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Soft Agar Techniques
276
V. SINGLE CELL ISOLATIONS AND CLONING
but nevertheless the facts remain that under the conditions of these experi ments oxygen concentrations of 20% as found in air were inhibitory to the growth of clones. The possibility arises that many basic phenomena of tissue culture, including the production of viruses and hormones in cell cultures, may be enhanced by the use of low oxygen tension gas phases.
CHAPTER
Ian
7
Macpherson
Cells derived directly from trypsinized normal animal tissues or from primary or low passage culture do not multiply in liquid or semisolid suspension culture. This characteristic is retained to some extent by certain cell lines that have ac quired a higher degree of autonomy in culture (e.g., BHK21, 3 T 3 ) . These cells 1 2 3 also respond to growth controls in vitro including contact inhibition of move 4 inhibition of growth, topoinhibition, and serum de ment, density-dependent pendence. Cell lines that have been carried in vitro through many generations, cancer cells, and cells transformed by oncogenic viruses all multiply to a greater or lesser extent in suspension culture. The acquisition of the ability to grow in5 suspension has been utilized in a selective assay for virus-transformed cells. If cells that do not multiply in suspension are infected with a tumor virus and cultured in soft agar medium only transformed cells grow progressively to form colonies. This technique may also be used as a convenient method for cloning. Colonies may be picked from the agar with pipettes and used to initiate conventional cultures. The selectivity of agar suspension culture is due to the inhibitory effect 6 of acidic and sulfated polysaccharides in most agars on normal cell multiplica tion. Normal cells also require extension on a solid substrate in order to undergo cell division. Agar medium in which the polyanions have been complexed with DEAE-dextran is capable of supporting at least limited multiplication of some
1 2M. Abercrombie and J. E. M. Heaysman, Exp. Cell Res. 6, 293 (1954). 3M . G. P. Stoker and H. Rubin, Nature (London) 215, 171 (1967). 4R . Dulbecco, Nature (London) 227, 802 (1970). 5R . R. Biirk, Nature (London) 212, 1261 (1966). 61 . Macpherson and L. Montagnier, Virology 23, 291 (1964).
L . Montagnier, Ciba Found. Symp. on Growth Control in Cell Cultures p. 33 (1971).
7. Soft Agar
277
Techniques
67 cell lines unable to grow in untreated a g a r . Agarose, which is agar with most of the large charged molecules removed also supports the growth of some 68 the addition cells. Conversely an increase in the concentration of poly anions by of dextran sulfate reinforces the inhibitory effect of the agar g e l . A basic technique for the preparation of soft agar cultures suitable for the 5 7 9 4- 2here. assay of virus transformation in cell lines such as BHK21 is described Factors affecting the growth of cells in agar are listed in Table I . " ' Modi fications for special purposes can be achieved, e.g., by the substitution of agarose for agar. Additional promotors or inhibitors can be applied within these general procedures. I f the technique is to be used for the production of clones, then a number of promoters should be tested in the medium to find the optimum conditions of culture, e.g., the basic method with the addition of DEAE-dextran to a level that promotes the maximum number and size of colonies in medium plus feeder cells would be an appropriate starting point. If this was unsatisfactory then other additions such as insulin, purines, or collagen could be tested. When the medium is to be used for selective culture, the "background" growth of the untransformed or untreated cells can be sup pressed to a suitable level by the addition of dextran sulfate. Since mycoplasma infection of cells may promote their growth in soft agar, care should be taken to control for this factor by testing the effect of broad-spectrum antibiotics on growth in agar. An additional advantage of the soft agar method is its use for producing diffusion gradients in the medium. This can be achieved by placing filter paper disks soaked in the test material in the dishes before adding the agar base. 25 Growth in agar medium is correlated with malignant potential in animal cells. Variants of BHK21/13 cells resistant to dextran sulfate are highly tumori-
7 81 . Macpherson, unpublished data. 9L . Montagnier, Bull. Cancer 57, 13 ( 1 9 7 0 ) . 110. Macpherson, J. C e l l S c i . 1, 145 ( 1 9 6 6 ) . R. M. McAllister, M. O. Nicolson, A. M. Lewis, I . Macpherson, and R. J . Huebner,
11 Virol. /. Gen.
4, 29 ( 1 9 6 9 ) .
