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Unformatted text preview: JOURNAL OP BACTERIOLOGY, Oct., 1966 Copyright @ 1966 American SocietyforMicrobiology Vol.92,No.4 PrintedinU.S.A. Measurement of Size Distributions of BacterialCells R. J.HARVEY AND ALLEN G. MARR DepartmentofBacteriology,UniversityofCalifornia,Davis, California Receivedforpublication31May 1966 ABSTRACT HARVEY,R.J.(UniversityofCalifornia,Davis),ANDALLENG. MARR.Measure- ment ofsizedistributions ofbacterialcells.J. Bacteriol.92:805-811. 1966.-Ap- paratusfortheautomatic determinationofthe volumedistributionofparticlesby measurement oftheamplitudeofpulsesgenerated ina Coultertransducer isde- scribed.Distributionsofvolumeestimatedbydirect measurementofpulseampli- tude are distorted by coincidence. Differentiationand integration ofthe pulses followedbyautomaticpulse-heightanalysispermitprecisemeasurementofvolume oflatexspheresandofbacteriaoverarangeofat least0.25to20,l.Theapparatus isalsocapableofaccuratedeterminationof particleconcentrationoverawiderange. Otheradvantagesarethespeedofbothmeasurementanddata processing. The technique of determiningparticlesizeby measuring the changein resistanceas a particle movesthroughasmallchannelfilledwithelectro- lytewas developed by Coulter (U.S. PatentNo. 2,656,508,1953).Kubitschek (2,3)modified the apparatus of Coulterto measure the volumesof bacterialcells. No adequate theory relates the volume and shapeoftheparticlepassingthroughtheaperture ofthe Coulter transducer to the amplitudeand shapeoftheresultingelectricalpulse. Hence,the validity of measurements of particle size by a systemutilizingthistransducerrequiresempirical evaluation.We haveusedthedistributionofsize measured by electron microscopyas a primary standard againstwhich the performance of the electronicapparatus was measured. MATERIALS AND MErODS Preparationofsamplesformeasurement.Escherichia coli strain ML30 was grown in glucose minimal mediumat30C. For electronicmeasurement of size, samples of liquid cultures were diluted in 0.85% NaCl containing0.04% formaldehydeto give a final concentration ofabout 2 X 106cellsper milliliter. For measurement of size by electron microscopy, 0.1%formaldehydewasaddedtoa sampleof culture, which was then centrifuged, washed once in 0.04% formaldehyde, and suspended in a solution con- taining0.04% formaldehyde and 0.1% serum albu- min to give a concentration of 2 X 108 cells per milliliter. The suspgnsion was sprayed onto Form- var-coated grids and air-dried. Polyvinyltoluene latex spheres with nominal diameters of 0.796, 1.305, 2.051, and 3.49 , were obtained from Dow Chemical Co., Midland, Mich. Sampleswerepreparedforelectronicmeasurement of size as described above and were sprayed directly onto Formvar-coated gridsforelectron microscopy....
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