The various methods to detect bacteria and assess the level of microbial activity in water.
The importance of preventing microbiological contamination and growth of the water used in pharmaceutical manufacturing processes cannot be overstated, and a regular testing regime is essential to ensure that the purification techniques employed to minimize the proliferation of micro-organisms are operating at optimum levels.
Assessing the Level of Microbial Activity
The first challenge in assessing the level of microbial activity in water is to determine exactly what constitutes a viable organism. The conventional definition suggests that a viable organism should be capable of living, able to live on its own, able to reproduce, or able to carry out normal cellular functions, but many bacteria would fall outside these norms when tested with conventional culture-based systems.
An organism that can reproduce is clearly viable, and for this reason bacterial culture remains the ‘gold standard’ when it comes to assessing viability. However, this approach may be too simplistic to guarantee accurate and reliable test results in all circumstances. Growth depends on many factors, including nutrients, pH, osmotic conditions and temperature, and the ability to reproduce is dependent on the characteristics of the specific organism and of the medium in which it is placed. Some bacteria that may not be able to replicate on artificial media, for example, may regain that ability once inside an animal host where conditions are more amenable to reproduction.
Tryptic Soy Agar (TSA) at 37°C is the culture medium most commonly used in the pharmaceutical industry, but in general this detects far less than Reasoner’s 2A agar (R2A) at 22°C or yeast extract agar (YEA) at either temperature. Furthermore, it may not detect those bacteria that might cause spoilage of the product during storage because they will grow under refrigeration conditions but not at body temperature. Careful choice of the appropriate culture method is therefore essential as different combinations of nutrients and temperature select for different groups of organisms.
Detecting Bacteria: Alternative Methods
In an attempt to circumvent the shortcomings of culture, alternative methods of detecting viable but non-culturable (VBNC) bacteria have been developed, but these too have their limitations. The results are always dependent on the method of detection and the viability marker that is being used. Therefore, the selection of an alternative method should take the sample type and the number of anticipated bacteria present into consideration.
A common technique applied to the detection of VBNC bacteria is fluorescence microscopy, yet a wide variation in results can be seen on identical samples. The inference is that either some bacteria are lost during the preparation of the sample for microscopy or that the staining methodology used is not as effective as it could be.
Flow cytometry is another technology used to check for the presence of viable bacteria in water samples, but this is less suitable for use in the pharmaceutical manufacturing sector because it is most applicable to samples with relatively high microbial counts, such as potable water.
Of greater relevance to the pharmaceutical industry is solid phase scanning. Table 1 shows that in samples comparing two different media incubated at both 22°C and 37°C, fewer bacterial cells were detected using yeast extract agar medium than with R2A, which is a medium specifically designed for low nutrient environments. However, although significantly more bacteria were counted via solid phase scanning, the relationship between the number of organisms detected by culture or by laser scanning was inconsistent. The fact that the commonly used yeast extract agar detected fewest bacteria clearly demonstrates the impact of the choice of test method on the results.
Method |
0-10 |
11-100 |
101-1000 |
1000+ |
Scanning (FDA) |
7.5 |
20.9 |
40.2 |
16.8 |
R2A 22°C |
35.5 |
39.3 |
21.5 |
2.8 |
R2A 30°C |
43.9 |
40.2 |
13.1 |
2.8 |
YEA 22°C |
28.9 |
31.0 |
10.3 |
0 |
YEA 37°C |
92.5 |
7.5 |
0 |
0 |
Table 1: Plate counts and laser scanning. Results presented as the percentage of total samples examined that fell into each category.
Polymerase chain reaction (PCR) in combination with intercalating dyes is a more recent technique for the detection of viable bacteria. PCR will detect anything that contains DNA, regardless of its viability, so the dye is used to offer a degree of discrimination. The membrane of a damaged cell is non-selective in terms of what it allows to pass across it, whereas the membrane of a living cell will exclude intercalating dyes. When the dyes enter damaged cells, they intercalate between the DNA bases, preventing nucleic acid replication from taking place—thereby distinguishing between live and dead cells.
