Experiment-2

Aim: Cleaning and sterilization of glasswares and laboratory sterilization

Theory: Cleaning is a process to remove all dirt and solid material adhering to the surface and
dried before subjecting the material for sterilization.

Cleaning of New Glassware

1. Place the articles- test tubes, Petri dishes, conical flasks in suitable container preferably
stainless steel and cover them with 1% solution of trisodium phosphate and heat to boiling.

2. Remove all the glassware from solution.

3. Rinse with tap water and immerse in 1% HCl to neutralize the alkalinity.

4. Rinse again in tap water and lastly in distilled water.

5. The glassware is allowed to dry and then sterilization is done.

Cleaning of Used Glassware

1. Empty the contents of glassware, rinse in cold water, then in warm water (50- 55ºC).

2. Then washing can be done either with 1% detergent solution or 1% tri sodium phosphate
solution.

3. Wash with tap water, rinse with distilled water.

4. Dry and sterilize.

Sterilization

Sterilization is a process of making an article, surface or medium free from any type of
microorganisms. The concept was introduced by L. Pasteur. Sterilization is one of the most
important steps for cultivation, isolation and study of microorganisms in the laboratory.

I Physical Methods of Sterilization

There are many kinds of approaches done to sterilize the material.
(i) Moist Heat: Culture media and water are sterilized by using moist heat i.e. steam under
pressure. It is done through autoclaving.
Autoclaving (steam under pressure) or pressure cooker: Autoclaving is the most effective and
most efficient means of sterilization. All autoclaves operate on a time/temperature relationship.
The usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes. Autoclaving is
ideal for sterilizing biohazardous waste, glassware, many types of microbiological media, liquids
etc.
(ii) Dry Heat: Dry heat is produced by a hot air oven. Glasswares like pipettes, flasks, petri dishes,
o o
etc., are sterilized in an oven at 160 /2hours or 170 /1hour .Care should be taken to remove
these apparatus/ instruments only when the temperature cools down, otherwise the glassware
will break.

(iii) Incineration: It is a process of killing the microorganisms by using flame (heat), hence also
called flame sterilization. It is done for sterilization of inoculation loop, forceps etc. by
inserting an inoculation needle, inoculation loop and points of forceps into the flame of a
Bunsen burner (spirit lamp) till it becomes red hot. The microorganisms present on the
surface of the needle are destroyed. In addition, sterilize the mouth of culture tubes, glass
slides etc., through flaming i.e. bringing these near the vicinity of flame of the burner only
for a second.
(iv) Radiation: Normally ultra violet (UV) radiation is used in inoculation chamber or laminar air
flow. Expose the working area under UV radiation half-an-hour or so prior to start the work.
The source of UV radiation is generally UV lamp or UV tubes enclosed in quartz because
glass will not transmit UV radiation. The radiation emitted by UV source cause damage to
 cells by hydration and thymine dimmer formation and produce lethal effects. Care should be
taken not to see the UV radiation with naked eyes.

II Chemical Methods of Sterilization

There are several chemicals used for sterilization of glassware, working table, hands etc. for
microbial work.
(i) Alcohol: Ethanol or iso-propanol (70%) is used to sterilize the working tabletop, inoculation
chamber, etc.
(ii) Aldehyde: Generally laboratory is fumigated with formaldehyde when the number of
contaminants increases.
(iii) Inorganic chemicals: Chemicals like sodium hypochlorite (10%) or calcium hypochlorite
(10%) are used as disinfectants. Dip the materials to be disinfected in the solution for 1
minute. Take out the material, transfer into sterilized distilled water and wash properly.
Again repeat the process of washing for 5-8 times to remove the traces of chemicals. Blot
dry and inoculate the same or use as required.