Stanford Neurospora Methods

FROM Neurospora Newsletter 4:21-25

The methods listed below are extracted from a working notebook in the laboratory of D. Perkins, Department of Biological Sciences, Stanford University. This laboratory has been shared at various times by E. Barry, B. Maling, P. St. Lawrence, N. Murray, W. N. Strickland, D. Newmeyer, B. N. BoleGowda, J. Mayo, J. R. Singleton, K.S. Hsu, H. R. Cameron, and C. Ishitani, all of whom have contributed. Methods have been collected from various published and unpublished sources, many of them in other laboratories, and in most cases it would be difficult to trace and assign credit properly for the original source or for modifications. Inasmuch as we ourselves refer constantly to the notebook, we feel that these extracts from it may be sufficiently useful to others, especially in laboratories not already geared to using Neurospora, to warrant making them available, even though undocumented or incompletely documented. This note has not been checked with most of the people involved; it was prepared by N. Murray and D. Perkins. If the consensus is that this sort of summary is worthwhile, additional methods may be described in future issues.

1) Standard strains: Two Oak Ridge wild types from F. J. de Serres and H. E. Brockman are used as standards for inbreeding and reference. These are 74-OR23-1 A (FGSC No. 986) and 74-OR8-la (FGSC No. 532). They were derived from St. Lawrence 74A (see NN 2:25), are homozygous for heterokaryon compatibility factors, and are cytologically favorable. Two fluffy strains (Linkage group II) that originated spontaneously in St. Lawrence background are used for sex-tests and for scoring aberrations according to defective spores projected. These strains are highly fertile as protoperithecial parents and develop perithecia rapidly and simultaneously. Their main convenience is that no conidia are produced, so that contamination risks are minimized. The strains are: flA (flp: FGSC No. 295), and fla (P605: FGSC No. 297).

2) Crosses: The synthetic cross medium (SC) is basically that of Westergaard and Mitchell (1947). Biotin concentration has been increased 100-fold (it probably makes no difference (and phosphate is supplied as K2HP04 and KH2PO4 rather than as the monobasic salt alone. pH is 6.5 without adjustment.

For 1 liter of SC:

KNO3                     1.0 g
KH2PO4                   1.0 g
MgSO4.7H20               1.0 g
NaCl                     0.1 g
CaCl2                    0.1 g
biotin                   0.5 mg
trace element solution   (see below)
Trace element solution for SC: We have been using metals in the amounts B 0.01 mg, Cu 0.1 mg, Fe 0.2 mg, Mo 0.02 mg, and Zn 2.0 mg per liter final medium (see Beadle and Tatum 1945, Am. J. Botany 32:678-686). In the future, we propose to use for SC the trace element solution given by Vogel for Medium N, in the amount 0.1 ml TE solution per liter SC. This will provide the metals in approximately the same concentration, but it differs in containing citric acid.

SC is conveniently prepared as a concentrated stock solution (2x). The unautoclaved stock is stored at 5 C, with 2 ml/l chloroform as preservative. Sucrose (2%) and agar (2%) are added before autoclaving. There is normcily some cloudiness from precipitation after autoclaving.

All crosses are routinely carried out at 25°C, in 15 cm tubes, or in Petri dishes. Random ascospores are usually not isolated until at least 18 days after fertilization because germination or allele ratios are not good before this time. Ascospores are heatshocked (30 min. 60°C water bath) without further aging after isolation; no hypochlorite is therefore necessary to kill conidia or hyphae. The ripening of ascospores may be speeded by moving crosses from 25° to 30°C for a few days, beginning after ascospores have started to be shot from the perithecia.

Cornmeal agar (Difco) may be used for crosses under special circumstances (e.g., when crosses are unsuccessful on SC). It should be noted that Difco lists two cornmeal agars, with glucose (B114), and without (B286). We have used B114.

3) Minimal medium: Medium N is made up as a 50x stock solution as described by H. Vogel [(1956, Microb. Genet. Bull. 13:42-43) see section 12 below], with the following added precautions: In preparing the 50x stock: Do not beat. Add components sequentially. Make certain that everything is dissolved before adding next component (conveniently accomplished in a large flask and a shaker or with a mechanical stirrer).

