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All the glass wares which are used for tissue culture should be clean and reserved
for that purpose alone. When glass wares are used either for storage or for
culture. The problem of contaminants leaking out into media or regents remain
and absolute cleanliness is therefore essential.


Ne glass wares, glass wares used in non-infectious work, glassware used in

infectious works, filter, assembly, potassium dichromate sulphuric, acid, boiling
pan, soap, detergent, bottle brushes, distilled water, cotton wool, gauge cloth,
cotton, papers aluminum foils, scissors, automatic pipette cans autoclave, hot air


1.washing of new glass wares

2. clean the dust of new glass wares with the aid of a piece of cloth.

3.till the bottles, beakers with dichromate solution (potassium dichromate and
sulphuric acid in the mixture of 63gm of potassium dichromate and 35ml distilled
water cool it then add conc. sulphuric acid to mater in 1 liter.

4.Also immersed the other glass ware like pipette, burette, test-tube, beaker, etc.
in the dichromate solution an kept it immersed over night

5.remove the dichromate solution on the next day(this solution can be reused still
it changes it’s color
6.rinise the glass ware with clean water for several time and keep these glass
ware immersed in clean water over night and then follow the further steps for
further cleaning and washing

II) washing of glass ware used in non-infectious work

1. collect the glass ware, keeping soaped water/detergent water(e.g labotine)

2.the pan should not be more than 3 quarter filled

3.sort out the glass ware boil in water containing detergent for 10 min

4. allow the pan to cool till the water is Luke warm and then clean different atoms
as follows:

a)tube, flask, bottles scrub well mechanical or hand brush

b)pipette-rinse for half and hour in the pipette rinse or pipette washer

c)needles check the point and sharpen it if necessary clean the needle with style

d)syringe is – scrub well with brush the pistol and barrel working smoothly

5.rinse the glassware in hot water or boil it in clean tap /distilled water

6. rinise several in cold water

7.rinse 3 times in distilled water

8.soak in distilled water minimum for 2hour or longer or overnight if necessary

9.drain the water and dry in hot oven at 60-110c

10.remove the glassware for packing when it is cooled.

III) Washing of glass ware used in infectious work

1.deconataminate by boiling it for 10 min in a pan if necessary auto clave it

2.allow to cool and follow the steps as described for uninfected glass ware

3.plastic ware used in infectious work

1.imrsed the plastic plate which are contaminated in 10% sodium hypocloride or
potassium hydrochloride for ½ hr

2.remove the plate after ½ and hour and immerse in 3+hypoclorate solution For
at least 2 hours if necessary over night also.

3.Rinise with clean water .

4.imred in detergent water for a min for 2 hr or longer or overnight if necessary.

5. rinsed with clean water under pressure.

6.imresd in distilled water for 2 hr.

7.rinsie 3 times or more times with distilled water.

8.drain the water ,allows to dry in a dust free area.

9.after drying sterilize under uv light for 10 min at a distance for 12 inch of the

Filter assembler: the assembly.

2.clean the intermediate part of filter assemblies by scrubbing with brush and

3.rise it with clean water for 10 times or with distilled water for 5 times.

4.allow to drain, dry in a clean dust free place.

Ensure that the filter paper in interact.

6.assemble the filter paper and make it ready packing & sterilize.
Experiment no -3

Packing of glassware

1.inspect the glassware for marks, patches etc.

2.discard the glassware which show marks & patches.

3.all glassware’s are removed from the oven after overnight.

4.if there is any deposit, patch or marks on any container send it back for on
second wash.

5.the bottles with lid are loosely sorrowed

6.the plastic tips are loaded into tip holders and holders are wrapped in paper .

7.plug the upper end of the pipettes tightly with cotton wool(non-absorbent )

8.the conical flasks ,measuring cylinder ,tubes etc should be plugged with cotton
wool& envelope with cotton gauge.

