Plunge freezing

Plunge freezing Plunge specimen into a container of cryogen with forceps cell suspensions can be adhered to coverslips coated with poly-L-lysine design of cryogen baths is important (27) baths are cheaper than other freezing methods... 

In contrast transmission electron microscopy (TEM) can in skilled hands yield detailed quantitative data on pore structure, and can even provide valuable information on the wet state of resins by plunge freezing such samples and microtom-ing on a cold stage [105]. To obtain quantitative information it is necessary to use advanced image analysis methodology which is extremely powerful [106]. Unfortunately the approach is time consuming and costly and can rarely be applied routinely in morphology studies. 

Figure 11.8. Illustration of sample preparation for cryo-TEM imaging. Parts (a)-(d) show specimen preparation for sample freezing (e) shows plunge freezing apparatus with temperature and humidity control and ( ) a typical image obtained by cryo-TEM. Copyright 2006 Nestec Etd.

M. J. Costello, R. Fetter, and J. M. Corless, Optimum conditions for the plunge freezing of sandwiched samples, in Science of Biological Specimen Preparation (O. Johari, ed.), SEM Inc. AMF O Hare, Chicago, pp. 105-115. 

A frozen-hydrated state is often useful to observe cells. After culturing the cells directly on a Si substrate, wash the cell surface with distilled water to remove the culture medium and then plunge freeze the entire construct into liquid C2H6 in order to minimize ice crystallization which can damage molecules. While with a cooled SIMS measurement with liquid nitrogen, ice... 

If the reaction is accompanied by a rise of temperature, even if no minimum is specified in the directions, accidents may often be prevented by keeping a pot of ice water or freezing mixture at hand, into which the vessel may be plunged in time to prevent boiling over or decomposition. If gases such as hydrobromic acid, for example, are evolved, the reaction should be carried out under the hood, and the vapors led into a flask of water by a tube terminating above the surface. 

During this time the solution changes from gold to dark reddish brown indicating the formation of Fe hydroxy-polymers. No precipitate should form. Cool rapidly by plunging into ice water, transfer to a dialysis bag and dialyse for at least three days, changing the water several times each day. Collect the product and freeze dry it. 

Flash freezing was pioneered by Haas and Rossmann [208] in their early studies on lactate dehydrogenase at 198 K (—75°C) and was also used in the recent studies on myoglobin at 80 K (- 193°C) [203]. In this method the crystals are plunged into either... 

A preferred method for freezing samples is to use strips of aluminum foil (7- to 8-mm wide and 40-mm long) with an identification pressed in at one end with a pencil and the sample sitting on the other. Remove the tissue from the 20% sucrose solution, blot on a piece of paper towel, and set on the other end of the foil. Immediately with a forceps, plunge the foil strips with the tissue into the cold isopentane (Fig. 4.2). The tissue will stick to the foil and it can be placed in a vial in the liquid nitrogen bath and is subsequently stored in a -70°C freezer in individual small tubes. For sectioning, the frozen tissue is mounted later in O.C.T. on a chuck. 

Fig. 4.2 Method of freezing tissue. To freeze tissue rapidly, the amount of material frozen needs to be as small as possible. Freezing tissue with no liquid is best. On a strip of aluminum foil, write the sample information, place a blotted price of tissue on the foil, and plunge in isopentane. Freezing tissue surrounded by liquid has a slower rate of freezing, but this method is sometimes used. Place a piece of tissue in a mold, cover with O.C.T., and plunge in isopentane...

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