APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1976, p. 509-513 Copyright ©D 1976 American Society for Microbiology
Vol. 31, No. 4 Printed in U.S.A.
Substitute for Agar in Solid Media for Common Usages in Microbiology NED WATSON AND DAVID APIRION* Department of Microbiology and Immunology, Washington University Medical School, St. Louis, Missouri 63110
Received for publication 1 December 1975
The potassium salt of carrageenan was found to be an adequate replacement for agar in solid bacteriological media. The common microbial genetic techniques, such as purifying colonies by streaking, replication tests, and titration of cultures, were carried out successfully with a number of mutant strains of Escherichia coli.
The use of solid culture media has been of fundamental importance to microbiological research since the late nineteenth century. Desirable qualities of a solidifying agent for media include solidity over the temperature range of bacterial growth, resistance to digestion by bacteria, lack of syneresis, transparency, and the ability to form a reversible colloid. The medium must be firm enough to allow the carrying out of common techniques such as streaking out cultures, plating, and replication. In addition, it is helpful if the gelation agent is relatively inexpensive and easily obtained. Since Koch (7) first introduced agar as a gelifying agent in bacterial media (6), it has become the primary material for solid media throughout the world. However, increased cost and recent shortages have made a more readily available substitute for agar desirable. During World War II attempts were made to find a substitute for agar in solid bacterial media. A report appeared that extracts from Irish Moss can substitute for agar in bacteriological culture media (10). Since two carrageenan (Irish Moss) salts are commercially available now, and since present needs in a microbial genetics laboratory are somewhat different from those that existed in 1943, we decided to test a number of commercially available gelifying agents as substitutes for agar. Our experiments suggest that the potassium salt of the sulfate polysaccharide, carrageenan, is an adequate substitute for agar in most common genetic manipulations of Escherichia coli. MATERIALS ANI) METHOi)S Bacterial and phage strains. Bacterial strains are described in Table 1. Bacteriophages P1, T4, and T7 were from our own stocks. Media. Media were prepared as described by Apirion (1) except for substitutions of the various gelation materials.
Gelation materials. The agar used was Difco agar. Gelatin types I and II, carrageenan calcium salt, and carrageenan potassium salt were from Sigma Chemical Co. (St. Louis, Mo.).
RESULTS Gelling properties. The materials tested by us for their gelling properties included gelatin types I and II and carrageenan calcium and potassium salts. Various concentrations of these materials (1.0, 1.5, 2.0, and 4.0%) were prepared, autoclaved, and allowed to solidify in small depression plates (Table 2). They were then incubated at 25 and 45 C. Both types of gelatins gave too soft a consistency at all concentrations to warrent further investigation. All concentrations of the Ca2+ salt of carrageenan were rather firm at room temperature, but none were firm at 45 C. On the other hand, all concentrations of the K+ salt of carrageenan were adequately firm at room temperature, and the 2 and 4% concentrations were also firm at 45 C. It was later observed, when attempting to plate cells by spreading them on top of the medium, that the Ca2+ salt was too soft even at 37 C for such a purpose. Like agar, carrageenan has to be melted to obtain a uniform suspension. Replication tests. To test the K+ salt of carrageenan as a medium support, master plates were made with rich medium containing agar (1.5%) and rich medium containing carrageenan (2.0%) with eight different E. coli strains in common use in our laboratory at that time (Table 1). No differences were observed in the firmness of the plates. After overnight incubation at 37 C the patched colonies grew similarly on both master plates; in fact, the plates with carrageenan were clearer and the colonies were more readily distinguishable. (Later, when we used a different lot of carrageenan K+ salt, also from Sigma, plates were turbid and 509
510
WATSON AND APIRION
APPL. ENVIRON. MICROBIOL.
did not support growth as well. This was probably due to a difference in purity between the two lots, but we were unable to get any information on the differences between the two lots.) The master plates were then replicated to a variety of media containing either agar or carrageenan and were incubated at various temperatures (Table 3). The test plates included rich medium, minimal medium with a variety
of specific additions, and different sugars and indicator plates. The results were almost identical using carrageenan K+ salt plates as compared with agar plates (Table 3). We purified colonies of different strains by streaking them on rich medium plates containing either 1.8% agar or 2% K+ carrageenan and found that both sets of plates gave us equally adequate results. The carrageenan plates were firm enough to
TABLE 1. Strains used Strain
D10 A19 AB301 N141
N2048 N2053 N2089 N2090 a
Sex and markers
Source or derivation
Hfr P021 metBI rna-10 rel-1 (A) Hfr P021 metBI aux rna-19 rel-I (X) Hfr P021 metBI rel-1 (X) F-lac-1 xyl mal-141 gal-2 thi arg-3 his-4 ilv-158 pro-2 F- metBI his rna-19 rnc-105 F- like N2048, but rnc+ F- thi-1 argAl nadB4 gal-6 malAl XRxyl 7 ara13 mtl-2 str-9 tonA2 supE44 F- like N2089, but rnc-105
NGa from AB301 (5) NG from AB301 (5) (1, 5) Spontaneous mal- derivative of strain AB774 (2) (3, 4) (3, 4) Lac+ derivative from strain N2076 (ref. 4) Lac' derivative from strain N2077 (ref. 4)
NG, Nitrosoguanidine.
