AEM Accepted Manuscript Posted Online 12 June 2015

Appl. Environ. Microbiol. doi:10.1128/AEM.01327-15

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Beyond agar: gel substrates for microbial growth experiments with

2 improved optical clarity and drug efficiency and reduced autofluorescence

4 Philipp A. Jaegera, Cameron McElfreshb, Lily R. Wongc, Trey Idekera#

6 Departments of Bioengineering and Medicine, University of California San Diego,

7 La Jolla, California, USAa

8 Nanoengineering Program, University of California San Diego, La Jolla,

9 California, USAb

10 Bioengineering Program, University of California San Diego, La Jolla, California,

11 USAc

12
#
13 Address correspondence to Trey Ideker, tideker@ucsd.edu

14

15 Running title: Alternative gel substrates for microbial growth

16 ABSTRACT

17 Agar, a seaweed extract, has been the standard support matrix for microbial

18 experiments for over a century. Recent developments in high-throughput genetic

19 screens have created a need to re-evaluate the suitability of agar for use as

20 colony support, as modern robotic printing systems now routinely spot thousands

21 of colonies within the area of a single microtiter plate. Identifying optimal

22 biophysical, biochemical, and biological properties of the gel support matrix in

23 these extreme experimental conditions is instrumental to achieving the best

24 possible reproducibility and sensitivity. Here we systematically evaluate a range

25 of gelling agents using the yeast Saccharomyces cerevisiae as a model microbe.

26 We find that carrageenan and phytagel have superior optical clarity and reduced

27 auto-fluorescence, crucial for high-resolution imaging and fluorescent reporter

28 screens. Nutrient choice and use of refined noble agar or pure agarose reduce

29 the effective dose of numerous selective drugs by >50%, potentially enabling

30 large cost savings in genetic screens. Using thousands of mutant yeast strains to

31 compare colony growth between substrates, we find no evidence of significant

32 growth or nutrient biases between gel substrates, indicating that researchers

33 could freely pick-and-choose the optimal gel for their respective application and

34 experimental condition.

2
35 INTRODUCTION

36 Single-cell organisms such as bacteria and yeast have been used extensively to

37 study genes and genome organization, proteins and protein interactions,

38 biological pathways, cellular structure, and to address numerous other

39 fundamental biological questions (1, 2). Many microbes, especially the species

40 Escherichia coli and Saccharomyces cerevisiae, combine numerous beneficial

41 properties, which make them ideal model organisms: easy laboratory cultivation,

42 easy genetic manipulation, short generation time, and safe handling. Microbial

43 cultures can be maintained in liquid growth media or on solid/semi-solid gel

44 substrates (3). Liquid colony maintenance allows the microbial cultures to expand

45 rapidly and is used in screens that rely on optical density measurements to

46 extract kinetic growth parameters or use downstream liquid-based assays such

47 as flow cytometry or high-throughput microscopy (4-7). While throughput in liquid

48 is generally bound to a maximum of 96 or 384 samples per plate, solid-substrate

49 colony maintenance has the advantage that individual cell clones can be readily

50 separated and that colony parameters such as color, shape, structure, and size

51 can be extracted easily using digital image acquisition (8-10). Additionally,

52 advances in robotic pinning devices allow for much higher throughput on solid

53 substrate, with close to 25,000 colonies possible on a single microtiter footprint-

54 sized gel plate (11). This much higher density has led to solid-substrate

55 screening being the method of choice for many current high-throughput

56 applications (12-14).

57

3
58 Historically, agar has been the predominant gelling agent in microbial research.

59 Agar is a mixture of polysaccharides from the cell wall of the marine red algae

60 Rhodophyta, where it provides flexibility and strength (15). The Japanese

61 innkeeper Minoya Tarozaemon discovered agar in 1658, when a seaweed jelly

62 dish accidentally froze when left out overnight and subsequently dried to white

63 powder in the morning sun. He observed that this powder could be re-dissolved

64 in warm water and led to a clearer jelly than before (16). Fannie and Walther

65 Hesse – assistant to the famous German microbiologist Robert Koch –

66 introduced the use of agar to microbial research in 1882 after realizing the

67 superior gelling qualities of agar over the previously used gelatin (17). Agar

68 exhibits hysteresis, reflected in its ability to gel (32 ºC to 40 ºC) and melt (~85 ºC)

69 at very different temperatures, making it much more suitable for microbial

70 research than the low-melting gelatin (25 ºC to 30 ºC) (16). Additionally, agar is

71 indigestible by most organisms.

72

73 Thus, Bacto agar (a proprietary modified agar with “optimal” magnesium,

74 calcium, iron and copper concentrations) - has become the de facto standard

75 gelling medium. However, Bacto agar’s opaque gel appearance and batch

76 variability call into question whether agar is indeed the optimal gel matrix for

77 state-of-the-art high-throughput screening techniques. Additionally, agar is not

78 necessarily readily compatible with all media formulations (18). Finally,

79 laboratories working with solid media screens routinely consume thousands of

80 agar plates in a single experiment. It may thus be worthwhile to identify suitable,

4
 81 more cost effective gelling agents with identical or better optical and growth

82 properties than standard agar. In this regard, Agar is certainly not the only gel

83 forming substance. Other seaweed-based gelling agents include alginic acid

84 sodium salts and carrageenans; gellan gum (Phytagel / Gelrite) is produced by

85 bacteria; and various bean and seed extracts, which also have gel-forming

86 properties (i.e. guar gum). However, a controlled parallel comparison of different

87 gelling agents for their ability to serve as high-throughput microbial substrates is

88 lacking.