1% sucrose is used as standard carbon source.

Difco now lists three varieties of Neurospora minimal (0324-15, 0460-15, 0817-01), as well as a Neurospora complete (0321-15; see N.N. 1:13). We have not used these ourselves, but have suggested them for convenience to school teachers or students who inquire about doing Neurospora projects.

4) Color coding of media: Media containing various supplements may be readily coded prior to plating or tubing by the use of Schilling food color (McCormick and Co., Inc., Baltimore and San Francisco). This is added to the medium before autoclaving at approximately 0.05 ml dye per 100 ml medium. The dyes are available from grocery stores in red, orange, yellow, green and blue, and come as 4% solutions in distilled water plus propylene glycol. In our experience these are biologically inert, stable and nontoxic. (Their use was introduced here by P. St. Lawrence).

5) Agar substrate for manipulation and isolation: For the benefit of workers in other countries, it should be mentioned that 4% agar is routinely used here for ascospore manipulation and isolation because 4% is near the limit of solubility for relatively pure agars such as Difco. Our small experience with crude agars suggests that much higher concentrations of them are required to produce an equally stiff gel. This may explain the difficulties mentioned by Prakash (NN 3:11).

6) Stock solutions of supplements:

                    Amount per ml  Stock solution used 
Supplement          stock solution per 100 ml medium

p-aminobenzoic acid    0.4   mg.        0. 5 ml.
choline chloride       2.0              1.5
inositol               5.0              1.0
nicotinamide           5.0              0.2
Ca-pantothenate        1.0              1.0
pyridoxine HCl         1.0              1.0
thiamine               1.0              1.0
L-arginine            40.0              1.25
L-histidine HCl       25.0              2.0
DL-homoserine         10.0              2.0
indole                 1.0              2.0
L-leucine              5.0              4.0
L-lysine              20.0              2.5
L-methionine          10.0              5.0
L-phenylalanine       10.0              2.0
L-proline             10.0              5.0
sulfanilamide          3.4              1.0
L-threonine            5.0              2.0
L-arginine            20.0              2.5
     + L-lysine       40.0    
L-isoleucine           3.0              3.0
     + L-valine        7.0
Individual supplements are dissolved in water and stored at 5°C over chloroform. Adenosine, adenine sulphate, adenylic acid, uracil, and tyrosine are not sufficiently soluble to allow the use of concentrated stock solutions. Adenosine is used at 0.5 mg/ml, uracil at 1.0 mg/ml, and tyrosine at 0.4 mg/ml.

As a rough general rule, we use per ml final medium about 0.2-0.5 mg amino acids, 10 micrograms vitamins, and 0.2-0.5 mg purines and pyrimidines. The concentrations provided are in many cases greater than indicated in the literature as necessary to provide maximum growth in flask assays. The higher concentrations are in a number of cases necessary in crossing and germination media in order to obtain optimal ripening and germination of auxotrophic segregants, e.g., in the case of nic mutants, good allele ratios are attained only when supplement in crossing medium approaches 40 µg/ml nicotinamide--several times the amount necessary for optimal growth (P. St. L.). Even when wild type is used as protoperithecial parent, and nic- to fertilize, recovery of nic- segregants is better when SC is supplemented.

7) Mating-type tests: Pairs of standard Petri plates, each containing about 15 ml SC agar, are inoculated in the center with the fluffy tester strains. Plates are wrapped in paper towels and incubated 7 days at 25° before tests are made. Plates are marked on the bottom into suitable sectors, and a small inoculum of each culture to be tested is spotted onto a marked sector. Tests are made on both mating types. The plates are reincubated at 25°C and may be scored for the presence of perithecia after 48-72 hours. If spore patterns are to be checked, the lids may be secured with plastic tape, and plates inverted. (See B. R. Smith, NN 1:14, for a similar method using the mutant spray for mating type tests.)