12P. H. Black, V i r o l o g y 28, 760 ( 1 9 6 6 ) .
R. M. McAllister and I . Macpherson, / . G e n . V i r o l . 4, 29 ( 1 9 6 9 ) . "14I . Macpherson, S c i e n c e 148, 1731 ( 1 9 6 5 ) . 1 H. 5 Rubin, E x p . C e l l R e s . 4 1 , 138 ( 1 9 6 6 ) . 16J . Zavada and I . Macpherson, Nature ( L o n d o n ) 225, 24 (1970).
1 M. 7 G. P. Stoker, Nature (London) 218, 234 ( 1 9 6 8 ) . 181 . Macpherson and W. Russell, Nature ( L o n d o n ) 210, 1343 ( 1 9 6 6 ) . 1 I.9 Macpherson and A. Bryden, E x p . C e l l R e s . 6 9 , 240 ( 1 9 7 1 ) . 201 . Macpherson, this volume, Section V, Chapter l . B . G. D. Clarke, M. G. P. Stoker, A. Ludlow, and M. Thornton, Nature 798 2( 119 7 0 ) . G.
22 Cultures,
D. Clarke and
M. G. P. Stoker, Ciba
Found.
Symp.
p. 17 ( 1 9 7 1 ) .
on Growth
( L o n d o n ) 227, Control
23 H. Otsuka, / . C e l l S c i . 10, 137 ( 1 9 7 2 ) . 2 E.4 Tjotta, M. Flikke, and O. Lahelle, Arch. G e s . V i r u s f o r s c h . 23, 288 ( 1 9 6 8 ) . 25F . K. Sanders and J . D. Smith, Nature ( L o n d o n ) 227, 513 ( 1 9 7 0 ) . T. Kakunaga and J . Kamahora, B i k e n J. 11, 313 (1968).
in
Cell
278
V. SINGLE CELL ISOLATIONS AND CLONING TABLE I Factors Affecting Growth in Soft Agar Medium Factors Promotors 1. Transformation by oncogenic viruses (a) Polyoma (b) SV40 (c) Adenoviruses (d) Rous sarcoma virus (e) Murine sarcoma virus 2. Abortive transformation by polyoma virus 3. Infection with mycoplasma 4 4. Addition of X-irradiated or mitomycin Ctreated cells as feeders (5 X 10 /culture 6 in top layer) 5. DEAE-dextran (mol. wt. 2 X 10 ) added to complex acidic and sulfated polysaccharide in Difco Baeto-Agar (approx. 5 /ig/ml) 6. Insulin about 0.1 to 1.0 M g/nri 7. Serum (a) Increased levels up to 1 5 % (b) Pig 8. Conditioned medium 9. Collagen 10. Purine derivatives
5 7 Inhibitors 1. Dextran sulfate (mol. wt. 5 X 10 -4 X 10 ) up to 50 //g/ml 2. Heparin, up to 50 Mg/ml
References
5 11 10, 12 13, 14 15 16 9, 17 18, 19 6, 7 7, 20 21 22 23 6, 24 6 6, 21 21
26 genie and also have a reduced serum requirement for growth. However, the correlation is not absolute and some lines of cells that712 grow well in agar containing sulfated polysaccharides are nontumorigenic. ' Agar suspension culture may be used to grow tumor cells from trypsinized tumors and suppress the growth of stromal fibroblasts. METHODS
Preparation of 1.25% Agar Stock. Add 12.5 g of Difco Bacto-agar to about 800 ml of cold water (tissue culture quality). Boil to dissolve the agar and make up to 1 liter with hot water. Dispense 80-ml amounts ( ± 1 or 2 ml) of hot agar in screw-capped bottles (approx. 250-ml capacity). Sterilize by auto claving at low pressure with the screw caps loosened. Cool to room tempera ture, tighten caps, and store at room temperature. For use, melt agar in boiling water checking to see that it does not contain any residual pieces of unmelted agar. Cool bottles briefly at room temperature (to prevent cracking) before transferring them to a water bath at 44°C. Ensure that the level of the water