89

90 Here, we systematically evaluate the properties of potential replacements for

91 Bacto agar for use in microbial experiments. A number of gel-forming substances

92 are considered (Table 1): Agarose is agar that has been purified to remove

93 agaropectin (a poorly defined, complex polysaccharides with sulfate groups)

94 removed (16). It has been used as a microbial substrate (19-22) but is much

95 more commonly used as matrix for gel electrophoresis (23). Noble agar is a

96 bleached and washed derivative of agar resulting in a whiter gel (24),

97 occasionally used as growth substrate (25, 26). Carrageenan is a ubiquitous

98 emulsifying and gelling agent used extensively in food science and

99 pharmaceutical research (27) and has been investigated as substrate or

100 immobilizer for yeast and microbial cells (28, 29). Phytagel forms very hard and

101 clear gels widely used in plant sciences to study root development and as a

102 seedling growth substrate (30). It is produced in a controlled fermentation

103 process (31, 32). Phytagel has found use as a microbial growth medium (33),

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104 especially for thermophilic microbes (34). Alginic acid has numerous industrial

105 applications as gelling agents and thickeners, in culinary arts and in

106 pharmaceutical sciences (35). It is widely used to encapsulate and immobilize

107 cells (36, 37) and has been explored for use in protoplast culture (38, 39). Guar

108 gum has many industrial and culinary applications as a thickener, emulsifier, and

109 coating substance (40) and has been suggested as a microbial growth substrate

110 (41). Gelatin has many industrial and culinary applications (42) and was used as

111 microbial growth medium before the introduction of agar (43). It still finds use in

112 culturing certain microbes, for example to test for external protease activity (44).

113 Very recently, bacterial cellulose has been suggested as a promising microbial

114 cell culture substrate (45), however due to the lack of commercial availability, we

115 did not test this gelling agent.

116

117 MATERIAL AND METHODS

118 Gel preparation, selection markers, and media

119 The following gelling reagents were used: Agarose (#20-102, Lot #LF45120012,

120 Genesee Scientific, San Diego/CA), Bacto agar (#214040, Lot #4202919, BD

121 Biosciences, San Jose/CA), Carrageenan (C1013, Lot #SLBK3896V, Sigma-

122 Aldrich, St. Louis/MO), Noble agar (A5431, Lot #SLBJ3882V, Sigma-Aldrich),

123 Phytagel (P8169, Lot #SLBK1372V, Sigma-Aldrich), Alginic acid (A0682, Lot

124 #O81M1093V, Sigma-Aldrich), and Guar gum (G4129, Lot #SLBH5231V, Sigma-

125 Aldrich). Supplemental reagents and media were Bacto yeast extract (#212720,

126 BD Biosciences), Bacto peptone (#211820, BD Biosciences), Magnesium sulfate

6
127 (M7506, Sigma-Aldrich), Difco Dextrose/Glucose (#215520, BD Biosciences),

128 Difco Yeast nitrogen base without amino acids (#291920, BD Biosciences) and

129 Difco Yeast nitrogen base without amino acids and ammonium sulfate (#233520,

130 BD Biosciences). Synthetic complete (SC) or SC-dropout media were prepared

131 following standard procedures using amino acids from Sigma-Aldrich. If

132 indicated, selective pressure was maintained using geneticin (G418, KSE

133 Scientific, Durham/NC), nourseothricin (clonNAT, Werner BioAgents, Jena,

134 Germany), S-(2-Aminoethyl)-L-cysteine hydrochloride (S-AEC, A2636, Sigma-

135 Aldrich), or L-(+)-(S)-Canavanine (Can, C9758, Sigma-Aldrich) at the indicated

136 concentrations. Gelling, supplemental, and media reagents were mixed in ddH2O

137 and autoclaved for 15 min at 121 ºC before use; selective drugs were added after

138 the liquid gel solution cooled to below 60 ºC in a water bath. Data for the range of

139 typical element composure of agarose, Bacto agar, noble agar, and Phytagel

140 were obtained from Difco (24) and for carrageenan (potassium, sodium, calcium

141 only) from the Sigma-Aldrich website. Additional, lot-specific cation concentration

142 information was obtained from the Certificates of Analysis from BD Biosciences

143 and Sigma-Aldrich respectively.

144

145 Colony imaging

146 White-light images of gels and yeast colonies were acquired using a digital

147 imaging setup described previously (11) with a commercially available SLR

148 camera (18 Mpixel Rebel T3i, Canon USA Inc., Melville/ NY) with an 18–55 mm

149 zoom lens. We used a white diffusor box with bilateral illumination and an

7
150 overhead mount for the camera in a dark room. Colony information was collected

151 after images were normalized, spatially corrected, and quantified using a set of

152 previously published custom algorithms, aka ‘‘The Colony Analyzer Toolkit’’ (11).

