Colvin, H. J.

Munkres, K. D.

English

Kansas State University

Fungal Genetics Reports Volume 20 Article 31 Procedure for measurement of logarithmic growth H. J. Colvin University of Wisconsin K. D. Munkres University of Wisconsin 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 Colvin, H. J., and K.D. Munkres (1973) "Procedure for measurement of logarithmic growth," Fungal Genetics Reports: Vol. 20, Article 31. https://doi.org/10.4148/1941-4765.1839 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. Procedure for measurement of logarithmic growth Abstract Measurement of logarithmic growth This technical note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol20/iss1/31 TECHNICAL NOTESColvin,H.  J. and K. D.Munkres.  Procedure Three main conditions that ore  neccrrory  for logarithmic growth of Neurorporofor measurement  of logarithmic growth.are: I ) Iorge inoculum  ( L 1 x 106 conidia/ml  ), 2) odequote aeration, and  3)disperse growth (Davis and de Serres  1970 Methods in Enzymol. 170:79).  Giventhere conditions, the cultivation  of o large number of cultures in one experimentis expedited by the use  of small  flasks  and  rmoll  volumes of medium. With small cultures, however, one must  use micrwnolyticaltechniques for the meawrement  of growth. Moreover, the meawrement  of growth throughout the cycle requires  the occurate col-lection and  weighing of conidio and germinated conidio (II well as  mycelio. Finolly,  since the drying of the cells moy be detri-mental to cell functions such  os respiration or enzyme activities, one moy wish to cultivate replicate flasks - some for dryweight meowrements  and  others for meowrements  of other cell functions. Hence, the cell dry weights from replicate flasksshould  be highly reproducible.We shall describe o procedure which appeorr  to meet  all of the above  criteria. Small  flasks  (25 ml) containing 10 ml of themedium ore  used. With a New Brunswick shaker,  108  flasks con  be used in one experiment. To insure that the myceliol growthwill be disperse rather than  “clumpd”,  we remove myceliol  frogmentr from the conidiol inoculum  and two+  the inner surface ofthe flasks with o silicone reagent to prevent the mycelia  from sticking to the glass. Uniform inoculum  sizes are obtained bymeasurement  and  odjustmcnt  of optical density. Adequate aeration is achieved by rofory  shaking of the cultures a&  by the useof loose cops on the flasks  rother  than cotton plugs. The entire cell miss  in a flask is collected, dried, and  weighed on D taredfilter. This  sampling technique, unlike previously described ones (Davis and de Serres  1970),  does not require addition.1  hond-ling of the cells after they are collected.Moteriolr  and methods: Media;  E’thI er  minimal medium (Fries (Davis ond de Sever  1970)) or on enriched medium (YEGCE-Howell, Zuiches  and  Munkrer  1971 J.&II  Biol.50:721  ) were supplemented with 2% glucose. There medio were solidified with1.5% ogar.  Growth; Conidio of wild-+ype N. crassa  (RL 1256A.  a derivative  of 74A) were obtained from 7-14  day-old rlonts.Conidio were washed  from the slants  with IiFidmedium  and filtered through gloss  wcczl. The optical density of the conidiolrvrpenrion  et  550 nm was  adjusted to 0.3-0.4 by dilution with the medium. Ten ml of conidiol ruspenrion  was  added to each  ofa series of sterile siliconized  25 ml Erlenmeyer flasks. The floskr  were  incubated ot 30°C in o new Brunswick rotary shaker(Model G-25 ) at  270 rpm on o platform No. AG-50 (The platform would occommodote  108  flasks.  ) Me~ruremenh of oxygentension in the culture medium with on oxygen electrode indicated that  aeration WOI odequote. During the early logarithmicgrowth phase  the medium was  fully saturated with oxygen and at  least  50??  