Microbiologic examination may include the following steps or techniques. First of all is the direct examination of the specimen to find out its gross pathology. Direct examination may include microscopy, which may help in identifying the microorganism. Specific microbial antigen can be revealed through procedures like staining the specimen with immuno-peroxidase, immunofluorescence, or with other immunoassays. On the other hand, the DNA or RNA sequences that are genus or species specific can be recognized through the use of genetic probes.

Another technique in microbiologic examination is by doing culture. This procedure requires specialized media for the specimen to grow. A nonselective (noninhibitory) media lets a wide variety of microorganisms to grow, while a selective media permits only selected microorganisms because of the inhibitory substances that it contains. A preliminary microbial identification can be done by the utilization of the morphology of the colony or of the cellular component. It is also necessary in microbial identification the observation of the growth patterns of the specimen under a variety of conditions and other important processes inside the cells.

A serodiagnosis or immunodiagnosis is done to diagnose a certain disease on the basis of the interaction between the antigens and antibodies present on the blood serum. Moreover, an antimicrobial susceptibility test is done to find out if the microorganism is vulnerable to certain antimicrobial agents.

Specimen Selection, Collection and Processing

In microbiologic examinations, the selected specimen must be relevant to the disease that is being studied. The amount of the collected specimen should be sufficient to permit a thorough and complete microbiologic examination. The quantity of microorganisms that is present in a certain measure of the specimen is highly variable. Swabs is one of the very common method in specimen collection, however it usually produces minute specimen to allow a precise microbiologic examination. Thus, it must be only utilized in collecting samples coming from the mucous membranes and the skin.

Both the skin and the mucous membrane are a home to a huge and wide variety of indigenous flora, thus all efforts must be done to eradicate or lessen the contamination of the specimen on the collection process. Many approaches can be applied to avoid contamination. One of this is the disinfection of the skin before aspirating or incising a lesion. Or maybe, the contaminated area can be avoided in taking samples. One example of that procedure is a transtracheal puncture and aspiration of lower respiratory secretions that is usually utilized to identify mycobacterium tuberculosis in patients. It is really impossible to have a specimen that is free of contamination, thus a decontamination technique must be used such as a cultures that has selective media or quantitative media.

Those specimens obtained though the invasive techniques, such as intraoperatively, needs special attention. The quantity of the tissue collected for histopathologic and microbiologic examination must be enough to conduct the following examinations thoroughly. Histopathologic examination is necessary to differentiate between neoplastic lesions as to that of inflammatory lesions and also those acute inflammations from the chronic ones. Histopathologic examination is fundamental in the diagnosis of a certain disease and it can also guide the microbiologic microscopy that should be done on the specimen. One example of this is, if in the histopathologic examination, a caseous granuloma is discovered, then the microbiologic examination that should be done of\n the specimen must include cultures for mycobacteria and fungi. Moreover, the surgeon should also take numerous samples that came from a single big lesion or from many smaller lesions that will be used in microbiologic examinations. In the event that there is an abscess present, the surgeon must take samples of pus in milliliters and also of the abscess wall for the same purpose of microbiologic examination. It should be remembered, however, that the swabs should always be kept out of the operating room.

Moreover, samples should be taken before dispensing antibiotics only if possible. Furthermore, an open line of communication between the clinician and the microbiologist should be established to make sure that the proper specimens are collected and that they are properly examined for the effective and efficient microbiologic examination of the specimen that will result to an accurate and precise diagnosis of a disease.

The growth of microorganisms inside cultures can be identified though the manifestation of turbidity, the formation of gas, or the distinct colonies in broth or agar that can be seen by the naked eye, the cytopathic effects of certain microorganism in the culture, or perhaps by discovering certain antigen or nucleotide in samples, the culture medium or he culture system itself, which is exclusive only to a certain genus or species.

