A Micro-nianipulalion Method for the Preparation of Calibrated Microdroplets 



127 



swelling and the retention of contents increases their 

 sedimentation rate. The medium has no biochemical 

 disadvantages; it does not significantly inhibit any 

 enzymes. 



Using this medium particles (tig. 2, inset) have 

 been seen in light mitochondrial fractions which are 

 similar to those described by Kuff <:'/ al. (6). They arc 

 smaller and denser than mitochondria; they have 

 only a single external membrane and a rather granu- 

 lar interior. Duve (4), from the biochemical evidence 

 of enzyme distributions in light mitochondria frac- 

 tions, has postulated a new cytoplasmic particle, the 

 lysosome in which certain hydrolytic enzymes would 

 be contained. 



Discussion. — These results demonstrate how elec- 

 tron microscopy can help the biochemist by giving 

 him an improved method of controlling the purity 

 of the cell fraction. Moreover they also demonstrate 

 how the methods may be improved so that the frac- 

 tions may be obtained not only in a greater state of 

 purity, but also less damaged by the separation. 



The method is of equal importance to the electron 

 microscopist, for it is probably the only way in 

 which he will be able to determine the chemical 

 nature of the fine structures he sees. It is unlikely 

 that histochemistry will ever be possible in electron 

 microscopy, to the same extent as it is in light 

 microscopy. The electron microscopist must there- 



fore separate the structures he observes without 

 damaging them, and then chemically analyse them. 

 The techniques of separation by centrifugation are 

 examples of such a general method. 



This invcstig;ilion has been supported by grants to 

 the Chester licatty Research Institute (Institute of Cancer 

 Research: Royal Cancer Hospital) from the British 

 Hmpire Cancer Campaign, Jane Coffin Chils Memorial 

 Fund for Medical Research, the Anna Fuller Fund, 

 and the National Cancer institute of the National Insti- 

 tutes of Health, U.S. Public Health Service. 



The authors are particularly grateful to Mr. K. G. 

 Moreman for supplying the illustrations. 



References 



1. BiRBECK, M. S. C. and Rhd, H.. Biodiim. et Biophys. 



Ada 20, 419 (1956). 



2. — J. Biophys. Biochcni. Cylol. (1937, in press). 



3. Davison, P. F. and Mfrcfr, L. H., Exptl. Cell Research 



II, 237 (1956). 



4. DuvE, C. DE, Pressman, Ei. C, Gianftto, R., Wattiaux, R., 



and Ai'PLFMan, F., Bioihem. J. 60, 604 (1955). 



5. HoGEBOOM, G. W., S( hnfidfr, W. C, and Palade, G. E., 



/. Biol. Cheiii. 172, 619 (1948). 



6. Kuff, E. L., Hogfboom, G. H., and Daiton, A. J., /. 



Biophys. Biochem. Cytol. 2, 23 (1956). 



7. Palade. G. E., J. E.xptl. Med. 95, 285 (1952). 



8. Philpot, J. St. L. and Stanier, J. E., Biochem. J. 63, 



214 (1956). 



9. Witter, R. F., Watson, M. L., and Cotione, M. A., 



J. Biophys. Bioehem. Cylol. I, 127 (1955). 



A Micro-manipulation Metho(J for the 

 Preparation of Calibratecd Micro(droplets over the Range of 10^-10'^ ml 



in Electron Microscopy 



Irene Sugar 



Dept. for Electron Microscopy of the Institute for Measurement and Instrumentation of 

 the Hungarian Academy of Sciences, Budapest 



I HE electron microscope is a research tool that can 

 in principle make it possible to estimate the concen- 

 tration of particles of colloidal dimensions in colloi- 

 dal systems. A condition for this is to find a method 

 to measure the volume which corresponds to a 

 counted number of particles. 



Riedel and Ruska (3) and Backus and Williams ( 1 ) 

 gave different solutions of this problem, by applying 

 internal standards in micro-droplets sprayed on to 

 the specimen supporting film. The method of Backus 

 and Williams, using uniforinly sized polystyrene 

 latex as a standard can be applied in a more general 

 way than that of Riedel and Ruska using salt crystals 

 for this purpose. In investigating molecules of small 

 particle size, it was observed that uncertainties may 

 arise from the fact that the droplets are so to say 

 quantized for the standard but remain statistical for 



the material and it is difficult to obtain good corre- 

 lation between counts of particles and the number of 

 latex spheres in various droplets. 



In this paper we present a micromanipulation 

 method for preparing measured micro-quantities. 

 This requires considerably more technical work than 

 the spraying method but does not rely on an internal 

 standard. Depending on the magnifications used in 

 the electron microscope, we can in a reproducible 

 way choose the proper micro-volume between 10""- 

 10-'" ml. 



The separation of the micro-volumes and their 

 placing onto the specimen holder takes place with 

 the use of micro-pipettes whose internal diameter is 

 only a few microns, in the optimal case 2-3 microns. 

 These pipettes are made with the microforge of de 

 Fonbrune (2) but his original method has been 