saturated  in late log ond early stotionory phase.Growth flasks; Erlenmeyer  flasks  (25 ml ) were  treated twice with D 1% solution of dimethyl-dichlororilane  in benzene ot6O’C (Sigma Chemical Co. ). The flasks were dried at W’C  for 30 min after each treatment with this solution. Metol  caps(22 mm 0. D.) (Motheron Scientific Co., Cot.No.61756-45)with  a l/4an  cotton layer in the top were used  os aseptic coversfor the flasks. Filtration; Cells were horverted  by vacuum filtration  with an apparatus consisting of a Gooch  porceloin crucible(Caor’r  No.Z7002),ppoper  filter, D 125 ml vocwm  flask,  and  a crucible holder. The latter was  constructed by cutting o l/4in. hole in o serum  battle stopper (5/B in.dio.)  and inserting a I.5  in. piece of IO  mm glass  tubing in the smaller  end of thestopper. The crucibles were worhed  serially in nitric acid, top water, ond distilled water  end  dried. The filter pods were eitherWhatmon no. 1 or 41  paper. The former war used  for the collection of cells during the first four hours of growth and  the Iotterfor oil subsequent timer. Filter papers of 17  mm dia.  were cut with the aid of a device resembling o cork borer. Filter paperswere placed in the crucibles and  both were  dried overnight in on oven ot 9O’C.determined to within f 0. I mg.The tared weight of crucible and paper  wasAll of the cells from a growth flask  were collected with the filtration device andwashed  on the filter with distilled water. Cells, filter paper and crucible were driedin on oven overnight at  90°C,  cooled to ream  temperature, and weighed with D MettlerH-16 balance to within l O. I mg. Net cell dry weight was  calculated as  the differencebetween gross and  tare  weights.Special care  in the collection of conidio is required. The use  of Whatmon I1 paperrather than *41  prevcntr  the conidio from parsing through the filter. #4l paper, how-ever, WOI more suitable for the collection of mycelia  than II  because the mycelio clogthe pores in the latter and the filtration  proceeds more slowly. Even with the I1 paper,however, the conidiol suspension tends to flow over the edge ond under the filter pad.This problem con be avoided by slowly pipetting  the conidiol suspension to the centerof the pod with D Pasteur  pipette.Results wd  Dircurrior: The rewlh  of two typical experiments ore shown in Figure 1.Logarithmic growth occurs  for about 5 hr with a mars  doubling time of 2 hr. The coef-ficient of variation  of dry weights from replicate flasks  was  10% during the first 4 hrand  i-3%  thereafter. The lotter opprooches  the vorionce  inherent in replicate mcosure-Figure I.  Growth of wild +ype  74A on YEGCE ----- and  Fries - medio.Doto points ore mean values  for 3 flasks. Barr  indicate 1 standard  deviationmerits of one sample. Grecrter reproducibility could probably  be achieved by the use of a microbalance. Serious deviations fromlogarithmic growth rates  occurred if the mycelia  clumped rclther  than remaining dispersed. Dry weights in the former case  were asr&h  a ZO-40%  lower than  in the latter. The clumping pottern  of growth was avoided by the removal of mycelial  fragments fromthe conidial inoculum  and by coating the inner surface of the culture flak  with dimethyl-di-chlorosilone.This work was supported by NIH grant GM 15751.- - - Loboratories  of Molecular Biology and Genetics, University of Wisconsin, Madison, Wisconsin 53706.Creighton,M.  0. and J. R. Trevithick. To overcome the necessity of constant passage  in either liquid or solid medium of theMethod for freezing slime.shin FGSC”326  (fz;  sg; a-1,  arg-I, cr-I, au), a  slime variant of N. crrzso,  freezingwas studied as a hoTdiG  method.