TABLE 2. Gelling characteristics of gelatin and carrageenan" Gelatin type I
Gelatin type II
Carrageenan Ca+ salt
Carrageenan K' salt
25C 45C 25C 45C 25C 45C 25C 45C 1.0 Soft Soft Soft Soft Firm Soft Firm Soft 1.5 Soft Soft Soft Soft Firm Soft Firm Soft 2.0 Soft Soft Soft Soft Firm Soft Firm Firm 4.0 Soft Soft Soft Soft Firn Soft Firm Firm Two types of gelatin and two salts of carrageenan were mixed with distilled water in the concentrations indicated above and autoclaved. Plates were poured and allowed to solidify at room temperature for 1 day. The plates were then incubated for another day at the temperatures indicated above. The consistency of the gel was tested by visual observation or by streaking on the top of the plates with a bacteriological loop. TABLE 3. Replication tests on agar and carrageenan platesa Rich medium
MM
MM MM MM MM MM Lac Lac Lac -Arg -His -Met -Nic -Pro MM EMB Tet Strain 300 370410430450370430 (37 C) (37 C) (37 C) (37 C) (37 C) (37 C) (37 C) |(37 C) 30AC 37C 41AC43CC 45 C 37 C 43A C C
AIC
AIC|
A C A C A C A C C1 A |C JA A |C A|C| A |C A C A |C |A |C + + + + + + + + + + + + + + A19 + + + + + ++ + + ++ ++ + + AB301 + +++++++++++++ + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + D10 ++ ++++++++++++++ + + N141 + + -4-. N2048 + ± + ± ± ± ± ± - + + + =+_ + + 0 + + + + + + +± + + ++ ±N2053 + + + + + ± + ± ± + + ++ + + + + - + + + + N2089 + + + + + + + + + + + + + + - - + + + + + + + + + + 4+ + -++ ++ + + - + + + + N2090 + + + + + + + + + + " Two sets of plates were used in this experiment. In columns designated A, plates contained 1.8% agar as the gelation agent; and in C, plates contained 2.0% carrageenan as the gelation agent. All strains were grided on two rich medium master plates, one containing agar and the other carrageenan, and incubated at 37 C overnight. The master plates were then replicated to a set of plates as indicated. The plates were incubated at various temperatures and scored after 1, 2, and 3 days; the 2-day scoring is shown. Minimal medium (MM) contained 0.2% glucose and 50 jig each of thiamine, L-arginine, Lhistidine, L-isoleucine, L-valine, methionine, nicotinic acid, and L-proline per ml. In Lac MM, 0.2% lactose was substituted for glucose. Lac EMB plates contained lactose and eosin and methylene blue (1), and Lac Tet plates were lactose tetrazolium plates (3). Symbols: +, good growth, _, fair growth; -+, poor growth, +, very poor growth; -, no growth. On indicator plates: +, indicates positive test; -, indicates negative test; 0, indicates cells failed to grow. (A number ofE. coli mutants fail to grow on EMB plates. Apparently eosin and/or methylene blue are toxic for them.)