8) Auxanography: Conidial suspensions (e.g., 0.5 - 1.0 ml in a 75 or 100 mm tube) are made in sterile water. (The conidia should be well distributed by shaking the tube or by pipetting. ) A visibly turbid conidial suspension is plated in molten, 1.5% agar minimal medium, and thoroughly mixed by swirling the plate. When the medium has solidified, test substances (usually solids) are spotted at marked points (which are indicated on the base of the plate with wax pencil). A flamed microspatula is used. Tests can be read after 12-24 hours incubation at 34°C. With some strains, preincubation for a few hours before spotting may give better results.

9) Preservation of stocks by silica gel: The procedure is described in detail by Perkins, 1962, Canad. J. Microb. 8-591 (see Ogata, NN 1:13, for variations). 100 mm tubes are convenient; stock numbers are etched on tubes with a carborundum grinding tool ("Handitool ", Chicago Tool and Mfg. Co. Tubes are sealed with parafilm over a cotton plug, and stored at 5° in closed containers.

10) Cleaning of glassware: Alconox and Event are effective, nontoxic detergents. (Some domestic detergents leave a residue which is very toxic to Neurospora--at least they did so ten years ago. ) For acid cleaning an undiluted mixture is used consisting of 2 liters concentrated sulfuric acid (tech) plus 100 ml concentrated nitric acid.

11) Control of mites: Mite infestations are readily controlled (and prevented) by using a concentrated solution of Gammexane (Lindane; 1,2,3,4,5,6 Hexachlorocyclohexane) in 95% alcohol; this solution is painted on incubators, shelves, racks, containers, desk-tops, etc. where cultures are to be stored (or containers may be dipped in the solution).

A milder technique for control of mites

12) With the permission of H. J. Vogel, we include here his directions for preparing Medium N, as given in the Microbial Genetics Bulletin 13:42-43, 1956.

In 750 ml. distilled water, dissolve successively with stirring at room temperature (Mallinckrodt Analytical reagents are satisfactory):

Na3 citrate, 5 1/2 H20                 150 grams(NOTE, you may substitute 125 g Na3 Citrate 2 H2O)
KH2PO4, anhydrous                      250 grams
NH4NO3, anhydrous                      100 grams
MgSO4, 7 H20                            10 grams
CaCl2, 2 H20                             5 grams
Trace Element Solution (see below)       5 ml.
Biotin Solution (see below)              2.5 ml.
Add with stirring

The resulting total volume is about 1.00 liter. Chloroform (2 ml. ) is added as a preservative, and the 50 times strength medium obtained is stored at room temperature. For use, this medium is diluted 50fold with distilled water. The resulting single-strength medium is designated N; it has a pH of about 5. 8. Medium N is supplemented with a suitable carbon source such as sucrose (20 grams per Iiter), and the thus supplemented medium is sterilized by autoclaving.

The trace element solution (containing citric acid as a solubilizing agent) is made up as follows: In 95 ml. distilled water, dissolve successively with stirring at room temperature:

                  Citric acid, 1 H20         5. 00 grams
                  ZnSO4, 7 H20               5. 00 grams
                  Fe(NH4)2(SO4)2, 6 H20      1. 00 gram
                  CuSO4, 5 H20               0. 25 gram
                  MnSO4, 1 H20               0. 05 gram
                  H3BO3, anhydrous           0. 05 gram
                  Na2MoO4, 2 H20             0. 05 gram
The resulting total volume is about 100 ml. Chloroform (1 ml. ) is added as a preservative, and the trace element solution is stored at room temperature.

The biotin solution is prepared by dissolving 5.0 mg. biotin (Merck) in 50 ml. distilled water. The solution obtained is dispensed in test tubes and stored in the frozen state.

To prepare "complete medium, " supplement Medium N with a carbon source, 0. 5% yeast extract, and 0.5% N-Z-Case (Sheffield). To prepare "minimal " and "complete " slants, use 1% sucrose plus 1% glycerol as carbon source, and solidify with 1.5% agar.

Note that the above may not be cited in publication. It is designed to be used for information purposes only.

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Last modified 9/04 KMC