ASEPTIC TECHNIQUES

• LAMINAR FLOW FACILITIES OR STERILE

ROOMS PROVIDE A SUITABLE ENVIRONMENT,
• IF A NONSTERILE ENVIRONMENT IS USED, THE
RELIANCE ON ASEPTIC TECHNIQUES IS VERY
HIGH.
• THE WORK AREA SHOULD BE CLEAR,
SWABBED DOWN REGULARLY WITH 70%O
ETHANOL AND ALL THE EQUIPMENT USED BE
STERILIZED.

• CLEAN LABORATORY COATS ARE ALSO

ESSENTIAL.
• THE BASIC RULES FOR ASEPTIC TECHNIQUES:
• IF WORKING ON THE BENCH, USE A BUNSEN
FLAME TO HEAT THE AIR SURROUNDS THE
BUNSEN. OPEN ALL BOTTLES AND PERFORM
ALL MANEUVERS IN THIS AREA ONLY;
• SWAB ALL BOTTLE TOPS AND NECKS WITH
70%•ETHANOL TO CLEAN THEM BEFORE
OPENING; FLAME ALL BOTTLE NECKS AND
PIPETTES BY PASSING VERY QUIKLY THROUGH
THE HOTTEST PART OF THE FLAME. THIS IS
NOT NECESSARY WITH STERILE INDIVIDUALLY
WRAPPED, PLASTIC FLASKS AND PIPETTES;
• AVOID PLACING CAPS AND PIPETTES DOWN
ON THE BENCH; PRACTICE HOLDS BOTTLE
TOPS WITH THE LITTLE FINGER WHILE
HOLDING THE BOTTLES FOR POURING
PIPETTING;
• WORK EITHER LEFT TO RIGHT OR VICE VERSA,
SO THAT ALL MATERIAL TO BE USED IS ONE
SIDE AND, ONCE FINISHED, IS PLACED ON THE
OTHER SIDE OF THE BUNS BURNER. (THIS MAY
ALSO STOP THE OPERATOR USING THE SAME
REAGENT TWICE
• MANIPULATE BOTTLES AND FLASKS CAREFULLY.
• THE TOPS OF BOTTLES AND FLASKS MUST NOT
BE TOUCHED BY THE OPERATOR.
• TOUCHING OF OPEN VESSELS SHOULD ALSO BE
PREVENTED WHEN POURING.
• IF NECESSARY PRACTICE POURING FROM ONE
CONTAINER TO ANOTHER KEEPING A DISTANCE
OF 5 MM BETWEEN THE VESSELS;
• CLEAR UP SPILLS IMMEDIATELY AND ALWAYS
LEAVE THE WORK AREA CLEAN A TIDY.
•
• DISPOSE OF GLASSWARE IN APPROPRIATE
BINS AND DISCARD USED PLASTICWARE IN
MARKED POLYTHENE BAGS FOR
AUTOCLAVING OR INCINERATION,
• REUSABLE GLASSWARE OR PLASTICWARE
USED FOR INFECTIOUS WORK MUST ALWAYS
AUTOCLAVED BEFORE INCINERATION.
• RE-USABLE GLASSWARE SHOULD IMMERSED
IN DISINFECTANT WHILST AWAITING
TRANSFER TO AN AUTOICLAVE
• CHECKING AND PREVENTING FOR
CONTAMINATION
• CONTAMINATION OF CELL CULTURES
– BACTERIA,
– FUNGI
– MYCOPLASMA
– CROSS CONTAMINATION
• REGULARLY CHECKING ALIQUOT FROM CULTURE
FLASKS USING A MICROSCOPE CAN REVEAL SOME
TYPES CONTAMINATION,
• FOR EXAMPLE, YEAST AND FUNGI CAN BE SEEN EASILY.
• BACTERIA CAN BE GRAM STAINED OR PLATED OUT
ON BLOOD AGAR PLATES AND
• GENERAL CLOUDINESS OF MEDIUM ALSO INDICATES
YEAST OR BACTERIAL CONTAMINATION
•
• MYCOPLASMA IS MORE INSIDIOUS,
– THE MEDIUM IS NOT CLOUDY,
– THE ORGANISM CANNOT BE SEEN UNDER AN ORDINARY MICROSCOPE
AND
– MOST MYCOPLASMA SPECIES ARE VERY DIFFICULT TO GROW ON AGAR
PLATES.
• TESTING FOR MYCOPLASMA IS TIME CONSUMING BUT MUST BE
DONE REGULARLY BECAUSE CROSS CONTAMINATION VERY EASY.
• DNA STAINS OR MOLECULAR PROBES MAY BE USED TO DETECT
THEIR PRESENCE IN CELLS,
• THOSE CELLS WHICH TURN OUT TO CONTAIN DNA IN THE
CYTOPLASM MUST BE DISCARDED.
• MUCH OF THE CONTAMINATION COMES FROM THE CULTURE MEDIUM
AND ITS COMPONENTS OR FROM INADEQUATELY CLEANED GLASSWARE
• ROUTINE STERILITY CHECKS ON THE MEDIUM, SERUM AND NUTRIENTS
ISRECOMMENDED.
• TO DO THIS, A SMALL ALIQUOT OF THE MEDIUM SHOULD INCUBATED AT
ROOM TEMPERATURE AND ANOTHER SIMILAR ALIQUOT
SIMULTANEOUSLY INCUBATED AT 37 °C FOR UP TO ONE WEEK BEFORE
THE MEDIUM IS USED F CULTURING CELLS.
• IF THESE SMALL SAMPLES ARE FOUND TO BE CONTAMINATED, THE
MEDIUM SHOULD BE AUTOCLAVED AND DISCARDED.
• THE USE OF ANTIBIOTICS IN T MEDIUM HELPS TO CONTAIN THE PROBLEM
TO SOME EXTENT BUT IT SHOULD NEVER SEEN AS AN ALTERNATIVE TO
GOOD ASEPTIC TECHNIQUE AND CAREFUL MONITORING
• CONTAMINATED CULTURES SHOULD BE
DISPOSED INTO 2.5% HYPOCHLORITE
SOLUTION.
• MEDIA BOTTLES KNOWN, OR SUSPECTED, TO
CONTAMINATED SHOULD BE DISPOSED OF.
•
 BASIC PREVENTIVE MEASURES
• CHECKING STERILIZING PROCEDURES
(AUTOCLAVE AND OVEN PROCEDURES);
• CHECKING STERILITY OF LAMINAR LOW
HOODS;
• REGULAR CHECKS ON CULTURES;
• DISPOSAL OF CONTAMINATED CULTURES
• SUBSTRATE, CULTURE VESSELS AND
APPARATUS
• DISPOSABLE PLASTIC WARE CAN BE USED
BUT COSTLIER.
• GLASSWARE ARE ADEQUATE FOR MOST
PURPOSES.
 STERILIZATION OF GLASSWARE
• WASHED WITH NON-TOXIC DETERGENT,
FOLLOWING AN OVERNIGHT SOAK.
• THOROUGHLY RINSED IN TAP WATER
FOLLOWED BY DISTILLED WATER.
• GLASSWARE KEPT IN CONTAINERS OR
WRAPPED IN ALUMINIUM FOIL AND USUALLY
STERILIZED IN DRY HEAT AT 1600 C FOR 1 HR .
• SCREW CAPS ARE STERILIZED SEPERATELY.
 MEDIA AND REAGENTS