153 Digital images were cropped and assembled in Photoshop and Illustrator (CS5,

154 Adobe Inc., San Jose/CA) for publication. Fluorescent images of gels were

155 acquired using a custom fluorescent digital imaging setup. We used a

156 commercially available SLR camera (20.2Mpixel EOS 6D, Canon) with a 100 mm

157 f/2.8 macro lens (Canon) and a green band-pass filter (BP532, Midwest Optical

158 Systems, Inc., Palatine/IL). We used a 460 nm LED panels (GreenEnergyStar,

159 Vancouver BC, Canada) with a ¼ white diffusion filter (#251, Lee Filters,

160 Burbank/CA, USA) for 45º bilateral illumination (205560, Kaiser Fototechnik

161 GmbH & Co.KG, Buchen, Germany), and an overhead mount for the camera

162 (205510, Kaiser) in a dark room.

163

164 Spectral absorbance measurements

165 For spectral absorbance measurements 200 µl of gel were prepared as

166 described and poured into 96-well, UV-transparent microtiter plates (#3635,

167 Corning Inc., Tewksbury/MA) and allowed to solidify. Absorbance measurements

168 were taken in triplicates in sweeps from 350 nm to 750 nm on a SpectraMax 190

169 (Molecular Devices, Sunnyvale/CA).

170

171 Bioavailability screen in S. cerevisiae S288C/BY4741

8
172 To test how available nutrients are in the different media, gels were prepared as

173 described and poured into sterile rectangular high-throughput screening plates

174 (PlusPlates V2, Singer Instrument Co. Ltd., Watchet/Somerset, UK). Randomly

175 selected yeast deletion mutants (N = 1536) from the Yeast Knock-out Deletion

176 Collection [(46). YKO, GE Dharmacon, Lafayette/CO, USA] were grown on SC

177 media supplemented with G418 (100 µg/ml), and the plates were then pinned

178 using the Rotor HAD (Singer) onto the various gel substrates (w/o any selection

179 markers). Plates were grown at 30 ºC for 24 hrs, and colony sizes measured

180 using the white-light colony imaging station described above.

181

182 Bioavailability screen in diploid S. cerevisiae and S. paradoxus wildtype

183 strains

184 To test if non-standard laboratory or wild yeast strains are able to digest the gel

185 material and utilize it as a carbon or a nitrogen source, we prepared gels as

186 described and poured them in sterile 10 cm petri dishes. We then streaked out

187 12 yeast strains (6 S. cerevisiae and 6 S. paradoxus, see Supplemental Fig. 2)

188 onto each gel and recorded the qualitative growth and appearance of the

189 colonies after 72 hrs at 30 ºC. Strains were a gift from Scott Rifkin and initially

190 acquired from the National Collection of Yeast Cultures (Norwich, UK).

191

192 Gel strength measurements

193 Gel strength measurements were performed using a TA.XT2.plus texture

194 analyzer (Stable Micro Systems, Godalming/Surrey, UK). Gels were prepared as

9
195 described in 100 ml of ddH2O in a standard 100 ml Pyrex Griffin glass beaker.

196 After pouring, gels matured and solidified for 24 hrs at 4 ºC. Prior to the gel

197 strength measurements, gels were allowed to warm to room temperature. The

198 following settings were used for the TA.XT2: Pre-test Speed (5 mm/s), Test

199 Speed (1 mm/s), Post-Test Speed (5 mm/s), Target Mode (Distance), Distance

200 (2 mm), Time (5 s), Trigger Type (Force), Trigger Force (5 g). We used a

201 standard 12.7 mm diameter, flat, sharp edged plunger (TA-10; Texture

202 Technologies, Hamilton/MA) in the center of the gel. Gel strength reported is the

203 force [g] required to depress the gel by 2 mm. Measurements were recorded with

204 Exponent (Stable Micro Systems). Contact angle α was defined as the angle

205 formed between two lines drawn on the colony cross-section: The first line was

206 drawn from the point where the colony sides touch the gel surface on the left side

207 of the colony to the point where the colony sides touch the gel surface on the

208 right side of the colony; the second line was drawn from the point where the

209 colony sides touch the gel surface on the left side of the colony to a point at the

210 left side of the colony outline where the colony reaches its half-maximum colony

211 height.

212

213 Gel pH measurements

214 Gel pH measurements were performed with a double junction, flat bulb, gel-filled

215 epoxy electrode (A57184; Beckman Coulter, Brea/CA) in full contact with the gel

216 surface in triplicate. Gelling, supplemental, and media reagents were prepared as

217 described. A 10 ml sample of each gel were filled into 20 ml glass scintillation

10
218 vials (Wheaton, Millville/NJ), sealed, and allowed to harden overnight at 4 ºC.

219 Measurements of pH were performed on a PHI510 bench top pH-meter

220 (Beckman Coulter) after gels returned to room temperature and were allowed to

221 equilibrate with ambient air for ~1 hr.

222

223 Diffusion measurements

224 Using the fluorescent gel imaging station, diffusion was assessed by tracking the

225 spread of fluorescein sodium salt (FITC, F6377; Sigma Aldrich) in the gels.

