long because if the discharge tap is not opened, vacuum is created and media gets dehydrated.