26
G. D. Clarke, unpublished data.
7. Soft Agar
Techniques
279
bath is above the level of the agar in the bottle otherwise it may gel at the meniscus. Synthetic Medium ( 2 X ) . Prepare double-strength synthetic medium (i.e., with twice the normal concentration of all components). Sterilize the medium by filtration through Millipore membranes. Dispense in 80-ml amounts in screw-capped bottles. Store at 4 ° C for periods of up to 1 week. Serum. Millipore-filtered calf or fetal calf serum. Store frozen and heat at 56°C for 30 minutes before use. Tryptose Phosphate Broth. Autoclaved Difco Bacto tryptose phosphate broth. Store at room temperature. Preparation of Complete Agar Medium. Add 20 ml of serum and 20 ml of tryptose phosphate broth to the 2 X synthetic medium, warm to 44°C, and then add to the melted agar. Mix by swirling but avoid frothing and trapping air bubbles in the medium. Preparation of Agar Base Layers. Pipette 7 ml of agar medium into 5-cm Petri dishes and leave to set at room temperature. The dishes may be glass or plastic. The latter need not be of tissue culture grade since the cells do not come into contact with the surface of the dish. It is inadvisable to prepare the base layers more than an hour or so before use or to store them in the refrig erator, since fluid is exuded from the base layer and this prevents the adhesion of the top layer. The base layer permits the concentration of the cells in a thin top layer which facilitates the inspection of the plates by low-power microscopy. The base layer also provides the bulk of the nutrients for growth. Preparation of the Cell Containing Top Layer. When the agar suspension method is applied to the assay of virus-transformed colonies the cells may be infected in suspension or as monolayers which are subsequently resuspended with trypsin. In either case care must be taken to ensure that the suspension is free of cell aggregates. Some strains of small-plaque polyoma virus cause hamster cells to agglutinate when they are infected in suspension with high concentra tions of virus. I f this occurs, the virus should be adsorbed to monolayers. Fol lowing adsorption the cultures are fed with fluid medium and left for 1 or 2 hours to allow the virus to penetrate the cells. They may then be trypsinized. One volume of cell suspension in medium at 37°C is mixed with two volumes of 0.5% agar medium (as prepared above) at 44°C. Mixing is achieved by gentle swirling or by gentle pipetting once or twice. Large volumes should not be mixed at one time since it is difficult to maintain the agar medium fluid during pipettings. A 30-ml screw-capped bottle with a total of 15 ml of agar medium and cell suspension can be easily handled. The volume of the top layer is 1.5 ml. The optimum number of cells per culture depends on the type of cells used and the number of transformed colonies expected. I f large wellseparated colonies are required, fewer cells should be plated, e.g., for B H K 2 1 / 1 3
280
V. SINGLE CELL ISOLATIONS AND CLONING
4 5 the optimum number 3 of cells plated for transforma cells and polyoma virus tion assays is 5 X 1 0 - 1 0 cells per dish and 10 cells per dish when the method is used to isolate transformed colonies. Cultures are incubated at 37°C in a well humidified atmosphere of 5 to 10% C 0 2 in air. I f a suitable C 0 2 incubator is not available the dishes may be en closed in a gas-tight box or plastic bag containing a suitable C 0 2/ a i r mixture. Cells capable of dividing do so shortly after incubation has commenced and grow progressively to form colonies 0.1-0.2 mm in diameter in 7 to 10 days. I f cultures are to be incubated for more than 10 days they should be fed with 1 to 2 ml medium on the top layer. This can be changed by careful pipetting.
