Microscopy direct counting

In dark-field microscopy, the particles are only a blur no details are distinguishable at all. Some rough indication of the symmetry of the particles is afforded by the twinkling that accompanies the rotation of asymmetrical particles, but this is a highly subjective observation. However, the technique does permit the rate of particle diffusion to be observed. We see in Chapter 2 how to relate this information to particle size and shape. The number of particles per unit volume may also be determined by direct count once the area and depth of the illuminated field have been calibrated. This is an important technique for the study of coagulation kinetics, a topic we discuss in Chapter 13. 

Hennes, K. P., and Sutde, C. A. (1995). Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnol. Oceanogr. 40, 1050—1055. 

The basic approach with the axoneme-based analysis is to combine tubulin and axonema fractions and then to quench the reaction with glutaraldehyde. Samples of sufficient axoneme concentration are then added directly onto Formvar-coated sample grids for staining and electron microscopy. In some cases where the axoneme count is too low, samples may be sedimented onto grids by the method of Gould and Borisy (1977). With the methodology perfected by Borisy and Bergen (1982) samples can be taken as frequently as every 20 seconds, and the... 

As demonstrated by Yu and Standish [100] distribution transformations can lead to unacceptable errors, particularly with distribution laws with no limiting maximum or minimum sizes. Even direct transformation of a number count to a volume count for example is unacceptable, unless the distribution is narrow, the count is excessively high or special procedures are employed, as with cariying out volume counts in microscopy. ... 

Microscopy is a standard method for quantifying planktonic particulates. Counting by microscopy results in data, consisting of number concentrations of plankton species or other particulates, but it does not produce size distribution data. However, this method is direct and produces same information on particle shape, number, and type, which cannot be obtained by other means. 

One liter in situ samples directly taken on-site, were concentrated by the membrane filtration method for enumerating nannoplankton. A measured volume of a well-mixed sample (20 -100 mL, depending on the number concentration of algae) was filtered at a constant suction rate through a membrane filter having a pore diameter of 0.45 um. The algae collected on the membrane were identified and counted by means of optical compound microscopy. If the identification after membrane filtration is too difficult as a result of alteration of cell shape, the above mentioned settling chamber technique is used. 

The second method for obtaining the rate constant of flocculation is by direct particle counting as a function of time. For this purpose, optical microscopy or image analysis may be used, provided that the particle size is within the resolution hmit of the microscope. Alternatively, the particle number may be determined using electronic devices such as the Coulter counter or flow ultramicroscopy. 

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