Rehydrating ascospores to improve germinatio

Perkins, D. D.

Strickland, W. N.

Fungal Genetics Reports

English

W. N. Strickland

D. D. Perkins

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Rehydrating ascospores to improve germination

Kansas State University

Fungal Genetics Reports Volume 20 Article 39 Rehydrating ascospores to improve germination W. N. Strickland Stanford University D. D. Perkins Stanford University 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 Strickland, W. N., and D.D. Perkins (1973) "Rehydrating ascospores to improve germination," Fungal Genetics Reports: Vol. 20, Article 39. https://doi.org/10.4148/1941-4765.1847 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. Rehydrating ascospores to improve germination Abstract Rehydrating ascospores to improve germination This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol20/iss1/39 cr  sn  & and  CI  m csp-2+ derivatives each  hove useful  properties for the examination of individual colonies in o loge pop”-la&~  cr  m csp-Fzlonies  can be accurately replica plated with velveteen covered blocks. A single velveteen master  is usedto foithf;ilrpche  location of each colony onto ten or  more additional plates  by transfer of conidia. There secondary platercan contain media which test directly the properties of the transferred conidio, or which test the properties of colonies that growfrom the transferred conidia. Plate cultures of confluent CT  sn  crp-2+  colonies are also on excellent source  of macroconidio.- -One 9 cm culture yields ca.  5 x IO9  eonidio.  On  the other hand, conidiating  colonies of CT  m eon  plates con be exposedindividually to ony “tert’Tmedium,  in situ, by adding the medium in D soft agor  overlay.- - ?&<verloy  is poured without disturbingthe chains of conioined  conidia. Therefore, cross contomimtion  of colonies, via  freed conidia, is minimized.We have capitalized on the described properties of there stocks to isolate single gene mutants which lock NAD(P)  glycohydrolareactivity  (EC 3.2.2.6) . ( Supported in part by UCLA Medical Sciences Research Fund to P. T.Cahen  and on NSF grant to R.W.Siegel)- - - Department of Biology, University of California, Los Angeles, California 90024.Selitrennikoff,C.  P. and R. E. Nelson. A screening A rapid and simple method for the detection of cultures  defectivetechnique for the isolation of mocroconidiation  mutants. for the development of wild-type macroconidia is presented. Thismethod provides more efficient detection of mutants  than  microsco-pic observations previously  used in this lob. More  importantly, mutontr blocked late in the process  of conidiotion ore not easilyrecognized in the course of routine macroscopic  examination; the method described here Permits the discrimination between thereand wild types.Cultures ctre  grown  in cotton plugged tuber (7 cm x 1 cm) containing I ml Vogel’s N + 1.5% agor for 3-5 days in the light at35’C. Each tube is then inverted and given D single sharp  tap agoinst  the metol  light shade  of a fluorercent  lomp. The lampprovider a bright light source IO that any conidia mechanicolly  freed are visualized as a cloud of porticles falling  from the aerialhyphal mass  towards the cotton plug.As on  example of the power of the method, a single isolate which Produced very few freed cooidio was  readily detected amongE 3500  tubs cultures started from mutagenized  74-ORE-10  conidio (see Selitrennikoff  1972 Neurorpom  Newrl.  19: 23). Inagreement, microscopic examination (600X) hI owed that this culture producer  chains of conidio and, relatively rarely, individualconidia. Genetic analysis  demonstrated that the phenotype is due to a single  gene mutation, e  (conidial  reparation defec-tive, allele *37),  which is tightly linked to erg-3  on IL. Detailed obrervotionr of& and aconidiol  strains  will be reportedelsewhere. It moy be noted that the method hor proved useful  for the detections  of similar mutants in ouxotrophr grown on oppro-priately  supplemented media.(Supported in port by on NSF Prsdoctorol  Traineeship  to CPS and  an  NSF Grant to R. W. Siegel ).- - - Department of Biology, University of California,  Los Angeles, California 90024.Smi+h,B.  