Liquid scintillation counting of discrete samples is conceptually straightforward. A sample is mixed with an appropriate volume of scintillation cocktail, and the mixture is placed in an LSC vial and counted. For some samples no additional steps are required, but in many situations samples must be processed to avoid artifacts. The most common causes of artifacts are static electricity counts, chemiluminescent counts and color quenching. Protocols for sample preparation to maximize the efficiency of counting and minimize background are given below. These protocols can be readily adapted to a variety of samples. Following this section, the preparation and counting of several unique sample types are presented, as "special applications."

The best counting efficiencies are achieved when samples uniformly disperse into the cocktail to produce a clear, colorless, pH neutral emulsion. Uniform dispersion of the sample is achieved by selecting the appropriate cocktail formulation. Organic samples present no problem. With organic samples, the highest efficiency can be achieved in cocktails which contain no emulsifiers and which are only suitable for organics. Organic samples, however, can be successfully counted in emulsifying cocktails, and often the convenience of using one cocktail for all applications outweighs any loss in efficiency.

Aqueous samples present more of a challenge. The choice of cocktail will depend upon the balancing of sample holding and efficiency. It is a good idea to choose a cocktail which can hold at least 10% more sample than you intend to add, as sample capacity may be strongly affected by temperature or sample components.

Sample Neutralization (Elimination of chemiluminescence)

The neutralization of strongly alkaline samples is necessary to avoid chemiluminescence. Neutralization can be accomplished by the addition of acetic acid. If the sample contains a high concentration of alkali, the addition of acetic acid may increase the overall salt content beyond the capacity of the scintillation cocktail. In such situations, the sample will need to be diluted prior to the addition of cocktail. If neutralization is not practical, samples may be left to stand 1-3 hours, or in some cases, overnight, before counting. This allows time for the chemiluminescent reaction to run its course and die out. If chemiluminescence is suspected, samples should be counted repeatedly at intervals of greater than 1 hour until a stable reading is obtained.

Decolorizing

Achieving a colorless solution of sample in cocktail is generally not problematic. Many samples are colorless, or contain so little color that dilution into the scintillation cocktail gives an essentially colorless solution. In those cases where samples are deeply colored, particularly when the sample absorbs in the region of 300-400 nm, where scintillation phosphors emit, several decolorizing protocols are available. As visible color often depends upon long conjugated polyene systems, strong oxidants are used to "bleach" the samples. Samples can be treated quite harshly prior to counting, because chemical changes to the labeled compounds will not alter the number of DPM emitted.

Decolorizing LSC Samples with Ultraviolet Light

Ultraviolet irradiation is often effective in bleaching visibly colored samples. The optimal wavelength, intensity and time must of course be determined for each sample. In many cases, exposing samples to sunlight for 1-2 hours is sufficient. Bleaching by UV has a great advantage over other methods - nothing is added to the sample, avoiding the potential quenching or chemiluminescent effects of other bleaching agents.

Decolorizing LSC Samples with Hydrogen Peroxide

Hydrogen peroxide is a strong oxidant and a very effective bleaching agent. It is inexpensive, easy to work with, and miscible with aqueous samples. The only disadvantage to using H₂O₂ is that it decomposes to produce molecular oxygen, which is an effective quenching agent. Samples must be heated to drive off the O₂ following H₂O₂ bleaching, to ensure reproducible results.

1. Mix 0.1 - 0.3 ml of 30% H₂O₂ with 1ml sample
2. Incubate 1 hour at 50°C and shake occasionally.
3. Cool to room temperature, add scintillation cocktail and count.

Decolorizing LSC Samples with Benzoyl Peroxide

Samples which are not soluble in water or tissues which have been dissolved in organic solubilizers, can be bleached with benzoyl peroxide.

1. Dissolve 1g benzoyl peroxide in 5 ml toluene - heating to 60°C may be required. Filter solution if cloudy. (Caution: Toluene has a flash point of 7°C. Heating must be carried out in a spark-free fume hood to avoid an explosion hazard.)
2. Add 2 ml benzoyl peroxide/toluene solution to 1 ml sample.
3. Incubate for 30 minutes at 50°C.
4. Cool to room temperature, add scintillation cocktail and count.

NEXT TOPIC: Counting Samples from TLC Plates by LSC