Microbial detection methods

The fact that pathogenic microorganisms can be present in low numbers often hampers their detection. Conventional cnlturing may take several days and underestimate the microbial load. Therefore, more rapid and sensitive microbial detection methods wonld be nsefnl for many applications to complement or replace these traditional cnltnre methods. As SPC is a fast and sensitive tool to detect low numbers of microorganisms, it has been used for the detection and quantification of several important species. 

Some bioreactor systems must be completely protected from microbial contamination, meaning that not a single alien bacterium or virus particle can be allowed to penetrate the system. Reliable and economical systems need to be developed to achieve this level of contamination prevention. Along with the need for prevention is the need to be able to detect contamination at a level of a few microorganisms in a hundred kiloliters of medium. This degree of detection is not yet achievable. Research could vastly improve the crude detection methods that are used today. 

All cell culture-based methodologies are essentially limited when studying whole microbial populations, since the dominant proportion of microbial biomass of soil, rhizosphere and rhizoplane, and other environments cannot be cultured on standard laboratory media (114). To obtain information on the composition and activity of the nonculturable fraction and to aid the study of the culturable fraction, direct detection methods are needed. 

Egawa et al. [103] by using a microbial assay method reported that miconazole was not detected in the blood after human subjects were given 100 mg tablets of miconazole intravaginally for 14 days. Six species of 12 strains of human vaginal Lactobacillus were insensitive to miconazole. 

Application of some kind of sample treatment may have the potential to improve substantially the detection of certain antibacterials in milk by microbial routine methods (59). Treatment, for example, of milk samples with ammonium oxalate solution prior to analysis can lead to lower limits of detection of tetracyclines by both microbial inhibition and microbial receptor assays. This is due to the fact that tetracycline residues tend to form chelates with divalent cations and bind to proteins, which reduce their antibacterial efficacy. However, the oxalate treatment causes splitting of complex and/or protein bonds without increasing the detection limits of other antibacterials commonly used in dairy cows. 

A comparison was made between this approach and the microbial inhibition method (Bacillus stearothermophilus disk assay according to [77]). No significant differences were found for AMO and AMP residues in milk within the reliable detection range of the microbial inhibition assay. The LC method was found more sensitive than the microbial inhibition method for residues lower than 10 yUg/L (78,79). 

The term bioburden refers to the amount of microbial flora that can be detected on an item or surface or in a solution. The microbial recovery method used depends on the type of material being evaluated. Aerobic bioburden counts in parenteral solutions are obtained by conducting the total aerobic count and total yeast and molds count as specified in the USP microbial limits test (<61>) or a equivalent compendial test. Alternatively, a modified membrane filtration technique can be used to allow filtration of larger volumes of solution to assess bioburden recoveries where sample results are expected to contain a negligible number of microbial flora from the overall solution. 

Bioassay is the quantitative method in which the endpoint is an observable effect on a biological system or an organism. The classical approach to microbial detection involves the use of differential metabolic assays (monitored colormetrically) to determine species type in the case of most bacteria, or the use of cell culture and electron microscopy to diagnose viruses and some bacteria that are intracellular parasites. Samples taken from the environment, such as soil and water, and most clinical samples must be cultured in order to obtain sufficient numbers of various cell types for reliable identification. The time required for microbial outgrowth is typically 4-48 hours (or two weeks for certain cases, such as Mycobacterium tuberculosis). Furthermore, bacterial culture suffers from an inherent drawback cells that are viable may not be culturable, because they possess unanticipated nutritional requirements as a result of genetic mutation. 

Japanese workers are at the forefront in the search for physiologically active microbial metabolies. Their detection methods are worthy of note for lack of satisfactory testing methods has long hampered this search. Umezawa and his co-workers used in vitro enzyme inhibition tests to detect fusaric acid, pepstatin, chymostatin, and the leupeptins. Nigrifactin was discovered by Terashima and co-workers by seeking in experimental fermentations, materials having the chemical properties of alkaloids. 

The exquisite dereplication ability of the mammalian immune systems to identify and remove unwanted xenobiotics has been utilized by some natural products chemists. Application of antibody-based detection methods for natural products have thus far been limited to those natural products that are of significant economic interest, either as agricultural contaminants or as potential pharmaceutical constituents. However, the development of more readily available microbially derived antibody fragments (58) and the advent of various methods for increasing their affinity (59) augur well for more widespread use of these technologies for dereplication in the future. Of the numerous reports of the use of this technique of antibody-based detection to identify natural products and related family members, two representative examples follow. 

Pikkemaat MG, Oostra-van Dijk S, Schouten J, Rapahini M, van Egmond HJ, A new microbial screening method for the detection of antimicrobial residues in slaughter animals The Nouws antibiotic test (NAT-screening), Food Control 2007 19 781-789. 

Pikkemaat, M.G., 2009. Microbial screening methods for detection of antibiotic residues in slaughter animals. Anal. Bioanal. Chem. 395, 893—905. 

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