COUNTING COLONIES
Colonies are examined and counted unfixed and unstained with the aid of a plate microscope ( 2 5 - 5 0 X magnification). Cultures may be left for several days in the refrigerator before being examined. Occasionally normal cells undergo a few divisions and form small colonies. By examining control cultures it is possible to determine the size of colony that should be considered as "background." The longer cultures are incubated the more obvious this distinc tion becomes. Transformed cell colonies increase in size more rapidly than the "abortive" normal cell colonies. A more certain method of classifying colonies is to use an image-shearing eyepiece when counting (manufactured by W. Watson & Sons, Barnet, Herts., England). This splits the image of a colony into green and red components. The eyepiece may be calibrated for a particular colony size, say 0.1 mm. Colonies of this size present two images, just touching. Colonies larger than 0.1 mm in diameter will have overlapping images and colonies smaller than 0.1 mm will have separated images. In cultures with many colonies a convenient method of counting is to lay a small coverglass (e.g., 5 X 20 mm) on the top layer surface and make a sample count of the colonies below the coverglass. A grid may be ruled on the coverglass with a ball-point pen.
SUBCULTURING COLONIES
Colonies may be removed from the agar with finely drawn Pasteur pipettes. The whole colony is transferred to a tube containing 1 ml of medium and pipetted until it breaks into several fragments. It is essential to remove the agar from the colony, otherwise the cells will be prevented from migrating onto the glass and dividing. A true assessment of the type of cells in colony isolates can only be obtained if the initial growth is trypsinized and subcultured. The whole cell growth from an agar suspension culture can be harvested by pipetting the top layer off and pipetting in medium to free the colonies from the agar. The colonies may then be cultured directly on glass or trypsinized.
V. SINGLE CELL ISOLATIONS AND CLONING
but nevertheless the facts remain that under the conditions of these experi ments oxygen concentrations of 20% as found in air were inhibitory to the growth of clones. The possibility arises that many basic phenomena of tissue culture, including the production of viruses and hormones in cell cultures, may be enhanced by the use of low oxygen tension gas phases.
CHAPTER
Ian
7
Macpherson
Cells derived directly from trypsinized normal animal tissues or from primary or low passage culture do not multiply in liquid or semisolid suspension culture. This characteristic is retained to some extent by certain cell lines that have ac quired a higher degree of autonomy in culture (e.g., BHK21, 3 T 3 ) . These cells 1 2 3 also respond to growth controls in vitro including contact inhibition of move 4 inhibition of growth, topoinhibition, and serum de ment, density-dependent pendence. Cell lines that have been carried in vitro through many generations, cancer cells, and cells transformed by oncogenic viruses all multiply to a greater or lesser extent in suspension culture. The acquisition of the ability to grow in5 suspension has been utilized in a selective assay for virus-transformed cells. If cells that do not multiply in suspension are infected with a tumor virus and cultured in soft agar medium only transformed cells grow progressively to form colonies. This technique may also be used as a convenient method for cloning. Colonies may be picked from the agar with pipettes and used to initiate conventional cultures. The selectivity of agar suspension culture is due to the inhibitory effect 6 of acidic and sulfated polysaccharides in most agars on normal cell multiplica tion. Normal cells also require extension on a solid substrate in order to undergo cell division. Agar medium in which the polyanions have been complexed with DEAE-dextran is capable of supporting at least limited multiplication of some
1 2M. Abercrombie and J. E. M. Heaysman, Exp. Cell Res. 6, 293 (1954). 3M . G. P. Stoker and H. Rubin, Nature (London) 215, 171 (1967). 4R . Dulbecco, Nature (London) 227, 802 (1970). 5R . R. Biirk, Nature (London) 212, 1261 (1966). 61 . Macpherson and L. Montagnier, Virology 23, 291 (1964).