+T+ -T-
-
+T+ +I+
VOL. 31, 1976
SUBSTITUTION FOR AGAR IN BACTERIAL MEDIA
permit convenient streaking of the bacterial cells with a loop. Plating efficiency. We compared the plating efficiency of a single standard E. coli strain, D10, on regular minimal medium and on similar media in which either the Ca-+ or K+ salt of carrageenan was substituted for agar. A culture was grown overnight in liquid rich medium, washed and diluted in dilution buffer, and plated at 37 and 43 C in minimal medium gelled by agar or carrageenan (Table 4). Even at 37 C the Ca2+ salt of carrageenan was soft and difficult to work with and supported growth rather poorly. However, the plates with the K+ salt of carrageenan gave colony counts similar to the agar plates. In fact, the colonies TABLE 4. Plating efficiency of D10 on agar and various carrageenan mixtures" 37C 43C No. of Size No. of Size cells (mm) cells (mm) 4.9 x 109 1.1-2.5 3.9 x 109 2.2-3.2 Agar, 1.8% Carrageenan Ca2+ 2 x 107 2.5-3.5 _ salt, 3% Carrageenan K+ 6.3 x 109 2.6-3.5 6.0 x 109 2.5-4.5 salt, 2% Carrageenan K+ 5.8 x 109 2.2-3.2 5.6 x 109 2.5-4.0 salt, 3% a A liquid culture of D10 was grown overnight at 37 C in rich medium with agitation, washed once in dilution buffer, and diluted in the same buffer. One-tenth-milliliter volumes were then plated onto minimal medium plates containing 50 ,ug of methionine per ml using the various gelation agents as indicated. The plates were incubated for 2 days at the temperatures shown. Colonies were counted and the results from various dilutions were averaged and expressed per milliliter of original culture. The dashes indicate no colonies observed. The size of the smaller and larger colonies was determined by averaging the diameter of 6 to 10 well-separated colonies on the plates; the size of both types of colonies is shown. Gelation
511
appeared sooner and grew larger than those on the agar plates. The K+ salt of carrageenan also appeared to give slightly better viability. In another set of experiments, strains D1O and N2089 were plated on rich and minimal medium supported by agar or K+ carrageenan (Table 5). The numbers of colonies were comparable at all temperatures in rich medium and at 37 and 43 C in minimal medium. At 45 C similar results were obtained using rich medium. Using minimal medium, carrageenan seems to have the edge. One of the strains tested, D10, failed to form colonies on minimal agar medium, and the other strain tested, N2089, grew faster on the minimal carrageenan plates (see Table 5). Growth of phage (plaque formation). For many purposes, when working with bacteria and bacteriophages, it is useful to use a soft agar medium as a top layer. The K+ carrageenan that we tested did not seem suitable for this type of medium, since an 0.7% K+ carrageenan medium lost its reversible colloidial properties. Therefore, we tested whether it is possible to use carrageenan plates as a support for growth of bacteriophage when the bottom layer is made with carrageenan and the top layer (soft) with agar. Using this technique we found comparable plaque-forming ability using rich medium plates containing either agar or the K+ salt of carrageenan for the titration of P1, T4, and T7 bacteriophages (Table 6). DISCUSSION The experiments described here suggest that agar can be substituted successfully by the K+ salt of carrageenan in microbiological media. Whereas the experiments presented here were carried out with various mutants of E. coli, it is
TABLE 5. Plating efficiency at various temperatures on rich and minimal medium containing agar or carrageenan K+ salt, Viable cell counts/ml (x 109)
Medium
D10 37C
43C
N2089 45C
37C
43C
45C
5.2 5.5 4.9 0.8 1.0 Rich, 1.8% agar 1.1 1.0 1.1 4.7 4.8 5.6 1.3 Rich, 2% carrageenan 0.8 1.0 5.2 3.7 _b 0.8' Minimal, 1.8% agar 5.2 3.0 1.0 5.1 1.0 Minimal, 2% carrageenan 0.5d Strains D10 and N2089 were grown overnight in liquid rich medium at 37 C with agitation, washed once in dilution buffer, and diluted 10-" and 2 x 10-7 in the same buffer. One-tenth-milliliter aliquots were plated onto rich or minimal medium plates containing the gelation agent as indicated. The minimal medium plates for D10 contained 50 ,g of methionine per ml, whereas those for N2089 contained 5 gg of nicotinic acid and thiamine per ml and 50 ,ug of arginine per ml. The plates were incubated for 2 days or as indicated at the temperatures shown. Colonies were counted and averaged as in Table 4. b No colonies were observed after 4 days of incubation. It was necessary to incubate plates for 4 days in order to score them. d It was necessary to incubate plates for 3 days in order to score them. "
512
APPL. ENVIRON. MICROBIOL.
WATSON AND APIRION
TABLE 6. Titration of three bacteriophages on agar and carrageenan platesa No. of phage/ml Bacteriophage
Agar
Carrageenan K+ salt
2.9 x 109 P1 2.7 x 109 T4 10.3 x 108 8.4 x 10 5.2x108 3.7x108 T7 a Bacteriophage stocks were diluted, and similar volumes were added to prewarmed tubes containing approximately 108 E. coli cells (strain D10) and appropriate ions necessary for phage attachment. The soft agar medium was then poured onto rich medium plates containing the indicated gelation material, allowed to harden at room temperature, and incubated overnight at 37 C. Plaques were then counted; results are expressed as phage per milliliter of phage stock solution. The morphology of the plaques of each phage was similar on both sets of plates.
