resulted in a changed yeast-nitrogen base without amino acid which is .... vitamins were essential for growth of Saccharomyces: biotin, pantothenic acid and.
Journal o f Microbiological Methods 4 (1986) 2 8 7 - 291
287
Elsevier J M M 00141
Cheap and simple yeast media Ben J.M. Z o n n e v e l d Genetisch Laboratorium, Kaiserstraat 63, 2311 GP Leiden (The Netherlands) (Received 24 May 1985) (Revised version received 11 November 1985) (Accepted 20 November 1985)
Summary A defined minimal medium has been described for Saccharomyces. The nitrogen base is more than twenty times cheaper than the c o m m o n l y used medium, it is of simpler composition and uses 3 0 - 5 0 % less agar. For mutants a richer medium is necessary. The complex medium proposed here contains only half the a m o u n t of yeast extract and a quarter of the a m o u n t of peptone, yet leads to nearly the same a m o u n t o f growth and also saves on agar.
Key words:
Saccharomyces - media
Introduction There has been an explosive growth of research on yeasts. It is therefore surprising that more attention has not been given to develop defined media for their growth. Instead of the c o m m o n l y used media for Saccharomyces [1-3] it is possible to prepare media with considerable savings in time and money. The defined minimal medium described here is based on the formula of Wickerham [2] that was developed for classification of yeast. The components for this medium were evaluated and this resulted in a changed yeast-nitrogen base without amino acid which is twenty times cheaper than the commercial Wickerham medium. Materials and Methods
Test organisms Saccharomyces cerevisiae o~-his 1 and c~-his 4 obtained from CSH New York are mainly used for experiments but many other strains used subsequently show the same results. Growth conditions The media were tested as liquids with 30 ml/300 ml flask. They were inoculated
0 1 6 7 - 7 0 1 2 / 8 6 / $ 3 . 5 0 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
288 with 40 × 106 cells from an overnight culture per flask and shaken with a gyratory shaker at 200 cpm at 30 °C. Growth rates were measured as an increase in turbidity at 618 nm after 8, 16, 24 and 48 h. Growth was also tested on agar plates by comparing colony sizes after 3 days. Nearly 200 different media were tested. Results and Discussion
Minimal medium for Saccharomyces Many types of media were prepared by changing the concentration of one c o m p o n e n t at a time. The best minimal medium for the growth o f Saccharomyces cerevisiae is given in Table 1. For comparison this new medium (MY) is compared with the composition of the Yeast Nitrogen Base (YNB) of Wickerham. In Fig. 1 growth curves are shown for MY and YNB based medium with 2% glucose.
The salt solution Compared with the Wickerham medium both KC1 and NaC1 have been omitted. This does not have any effect on growth, which is not surprising since both sodium and chloride are, if required, used in very small quantities and are provided by the trace elements in the solution. Potassium is in large excess in KHzPO4; CaC12 is omitted from the salt solution and added to the trace elements solution. The amount TABLE 1 MINIMAL MEDIUM M E D I U M (YNB)
(MY)
FOR
Medium (MY) (per liter) STV
Glucose K O H (5 N)
Difco agar
10 10 1 10
ml g ml g
SACCHAROMYCES
COMPARED
WITH
WICKERHAM'S
Salt solution
( 1 2 5 x , g/800 ml)
(lOOx, g/I)
KH2PO; MgSO~ • 7HzO (NH4),SO~ NaC1
MY 100 50 100 0
YNB 100 50 600 10
STV (100 x )
Salts sol. Trace elements sol. Vitamin sol.
800 ml 100 ml 100 ml
Trace element sol. ( 1 0 0 0 x , g/I) HCI conc. H~BO~ CaCI2 • 2 H : O ZnSOa • 7 H : O CuSOa - 7 H 2 0 MnCI: - 4 H , O KJ FeCL NaMoOa
M Y 2 ml 0.5 10 0.4 0.04 0.4 0.1 0.2 0.2
Vitamin sol. ( 1 0 0 0 x , g/I) YNB 0 5 100 4 0.4 4
1 2 2
Biotin Myo-inositol Ca-pantothenate Pyridoxine HCI Thiamine Nicotinic acid p-Amino benzoic acid Riboflavin Folic acid
M )" 0,02 2 4 4 4 4 4 0 0
YNB 0.02 2 4 4 4 4 4 2 0.02
289 1 0
'-
0
0
12
lO
// £ co ILl
4
2S
0
0
8
i
i
16
24
i
I
32
T I M E (h)
Fig. 1. Gro~vth of Saccharomyces cerevisiae ~ His 1 in different liquid media. 40 x 106 cells were inoculated in 30 ml of medium and the increase in absorbance measured at 618 nm at the times indicated. The curves are for YPD ( o ), CY ( • ), MY ( • ) and Wickerham medium ( ,, ) all with 2% glucose.