9.cover the plugs of individual tubes , flasks etc with aluminum foil

10.the tubes can also be wrapped in bunch as per requirements.

11.always use brown paper for packing

12.sort out the pipettes & pack them in their respective conc.

13.bottem of the can should have cotton beds which should be changed at each

14.use aluminum foil for covering sharp points or they may tear the brown paper

15.the containers of aluminum or stainless steel should be used of appropriate

sizes for tubes pipettes etc.
16.the tube should be placed in an inverted position.

Sterilization of glassware’s

Types of sterilization

There are 2 types of sterilization

a)dry heat sterilization

b)moist heat sterilization

a.dry heat sterilization

1.keep the equipment to be sterilize in hot air sterilizer oven not overload, leave sufficient gap between material for free movement of
hot air

3.sterilise at 160c for 1½an hour. precaution should be taken that the
temperature should not exceed 160c

4.check the steri . indicator tape color to ascertain that each container is

5.first of all attain the dry air temperature above 155c for more than 30min

6.resterilise the material if there is any doubt but fix fresh steri. Tape to the

7.the following articles should not be sterilized by dry heat.



5.screw caps, syringes

6.filtration assembly

b.sterilisation by steam under pressure(autoclaving)

1.all solutions except heat labile solution can be steri. By steam under pressure

2.the pressure is variable depending upon solution it is variable as steam at 15lbs

&temp. is 120c or 10lbs & 115c is essential to check the temp.

4.rubber stopper, corn wall syringes filters otter instrument with rubber fittings
should be sterilized by autoclaving at 15 or10lbs at 120c or115c the material in autoclave care should be taken to losen the screw and
mark the mesious level

6.two glass surfaces should be not touch each other

7.close the autoclave cover & lock it securely

8.check the adjacent of the steam pressure

9.ensure that air is evacuated & replaced by steam

10.check the temp record the time & sterilize it for 15 min as per requirement

11.switch off the autoclave & allow to cool down the autoclave make the screw cop tight

13.check the color of stir. Indicator tap & meniscus the sol.

Sterilization by UV light-

The plastic equipment can be sterilized by exposure to UV for ½ an hour.

Experiment no :-6

Preparation and sterilization of animal culture media

Aim:-to study preparation and sterilization of animal culture media.


Medium is the most important in cell culture ideally the medium is an accurately
defined mixture of chemical substances .animal cell require the chemically
defined media to be supported with some mutual product e.g. serum. Tissue
extracts etc different all types different media.

The media required for animal culture in vitro must be prepared to contains

Certain factor necessary for survival of cell, cell growth and multiplication. These
are carbohydrate source, in organic irons ,gases, vitamins, hydrogen conc.etc

All these factors should be in aseptic condition through the cell culture media.

Media contain gin heal sensitive component are filter separately and then added
prior peruse this filtration must be under positive pressure of an inner gas like
nitrogen, it accerlate the filtration process in the


The standard powder media ,distilled wear ,sodium bicarbonate co2 source
phenol sterinement filter assembly attach with succession assembly or pressure
assembly, sterile bottle, laminar flow cabinet etc.

1. Take the required quantity of powder as per media.

2. Dissolve in appropriate volume of double distilled water by gentle stirring
at room temp and make it final volume.
3. Stir until resolve
4. For all medium except, laborite 15, add 1.5gm of sodium bicarbonate/liter.
5. Add essential quantizes of media supplement adjust the ph till the desired
ph is obtained.
6. It can be detected with the help of phenol red indicator which is present all
media except insect cell media, useful for visual assessment of ph.
7. Conform all ingredients have entered in the solution and no salt or
precipitates are observed
8. Filter the media through 0.2 micro mille pore with sterile membrane with
filter assembly.
9. Aliquot in sterile bottle in laminar flow cabinet. Incubate the aliquot of
media to check sterility of media at 37 c for 72 hours.
10. Also check the effect of serum if present in the medium on a maintain cell
line if it shows no adverse effects then this media can be stored for use.