quite likely that K+ carrageenan could serve as a useful gelation agent for a large variety of solid culture media in common use in microbiological work. We did encounter several problems. At lower concentrations, such as in a soft agar type of preparation, the ability to form a useful reversible colloid for phage manipulations was unsatisfactory compared to agar. This probably can be overcome by varying the K+ ion concentrations (8, 11, 12; J. H. Brewer and C. B. McLaughlin, Bacteriol. Proc., p. 25-26, 1953; A. Frieden and S. J. Werbin, U.S. Patent 2,427594, 1947). The Ca2+ salt of carrageenan generally had unsatisfactory gelling properties. This too might be remedied by varying ionic concentrations in the medium or by chemical modficiations of carrageenan; for example, by crosslinking or by the addition of hydrophillic groups (Paul Hartman, personal communication). The most important drawback encountered was a vast difference in the properties of different lots of the K+ salt tested. For instance, the lot (Sigma 71C-90019) we used in the experiments reported here gave us very reproducible results. On the other hand, another lot (Sigma 94C-0281) gave us only 12% of the colonies at 37 C and no colonies at 43 C when comparing colony-forming ability on minimal medium with agar plates of the same medium. The plates prepared from this lot were also quite turbid, indicating a difference in purity. Unfortunately, we were unable to get any information from the distributor as to the relative purity of the two lots or who was their manufacturer(s). Therefore we could not determine the basis for the variability observed with different lots. On the basis of our observations it is clear
that the K+ carrageenan can be used for most bacteriological purposes as a base for solidifying various media. We used it successfully for. preparation of all our routine media, which include minimal media with various sugars, rich medium, and indicator media (tetrazolium and eosin methylene blue). On such media we performed the four common manipulations of bacteria in a microbial genetics laboratory: streaking out cells (purification), replication from master plates to various other solid media, testing efficiency of colony formation from single cells, and using bacteria to clone bacteriophage by the plaquing technique (in this case only the bottom layer of medium contained K+ carrageenan). We found no instance in which the carrageenan could supply amino acids or sugars in the E. coli strains tested. Thus it seems that it cannot be utilized by E. coli and probably is suitable to be used for supporting media for many other bacteria as well (10). The K+ carrageenan used here was quite firm at 2% concentrations over the entire temperature range for growth of E. coli and was even more transparent than agar. Although syneresis is given as one of the major drawbacks to using carrageenan in bacteriological media (9-11), we did not observe this phenomenon in any of the batches of carrageenan used. Even after several months of storage in a cold room, carrageenan plates were as dry and firm as agar plates. Carrageenan is a polysaccharide extracted from the red marine algae Chondrus crispus, which is found in great abundance along the east coast of the United States and the western coast of Europe. The alga grows along the shore 1 to 14 feet (ca. 30.5 to 426.7 cm) below ebb tide and is easily gathered by hand picking or raking (11). On the other hand, Gelidium amansii, the principal source for agar, is found primarily in restricted areas in the Pacific Ocean on submerged rocky ledges and is difficult to harvest, requiring calm weather and often diving gear (11). Whereas, according to manufacturers, it is becoming increasingly difficult to obtain sufficient amounts of the algae from which agar is produced, there should be little problem obtaining a sufficient supply of C. crispus. Moreover, the cost of the carrageenan per pound that we tested was less than one-sixth of the price of agar. Therefore, we conclude that the K+ salt of carrageenan could become a desirable substitute for agar in various microbiological usages. ACKNOWLEI)GMENTS This work was supported by Public Health Service grant GM-19821 from the National Institute of General Medical Sciences and grant CA-15389 from the National Cancer Institute.
VOL. 31, 1976
SUBSTITUTION FOR AGAR IN BACTERIAL MEDIA LITERATURE CITED
1. Apirion, D. 1966. Altered ribosomes in a suppressor strain ofEscherichia coli. J. Mol. Biol. 16:285-301. 2. Apirion, D. 1967. Three genes that affect Escherichia coli ribosomes. J. Mol. Biol. 30:255-275. 3. Apirion, D., and N. Watson. 1974. Analysis of an Escherichia coli strain carrying physiologically compensating mutations one of which causes an altered ribonuclease III. Mol. Gen. Genet. 132:89-104. 4. Apirion, D., and N. Watson. 1975. Mapping and some characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid. J. Bacteriol. 124: 317-324. 5. Gesteland, R. F. 1966. Isolation and characterization of ribonuclease mutants of Escherichia coli. J. Mol. Biol. 16:67-84. 6. Hitchens, A. P., and M. C. Leikind. 1939. The introduc-
7.
8.
9.
10. 11.
12.
513
tion of agar-agar into bacteriology. J. Bacteriol. 37:485-493. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin. Wochenschr. 19:221-230. Rice, F. A. H. 1946. The effect of solvent and temperature on the viscosity of the polysaccharide of Irish moss and the effect of solvent on its initial gelation. Can. J. Res. Sect. B 24:12-19. Stoloff, L. S. 1943. The agar situation. Fish. Mark. News 5:1-5. Walker, A. W., and A. A. Day. 1943. Extracts from Irish Moss as a substitute for agar in bacteriological culture media. Food. Res. 8:435-443. Whistler, R. L., and C. L. Smart. 1953. Polysaccharide chemistry. Academic Press Inc., New York. Zabik, M., and P. Aldrich. 1965. Effect of selected anions of K salts on the gel strength of carrageenan high in the kappa fraction. J. Food Sci. 30:795-800.
