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.

Another important consideration in observing successfully biological phenomena that are happening within living cells is that the cell line selected for investigation must exhibit the needed morphological and physiological properties to show clearly the concept that you arte interested in. For example, in investigations that focus on mitosis, several cell lines are not suitable for imaging because of the fact that cells undergoing mitosis become spherical and it usually detaches from the substrate. Alternatively, cell lines that stay flat and connected to the substrate throughout mitosis are considered to be far more excellent in producing very fine details of the mitotic spindle in the event of cell division. Rat kangaroo kidney cells can be very useful to studies concerning mitosis. This cell line has a low level of transfection efficiency but it only has a small quantity of chromosomes that are far easier to spot and differentiate under the microscope. Moreover, other kidney lines like that of the pig and the African green monkey stay attached in the occurrence of mitosis and that it is much easier to be transfected. Even if both the cell lines of the pig and the African green monkey contains have more number of chromosomes as compared to the rat kangaroo, yet their high transfection efficiency and ease in imaging make them superior alternative for doing observations of mitosis. Cells that remain flat during cell division, beside from being helpful in the observation of the mitotic spindle, can also show the distribution of other cellular organelles and components such as the mitochondria, cytoskeletal elements, Golgi apparatus, and the endoplasmic reticulum.

A genetic or gene probe is a piece of DNA or RNA that is labeled with certain chemical or radioactive material that will attach itself to a specified gene or it is utilized as a tag in order to detect and extract that gene. This kind of probes has originated on the identification of the unique sequence of the nucleotide within the DNA or RNA of a microorganism. If that unique sequence of the nucleotide is found, it is then extracted and introduced into a cloning vector, and then, it is altered to become an E. coli to generate numerous copies of the probe. The sequence that is in the cloning vector (plasmids) is reextracted and is then marked with a certain radioactive or chemical substance for diagnostic use.

The utilization of gynecology microscopes and inverted tissue-engineering microscopes in the identification of infectious illnesses has been improved further by the advent of techniques that is concerned with gene amplification, such as PCR or polymerase chain reaction. This kind of technique has been very beneficial in the identification of infectious diseases that are caused by microorganisms that are very hard to culture such as HIV virus and those that have been cultured unsuccessfully like the Whipple’s disease bacillus.

In several instances, the cause of a certain infection can be identified by extracting and culturing the microorganisms either in an artificial media or in a living host and viewed through a high-power compound trinocular microscope. Both bacteria and fungi can be cultured using either liquid artificial media (broth), or by solid artificial media (agar). The liquid media gives better sensitivity for the extraction of small quantity of microorganisms, but recognition of diverse cultures growing in the liquid media needs a subculture using a solid media so that colonies can be extracted and be dealt with separately for it to be classified accordingly. And besides, the growth that occurs in a liquid media cannot normally be quantified. The solid media, on the other hand, produces isolated colonies which can later be quantified if needed and can be classified, even if it is less sensitive that the liquid media. Furthermore, several genera and species can be identified based on the morphology that the specific colony is exhibiting.

In several cases, the differential carbohydrate fermentation capabilities of different microorganisms can be taken advantage by including carbohydrates and its matching pH indicator on the medium. That kind of media is called differential media and it is usually used in isolating enteric bacilli. Moreover, the diverse genera of the Enterobacteriaceae could be classified supposedly by the utilization of the color as well as the morphology exhibited by the colony.

The culture media is made selective by way of including substances like antimicrobial agents that inhibit certain flora while still letting specific microorganisms that are resistant to these kinds of inhibitors to grow. An example of this selective culture media is the Thayer-Martin medium that is utilized to extract Neisseria gonorrhoeae, contains a variety of substances such as vancomycin, colistin, trimethoprim-sulfamethoxazole, anisomycin which in turn hinders the growth of Gram-positive bacteria, most Gram-negative bacilli, Proteus species, and fungi respectively. The microorganism, Neisseria species, N. gonorrhoeae and N. meningitidis, are not normally affected by the incorporation of those antimicrobial agents in the medium.

The quality of bacteria in a certain specimen can be utilized to identify the existence of an infection. For instance, there might be a little amount of bacteria (about < 103 colony forming units per ml) from urine specimens that came form healthy and normal women, except for other considerations, these characterizes the indigenous flora found in the urethra and the periurethral region. In contrast, an infection of the bladder or cystitis, and the kidney or pyelone-phritis is typically associated with a number of bacteria of about > 104 colony forming unit per ml. Because of this, urine specimens must always undergo quantitative cultures. A semiquantitative streak method over the agar would suffice for other specimens. However, for quantitative cultures, a certain volume of specimen is placed over the agar surface and the quantity of colonies per ml is predicted. In a semiquantitative culture, an unspecified volume of specimen is spread to the agar and it is then diluted by being spread out from the inoculation site with the use of a sterile bacteriologic loop. The amount of growth that can appeared on the agar is then describe semiquantitatively as few, moderate or many (1+, 2+, or 3+), which relies on hoe far the colonies appear form the inoculum site. A microorganism that has grown as far as all the spread area would be describe as many or 3+.

Chlamydia and other viruses can be cultured into cell culture systems, however, the extraction of a virus needs inoculation into animals such as primates, suckling mice, hamsters, or guinea pigs. Rickettsiae can be extracted but with added difficulty and with some risk for laboratory workers in animals or embryonated eggs, thus, the identification of this kind of infection is usually done serologically. There are some other viruses that cannot be isolated into cell culture systems like hepatitis viruses; hence, there identification relies on the recognition of hepatitis virus antigens or antibodies.

Cell cultures are normally incubated at a temperature range from 35 to 37°C with an atmosphere that consists of air, air added with carbon dioxide by about 3 to 10 percent, reduced oxygen for microaerophilic conditions, or no oxygen at all for anaerobic bacteria, these kinds of specimens are generally inoculated into a variety of general purpose, differential and selective media that are then placed under incubation under both aerobic and anaerobic conditions.

The period of incubation of each culture systems differs with the growth characteristics exhibited by the microorganism in the culture. Most of the aerobic and anaerobic bacteria would exhibit growth overnight, whereas some species of mycobacteria needs as much as 6 to 8 weeks.