The choice of procedure is also likely to be influenced by the concentration of cells within the sample. For example, for flow cytometry typical sample sizes are of the order of 100µl, whereas with solid phase laser scanning, samples that are liters in size can be run, although the filtration step may cause a degree of difficulty. The number of cells that might be expected to be present and the desired limit of detection will be key factors in making the final choice.
Other factors to take into account when detecting most bacteria, including coliforms, is the format and nutritional content of the medium. Liquid media of low nutrient content may give rise to significantly better recovery of microorganisms that have been living in water systems.
Table 2 compares samples prepared and tested in three ways: membrane filtration onto a solid medium, a liquid enrichment method with the same medium, and Colilert®, a low nutrient liquid medium. More organisms were detected using liquid enrichment than the solid medium, despite the fact that it was the same medium in both cases, while Colilert picked up significantly more of both E. coli and total coliforms. Clearly, a low nutrient medium is required for better detection as too high a concentration of nutrients can impede recovery of nutrient stressed organisms.
Method |
# samples E. coli recovered |
# samples total coliforms recovered |
Membrane filtration (MLSA) |
16 |
43 |
MPN (MLSB) |
22 |
49 |
Colilert |
27 |
72 |
Table 2: Recovery of E. coli and total coliforms.
In similar studies, the low nutrient Pseudalert® system picked up many more positive samples with Pseudomonas aeruginosa than either membrane filtration or liquid enrichment. Yet membrane filtration remains the primary technique used for water analysis, whether in the mains water supply, or in the pharmaceutical or food industry. In both cases a liquid medium with low nutrient levels puts the bacteria under less stress, making them more likely to grow and be detected.
When it comes to heterotrophic plate counts (HPC), which give a total count for viable bacteria, the media used are typically TSA, YEA, or R2A. Using a similar low nutrient, liquid-based system can avoid at least some of the issues caused by the very different nutrient levels. The simple HPC for Quanti-Tray® test procedure allows 100 ml test samples to be run, without the need for membrane filtration, with growth detected by the cleavage of fluorochromes. Enumeration is a simple case of counting fluorescing positive wells, which corresponds to a most probable number (MPN) of total heterotrophic organisms in the original sample.
Table 3 shows the results of a comparison test in which 44 samples were run at each of 22°C and 37°C, on two different media—the richer TSA that is commonly used in pharmaceutical water testing and the marginally less nutrient-rich YEA—and also low nutrient HPC for Quanti-Tray. When YEA is compared with YEB (its liquid medium counterpart) and Quanti-Tray, the liquid version picks up a comparable number to Quanti-Tray.
Method |
22°C (mean count) |
37°C (mean count) |
TSA |
1 |
2 |
YEA |
17 |
3 |
YEB |
25 |
8 |
QuantiTray |
26 |
9 |
Table 3: Comparison of liquid and solid media techniques for HPC (44 samples).
Solid vs. Liquid Media
Generally speaking, despite the fact that solid media continue to predominate, liquid media are more likely to detect bacteria in water samples. The reasons for this are still unclear, and if the bacteria have been damaged—for example, by exposure to a sub-lethal dose of disinfectant—the benefits of using a liquid medium are further enhanced.
One possible reason is that a liquid medium imposes less stress on the bacteria being tested for. A liquid medium is likely to reach the incubation temperature more slowly. Ideally, the sample would be incubated at the same temperature as when it was taken. Similarly, liquid media do not subject the bacteria to the levels of physical shock occasioned by the use of solid media. For example, using a solid medium instantly exposes the bacteria to atmospheric oxygen at a 20 percent concentration that they would not experience in the water supply, and this alone may prove toxic to some organisms.
In conclusion, although culture remains the ‘gold standard’ test method for detecting bacteria in water samples, the results can be optimized by the use of low nutrient, liquid media in low temperature conditions rather than using solid media. Consideration should also be given to the organisms most likely to be present and the most appropriate test methods and conditions selected accordingly.
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