o f (NH4)zSO4 needed is related to the c o n c e n t r a t i o n o f glucose in the m e d i u m . The a m o u n t of (NH4)2SO4 is reduced from 6 to 1 gram per liter as this is more t h a n sufficient for a m e d i u m with 2 % glucose as c o m m o n l y used. This can easily be shown by a d d i n g 1 ml 5 N K O H to the used culture m e d i u m . If there is still free a m m o n i u m the expelled vapor will give a basic reaction with a prewetted strip o f p H paper. So only three salts are left in the salt s o l u t i o n a n d this enables o n e to make a 125 times c o n c e n t r a t e d , clear solution. Trace e l e m e n t s o l u t i o n
A 1000 times c o n c e n t r a t e d stock s o l u t i o n is prepared. Starting with the c o n c e n t r a t e d HCI one o b t a i n s a completely dissolved preparation. It is convenient to make a 10 times more c o n c e n t r a t e d stock s o l u t i o n ( - I 0 0 0 0 x ) which is also completely dissolved. T h e end c o n c e n t r a t i o n o f trace elements is a b o u t l0 times lower t h a n in the m e d i u m o f W i c k e r h a m , but it is still a b o u t 5 times more t h a n that
290 recommended by Fink [4]. Even diluting the trace elements 10 times more than proposed does not seem to have any effect on growth. The CaC12 is also included in the trace element solution at a 10 times lower concentration than in the Wickerham formula. No effort was made to change the amount of the different trace elements relative to each other.
Vitamins All nine vitami,ns were tested in many combinations. It turned out that three vitamins were essential for growth of Saccharomyces: biotin, pantothenic acid and myoinositol. Four others are not essential but lead to a somewhat faster growth, i.e., pyridoxine, thiamine, p-aminobenzoic acid and nicotinic acid. Riboflavin and folic acid do not affect growth and have been omitted. The 1000 times concentrated stock solution can be kept at 4 °C for several months.
Glucose For the plate cultures and overnight 5 ml liquid cultures 1°70 glucose is sufficient and gives the same result as 2°70 glucose. Only if high density liquid cultures are needed or liquid cultures are grown for longer than 16 h should 2°7o glucose (MY2) be used. This, of course, also depends on the size of the inoculum.
Agar; p H Agar is a polymer that easily loses its gelling properties at acidic pH. The usual solution to this problem is to take higher amounts of agar such as 1.5 or 2 % so that there is still sufficient gelling capacity. A much cheaper but rarely used second solution to this problem is to increase the p H of the prepared medium in this case from p H 4.5 to 6.3 by adding some KOH. This results in agar cultures with a consistency comparable to the 2°70 agar cultures at the lower pH. Above that yeast grows even faster at the higher pH on plate cultures. As seen in Fig. 1 there is virtually no difference in the growth curves for MY and the Wickerham medium for shaken liquid cultures.
Complex medium for Saccharomyces Apart from a minimal medium a complex medium is needed on which most mutants are able to grow (Table 2). Several components of the commonly used YPD medium are in large excess. TABLE 2 C O M P L E X M E D I U M (CY) FOR S A C C H A R O M Y C E S Bacto yeast extract Bacto peptone Glucose KOH (5 N) Difco agar
5 5 10 0.1 10
g/l g/l g/l ml/l g/l
291 Compare only the amount of nitrogen in MY (1 g (NH4)2SO4/1) with the same ratio of nitrogen containing amount of peptone (20 g peptone/1 for YPD). Moreover, the yeast extract (10 g/l for YPD) also contains a fair amount of nitrogen. Both peptone and the yeast extract have been reduced to 5 g/l. An alternative is 1°70of yeast extract and omitting the peptone altogether. In both cases the results are comparable with the usual prepared YPD plate cultures. For liquid cultures it is better to double all ingedients (CY2). In Fig. 1 a higher maximum is reached for YPD due to the extra carbon in peptone and yeast extract. Also here it possible to use less agar by adding some KOH. General remarks All ingredients are in highly concentrated solutions. This enables one to prepare sterile liquid media by pipetting the necessary amounts into sterile water. It is convenient to prepare a 100 times stock solution of salts, trace elements and vitamin (STV 100x). This can be kept at 4 ° C for a long time. All ingredients can be autoclaved together. However if one wants to prevent browning of the medium (due to the reaction of ammonium with glucose) the glucose should be added after autoclaving. Another advantage of the CY medium is that adel and ade2 mutants become red on this medium within three days, which enhances color assays considerably, if compared with the usual minimal medium with a low amount of adenine, adel and ade2 petites stay white on this complex medium but turn red if 2% glucose is used instead. To avoid petites, 2°7o glycerol (CYG) instead of glucose can be used and addition of 2°/o ethanol (CYGE) enhances growth. To distinguish petites from grandes 0.1% glucose is added to the glycerol medium (CYGD). It should be added that replacing the (NH4)2SO a with 1 g asparagine per liter, or just adding an extra 1 g asparagine results in a better growth and higher biomass production. This may be partly related to the fact that the sulfate left after exhaustion of the ammonium acidifies the medium; this effect is missing with asparagine (or ammonium tartrate). Also Schizosaccharomyces pombe grows as well on these media as the usual Schizosaccharomyces media (YEA,MSA) [5]. References 1 Sherman, F., Fink, G.R. and Lawrence, C.W. (1974) Methods in Yeast Genetics. (Revised Ed., 1983), pp. 61-64, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 2 Wickerham,L. J. (1946) A critical evaluation of the nitrogen assimilation tests commonlyused in the classification of yeast. J. Bacteriol. 52, 293 - 301. 3 Difco Manual (1953) pp. 250-254, Difco Laboratories, MI. 4 Fink, G.R. (1970) The biochemical genetics of yeast. Methods Enzymol. 17A, 59-78. 5 Egel, R. (1976) Rearrangements at the mating type locus in fission yeast. Mol. Gen. Genet. 148, 149- 158.
