Bray, D.

Kapoor, M.

Fungal Genetics Reports

English

Kansas State University

Fungal Genetics Reports Volume 11 Article 17 A method for disrupting conidia of Neurospora M. Kapoor D. Bray 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 Kapoor, M., and D. Bray (1967) "A method for disrupting conidia of Neurospora," Fungal Genetics Reports: Vol. 11, Article 17. https://doi.org/10.4148/1941-4765.1982 This Spores 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. A method for disrupting conidia of Neurospora Abstract Method for disrupting conidia This spores is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol11/iss1/17 Graham, J. D. The use  of malachite green todetermine disruption of conidia by sonificatian.Our laboratory has  been studying the effects of ultraviolet  lightupon the ribonucleic  and deoxyribonucleic acids of Neurorporo crossa.The effects of UV on the rote of synthesis of DNA &d its effects on thenuclwsider  and nucleotider  are being explored. The most difficultbarrier to this  work has  been the resistance of the conidiol case  to most simple method5  of disruption. We found that normal acidhydmlysir had little or no effect on the strain under study, to the extent ‘hot ot leort 20% of the conidia were viable after o IO-minute hydrolysis with concentrated HCI.  Homogenization war 01x)  unsuccessful. The criterion for a successful  conidial dirrup-tion method in this study  was 100% disruption of the conidia without destruction of the primary  structure of the DNA moleculeand  the individual nucleosides  and nucleotider.  Cellular viability and integrity (as opposed to conidiol ) was not essential. Thechoice of ultmsound  over liquid nitrogen or carbon  dioxide freezing was  made on the basis of ease  of handling.Disruption was produced with a microprobe sonifier (Branson  Instruments, Inc. ). The conidia were suspended in IO ml ofwater (5 x 106  cells per ml) in o thick glass  tube, which was  immersed in ice water. The microprobe of the sonifier was placedin the solution to a depth of I l/2  cm. The sonifier was tuned to intensity level 7 and the current to between 4 and  5 amperes.The conidiol suspension was  sonified  for 60 seconds.In order to determine the success  of the disruption technique, a malachite green stain  was  used. The conidiol suspension  wassmeared  on o slide, cleaned with chromic acid solution before use. The slide was  air-dried and the cells fixed to the slide byporring it over  o flame several  times. The fixed slide was placed  on top of o boiling water bath and covered with a piece offilter paper.  A 5% malachite green wlution  WQI dripped onto the filter paper, keeping the paper thoroughly moistened for thefive-minute staining period. The slide was  rinsed with distilled water.  Counterrtoining,  if desired, was  done with 0.4 molarsafronin  for 45 seconds. Staining by this method was  strong and quite acceptable for purposes of visualizing the extent of con-idial  disruption. Absence of intact conidio and the presence of malachite green-stained frogmen‘s were  used  01  indications ofconidial disruption. If intact conidio were present, disruption was  less  than complete. In repeated trials, it was found to bemore  than 96% complete.Further studies showed that the DNA isolated  was highly polymerized. Also, nuclwtide  ond nvcleoride spectra  and Rf’rcorresponded  well with the litemture  rtondardr. One of the primary odvontager of this method was  the speed and simplicityof its use. It was  possible to prepare the conidiol suspension, snnify,  and prepare and stain  the slides all within 15 minutes._ _ _ Department of Biological Sciences, Kent State University, Kent, Ohio 44240.Kapoor, M. and D. Bray. A method for disrupting The following method is routinely used  by us for counting andconidio of Neurorpora crassa.dirrupting  the conidicr  for enzyme extraction. A conidial suspensioncontaining 2 - 3 x I07 conidio per ml is prepared in buffer (phosphateor tris  0.05 M, 5 x IO-4  M in EDTA and 10-d  M in 8-mercoptoethonol).The number of conidia is estimated by determining the absorbance  of the suspension with a Klett-Summerron  photoelectric color-imeter, using  ~1 blue filter. A rtandord was  prepared initially by correlating  the Klett reading with the number of conidia, calcu-lated  from colony counts  obtained by se&l  dilution followed by plating on sorbore  media. A linear relationshi between the num-ber of conidio and Klett reading is realized up to on obrorbance  of 300 Klett units (equivalent to about 6 x IO P  ”conadra). Sus-pensions at concentrations higher than thir do not fall within the linearportion of the standard curve. Our standard ho3 been prepared for con-idio harvested from cultures grown at 28X,  for 6 daxyI,  using  strainsp (Y8743m)  (FGSCt38)  and  wild type 790  (FGSC 533). IS. _Conidio are  disrupted by treating a suspension containing rat  nvxethan 2.1 x 107 conidia per ml (Klett reading of 100) with a Branson,q,..  _model S-75 ronifier  cell disruptor. Figure I illustroter  the quantityxof soluble protein libemted as a function of time of insonation  of5g . . _conidia of rtroin Y8743m.  The rigmoid shape of the curve  is due to Zthe release of one fraction of protein immediately  following the dir- .zruption of the conidiol wall, while that associated with the sepuro- Qr-tion of protoplasmic material from the wall  is released ot a rubse- :quent rtoge.  As much a 95% of the soluble protein is liberated 2within 15  minutes. Ilo_ fConidial  ATPare (Mg*-requiring)  is liberated within the first5 minutes of insonotion, whereas this  treatment yields only &ut/a.-40% of the tot.1 soluble protein. - - - Department of Biology,University of Calgary, Calgary,  Alberta, Canada.Ir , #1  I‘ 1.Time (min )

A method for disrupting conidia of Neurospora

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