Vol. 31, No. 4 Printed in U.S.A.
Substitute for Agar in Solid Media for Common Usages in Microbiology NED WATSON AND DAVID APIRION* Department of Microbiology and Immunology, Washington University Medical School, St. Louis, Missouri 63110
Received for publication 1 December 1975
The potassium salt of carrageenan was found to be an adequate replacement for agar in solid bacteriological media. The common microbial genetic techniques, such as purifying colonies by streaking, replication tests, and titration of cultures, were carried out successfully with a number of mutant strains of Escherichia coli.
The use of solid culture media has been of fundamental importance to microbiological research since the late nineteenth century. Desirable qualities of a solidifying agent for media include solidity over the temperature range of bacterial growth, resistance to digestion by bacteria, lack of syneresis, transparency, and the ability to form a reversible colloid. The medium must be firm enough to allow the carrying out of common techniques such as streaking out cultures, plating, and replication. In addition, it is helpful if the gelation agent is relatively inexpensive and easily obtained. Since Koch (7) first introduced agar as a gelifying agent in bacterial media (6), it has become the primary material for solid media throughout the world. However, increased cost and recent shortages have made a more readily available substitute for agar desirable. During World War II attempts were made to find a substitute for agar in solid bacterial media. A report appeared that extracts from Irish Moss can substitute for agar in bacteriological culture media (10). Since two carrageenan (Irish Moss) salts are commercially available now, and since present needs in a microbial genetics laboratory are somewhat different from those that existed in 1943, we decided to test a number of commercially available gelifying agents as substitutes for agar. Our experiments suggest that the potassium salt of the sulfate polysaccharide, carrageenan, is an adequate substitute for agar in most common genetic manipulations of Escherichia coli. MATERIALS ANI) METHOi)S Bacterial and phage strains. Bacterial strains are described in Table 1. Bacteriophages P1, T4, and T7 were from our own stocks. Media. Media were prepared as described by Apirion (1) except for substitutions of the various gelation materials.
Gelation materials. The agar used was Difco agar. Gelatin types I and II, carrageenan calcium salt, and carrageenan potassium salt were from Sigma Chemical Co. (St. Louis, Mo.).
RESULTS Gelling properties. The materials tested by us for their gelling properties included gelatin types I and II and carrageenan calcium and potassium salts. Various concentrations of these materials (1.0, 1.5, 2.0, and 4.0%) were prepared, autoclaved, and allowed to solidify in small depression plates (Table 2). They were then incubated at 25 and 45 C. Both types of gelatins gave too soft a consistency at all concentrations to warrent further investigation. All concentrations of the Ca2+ salt of carrageenan were rather firm at room temperature, but none were firm at 45 C. On the other hand, all concentrations of the K+ salt of carrageenan were adequately firm at room temperature, and the 2 and 4% concentrations were also firm at 45 C. It was later observed, when attempting to plate cells by spreading them on top of the medium, that the Ca2+ salt was too soft even at 37 C for such a purpose. Like agar, carrageenan has to be melted to obtain a uniform suspension. Replication tests. To test the K+ salt of carrageenan as a medium support, master plates were made with rich medium containing agar (1.5%) and rich medium containing carrageenan (2.0%) with eight different E. coli strains in common use in our laboratory at that time (Table 1). No differences were observed in the firmness of the plates. After overnight incubation at 37 C the patched colonies grew similarly on both master plates; in fact, the plates with carrageenan were clearer and the colonies were more readily distinguishable. (Later, when we used a different lot of carrageenan K+ salt, also from Sigma, plates were turbid and 509
510
WATSON AND APIRION
APPL. ENVIRON. MICROBIOL.
did not support growth as well. This was probably due to a difference in purity between the two lots, but we were unable to get any information on the differences between the two lots.) The master plates were then replicated to a variety of media containing either agar or carrageenan and were incubated at various temperatures (Table 3). The test plates included rich medium, minimal medium with a variety
of specific additions, and different sugars and indicator plates. The results were almost identical using carrageenan K+ salt plates as compared with agar plates (Table 3). We purified colonies of different strains by streaking them on rich medium plates containing either 1.8% agar or 2% K+ carrageenan and found that both sets of plates gave us equally adequate results. The carrageenan plates were firm enough to
TABLE 1. Strains used Strain
D10 A19 AB301 N141
N2048 N2053 N2089 N2090 a
Sex and markers
Source or derivation
Hfr P021 metBI rna-10 rel-1 (A) Hfr P021 metBI aux rna-19 rel-I (X) Hfr P021 metBI rel-1 (X) F-lac-1 xyl mal-141 gal-2 thi arg-3 his-4 ilv-158 pro-2 F- metBI his rna-19 rnc-105 F- like N2048, but rnc+ F- thi-1 argAl nadB4 gal-6 malAl XRxyl 7 ara13 mtl-2 str-9 tonA2 supE44 F- like N2089, but rnc-105
NGa from AB301 (5) NG from AB301 (5) (1, 5) Spontaneous mal- derivative of strain AB774 (2) (3, 4) (3, 4) Lac+ derivative from strain N2076 (ref. 4) Lac' derivative from strain N2077 (ref. 4)
NG, Nitrosoguanidine.