L . Montagnier, Ciba Found. Symp. on Growth Control in Cell Cultures p. 33 (1971).
7. Soft Agar
277
Techniques
67 cell lines unable to grow in untreated a g a r . Agarose, which is agar with most of the large charged molecules removed also supports the growth of some 68 the addition cells. Conversely an increase in the concentration of poly anions by of dextran sulfate reinforces the inhibitory effect of the agar g e l . A basic technique for the preparation of soft agar cultures suitable for the 5 7 9 4- 2here. assay of virus transformation in cell lines such as BHK21 is described Factors affecting the growth of cells in agar are listed in Table I . " ' Modi fications for special purposes can be achieved, e.g., by the substitution of agarose for agar. Additional promotors or inhibitors can be applied within these general procedures. I f the technique is to be used for the production of clones, then a number of promoters should be tested in the medium to find the optimum conditions of culture, e.g., the basic method with the addition of DEAE-dextran to a level that promotes the maximum number and size of colonies in medium plus feeder cells would be an appropriate starting point. If this was unsatisfactory then other additions such as insulin, purines, or collagen could be tested. When the medium is to be used for selective culture, the "background" growth of the untransformed or untreated cells can be sup pressed to a suitable level by the addition of dextran sulfate. Since mycoplasma infection of cells may promote their growth in soft agar, care should be taken to control for this factor by testing the effect of broad-spectrum antibiotics on growth in agar. An additional advantage of the soft agar method is its use for producing diffusion gradients in the medium. This can be achieved by placing filter paper disks soaked in the test material in the dishes before adding the agar base. 25 Growth in agar medium is correlated with malignant potential in animal cells. Variants of BHK21/13 cells resistant to dextran sulfate are highly tumori-
7 81 . Macpherson, unpublished data. 9L . Montagnier, Bull. Cancer 57, 13 ( 1 9 7 0 ) . 110. Macpherson, J. C e l l S c i . 1, 145 ( 1 9 6 6 ) . R. M. McAllister, M. O. Nicolson, A. M. Lewis, I . Macpherson, and R. J . Huebner,
11 Virol. /. Gen.
4, 29 ( 1 9 6 9 ) .
12P. H. Black, V i r o l o g y 28, 760 ( 1 9 6 6 ) .
R. M. McAllister and I . Macpherson, / . G e n . V i r o l . 4, 29 ( 1 9 6 9 ) . "14I . Macpherson, S c i e n c e 148, 1731 ( 1 9 6 5 ) . 1 H. 5 Rubin, E x p . C e l l R e s . 4 1 , 138 ( 1 9 6 6 ) . 16J . Zavada and I . Macpherson, Nature ( L o n d o n ) 225, 24 (1970).
1 M. 7 G. P. Stoker, Nature (London) 218, 234 ( 1 9 6 8 ) . 181 . Macpherson and W. Russell, Nature ( L o n d o n ) 210, 1343 ( 1 9 6 6 ) . 1 I.9 Macpherson and A. Bryden, E x p . C e l l R e s . 6 9 , 240 ( 1 9 7 1 ) . 201 . Macpherson, this volume, Section V, Chapter l . B . G. D. Clarke, M. G. P. Stoker, A. Ludlow, and M. Thornton, Nature 798 2( 119 7 0 ) . G.
22 Cultures,
D. Clarke and
M. G. P. Stoker, Ciba
Found.
