Hintsandprecautionsforthecare,feedingandbreedingofNeurospora

Some of these notes describe what may be established routines in older laboratories, but are either unpublished or published in inaccessible places or scattered through the old literature, and thus may not be known to those just beginning to use Neurospora. Others may be practices unique to our lab. The attempt here is to supplement compilations of methods such as Davis and de Serres (1970 Methods in Enzymology 17A:79-143) and the older Stanford Neurospora Methods (1963 Neurospora Newsletter 4:21-25). Hints and precautions regarding particular mutants or categories of mutants will also be found in individual entries in Perkins et al. 1982 Microbiol. Rev. 46:426-570 (referred to below as "Compendium"). Statements apply to N. crassa unless another species is indicated. Neurospora Newsletter is abbreviated N.N. All temperatures are degrees°C.

This document may be browsed or one can jump to any of the headings described below:

Crossing 
Tips on handling ascospoes 
General Laboratory Practices 
Stockkeeping 
Mutagenesis and enrichment 
Tips for encouraging colonial growth 
Solutions and media 
Handling Heterokaryons 

CROSSING

1. Temperature; glassware. Crosses are usually made at 25°C. Perithecia do not develop, or do so poorly, above 30°C. We routinely make crosses on SC slants in 18 X 150 mm tubes, using foam or cotton plugs. Push-on metal or plastic caps should be avoided with cross tubes because they result in more rapid water loss and are more prone to contamination during the extended incubation period. Crosses are made on petri dishes for specific purposes, as when shot asci are to be collected. But plates are more prone to contamination and desiccation than tubes, and less compact for incubation and storage.

2. Media and supplements. The synthetic crossing medium (SC) of Westergaard and Mitchell is most widely used. The need to adjust pH of SC to 6.5 can be avoided by substituting 0.7 g K2HP04 and 0.5 g KH2HP04 per liter for the monobasic salt in the original formula (1963 N.N. 4:21-25). The formula in Davis and de Serres, p. 86, incorporates this change. The trace elements used for Vogel''s medium N are suitable for SC at 0.1 ml per liter final volume; there is no need for a separate formula.

Russo, et al. (1985 N.N. 32:10-11) have modified Vogel''s Medium N for use as a crossing medium by reducing NH4NO3 tenfold. The modified Vogel''s can be made up as a 50X stock, whereas the Synthetic Cross formula of Westergaard and Mitchell allows only 2x.

High ammonium or amino nitrogen inhibits crossing. Keep amino acid, purine or pyrimidine supplements at a minimal level in crossing medium. If total amino acids do not exceed 0.3 mg/ml there is usually no problem with fertility. Usually the parent with the simplest requirements is preferred as female.

3. Use of per-1 to assure female parentage. Where the pedigree of a mitochondrial genome or plasmid is critical, and assurance is desired that only one parent is functioning as female, per-1 (type I) may be used. When per-1 is present in one parent, all perithecia should be black and no white perithecia should be present if the protoperithecial parent was per-1+, and vice versa if the protoperithecial parent was per-1-.

4. False perithecia. In some single-mating-type strains, protoperithecia may enlarge and become pigmented so as to resemble small perithecia. These "false perithecia" are devoid of beaks, asci, and ascospores, and unfertilized strains exhibiting them remain completely sterile. The strains can develop normal perithecia upon fertilization with the opposite mating type. False perithecia are characteristic of single-mating-type cultures of Neurospora tetrasperma and of the Kirbyville, Texas population of N. discreta. False perithecia are also encountered sporadically in some N. crassa genotypes - they are most commonly of mating type a. Some isolates of fl a (fluffy) genotype have tended to make false perithecia.

The false perithecia can be a nuisance for mating-type testing, and a cause of alarm if contamination is incorrectly suspected to be responsible. Ordinarily, continued complete sterility and failure to develop even after long incubation distinguish false from true perithecia, and there is no serious problem. False perithecia can be of serious concern, however, in crosses where legitimate true perithecia are barren as a result of duplications or of genes affecting meiosis and ascus development.

False perithecia are also an object of interest for those interested in sexual differentiation. For example, inactive mutants of mating type a show development of barren or false perithecia in abortive mating reactions with A or a testers (Griffiths et al. 1978 Genetics 88:239-254; 1982 Can. J. Genet. Cytol. 24:167-176).

ASCOSPORES. OBTAINING PROGENY

5. Ripening of ascospores. Ascospores are unripe when first shot, even though fully black. For good germination, ascospores should be aged 7-10 days at 25-30° (not 34°C) after shooting begins, before isolation.

6. Rehydration of ascospores. Ascospores from old cross tubes that are desiccated should be rehydrated before heatshock to avoid poor germination (Strickland and Perkins N.N. 20:34-35). This is most likely to be a problem with crosses in small tubes. Rehydration can be accomplished conveniently by adding water to the cross tube or by holding isolated ascospores overnight on moist fresh medium before heatshock. If ascospores are isolated to slants prior to heatshock, fresh tubed medium gives better germination that medium has partially dried down.

7. Ascospore viability and longevity. For ascospore maturation, some auxotrophs require supplementation of crossing medium even though they are heterozygous, recessive, and used as fertilizing parent. This is true of pan-2 (not pan-1), spe-1, and some nic, cys and met genes (see Compendium entries).

N. crassa ascospores may be stored in sterile water sealed in small vials without appreciable loss of viability for at least a year at room temperature and 18 months at 4°C (B.R. Smith 1973 N.N. 20:34). Mature ascospores of N. crassa also remain viable in ordinary cross tubes and can be germinated after many months storage at 5°C. In contrast, germination of N. tetrasperma ascospores is reported to decline after 19 days following simultaneous inoculation of A + a or after 11 days following inoculation of conidia from an (A + a) culture (Howe et al. 1966 Genetics 54:293-302).

When N. crassa perithecia become desiccated before all the asci within them have been shot, ascospores remain viable in the unextruded asci and show high germination months later when rehydrated and heatshocked. Intact linear asci may be extruded singly after perithecia that have been dried in this way are placed in liquid. Crosses made in 10 X 75 mm culture tubes often dry down before the perithecia have emptied their contents (P. St. Lawrence), and are conveniently stored for future use.

8. Heatshock. For heatshock, a 60°C water bath is preferable to a hot-air oven. (Greater latent heat, better heat transfer, more stable). Allow at least 30 minutes at 60°C to assure killing of vegetative cells with a water bath, double this for dry air. Cover the bath to assure killing of conidia on tube walls.

Never remove ascospores from 60°C and then return to 60°C. Once activated, ascospores are vulnerable to killing by heat.