The reaction below has been known since the 1940s and has been commercially exploited in its current format since the 1980s. Despite this relatively long history the pharmaceutical industry has been conservative in its uptake of the technique, even in the face of qualified encouragement, from some of the regulatory bodies.
Luciferase + Mg2+
ATP + D-luceferin + O2 ^^^^^^^ AMP + oxyluciferin + CO2 + Ppi +light
• The enzyme luciferase hydrolyses ATP in the presence of oxygen and magnesium to produce light at a wavelength of approximately 562 nm. The amount of light emitted is directly proportional to the amount of ATP present, ATP only being contained within living cells. As the amount of ATP in bacterial and fungal cells is relatively constant (regardless of genus/species) at 1.0 to 1.5 x 10-15g for the former and 1.0 to 1.2 x 10-14g for the latter, then the number of organisms present can be determined from the number of "light units" emitted during the above reaction.
In use, the sample in which organisms to be detected is treated to remove non-bacterial and fungal sources of ATP, preincubated to increase any organisms present, the ATP released from the cells, then the reaction initiated. The pre-incubation step can be just a few hours, but this obviously means that the number of organisms cannot be quantified, only their presence or absence determined. (This technique also offers the opportunity to subsequently identify organisms present following enumeration, as it can be considered nondestructive.) This technique is limited to pharmaceutical products with minimal to zero bioburden.
Literature would suggest that due to the efficiency of the light detection, the sensitivity of the method could be limited to the detection of not less than 100
cfu/ml. However, in our experience advances in the commercial application of the technique have increased this sensitivity to 2 to 10 cfus, and enumeration down to 1 cfu can be achieved .
The Steriscreen, Microcount and Pallcheck systems utilize this technique and the former has been extensively applied by one of the authors (C. Randell) to a wide range of pharmaceutical products including liquids, creams, ointments and suppositories with equal success. We have achieved regulatory approval in a number of E.U. countries, substituting the Steriscreen for the traditional method, with the caveat that, should microorganisms be detected, they would be quantified and identified where necessary using traditional methods. The time taken to achieve a result is generally 24 hours for bacteria and 48 hours for yeasts or fungi. The method has proved robust and reliable and very few products containing high ATP from nonmicrobial sources required further processing to remove the effect.
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