• WATER,
• SALTS,
• PEPTONE,
• TRYPTOSE, ETC. ARE AUTOCLAVED AT 121°C
FOR 20 MIN.
• SERUM,
• TRYPSIN,
• PROTEINS,
• GROWTH FACTORS, MUST BE STERILIZED BY
FILTRATION THROUGH 0.2-µM POROSITY
MEMBRANE FILTER.
• EACH FILTRATE SHOULD BE TESTED FOR
STERILITY TO AVOID FAILURE DUE TO
CONTAMINATION.
• AUTOCLAVING IS CHEAPER,
• LESS LABORIOUS AND
• UNIFORMLY EFFECTIVE.
• SO, AUTOCLAVING IS TO BE PREFERRED TO
FILTRATION AND AUTOCLAVABLE VERSIONS OF
THE MEDIA SHOULD BE USED.
• MOST OF THE MEDIA NOW COMMERCIALLY
AVAILABLE AREN USUALLY PRE-STERILIZED
 ISOLATION AND DISAGGREGATION OF
EXPLANTS
• CONTAMINATION OF ALL KINDS SHOULD BE
AVOIDED DURING EXPERIMENTAL
MANIPULATIONS.
• TISSUES SHOULD BE COLLECTED ASEPTICALLY
OR SHOULD BE STERILIZED AFTER REMOVAL.
• THE TISSUE IS WASHED WITH A SOLUTION
HAVING HIGH CONCENTRATION OF
ANTIBIOTIC—A BALANCED SALT SOLUTION
CONTAINING 100 UNITS OF PENICILLIN AND
0.5 MG OF STREPTOMYCIN OR NEOMYCIN PER
MILLILITER.