226 Gelling reagents were prepared as described and poured into sterile 10 cm petri

227 dishes. Gels were allowed to harden overnight at 4 ºC. The next day, gels were

228 warmed to room temperature and three circular holes were punched into the

229 center of the gel. 110 µl of fluorescein sodium salt solution (2 mM) were then

230 pipetted into the holes and allowed to diffuse into the gel. Images were acquired

231 every 5 min for a period of 12 hrs.

232

233 Drug-availability screen

234 To test the availability of common selection markers in the gel substrates, gels

235 were prepared as described [using SC-lys-arg+Mono sodium glutamate (MSG)

236 w/o (NH4)2SO4] and poured into sterile rectangular high-throughput screening

237 plates (PlusPlates V2, Singer) supplemented with increasing concentrations of

238 G418, clonNAT, S-AEC, or Can (0, 10, 20, 40, 100 [endpoint for S-AEC and

239 Can], 200, 400, 1000 [µg/ml]). The following test yeast strains (based on the S.

11
240 cerevisae strain BY4742/S288C) were grown to saturation in YPD medium

241 overnight:

242 A (XXX = his3Δ) & B (XXX = hoΔ): MATa his3Δ1; leu2Δ0; ura3Δ0; LYS2+;

243 can1Δ::STE2pr-SpHIS5; lyp1Δ::STE3pr-LEU2; XXX:: NatMX

244 C (YYY = his3Δ) & D (YYY = hoΔ): MATα; his3Δ1; leu2Δ0; ura3Δ0; met15Δ0;

245 LYS2+; CAN1+; LYP1+; YYY::KanMX

246 Twelve serial two-fold dilutions (1:1 to 1:211) of the overnight liquid culture were

247 pinned onto the respective gels, and colony growth was quantified after 24 hrs at

248 30 ºC by counting the total number of colonies with visible growth (X out of 12).

249 For example, if 12 G418 resistant colonies had grown up at a given concentration

250 of clonNAT and 12 clonNAT resistant colonies, then that would equate to 100%

251 viability relative to the resistant strain (12/12). If at another concentration of

252 clonNAT 4 G418 resistant colonies had grown up versus 12 clonNAT resistant

253 colonies, then that would equate to 33% viability relative to the resistant strain

254 (4/12).

255

256 High-throughput yeast screens

257 To assess the colony sizes of a large selection of yeast mutants with different

258 fitness on the gel substrates, we pinned a random selection of 1536 strains from

259 the Yeast Knock-out Deletion collection (46) (YKO, Dharmacon) onto standard

260 Bacto agar plates with 100 µg/ml G418 [SC+MSG w/o (NH4)2SO4]. From these

261 plates colonies were then transferred onto the indicate gel substrates [100 µg/ml

262 G418 in SC+MSG w/o (NH4)2SO4]. Colony sizes were correlated between Bacto

12
263 agar and all other substrates. Inter-plate correlations were calculated by

264 computing the Pearson’s correlation between same-strain colony sizes (N=1536

265 per plate). Detailed description of the 1536 colony array procedure

266 (Supplemental Fig. 1): The 1536 yeast strains were stored as liquid glycerol

267 stocks in 16 96-well microtiter plates. After thawing, yeast aliquots were

268 transferred onto rectangular Bacto agar (+YPD) gels with a standard microtiter

269 footprint (12.7 cm x 8.5 cm) using a disposable plastic pinning tool (“pads”) with

270 96 individual long pins in a pneumatically operated pinning robot (RoToR HDA,

271 Singer). The yeast colonies were grown as individual microcolonies (~0.2-3mm in

272 diameter). During successive rounds of overnight growth at 30 ºC and re-pinning

273 the colonies were then combined into higher and higher colony densities using

274 96 pin, and then 384 pin pads. Colonies were than replicated onto the various gel

275 substrates using 1536 pin pads. This process is extremely fast (1536 colonies

276 are replicate-pinned in under 30 s) and highly scalable (i.e. colonies can be

277 arrayed in higher or lower colony densities and transferred between conditions).

278

279 Autofluorescence measurements

280 Autofluorescence measurements were taken using the fluorescent imaging

281 station described above, using the average pixel intensity of a 20x20 pixel area in

282 the center of the jar as readout. Images of GFP-expressing yeast colonies were

283 all taken at identical camera settings and signal-to-noise ratios were calculated

284 based on the ratio between the low- or high-GFP signals after background

13
285 subtraction. The low/high GFP-expressing strains were were a gift from Randy

286 Hampton (UCSD) and based on the S. cerevisae strain BY4742/S288C:

287 Basal GFP fluorescence: ade2-101, met2, lys2-801, trp1::hisG, leu2Δ, his3Δ

288 200, ura3-52::URA3::HMG2-GFP

289 Increased GFP: ade2-101, met2, lys2-801, trp1::hisG, leu2Δ, his3Δ200, ura3-

290 52::URA3::HMG2-GFP, hrd1Δ::KanMX

291

292 RESULTS

293 After following previously published protocols (39, 41), we were unable to obtain

294 properly solidified gels when using alginic acid or guar gum, despite testing a

295 wide range of substrate concentrations and mixing methods (2-10%, data not

296 shown). Similarly, even high gelatin concentrations (16%) yielded only semi-solid

297 gels (47) and gelatin contains large amounts of arginine, lysine, and histidine (48)

298 that can be freed through yeast proteinase digestion (49) rendering it unusable

299 for auxotrophic selection. Consequently, alginic acid, guar gum, and gelatin do

300 not provide an acceptable gel quality for high-throughput screening and were

301 thus excluded from further analysis.