TABLE 2. Gelling characteristics of gelatin and carrageenan" Gelatin type I
Gelatin type II
Carrageenan Ca+ salt
Carrageenan K' salt
25C 45C 25C 45C 25C 45C 25C 45C 1.0 Soft Soft Soft Soft Firm Soft Firm Soft 1.5 Soft Soft Soft Soft Firm Soft Firm Soft 2.0 Soft Soft Soft Soft Firm Soft Firm Firm 4.0 Soft Soft Soft Soft Firn Soft Firm Firm Two types of gelatin and two salts of carrageenan were mixed with distilled water in the concentrations indicated above and autoclaved. Plates were poured and allowed to solidify at room temperature for 1 day. The plates were then incubated for another day at the temperatures indicated above. The consistency of the gel was tested by visual observation or by streaking on the top of the plates with a bacteriological loop. TABLE 3. Replication tests on agar and carrageenan platesa Rich medium
MM
MM MM MM MM MM Lac Lac Lac -Arg -His -Met -Nic -Pro MM EMB Tet Strain 300 370410430450370430 (37 C) (37 C) (37 C) (37 C) (37 C) (37 C) (37 C) |(37 C) 30AC 37C 41AC43CC 45 C 37 C 43A C C
AIC
AIC|
A C A C A C A C C1 A |C JA A |C A|C| A |C A C A |C |A |C + + + + + + + + + + + + + + A19 + + + + + ++ + + ++ ++ + + AB301 + +++++++++++++ + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + D10 ++ ++++++++++++++ + + N141 + + -4-. N2048 + ± + ± ± ± ± ± - + + + =+_ + + 0 + + + + + + +± + + ++ ±N2053 + + + + + ± + ± ± + + ++ + + + + - + + + + N2089 + + + + + + + + + + + + + + - - + + + + + + + + + + 4+ + -++ ++ + + - + + + + N2090 + + + + + + + + + + " Two sets of plates were used in this experiment. In columns designated A, plates contained 1.8% agar as the gelation agent; and in C, plates contained 2.0% carrageenan as the gelation agent. All strains were grided on two rich medium master plates, one containing agar and the other carrageenan, and incubated at 37 C overnight. The master plates were then replicated to a set of plates as indicated. The plates were incubated at various temperatures and scored after 1, 2, and 3 days; the 2-day scoring is shown. Minimal medium (MM) contained 0.2% glucose and 50 jig each of thiamine, L-arginine, Lhistidine, L-isoleucine, L-valine, methionine, nicotinic acid, and L-proline per ml. In Lac MM, 0.2% lactose was substituted for glucose. Lac EMB plates contained lactose and eosin and methylene blue (1), and Lac Tet plates were lactose tetrazolium plates (3). Symbols: +, good growth, _, fair growth; -+, poor growth, +, very poor growth; -, no growth. On indicator plates: +, indicates positive test; -, indicates negative test; 0, indicates cells failed to grow. (A number ofE. coli mutants fail to grow on EMB plates. Apparently eosin and/or methylene blue are toxic for them.)
+T+ -T-
-
+T+ +I+
VOL. 31, 1976
SUBSTITUTION FOR AGAR IN BACTERIAL MEDIA
permit convenient streaking of the bacterial cells with a loop. Plating efficiency. We compared the plating efficiency of a single standard E. coli strain, D10, on regular minimal medium and on similar media in which either the Ca-+ or K+ salt of carrageenan was substituted for agar. A culture was grown overnight in liquid rich medium, washed and diluted in dilution buffer, and plated at 37 and 43 C in minimal medium gelled by agar or carrageenan (Table 4). Even at 37 C the Ca2+ salt of carrageenan was soft and difficult to work with and supported growth rather poorly. However, the plates with the K+ salt of carrageenan gave colony counts similar to the agar plates. In fact, the colonies TABLE 4. Plating efficiency of D10 on agar and various carrageenan mixtures" 37C 43C No. of Size No. of Size cells (mm) cells (mm) 4.9 x 109 1.1-2.5 3.9 x 109 2.2-3.2 Agar, 1.8% Carrageenan Ca2+ 2 x 107 2.5-3.5 _ salt, 3% Carrageenan K+ 6.3 x 109 2.6-3.5 6.0 x 109 2.5-4.5 salt, 2% Carrageenan K+ 5.8 x 109 2.2-3.2 5.6 x 109 2.5-4.0 salt, 3% a A liquid culture of D10 was grown overnight at 37 C in rich medium with agitation, washed once in dilution buffer, and diluted in the same buffer. One-tenth-milliliter volumes were then plated onto minimal medium plates containing 50 ,ug of methionine per ml using the various gelation agents as indicated. The plates were incubated for 2 days at the temperatures shown. Colonies were counted and the results from various dilutions were averaged and expressed per milliliter of original culture. The dashes indicate no colonies observed. The size of the smaller and larger colonies was determined by averaging the diameter of 6 to 10 well-separated colonies on the plates; the size of both types of colonies is shown. Gelation