R. Storage of orcospores  in water. Arcorpres  may be stored in sterile water  for periods of up to a year atroom temperature ond up to eighteen months ot 4’C  without appreciableloss in viability. Even longer periods of storage  without great loss  in vio-bility  may  be possible, but no tests have been mode.Ascorpores  ore  horverted  by shaking crosses in sterile water,  followed by filtration through  two  layers  of cheese cloth to re-move large mycelial frogmentr. Suspensions are left to stand for 30 min to allow the orcospcres  to settle out.  Excess water,containing conidio, is pipetted off leaving a clean  suspension. For rtomge, l/2  oz. screw-copped bottler ore convenient.In a series of ten crosses of his-5 X py’-3 mutants  stored for I8 months ot 4”C,  the loss in viability varied between 2 and  12%with no detectable change in mlity due to genotype. It is not known whether arcosporer  of other genotypes survive  os wellwhen stored for long periods in water.- - - Deportment of Genetics, University of Aberdeen, Aberdeen A09 ZTN,  Scotland.Stricklond,W.  N. and D. D. Perkins. Rehydroting Arcorporer  from desricoted  crosses  germinate poorly if heat-shockedorcosparer  to improve germination.directly after  howesting  or isolation. The low germination con be over-come by rehydroting either in water  or on the surface of fresh ogar medium.We obtain good results, when isolating from old cross tuber, either by odd-ing sterile water to the tube 12 hours before isolating or by isolating the arcorporer  from the dehydrated cross tube to fresh slants,which are  then left overnight at room temperature (ZI’C)  b e oref subjecting them to heat-shock. (Longer Periods of rtomge  with-w t  refrigeration might result in sufficient mycelial growth that heat  shocking would not kill oil vegetative cells.)Quantitative  data on the effect of dehydration  and rehydrotion were published earlier (Strickland 1960 J. Gen. Micrabiol.22:585). We ore prompted to toll  attention  to the effect once ogoin  because  it doer  not seem  to be generally known, especiallyby those beginning to work with Neurorporo,  and because dehydration con seriously impair  efficiency if it i&unremedied. Inthe 1960 report, germination was  reduced to less than one-third after 27 days; this  was  restored to 97.5% by rehydration.Rehydration is not necerrory  when our standard  procedure for crosses is followed. Crosser  ore made on IO  ml slants  in largetuber. Spores ore ripe and germination is good  27 days  after first inoculation. At this  time the water loss  from evoporotion willhave shrunk  the slant  somewhat, but  not so OS  to derricote  the spores. Only  if irolotion  is delayed  until the agor has shrunk tohalf or less  ib original volume do we take steps to assure that orcosporer  are hydrated. With crosses  in small  tubes (IO  x 75 mm 1,drying out  occurs  quickly and rehydrotion  is more likely to be necessary._ _ _ Deportment of Biochemistry, University of Cope Town, P/Bog Rondeborch,  Cape Province, South Africa, and  Deportment ofBiological Sciences, Stanford University, Stanford,  California 94305.Westrum,  F. M. and  N. V. Vigfurson. The following method of N-methyl-N’-nitro-N-nitroroguclnidine muto-- -A method of NTG mutagenerir  of N. crosso.genesis  has  been used  to yield on ouxotrophic  mutant production rote 01- - high os 17%. This method is particularly useful  where other methods ofmutogenerir,  such as  UV  light, ore unovoiloble  or unodvirable. The wild-type s+roin  STA4, St. Lawrence stondord  74A was used for all  tests. The NTG is  first prepared by dissolving it in distilled waterto give o concentration  of 3 mg/ml. NTG is slightly insoluble in water  and the solution may  hove to be warmed slightly to oidin dissolving. Freshly prepared solution will give the best results, os  NTG deteriorates with age. (At 30 days it is less than50% OS  active  OS  fresh). A conidiol suspension is then mode by worhing  the conidio from o  slant  culture using sterile distilledwater,  filtering it through eight layers  of sterile  gauze,  and adjusting  it to o  final  concentration  of 1 x 106 conidio per CC.Two ml of the NTG solution is odded  to 100 ml of the I x 106 conidiol suspension, giving on NTG concentration of O.@5mg/mlof medium. This is allowed to stand 01  room temperature  for 30 minutes with periodic ogitotion.  