302

303 Gelling agents vary widely in their optical qualities. All agar-based gels

304 (agarose, Bacto agar, noble agar) result in gels with a hazy quality, while

305 carrageenan and Phytagel gels are much clearer (Fig. 1A). We quantified the

306 absorbance of the gels from near ultraviolet (350 nm) to near infrared (750 nm)

307 and found a peak absorbance in the UV range for these types of gels (Fig. 1B,C).

14
308 Agar-based gels exhibit dramatically stronger absorption and scattering in

309 comparison to both carrageenan and Phytagel, which can be easily appreciated

310 when looking at a gel plate with yeast colonies from the side (Fig. 1D).

311

312 Different gels are not a significant source of nutrients. One major concern

313 when evaluating new gelling agents is to unknowingly introduce nutrients into the

314 experiment. To test whether any of the gels are able to provide non-supplied

315 nutrients, we tested the growth characteristics of 1536 yeast strains from the

316 yeast deletion collection for their ability to grow on plain gel, gel supplemented

317 with only a carbon source (glucose), or gel supplemented with only a nitrogen

318 and vitamin source (yeast extract and peptone). We found no growth under

319 nutrient-limited conditions for any gel, indicating that they are not a significant

320 source of nutrients (Fig. 2A). To ensure that this observation was not an artifact

321 of the highly auxotrophic laboratory strains used in the yeast gene deletion

322 collection, we also tested the growth of 12 diploid wild-type S. cerevisiae and S.

323 paradoxus strains and observed similar results (Supplemental Fig. 2). To what

324 extent these results hold true for other, non-yeast microbes will have to be

325 determined on an individual case-by-cases basis.

326

327 Colony morphology differs between gels. All gels tested allowed the growth of

328 clearly identifiable, almost perfectly round yeast colonies. To identify potential

329 changes in colony morphology and specifically colony width and height, we

330 investigated colonies grown under nutrient-rich conditions more closely (Fig. 2A).

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331 Using intensity as a measure of colony thickness, we compared colony cross-

332 sections between the gels and found that agar-based colonies exhibit almost

333 identical colony shapes, while carrageenan and Phytagel grown colonies are

334 taller and have a steeper contact angle α between the edge of the colony and the

335 gel surface (Fig. 2B). This more vertical distribution of biomass leads to overall

336 smaller colonies that are consequently more widely spaced apart on carrageenan

337 and Phytagel (Fig. 2C).

338

339 Gel pH and gel strength. To investigate the biophysical properties that might

340 underlie the observed changes in colony morphology, we measured gel pH and

341 gel strength of the different gels used. While the plain gels differ quite widely in

342 their initial pH (~5 to 6.5), addition of buffering media, such as yeast extract and

343 peptone or SC, results in gels with a much more narrow pH range (6.1 to 6.3 and

344 4.2 to 5.2, Fig. 3A). These findings suggest that pH alone does not differentiate

345 the gel types.

346

347 Next, we measured gel strength (the force necessary to depress the gel surface

348 by 2 mm) and found it to vary greatly between gels (~200 g to ~1800 g, Fig. 3B).

349 Gel strength has previously been reported to influence the contact angle α

350 between yeast colonies and the gel surface (50), which could explain the much

351 steeper and higher colonies on Phytagel; however, it does not explain the

352 behavior of the carrageenan-grown colonies.

353

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354 Gel diffusion is dependent on gel concentration, not gel strength. Nutrients,

355 waste products, and drugs need to diffuse through the gel matrix to enable

356 colony growth. To assess if differences in diffusion exist between the gel types,

357 we performed a radial diffusion experiment using fluorescein isothiocyanate (Mw

358 = 389.38 g/mol) as a molecular tracer (Fig. 4A). We measured the diameter of

359 the diffusion disc at given intervals and observed that the diffusion kinetic is well-

360 described in terms of the Higuchi equation, which models the diffusion of drug

361 into gel as a perfect sink (51) [Fig. 4B]. The speed of diffusion was proportional to

362 the concentration of gelling substance used, but it was independent of gel

363 strength or substrate (Fig. 4C).

364

365 Drug bioavailability is affected by gel substrate. Microbial experiments

366 frequently rely on selective drugs to maintain certain cellular populations.

367 However, some differences of drug bioavailability in different gel media have

368 been reported (52). To assess if commonly used drugs are equally effective in

369 the different gelling agents tested, we prepared plates with increasing

370 concentrations of nourseothricin (clonNAT), geneticin (G418), S-(2-Aminoethyl)-

371 L-cysteine hydrochloride (S-AEC), and canavinine (Can). We then prepared

372 dilution series of liquid overnight cultures of strains sensitive or resistant to the

373 tested drugs and plated those onto the gels. Colony growth was evaluated

374 relative to the respective drug resistant strain (Fig. 5A). We found no difference

375 between his3Δ and hoΔ (data not shown) and therefore subsequently combined

376 these measurements.