Symp.
p. 17 ( 1 9 7 1 ) .
on Growth
( L o n d o n ) 227, Control
23 H. Otsuka, / . C e l l S c i . 10, 137 ( 1 9 7 2 ) . 2 E.4 Tjotta, M. Flikke, and O. Lahelle, Arch. G e s . V i r u s f o r s c h . 23, 288 ( 1 9 6 8 ) . 25F . K. Sanders and J . D. Smith, Nature ( L o n d o n ) 227, 513 ( 1 9 7 0 ) . T. Kakunaga and J . Kamahora, B i k e n J. 11, 313 (1968).
in
Cell
278
V. SINGLE CELL ISOLATIONS AND CLONING TABLE I Factors Affecting Growth in Soft Agar Medium Factors Promotors 1. Transformation by oncogenic viruses (a) Polyoma (b) SV40 (c) Adenoviruses (d) Rous sarcoma virus (e) Murine sarcoma virus 2. Abortive transformation by polyoma virus 3. Infection with mycoplasma 4 4. Addition of X-irradiated or mitomycin Ctreated cells as feeders (5 X 10 /culture 6 in top layer) 5. DEAE-dextran (mol. wt. 2 X 10 ) added to complex acidic and sulfated polysaccharide in Difco Baeto-Agar (approx. 5 /ig/ml) 6. Insulin about 0.1 to 1.0 M g/nri 7. Serum (a) Increased levels up to 1 5 % (b) Pig 8. Conditioned medium 9. Collagen 10. Purine derivatives
5 7 Inhibitors 1. Dextran sulfate (mol. wt. 5 X 10 -4 X 10 ) up to 50 //g/ml 2. Heparin, up to 50 Mg/ml
References
5 11 10, 12 13, 14 15 16 9, 17 18, 19 6, 7 7, 20 21 22 23 6, 24 6 6, 21 21
26 genie and also have a reduced serum requirement for growth. However, the correlation is not absolute and some lines of cells that712 grow well in agar containing sulfated polysaccharides are nontumorigenic. ' Agar suspension culture may be used to grow tumor cells from trypsinized tumors and suppress the growth of stromal fibroblasts. METHODS
Preparation of 1.25% Agar Stock. Add 12.5 g of Difco Bacto-agar to about 800 ml of cold water (tissue culture quality). Boil to dissolve the agar and make up to 1 liter with hot water. Dispense 80-ml amounts ( ± 1 or 2 ml) of hot agar in screw-capped bottles (approx. 250-ml capacity). Sterilize by auto claving at low pressure with the screw caps loosened. Cool to room tempera ture, tighten caps, and store at room temperature. For use, melt agar in boiling water checking to see that it does not contain any residual pieces of unmelted agar. Cool bottles briefly at room temperature (to prevent cracking) before transferring them to a water bath at 44°C. Ensure that the level of the water
26
G. D. Clarke, unpublished data.
7. Soft Agar
Techniques
279
bath is above the level of the agar in the bottle otherwise it may gel at the meniscus. Synthetic Medium ( 2 X ) . Prepare double-strength synthetic medium (i.e., with twice the normal concentration of all components). Sterilize the medium by filtration through Millipore membranes. Dispense in 80-ml amounts in screw-capped bottles. Store at 4 ° C for periods of up to 1 week. Serum. Millipore-filtered calf or fetal calf serum. Store frozen and heat at 56°C for 30 minutes before use. Tryptose Phosphate Broth. Autoclaved Difco Bacto tryptose phosphate broth. Store at room temperature. Preparation of Complete Agar Medium. Add 20 ml of serum and 20 ml of tryptose phosphate broth to the 2 X synthetic medium, warm to 44°C, and then add to the melted agar. Mix by swirling but avoid frothing and trapping air bubbles in the medium. Preparation of Agar Base Layers. Pipette 7 ml of agar medium into 5-cm Petri dishes and leave to set at room temperature. The dishes may be glass or plastic. The latter need not be of tissue culture grade since the cells do not come into contact with the surface of the dish. It is inadvisable to prepare the base layers more than an hour or so before use or to store them in the refrig erator, since fluid is exuded from the base layer and this prevents the adhesion of the top layer. The base layer permits the concentration of the cells in a thin top layer which facilitates the inspection of the plates by low-power microscopy. The base layer also provides the bulk of the nutrients for growth. Preparation of the Cell Containing Top Layer. When the agar suspension method is applied to the assay of virus-transformed colonies the cells may be infected in suspension or as