- - - -Eight-day cultures, grown  on  Vogel’:  agar medium (I .5%  agar)  with arginine (50 mg/lM) m medium ) was washed  down with aIstream of Vogel’s orginine  SSP  medium (Vogel’s arginine medium plus 2% sucrose, 10% sorbore,  Penicillin G Sodium lOOU/ml  ofmedium, dihydrostreptomycin  rulphate  lC@g/ml  of medium) with a sterile Pasteur pipette. The milky liquid was transferred to  0sterile  tube and  centrifuged I5 minutes at 2500 rpm.The supernatont  was discorded and the residue resuspended in the freezing fluid (Vogel’s arginine SSP plus dimethyl  sulfoxide,4:)  ), until a four-fold dilution of this suspension gave on absorbance reading  of I  .O  at 280 nm on D Beckman spectrophotometer.2 ml of the concentrated suspension was placed In sterile vials, sealed and quick-frozen, using liquid nitrogen. The viols werestored a+ -7O’C  until used. To date they have been held up to three months, but longer periods of holding time ore being tested.Thwring was done in warm water  and the contents of the vial were emptied into D liter of Vogel’s medium plus arginine and anti-biotics (Penicillin and Streptomycin in the concentrations noted above). No retardation of growth has been noted when the fro-zen and thawed cultures hove been compared to others maintained  by continual passage.- - - Deportment of Biochemistry, Health Sciences, University of Western Ontario, London, Ontario, Canada.Griffiths,A.  J. F. and 8. Sivak. UVtronsmissionthrough voriws clear films in mutation experiments.To reduce the risk of contominotion  in long, or student-operated UVexposures, it is desirable to use some form of cover on  the irradiatedromple.  Traditionally quartz has been used for this purpose. Thisreport indicates that some cheaper materials are iust  0s good. Thematerials tested were: plastic Petri plates (from a/s Nokre  plost,DK 4690,  Naslev,  Denmark ); “Saran  Wrap” (from Dow Chemicals,Ltd.,122  Arrow  Road, Weston,  Ontario); “Look  Roasting Film”(from Look Film Associates, Scarborough, Ontorio  ); and “8aggier”(from Colgate-Palmolive Co.,  Ltd., New York, N. Y. ).Strip of the clear  materials from various sources were fitfed  into.*the sample cwette  of o Unicorn SP-BOO UV spectrophotometer, so)~ ; that the beam passed at right angles through one thickness. The$ behavior of the various films is shown in the Figure, in which thej base  line follows the 100%  trrmsmittonce  line. Most of the commonly-- used UV tubes (e.g., Hanovia  BBA-45, Osram HNS 12) emit ot 254millimicrons. It-&n  be seen that of those materials tested, Baggierprovide the only material which will transmit most radiation of thiswavelength’.- - - Botany Department, University of British Columbia, Vancouver8, B.C., Canada.Newcombe,K.  D. and A. J. F. Griffiths.  Adiustable This technique basically follows that  of Perkins (Neurcspora  Newrl.platforms for collecting shot asci. 9: 11  ) with the following modifications: (I)  Crosses ore  made on  filterpaper stripr  in tubes containing liquid Westergoord  medium. The med-ium contains 0.2% sucrose,  compared to the usual  2%. This drasticallyreduces conidiotian,  while maintoiling high fertility, and therefore makes the use of fluffy unnecessary. Crosses ore initiated bythe simultaneous  introduction of each parent as D drop or two of conidial suspension. (This technique was introduced to  A. J. F. G.by F. J. de Serres).  (2) Low conidiation  and the use of filter paper permit the removal from the cross tube of all  the perithecio.The paper can be cut up and placed on slides which ore held inverted on  odjurtoble  platforms over the ogor collection slcbs.  Twomodels of platform hove been used. (See figures on following page ).MDdel I has  been used extensively for the routine collection of hundreds of asci. It consists of hvo tubing clomps (a), mountedon-c  stand 6) with rapid-hardening epoxy glue. The inverted slide bearing the perithecia is placed ocrws  the top, andthe slide bearing the agar collection block is placed across the two adjustable arms and racked up into close proximity to the de-hiscing  perithecia. Two such  devices may be mounted back-to-back on  each stand.Model II is a more recent design and permits adjustment in hvo dimensions by the use of sliding plastic shelves (c). The shelves

Procedure for measurement of logarithmic growth

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Procedure for measurement of logarithmic growth

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