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377

378 Can and S-AEC (toxic amino acid analogues) are largely unaffected by the gel

379 substrate in their ability to severely impair yeast growth (Fig. 5B,C). Surprisingly,

380 we observed a dramatic difference between gel types when we evaluated the

381 toxicity of the aminoglycoside antibiotics G418 and clonNAT. A commonly used

382 concentration for these antibiotics is 100 µg/ml. We found that agarose and noble

383 agar facilitate drug toxicity and kill sensitive yeast at concentrations as low as 10

384 µg/ml to 40 µg/ml (Fig. 5D,E). In contrast, Bacto agar, carrageenan and Phytagel

385 failed to completely kill sensitive yeast in the range up to 100 µg/ml (Fig. 5D,E),

386 prompting us to extend the tested concentration range as well as compare SC

387 and YPD based media (Fig. 5F,G). With YPD as a medium base, G418 on Bacto

388 agar does indeed exhibit complete toxicity at the standard 100 µg/ml, however,

389 SC requires 200 µg/ml for complete toxicity (Fig. 5F, even in the absence of

390 ammonium sulfate as used here). Similarly, G418 on Phytagel exhibits toxicity at

391 400 µg/ml with YPD and 1000 µg/ml with SC (Fig. 5F). Remarkably, we were

392 unable to induce any toxicity in the test range using G418 on carrageenan plates

393 (Fig. 5F). Using clonNAT as antibiotic resulted in similar findings (Fig. 5G).

394

395 Typically, G418 is used in media lacking ammonium sulfate, which has been

396 reported to impair G418 toxicity (53). We thus speculated that cation content of

397 the gel could play a role in the bioavailability of particular drugs. This observation

398 is in agreement with earlier reports of cation interference with aminoglycoside

399 antibiotics in Pseudomonas aeruginosa (54, 55), however gelling substrate and

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400 nutrient mixes as a potent cation source had so far not been appreciated. While

401 there is no evidence of significant ammonium content in the gel substrates, the

402 gels do vary multiple orders of magnitude in their potassium, sodium, calcium,

403 and magnesium contents (Fig. 5H), and the effective antibiotic dose is directly

404 related to the overall cation concentration of the final gel (Fig. 5I). These findings

405 suggest that natural mineral content or contaminations from the manufacturing

406 process of the gel substrates can have a marked influence on the effect of some

407 drugs (i.e. aminoglycoside antibiotics), but not others (i.e. amino acid analogues).

408

409 High-throughput array performance. To assess the suitability of the different

410 gels as substrates for microbial high-throughput screens, we pinned 1536

411 random mutant yeast strains from the yeast deletion collection onto standard

412 Bacto agar plates and compared the normalized colony sizes (‘fitness’) between

413 gel substrates. Six replicates of the same 1536 colonies on Bacto agar yielded

414 inter-plate correlations between 0.88 and 0.95 (Pearson’s correlation, Fig. 6A).

415 To account for gel strength differences observed in the gels (Fig. 3B), we

416 adjusted gel concentrations slightly to achieve similar gel strengths in the test

417 gels. Additionally, we tested two Phytagel mixtures: 1% Phytagel with 0.05%

418 MgSO4 and 0.5% Phytagel with 0.1% MgSO4. Inter-gel correlations were

419 between 0.85 and 0.96 (Pearson’s correlation, Fig. 6B). Overall, the mutant

420 fitness between gel substrates correlated very well. In the non-agar-based plates

421 we observed a slight trend to more centralized, less extreme fitness values, as

422 indicated by the off-diagonal least-square fits (Fig. 6C) and tighter frequency

19
423 distributions (Fig. 6D). These results suggest that there are no dramatic relative

424 growth differences between the evaluated gel substrates.

425

426 Fluorescent high-throughput applications. Numerous fluorescent marker and

427 reporter systems have been developed in bacteria and yeast (56-58), and it

428 would be highly desirable to utilize them in a high-throughput screening setting.

429 To evaluate the fluorescent properties of the gels, we imaged the gel jars with

430 various media supplements using a fluorescent imaging setup, revealing

431 substantial differences in autofluorescent properties (Fig. 7A). While the plain

432 gels themselves have little autofluorescence, once supplemented with media the

433 agar-based gels are about twice as autofluorescent as carrageenan or Phytagel

434 (Fig. 7B). This increased fluorescence is likely due to enhanced scattering in the

435 more opaque agar-based gels. Additionally, in SC based gels the effect may be

436 due to the absence of quenching compounds in the less defined, extract-based

437 YP-media (Fig. 7B). To assess the effects of gel autofluorescence on substrate

438 usability for fluorescent screens, we plated low and high green fluorescent

439 protein (GFP)-expressing yeast colonies on the different gels supplemented with

440 yeast extract, peptone, and glucose (Fig. 7C). While gel autofluorescence was

441 not additive to high or low colony signals, the background intensity differed widely

442 between substrates. Thus, it is possible for this background fluorescence to wash

443 out very low GFP signals, such that reduced autofluorescence may make colony

444 edge detection, size determination and ultimately effect quantification much

445 easier (Fig. 7D).

