Cultures in an embryonated hen’s egg
Important sites of growth for viruses, chlamydiae (bedsoniae), and rickettsias are the yolk sac and the embryonic membranes of the developing chick embryo. Bacteria are occasionally grown in this way and then examined using compound binocular microscopes.
Cultivation of microbes in cell cultures
Cell (tissue) cultures are composed of animal cells in a suitable substrate in which they multiply and grow. The cells may be supported on a solid or semisolid substrate such as fibrin, agar, or cellulose, or they may be suspended in a liquid. The cells used are taken from mammalian or fowl embryos selected adult tissues, such as the cornea, kidney, and lung, and minced tis¬sue of the cell source is inoculated, Pathogenic agents such as rickettsias (also chlamydiae), viruses that do not grow in lifeless media, and other bacteria multiply with ease when incorporated into cell (tissue) cultures. These can all be observed using a microscope such as a compound binocular microscope or a high-powered microscope (electron microscope).
Antibiotic susceptibility testing
The susceptibility (or resistance) of bacteria to different antibiotics may be determined by suitable laboratory procedures, usually designated as susceptibility tests. Two methods are used: the disk susceptibility test and the tube-dilution method. The first employs disks of filter paper impregnated with precise amounts of different antibiotics. A Petri dish is heavily inoculated with the test bacteria, generally from a pure culture. The disks are dropped onto the freshly inoculated surface, and the plate incubated for 24 hours. Meanwhile, as the bacteria grow, the antibiotic diffuses throughout the culture medium. If the bacteria are susceptible to the antibiotic, a zone of inhibition (no growth) encircles the disk. If the bacteria are unaffected by the drug, that is, resistant, growth will cover the area around the disk. Several antibiotics can be tested on one plate, and the method is simple and rapid. Results correlate well with the effectiveness of clinical treatment.
The United States Food and Drug Administration has set its stamp of approval on the standardized disk susceptibility test as precisely defined step by step in the Federal Register 37(191):20527-20529, September 30, 1972, which is a modification of the well-known Kirby-Bauer method introduced in 1966. In the official test, Mueller-Hinton agar (beef infusion, peptone, starch. and agar at a pH of around 7.4 is used for culture of test microorganisms, since it is low in interfering substances and it supports the growth of most pathogens for which evaluation is needed.
The second method of susceptibility testing uses a tube of culture medium containing both test organisms and a specified amount of a given antibiotic. The tube is incubated and growth quantitated. Since only one drug can be tested at a time, this method is used to determine the minimal inhibitory concentration (MIC), the least amount of antibiotic that completely inhibits the growth of the test bacteria.
With this sort of testing, microbes that can be usually seen under a microscope such as a compound binocular microscope are reported as being susceptible or resistant to a given antibiotic. There are also techniques whereby the amount of certain antibiotics can be determined in the blood. Automated methods of susceptibility testing are available with Maims of speeding up the process to approximately 3 hours while good correlation with the Kirby-Bauer method is maintained.