monolayers which are subsequently resuspended with trypsin. In either case care must be taken to ensure that the suspension is free of cell aggregates. Some strains of small-plaque polyoma virus cause hamster cells to agglutinate when they are infected in suspension with high concentra tions of virus. I f this occurs, the virus should be adsorbed to monolayers. Fol lowing adsorption the cultures are fed with fluid medium and left for 1 or 2 hours to allow the virus to penetrate the cells. They may then be trypsinized. One volume of cell suspension in medium at 37°C is mixed with two volumes of 0.5% agar medium (as prepared above) at 44°C. Mixing is achieved by gentle swirling or by gentle pipetting once or twice. Large volumes should not be mixed at one time since it is difficult to maintain the agar medium fluid during pipettings. A 30-ml screw-capped bottle with a total of 15 ml of agar medium and cell suspension can be easily handled. The volume of the top layer is 1.5 ml. The optimum number of cells per culture depends on the type of cells used and the number of transformed colonies expected. I f large wellseparated colonies are required, fewer cells should be plated, e.g., for B H K 2 1 / 1 3
280
V. SINGLE CELL ISOLATIONS AND CLONING
4 5 the optimum number 3 of cells plated for transforma cells and polyoma virus tion assays is 5 X 1 0 - 1 0 cells per dish and 10 cells per dish when the method is used to isolate transformed colonies. Cultures are incubated at 37°C in a well humidified atmosphere of 5 to 10% C 0 2 in air. I f a suitable C 0 2 incubator is not available the dishes may be en closed in a gas-tight box or plastic bag containing a suitable C 0 2/ a i r mixture. Cells capable of dividing do so shortly after incubation has commenced and grow progressively to form colonies 0.1-0.2 mm in diameter in 7 to 10 days. I f cultures are to be incubated for more than 10 days they should be fed with 1 to 2 ml medium on the top layer. This can be changed by careful pipetting.
COUNTING COLONIES
Colonies are examined and counted unfixed and unstained with the aid of a plate microscope ( 2 5 - 5 0 X magnification). Cultures may be left for several days in the refrigerator before being examined. Occasionally normal cells undergo a few divisions and form small colonies. By examining control cultures it is possible to determine the size of colony that should be considered as "background." The longer cultures are incubated the more obvious this distinc tion becomes. Transformed cell colonies increase in size more rapidly than the "abortive" normal cell colonies. A more certain method of classifying colonies is to use an image-shearing eyepiece when counting (manufactured by W. Watson & Sons, Barnet, Herts., England). This splits the image of a colony into green and red components. The eyepiece may be calibrated for a particular colony size, say 0.1 mm. Colonies of this size present two images, just touching. Colonies larger than 0.1 mm in diameter will have overlapping images and colonies smaller than 0.1 mm will have separated images. In cultures with many colonies a convenient method of counting is to lay a small coverglass (e.g., 5 X 20 mm) on the top layer surface and make a sample count of the colonies below the coverglass. A grid may be ruled on the coverglass with a ball-point pen.
SUBCULTURING COLONIES
Colonies may be removed from the agar with finely drawn Pasteur pipettes. The whole colony is transferred to a tube containing 1 ml of medium and pipetted until it breaks into several fragments. It is essential to remove the agar from the colony, otherwise the cells will be prevented from migrating onto the glass and dividing. A true assessment of the type of cells in colony isolates can only be obtained if the initial growth is trypsinized and subcultured. The whole cell growth from an agar suspension culture can be harvested by pipetting the top layer off and pipetting in medium to free the colonies from the agar. The colonies may then be cultured directly on glass or trypsinized.