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446

447 DISCUSSION

448 After more than one hundred years of heavy agar use in microbial research, we

449 have taken a second look at agar’s usefulness for today’s more advanced high-

450 throughput techniques. Certainly, Bacto agar is an established and versatile

451 growth substrate that rightfully enjoys widespread acceptance. However, given

452 the specialized needs of modern genetics screening experiments, we have found

453 that other gel substrates provide highly beneficial properties.

454

455 In particular, Phytagel and carrageenan exhibit vastly superior optical clarity to

456 agar-based gels, allowing for scatter-free through-gel image acquisition and

457 improved fluorescent imaging. Additionally, Phytagel and carrageenan lead to

458 smaller, better-defined colonies that enable higher colony densities, which may,

459 in turn, lessen the risk of colony fusions. This is a highly beneficial property when

460 pursuing super-high colony densities. Instead of depending just on gel strength

461 (50), a combination of biophysical properties (gel strength and/or cation content)

462 appear to determine colony morphology: harder Phytagel and softer carrageenan

463 have better-defined colonies and both are cation-rich. Agarose and noble agar,

464 on the other hand, are particularly low on salt contaminants, which appears to

465 increase drug activity for certain drug types. This effect is likely mediated through

466 binding of cations to the yeast cell wall, as receptor-mediated drug uptake is

467 unaffected; however the precise mode of action should be investigated further.

468 Whenever drug cost or solubility are an issue, the use of these low-cation

21
469 substrates should strongly be considered. For example, instead of the standard

470 100 µg/ml clonNAT required for selection on Bacto agar SC plates, one could

471 perform the same selection on agarose with 10 µg/ml. Finally, additional savings

472 can be realized, as many substrates are less expensive than Bacto agar. In

473 terms of biological gel-gene interactions, we find no evidence that switching from

474 Bacto agar to any of the other substrates introduces any significant bias in the

475 observed fitness of yeast mutants, further encouraging the use of these gel

476 alternatives.

477

478 ACKNOWLEDGEMENTS

479 Gel-strength measurements were performed with the support of Kylee Scholar at

480 the Experimental Food Science Lab (San Diego State University, San Diego/CA).

481 Wild-type diploid strains were a kind gift from Scott Rifkin and cultured with

482 support of Molly Burke (UCSD). Randy Hampton and Sonya Neal (UCSD) kindly

483 provided the GFP strains. National Institutes of Health awards GM084279 and

484 ES014811 supported this work. Some of this research was conducted while PAJ

485 was an Ellison Medical Foundation/AFAR Postdoctoral Fellow. The funders had

486 no role in study design, data collection and analysis, decision to publish, or

487 preparation of the manuscript.

488

489 CONTRIBUTIONS

490 PAJ designed the experiments. PAJ, CM, LW performed the experiments. PAJ

491 and TI wrote the manuscript.

22
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675 1:76–80.
676

27
677 FIGURE LEGENDS

678

679 Figure 1: Gel appearance and optical qualities. (A) Gels were prepared either

680 on their own (Plain) or with common media components added (Glu = glucose,

681 SC = Synthetic complete mix, YP = yeast extract and peptone; scale bar 10 mm).

682 An empty set of vials was added at the bottom to create comparable lighting

683 conditions for all gels (only lids are visible at the bottom). (B,C) To assess the

684 optical quality of gels with growth media, absorbance was measured from 350

685 nm to 750 nm (C: SC; D: YP). (D) Side views of typical high-throughput yeast

686 screening plates prepared with the different gels demonstrate the range in gel

687 clarity (same concentrations as in A; scale bar 4 mm). Yeast colonies are

688 hanging from the gel slab; differences in the optical path make colonies appear

689 twice (red arrows in the schematic).

28
690 Figure 2: Nutrient availability and colony characteristics. (A) Yeast colonies

691 were grown for 24 hrs on gels with varying degrees of nutrients to assess if the

692 gels release anabolites. Gels were prepared without any additional nutrients (Gel

693 only), with a carbon source (+Glu), with a nitrogen source (+YP), or with full

694 media (+Glu +SC and +Glu +YP). Orange crosshairs indicate colonies analyzed

695 in B. The same nine colonies are shown in all conditions (scale bar 4 mm). (B)

696 Cross-sections of colonies in B. α = Contact angle of the colony edge with the

697 surface. (C) Mean raw colony sizes ± SEM of 1536 mutant yeast strains grown

698 on the different gels (+Glu +YP) for 24 hrs. All mean colony sizes are significantly

699 different from each other at p < 0.001 except Bacto and Noble agar (One-way

700 ANOVA followed by Tukey comparison of all pairs).

701

702

29
703 Figure 3: Gel pH and strength. (A) Gel pH was measured under gel only

704 conditions or with different media components added (Glu = glucose, SC =

705 Synthetic complete mix, YP = yeast extract and peptone). (B) Gel strength was

706 measured in plain gels as the force necessary to depress the gel by 2 mm.

707

708

709

30
710 Figure 4: Diffusion characteristics. (A) A small fluorescent tracer molecule

711 was allowed to diffuse into the gel matrices for 12 hrs (scale bar 10 mm). (B)

712 Diffusion diameter was measured at the indicated time points. In agreement with

713 the Higuchi equation, diffusion is proportionate to the square root of time and

714 appears to be only determined by gel concentration (% w/v). (C) Relationship

715 between gel strength and the slope of the diffusion fit.

