Classical microbiological test methods frequently are divided into three general categories, based on the test function performed.
These categories are: presence or absence of microorganisms (e.g., pathogen detection, absence of objectionable organisms,
sterility testing), enumeration of microorganisms (e.g., bioburden testing); and identification of microorganisms. This classification
answers three specific questions: "Is something there?" (presence or absence); "How much is there?" (enumeration); and "What
is there?" (identification).
Table 1. (continued) Applications of Rapid Microbiological Methods
Classification systems for rapid methods are based on how the technology works: methods that measure the growth of microorganisms;
methods that determine the viability of microorganisms; methods that detect the presence or absence of cellular components
or artifacts; nucleic acid methods; traditional methods combined with computer-aided imaging; and combination methods.
Growth-based Technologies. These methods are based on the measurement of biochemical or physiological parameters that reflect the growth of the microorganisms.
Examples of these types of methods include: adenosine triphosphate (ATP) bioluminescence, colorimetric detection of carbon
dioxide production, measurement of change in head space pressure, impedance, and biochemical assays.
Viability-based Technologies. These types of technologies do not require microorganism growth for detection. Varying methods are used to determine if the
cell is viable, and if viable cells are detected, they can be enumerated. Examples of this type of technology include solid-phase
and flow fluorescence cytometry.
Cellular-component or Artifact-based Technologies. These technologies look for a specific cellular component or artifact within the cell for detection or identification. Examples
of these systems include: fatty acid profiles, mass spectrometry (i.e., Matrix Assisted Desorption Ionized-Time of Flight,
MALDI-TOF), enzyme linked immunosorbent assay (ELISA), fluorescent probe detection, and bacterial endotoxin-limulus amebocyte
lysate testing (LAL).
Nucleic-acid-based Technologies. These technologies use nucleic acid methods as the basis for operation. Examples of this type of technology include: deoxyribonucleic
acid (DNA) probes, ribotyping/molecular typing, and polymerase chain reaction (PCR).
Traditional Methods with Computer-aided Imaging. This approach involves using a classical method for most of the processing of a sample, and then using imaging software to
detect the growth earlier than methods requiring visual growth detection. In most cases, detection of growth using human vision
typically requires growth of 105 or 106 cells. Computer-aided imaging can detect much lower levels of cellular growth, e.g., less than 100 cells.
Combination Methods. This term is used to describe those systems that involve more than one methodology or test to achieve a final result, e.g.,
a system that tells whether an organism is present and is also capable of identifying the microorganism.
Adenosine Tri-Phosphate (ATP) Bioluminescence
Type of Technology: Growth-based.
Premise of Technology: ATP is present in all living cells. In the presence of the substrate D-luciferin, oxygen, and magnesium ions, the enzyme
luciferase will use the energy from ATP to oxidize D-luciferin and produce light. The amount of light or bioluminescence produced
can be measured by sensitive luminometers, and is proportional to the amount of ATP in the sample. The emitted light is usually
expressed as relative light units (RLU) rather than as direct estimates of microbial numbers. Vendors of these technologies
have conducted studies to show the correlation between RLU readings and approximate number of organisms. These standard curves
are used to translate the raw RLU data to more meaningful organism-quantification data. ATP bioluminescence reduces the test
time required in the traditional method by approximately one-third. ATP bioluminescence can be used to screen both filterable
and non-filterable samples.