Viability of Neurospora conidia from stock cultures on silica ge

Brockman, H. E.

de Serres, F. J.

English

Kansas State University

Fungal Genetics Reports Volume 1 Article 10 Viability of Neurospora conidia from stock cultures on silica gel H. E. Brockman F. J. de Serres Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Brockman, H. E., and F.J. de Serres (1962) "Viability of Neurospora conidia from stock cultures on silica gel," Fungal Genetics Reports: Vol. 1, Article 10. https://doi.org/10.4148/1941-4765.1024 This Technical Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. Viability of Neurospora conidia from stock cultures on silica gel Abstract Viability of Neurospora conidia from stock cultures on silica gel This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol1/iss1/10 tion. Thus, at the end of nuclear division, each of the sister nuclei receives what appears to be half of the original nucleolus. Both of these observations are still at variance with those published by Somers &al. (Genetics , %:801-810, 1960) who suggested (1) "the presence of a spindle" DT the "image of a spindle" and (2) that new nucleoli are formed during telophase. It is also worth men- tioning that with the Helly technique the writer has now observed the presence of a nuclear membrane. However, from the variously stained preparations studied with light microscopy, it is still difficult to determine whether this membrane remains intact throughout division or not. It must be emphasized that the crux of the problem still rests upon the question of how the sets of sister chromosomes (formerly called chromosomal filaments by this writer) sort themselves out without the aid of a spindle. This is not only a problem in Neurospora but also in the vegetative mitoses of other fungi studied to date. Brocknan. H.E. and F. J. de Serres.* Anhydrous silica gel has been used for the pre- Viability of Neurospora conidia from servation of Neurospora cultures by various stock cultures on silica gel. investigators for several years, and the ability to recover such cultures has been comparable to that following lyophilization (Perkins, unpublished observations). We have initiated experi- ments to determine whether the viability of conidia from such cultures is strain-specific and whether survival is enhanced by storing the silica gel cultures at 4oC. A description of the procedure, which is similar to that used by other investigators, is given below. Culture tubes (13 x 100 mm) with teflon-lined screw caps are filled approximately three- fourths full of 6-12 mesh anhydrous silica gel (Eagle Chemical Co., Inc., Industrial Canal, Mobile, Ala.), dry-sterilized at 16O'C for l-1/2 hours, and stored at 60°C until needed. These tubes later are chilled and kept in an ice bath until after the coni3ial suspension has been added. Three cc of Carnation instant nonfat dry milk (7.5 g/100 cc; autoclaved 10 minutes) is added to a 5-7-day-old culture slant (20 x 150 mm). The conidia are then sus- pended by placing the tube on a Vortex mixer, and 1 cc of the suspension is added to a cold silica gel tube. The tubes are stored at room temperature (23°C) for 7 days with the screw caps on loosely. The caps then are tightened and the tubes are stored over silica gel in Nalgene pans (12 x 5 x 4-l/2 inches) at 4°C. To assay conidial viability, a number of silica gel crystals are shaken into 1-2 cc of cold distilled water, the conidia are suspended with a Vortex mixer, the suspension is de- canted, and total conidia are counted in a hemocytometer. Various dilutions of the conidial suspension are plated in Westergaard's mediwn supplemented with 1.5% sorbose, 0.1% sucrose, 1.5% agar, and the necessary mutant requirements. This medium is autoclaved for the period of time required for maximm viability (de Serres, Kblmark, and Brockman, Nature, 193: 556, 1962). Plates are incubated at 30°C for 3-4 days, and the percentage of viable conidia is determined from colony counts. Table 1 shows the res~ults of an experiment in which a wild-type strain, 74A-OR, and a" ad-3 mutant strain, l-155-0011, were assayed for conidial viability immediately before add- ing the suspension to silica gel (0 hours) and 2 hours and later after adding the suspension. Viability decreases rapidly during the 7 days at room temperature and less rapidly during storage at 4oc. I" another experiment, the conidial suspension was added to two silica gel tubes--one treated as described in the above procedures and the other stored continually at room tem- perature. The viability of these strains after 183-224 days is given in Table 2. All strains are ad-3 mutants induced in wild-type strain 74A except 74-OR21-6a, which is hi-, nit-2, al-2, pan-2. Viability is better at 4OC than at room temperature in every strain, but the difference is much greater for some strains. There is also a considerable difference in viability at each temperature from strain to strain. In all platings the morphology and nrnwth characteristics of the colonies were strikingly uniform and indistinguishable from those grown from fresh vegetative cultures. We conclude that conidial viability is greater from silica gel cultures stored at 4OC than at room temperature and that the viability is high enough for routine stock preservation and for experiments utilizing stored conidia. Table 1 ,ge of Silica Viabilitv (%) Gel Culture 74A-OR l-155-0011 0 hours 75.0 74.8 2 hours 82.3 61.2 8 days 18.0 34.8 14 days 12.5 23.2 35 days 16.0 31.9 97 days 13.2 7.1 155 days 9.6 8.0 Table 2 - Age (days) of Silica Viability i%) Strain Gel Culture 4oc Room temp. (23X) 2-015-0095 223 5.1 0.09 2-015-0128 223 7.7 0.03 l-154-0022 224 6.9 2.9 2-016-0001 212 2.8 2.3 2-016-0006 212 31.7 0.1 l-234-0106 211 48.6 5.4 l-234-0084 211 29.3 0.4 2-017-0002 183 21.7 0.1 2-017-0006 183 10.1 2.1 '4-OR21-6a 211 57.6 16.2 *From Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Operated by Union Carbide Corporation for the U. S. Atomic Energy Commission. de Serres, F.J. A procedure for making heterokaryon tests in liquid minimal mediun.* Various difficulties encountered in attempts to increase (1) the ninnbers of pairwise combina- tions or (2) the length of the observation period in heterokaryon tests on the ad-3 mutants with the agar plate method (de Serres, Genetics 41: 668, 1956) made it desirable to develop an alternate technique. A method was needed that would permit (1) making a large number of pairwise combinations, in individual containers, so that observations could be nade easily over a number of weeks and (2) precise control over the inoculum density per unit area, or per unit volume, of mediwr so that replicate combinations would agree both in the time of formation of a heterokaryon and the rate of subsequent growth. The method that was developed makes use of the facts that baskets of test tubes can be "plugged" by wrapping the "nit completely in a double layer of Saran Wrap, and that tubes so plugged can be inoculated with suspensions of conidia in liquid minimal medium by means of a pipetting syringe fitted with a hypodermic needle. The two inoculation holes over each test tube effect such a sharp hlnnidity gradient that aerial growth of the hetero- karyon is confined in each tube to a region l-2 cm above the meniscus. The rate of evapora- tion under average conditions (N 23X, 30-50% relative humidity) is low enough so that with 2 ml of medium per tube observations can be made for long periods of time (30-45 days). Aerial contamination can be controlled by covering the top of each basket of tubes with a square of blotting paper. To prepare for this type of experiment we first autoclave wire baskets of 100- 75 x 100 mm test tubes (in a 10 x 10 grid) and then wrap the unit in a taut double layer of Sara" wrap. These are covered with a square of sterile blotting paper and packaged in groups of four in heavy-weight paper bags. The bags are placed in a drying over at 60°C for 60-72 hours to insure sterilization of all components and to make the Saran Wrap more taut. Hemocytometer counts are made on each conidial suspension (after filtration through a 

Viability of Neurospora conidia from stock cultures on silica gel

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