511
appeared sooner and grew larger than those on the agar plates. The K+ salt of carrageenan also appeared to give slightly better viability. In another set of experiments, strains D1O and N2089 were plated on rich and minimal medium supported by agar or K+ carrageenan (Table 5). The numbers of colonies were comparable at all temperatures in rich medium and at 37 and 43 C in minimal medium. At 45 C similar results were obtained using rich medium. Using minimal medium, carrageenan seems to have the edge. One of the strains tested, D10, failed to form colonies on minimal agar medium, and the other strain tested, N2089, grew faster on the minimal carrageenan plates (see Table 5). Growth of phage (plaque formation). For many purposes, when working with bacteria and bacteriophages, it is useful to use a soft agar medium as a top layer. The K+ carrageenan that we tested did not seem suitable for this type of medium, since an 0.7% K+ carrageenan medium lost its reversible colloidial properties. Therefore, we tested whether it is possible to use carrageenan plates as a support for growth of bacteriophage when the bottom layer is made with carrageenan and the top layer (soft) with agar. Using this technique we found comparable plaque-forming ability using rich medium plates containing either agar or the K+ salt of carrageenan for the titration of P1, T4, and T7 bacteriophages (Table 6). DISCUSSION The experiments described here suggest that agar can be substituted successfully by the K+ salt of carrageenan in microbiological media. Whereas the experiments presented here were carried out with various mutants of E. coli, it is
TABLE 5. Plating efficiency at various temperatures on rich and minimal medium containing agar or carrageenan K+ salt, Viable cell counts/ml (x 109)
Medium
D10 37C
43C
N2089 45C
37C
43C
45C
5.2 5.5 4.9 0.8 1.0 Rich, 1.8% agar 1.1 1.0 1.1 4.7 4.8 5.6 1.3 Rich, 2% carrageenan 0.8 1.0 5.2 3.7 _b 0.8' Minimal, 1.8% agar 5.2 3.0 1.0 5.1 1.0 Minimal, 2% carrageenan 0.5d Strains D10 and N2089 were grown overnight in liquid rich medium at 37 C with agitation, washed once in dilution buffer, and diluted 10-" and 2 x 10-7 in the same buffer. One-tenth-milliliter aliquots were plated onto rich or minimal medium plates containing the gelation agent as indicated. The minimal medium plates for D10 contained 50 ,g of methionine per ml, whereas those for N2089 contained 5 gg of nicotinic acid and thiamine per ml and 50 ,ug of arginine per ml. The plates were incubated for 2 days or as indicated at the temperatures shown. Colonies were counted and averaged as in Table 4. b No colonies were observed after 4 days of incubation. It was necessary to incubate plates for 4 days in order to score them. d It was necessary to incubate plates for 3 days in order to score them. "
512
APPL. ENVIRON. MICROBIOL.
WATSON AND APIRION
TABLE 6. Titration of three bacteriophages on agar and carrageenan platesa No. of phage/ml Bacteriophage
Agar
Carrageenan K+ salt
2.9 x 109 P1 2.7 x 109 T4 10.3 x 108 8.4 x 10 5.2x108 3.7x108 T7 a Bacteriophage stocks were diluted, and similar volumes were added to prewarmed tubes containing approximately 108 E. coli cells (strain D10) and appropriate ions necessary for phage attachment. The soft agar medium was then poured onto rich medium plates containing the indicated gelation material, allowed to harden at room temperature, and incubated overnight at 37 C. Plaques were then counted; results are expressed as phage per milliliter of phage stock solution. The morphology of the plaques of each phage was similar on both sets of plates.