716

31
717 Figure 5: Drug bioavailability. (A) Example of a drug bioavailability experiment.

718 Strains susceptible (his3Δ::kanMX) or resistant to clonNAT (his3Δ::cNAT) were

719 diluted (1:1 to 1:211) and spotted onto increasing concentrations of clonNAT

720 containing Bacto agar with SC plates (without ammonium sulfate). Example

721 images underlying data in (E). (B) Titration curve for Canavinine on SC plates.

722 Viability is measured relative to the number of colonies from a drug resistant

723 strain at the same drug concentration. (C) Titration curve for S-AEC on SC

724 plates. (D) Titration curve for G418 on SC. (E) Titration curve for clonNAT on SC.

725 (F) Titration curve for G418 in YPD. (G) Titration curve for clonNAT on YPD. (H)

726 Estimated element content of typical batches of the gel substrates based on

727 supplier specification. No data are available for carrageenan magnesium content.

728 Red crosses indicate lot-specific cation content based on Certificat of Analysis

729 (CoA) where available. (I) Relationship between the clonNAT dosage that kills

730 6/12 dilution colonies (y-axis) and the total cation content of the underlying gel

731 substrate (x-axis). Results are shown for SC media, except carrageenan for

732 which the YPD data point was used.

733

32
734 Figure 6: High-throughput array performance. (A) A random selection of 1536

735 mutant yeast strains from the deletion collection was grown in parallel on six

736 Bacto agar 2% plates. Correlation between colony sizes on the six “identical”

737 plates ranged from 0.88 to 0.95 (Pearson correlation). The mean colony size

738 from these six plates was set to be the “gold standard” for C and D. (B) Each of

739 the six plates was pinned onto a test plate with the indicated gel substrate, and

740 colony sizes were correlated. Gel concentrations were adjusted slightly to

741 provide similar gel hardness. (C) Scatter plots between the fitness of the

742 indicated gels (spatially corrected and plate mode normalized colony sizes) and

743 the Bacto agar “gold standard” (r = Pearson correlation). Grey line indicates

744 diagonal, orange line indicates least-square fit of the scatter data; colonies

745 smaller than 0.05 were excluded. (D) Histograms of the fitness values for each

746 gel substrate compared to the “gold standard”.

747

33
748 Figure 7: Autofluorescence properties. (A) Autofluorescence of the gels varies

749 widely. Gels were prepared either on their own (Plain) or with common media

750 components added (Glu = glucose, SC = Synthetic complete mix, YP = yeast

751 extract and peptone; scale bar 15 mm). An empty set of vials was added at the

752 bottom to create comparable lighting conditions for all gels (only lids are visible at

753 the bottom). (B) Autofluorescence intensity of the gels relative to Bacto agar +SC

754 +Glu (a common screening medium). (C) Examples of low and high GFP-

755 expressing yeast strains on the indicated gel substrates (+YP +Glu; scale bar

756 3mm). (D) Intensity cross-sections (green channel) on the various media to

757 exemplify signal-to-noise ratio of the different gel substrates from C.

34
758 TABLES

759 Table 1: Description of the different gelling agents tested in this study.

Setting Melting
Name Synonym Components Source Comments
Point Point
Repeating unit of
agarobiose: a disaccharide
Red algae (Gelidium Agar without the
Agarose made up of D-galactose ~36ºC ~88ºC
spp., Gracilaria spp.) agaropectin
and 3,6-anhydro-L-
galactopyranose
Optimized Fe, Cu,
TM Agarose (70%) plus Red algae (Gelidium Mg, Ca
Bacto agar Kanten, Agar-agar 32-39ºC 83-89ºC
agaropectin (30%) spp., Gracilaria spp.) concentration for
microbial growth
Agarose (70%) plus Red algae (Gelidium Washed and
Nobel agar Kanten, Agar-agar 32-39ºC 83-89ºC
agaropectin (30%) spp., Gracilaria spp.) bleached agar
1,3α-1,4β-galactans having
Red algae Requires vigorous
one (κ-), two (ι-) or three
Carrageenan Vegetable gelatin (Kappaphycus 30-50ºC 50-70ºC mixing to avoid
(λ-) sulfates per
alvarezii) clumping
disaccharide unit
Gellan gum, Bacterial substrate
Bacterium
TM Nanogel-TC, composed of glucuronic Requires presence
Phytagel (Pseudomonas 35-50ºC >70ºC 2+ 2+
Gelrich, Grovgel, acid, rhamnose, and of Ca or Mg
elodea)
AppliedGel, Gelrite glucose
Linear copolymer with
blocks of (1-4)-linked β-D-
mannuronate and α-L-
Alginic Acid Algin, Alginate Brown algae n/a n/a Did not gel
guluronate residues, linked
together in different
sequences or blocks
Linear chain of β 1,4-linked
mannose residues to which Guar or cluster bean
Guar Gum Guaran galactose residues are 1,6- (Cyamopsis n/a n/a Did not gel
linked at every second tetragonoloba)
mannose
Too low melting
Hydrolyzed collagen Cattle, chicken, pigs
Gelatin Gelatine 25-35ºC 25-35ºC temperature, amino
extract or fish
acid source

760

35