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When putting up a laboratory, many considerations should be addressed so that you can have a more precise and conclusive study. If you are looking for a room to be used in a live-cell imaging experiments, the first factor to look for is adequate ventilation. Ventilation is very essential in this kind of experiments since the mercury and xenon arc-discharge lamps used in this kind of experiments produces ozone, as well as the fumes that is generated by the organic solvent that is utilized in cleaning the optical surfaces and also in disinfecting the microscope stage. Enough space should be reserved about microscope equipments for proper ventilation and also for cleaning and disinfecting the floors, tabletops, benches, and other experiment apparatus. Some of the failure of equipments can be attributed to air intakes that have been clogged, that are too close to the floor and are out of reach. A live-cell laboratory needs to be carefully cleaned and disinfected, and it should be kept in order to minimize the levels of factors such as smoke, dust and other vapors that can decrease the efficiency of the performance of optical and electronic apparatuses. The microscope stage and the adjacent area should be occasionally wiped with a 70 percent ethanol and other ways to disinfect it and to decrease the occurrence of live-cell culture contamination by microorganisms. Culture media spills are a fact of live when doing live-cell experiments, they are unavoidable. If this thing occurs, the area should be cleaned right away and the adjacent area be disinfected thoroughly.

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Choosing a cell line that will be utilized for live-cell imaging experiments is determined upon by a number of factors. Included in these factors are the proposed biological observations that will be undertaken in the conduct of the investigation and the ability of the cell line to be marked with synthetic fluorophores. Moreover, the transfection efficiency or the efficiency of the cell line to absorb nucleic and protein material without the use of viral means and the tolerance of that certain cell line to the rigorous conditions presented by the culture chamber and the illumination used in the imaging procedure. If a certain cell line shows outstanding properties in one or more of the categories presented, it is expected to either perform marginally or completely fail in another. The BPAE line or the normal bovine pulmonary artery endothelial cells, for example, could be stained using synthetic fluorophores that is designed for live cell imaging, which in turn reveals complex details within he cellular structure with great clarity. However, that particular line has very low transfection efficiency, only about 5 percent, and it is quite vulnerable to long-term illumination at low light levels, in which other cell lines can withstand. On the other hand, RK-13 or rabbit kidney epithelial cells has a high transfection efficiency with a myriad of plasmids and it is invulnerable to high illumination levels, such as laser light, throughout time-lapse sequences that could last for days. Yet, this kind of cell line cannot be sufficiently stained with a lot of common synthetic fluorophores.

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Direct examination with the help of a phase-contrast or epi-fluorescence microscope of the specimens, frequently provides the most rapid indication of microbial infection. In addition to that, the combination of the latest techniques in immunologic, microscopy and hybridization has been very essential for a fast diagnosis of a certain disease or illness.

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Every era in medicine has been driven by one particular discipline which recognized an exciting new development occurring outside its own sphere as an opportunity for a quantum leap. Although this initial phase of integrating an unfamiliar dimension into a traditional medical dominion was seldom blessed with clinical success, retrospectively this era is always perceived as the grand epoch of the particular discipline. For instance, the integration of technical achievements in optics and microscopy field into a discipline like surgery, previously considered to be more a skillful art than anything else, made cardiac surgery possible, thus we have what is called as “microsurgery.” (more…)

In studies of cell transfer/cell traffic, experimental design and techniques are of paramount importance in drawing valid conclusions, because it is a low frequency event and occurs frequently in healthy individuals. Interpretation of studies employing gynecology microscopes (specifically, inverted tissue culture microscopes and embryo-transplant microscopes) for detection of microchimeric DNA requires knowledge of the sensitivity and specificity of the particular assay employed in addition to specific methodological information such as the number of DNA aliquots tested and number of times testing was conducted. Gynecology microscopes (specifically, inverted tissue culture microscopes and embryo-transplant microscopes) may be compromised by cross-reactivity of some Y-chromosome sequences with autosomal sequences yielding false-positive results during microscopy and live-cell observation. Nested gynecology microscopy techniques are nonquantitative and subject to greater risk of contamination than closed gynecology microscopes (specifically, inverted tissue culture microscopes and embryo-transplant microscopes) systems. Despite good sensitivity of assays targeting Y-chromosome multi-copy sequences, some Y-sequences vary in copy number between individual men so that only multi-copy Y-chromosome sequences that have a stable copy number between men should be used for quantitative purposes. (more…)