A romple  is then removed onddiluted to D concentration of 1 x IO3conidio/cc. One ml ond 0. I  ml of this concentration are both plated and spread on com-plete rorbose medium. The plater  we then incubated ot 25’C  for +wo  to three days. When colonies become apparent, replicaplating  to minimal  sorbore  may be utilized to rapidly isolate  the biochemical  mutontr. Mutants  are then characterized  andcrossed  with a  wild-type strain  to verify that  they are  truly mutants.The above concentrations ond recoin-Table 1. Effect of varying  NTG concentration on survival  and mutant  yield. mended times were orrived  ot through sev-NTG Concentration 0.06 mg/ml  medium 0. 12  mg/ml mediumerol  tests. The results  of there tests areExpowre  t i m e  ( m i n )  5 15 30 45 60 90 5 15  30 45 60 90combined in Table  1. We began the testsusing  on NTG concentration  of 0.03 mg/ml_ _% Survival 45 27 32 22 6 5 35 29 23 19  4 4medium. This  concentration produced mu-fonts,  but required from  60 to 90 minutes of% Mutant  yield I7  9 2 8 exposure time. The 0.06 mg/ml  concentro-__-- tion  was chosen over  the lower one becoureit ollowed  completing the work in o  muchshorter time. The 0. 12  mg/ml concentration,  while time-raving, doer not reduce the survival  percentage os  much os  might be ex-petted,  although it drorticolly  reduter  the percent mutant yield. The 30 minute time of exposure  was chore”  becoure of the highmutant yield and the convenience of the shorter time span. Additional tests  showed no increole  in survival  as  o  result  of washingthe NTG from the conidiol rurpenrion  with sterile  distilled water  prior to plating.- - - Department of Biology, Eortern  Washington State College, Chewy,  Worhington  99004.Kuloev,1.5.  and V.I.Melgunov.  Determinat ionof phosphorous  in N. craw extroctr.A method of total phosphorous determination in Nevrorporo  extracts  horbeen described by Hedmon (1969 Neurorporo  Newrl.  14: 10). who suggestedthe use  of a  method for total  orthophorphote  content estimation. However,like oil  other calorimetric  methods of phosphate determination in the aqueousphase, this one seemr +o be liable to error oriring  from some disturbing factors (see Berenblum  ond Chain  1938 Biochem. J.32: 295 1.berider  wh;:ch,  the time of interaction between molybdote and phosphorous compounds in solution was  very long (5-IOmin). Thus,the totol  qrthophosphote  content will be overrtated  because  of the well-known catalytic effect of molybdote on the hydrolysis oforganic phosphates. Therefore, we wish  +o turn Neurorporologirtr’  attention to another, more advantageous,  procedure of phosphatedetermination.The method adopted in our laboratory  is bored mainly  on Weil-Malherbe  and Green (1951 Biochem. J.49: 286) and Martin andDoty (1949 Analyt.  Chem. 21: 965 ) modifications of the extraction  method of phorphote  estimation introduced by Berenblum andChain (1938,  ib id.  1. The solutions  used ore: (I) mixture of isobutonol-benzene ( l:l, v/v). (2) 5% ommoniwn  molybdote in 4NH2S04,  prepared fresh doily by dilution of Itock  solution  of 10% ammonium molybdote in 8 N H2SO4.  (3) Stock solution of stowncu  chloride; 10 g  SnCI2  dissolved in 25 ml cow.  HCI, kept in o  brown gloss-rtoppered  bottle o+ O’C.  (4) Dilute rtonnour  chlor-ide solution; 0.25 ml cont.  solution diluted to IO ml with 1  N H2S04  (must  be made up fresh when required ). (5) Acid ethanol;IO  ml cont.  H2ZO4  i 490 ml abrolute  ethanol.Procedure: If the solution +a  be tested is strongly acid or olkoline,  it must first be neutralized to pH 7-0  with NoOH  or HCI.Then odd 6 ml irobutonol-benzene mixture and  1  ml 5% ammonium molybdate  in 4 N H2S04  to the test solution mode up to 5 mlin cz  gloss-rtoppered  test tube. Shake it immediately for 15 sec. With o  fine-tipped pipette connected too suction  flask, discardthe aqueous bottom layer  OI  completely 0%  possible. Then odd o  pinch of onhydrous  Na2SO4  to the test  tube and  rhoke it untilthe extract is cleared of any emulsified droplets. By means of a  syringe pipette, withdraw  2 ml and  transfer to o  second test  tube.Add 2 ml acid ethanol, 0. I ml dilute SnCI2  solution and  mix by shaking. After 10  min.  the intensity of blue color  may  be deter-

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Rehydrating ascospores to improve germination

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