quite likely that K+ carrageenan could serve as a useful gelation agent for a large variety of solid culture media in common use in microbiological work. We did encounter several problems. At lower concentrations, such as in a soft agar type of preparation, the ability to form a useful reversible colloid for phage manipulations was unsatisfactory compared to agar. This probably can be overcome by varying the K+ ion concentrations (8, 11, 12; J. H. Brewer and C. B. McLaughlin, Bacteriol. Proc., p. 25-26, 1953; A. Frieden and S. J. Werbin, U.S. Patent 2,427594, 1947). The Ca2+ salt of carrageenan generally had unsatisfactory gelling properties. This too might be remedied by varying ionic concentrations in the medium or by chemical modficiations of carrageenan; for example, by crosslinking or by the addition of hydrophillic groups (Paul Hartman, personal communication). The most important drawback encountered was a vast difference in the properties of different lots of the K+ salt tested. For instance, the lot (Sigma 71C-90019) we used in the experiments reported here gave us very reproducible results. On the other hand, another lot (Sigma 94C-0281) gave us only 12% of the colonies at 37 C and no colonies at 43 C when comparing colony-forming ability on minimal medium with agar plates of the same medium. The plates prepared from this lot were also quite turbid, indicating a difference in purity. Unfortunately, we were unable to get any information from the distributor as to the relative purity of the two lots or who was their manufacturer(s). Therefore we could not determine the basis for the variability observed with different lots. On the basis of our observations it is clear
that the K+ carrageenan can be used for most bacteriological purposes as a base for solidifying various media. We used it successfully for. preparation of all our routine media, which include minimal media with various sugars, rich medium, and indicator media (tetrazolium and eosin methylene blue). On such media we performed the four common manipulations of bacteria in a microbial genetics laboratory: streaking out cells (purification), replication from master plates to various other solid media, testing efficiency of colony formation from single cells, and using bacteria to clone bacteriophage by the plaquing technique (in this case only the bottom layer of medium contained K+ carrageenan). We found no instance in which the carrageenan could supply amino acids or sugars in the E. coli strains tested. Thus it seems that it cannot be utilized by E. coli and probably is suitable to be used for supporting media for many other bacteria as well (10). The K+ carrageenan used here was quite firm at 2% concentrations over the entire temperature range for growth of E. coli and was even more transparent than agar. Although syneresis is given as one of the major drawbacks to using carrageenan in bacteriological media (9-11), we did not observe this phenomenon in any of the batches of carrageenan used. Even after several months of storage in a cold room, carrageenan plates were as dry and firm as agar plates. Carrageenan is a polysaccharide extracted from the red marine algae Chondrus crispus, which is found in great abundance along the east coast of the United States and the western coast of Europe. The alga grows along the shore 1 to 14 feet (ca. 30.5 to 426.7 cm) below ebb tide and is easily gathered by hand picking or raking (11). On the other hand, Gelidium amansii, the principal source for agar, is found primarily in restricted areas in the Pacific Ocean on submerged rocky ledges and is difficult to harvest, requiring calm weather and often diving gear (11). Whereas, according to manufacturers, it is becoming increasingly difficult to obtain sufficient amounts of the algae from which agar is produced, there should be little problem obtaining a sufficient supply of C. crispus. Moreover, the cost of the carrageenan per pound that we tested was less than one-sixth of the price of agar. Therefore, we conclude that the K+ salt of carrageenan could become a desirable substitute for agar in various microbiological usages. ACKNOWLEI)GMENTS This work was supported by Public Health Service grant GM-19821 from the National Institute of General Medical Sciences and grant CA-15389 from the National Cancer Institute.
VOL. 31, 1976
SUBSTITUTION FOR AGAR IN BACTERIAL MEDIA LITERATURE CITED
1. Apirion, D. 1966. Altered ribosomes in a suppressor strain ofEscherichia coli. J. Mol. Biol. 16:285-301. 2. Apirion, D. 1967. Three genes that affect Escherichia coli ribosomes. J. Mol. Biol. 30:255-275. 3. Apirion, D., and N. Watson. 1974. Analysis of an Escherichia coli strain carrying physiologically compensating mutations one of which causes an altered ribonuclease III. Mol. Gen. Genet. 132:89-104. 4. Apirion, D., and N. Watson. 1975. Mapping and some characterization of a mutation in Escherichia coli that reduces the level of ribonuclease III specific for double-stranded ribonucleic acid. J. Bacteriol. 124: 317-324. 5. Gesteland, R. F. 1966. Isolation and characterization of ribonuclease mutants of Escherichia coli. J. Mol. Biol. 16:67-84. 6. Hitchens, A. P., and M. C. Leikind. 1939. The introduc-
7.
8.
9.
10. 11.
12.
513
tion of agar-agar into bacteriology. J. Bacteriol. 37:485-493. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin. Wochenschr. 19:221-230. Rice, F. A. H. 1946. The effect of solvent and temperature on the viscosity of the polysaccharide of Irish moss and the effect of solvent on its initial gelation. Can. J. Res. Sect. B 24:12-19. Stoloff, L. S. 1943. The agar situation. Fish. Mark. News 5:1-5. Walker, A. W., and A. A. Day. 1943. Extracts from Irish Moss as a substitute for agar in bacteriological culture media. Food. Res. 8:435-443. Whistler, R. L., and C. L. Smart. 1953. Polysaccharide chemistry. Academic Press Inc., New York. Zabik, M., and P. Aldrich. 1965. Effect of selected anions of K salts on the gel strength of carrageenan high in the kappa fraction. J. Food Sci. 30:795-800.
