Introduction

A major advance in our knowledge of cells came about with the ability to maintain them in continous culture. Prokaryotes have been cultured for a relatively long time, but eukaryotic cultures were first accomplished in the early 1900's (Harrison, 1 Carrel 2), with major advances being made only in the past two or three decades

The procedures employed during this exercise will examine several types of prokaryotic cultures as well as a eukaryotic suspension culture, and establish a culture of embryonic fibroblasts. The prokaryotic cultures and the eukaryotic suspension culture are "established" while the embryonic culture will be a "primary" culture. Chick embryos are used for this latter procedure because of the relative ease of culture, ease of obtaining embryos, and relative simplicity of their nutritional needs. Highly differentiated cells would be more difficult to establish and in some cases not possible at all (given current technology).

Prokaryotes

Prokaryotes are cells without nuclei and are generally considered to be more primitive than eukaryotes (cells with true nuclei). Typically, prokaryotes are easier to culture in a laboratory because most of them have less stringent nutrient requirements. For this lab we will utilize bacterial cultures grown in nutrient agar, an environment in which human pathogenic organisms are extremely unlikely to grow.

Bacteria may be examined by observing the living, unstained microbes in a wet mount (phase contrast or dark field illumination), by observing dead cells stained with dyes under bright field illumination, or by observing cells prepared for electron microscopy. Our procedures will use "fixed" (i.e. dead) cells stained for standard light microscopy.

When cells are grown, they will have specific growth characteristics, depending on the media, the temperature and the strain of cells utilized. For bacteria (and some algae, fungi and other eukaryotic tissue culture) it is possible to measure the growth of cell populations by calculating cell number or mass. With modern equipment and the proper computer software, this can be a completely automated analysis, that would include the specific morphological data as well (size, shape and density of colonies or individual cells).

Since bacteria are small, they are difficult to count through direct visualization, but can be counted if one makes an assumption that each bacterium is capable of forming an individual colony. The mass of a bacterial suspension can be deduced from the optical turbidity of a suspension.

Eukaryotes

By contrast to the simple broth cultures of E. coli, the nutrient requirements of even the simplest eukaryotic culture are complex. Refer to Table 12.1 for a comparison of the ingredients of Nutrient Broth and Minimum Essential Media (MEM), a typical eukaryotic media. Eukaryotes also require supplemental sources of materials, most often in the form of blood serum. Fetal calf serum is used extensively for this purpose, since it is readily available, and the fetal nature of the serum limits the presence of antibodies, which might negatively effect cell growth.

Our first procedure will involve the simple transfer of an established culture from a suspension culture. An aliquot will be removed from a commercially available cell line and transferred to prepared transfer vessels. Each day students will observe these transfer cultures with an inverted phase contrast microscope, and remove aliquots for cell counting with a hemacytometer. Simultaneously, they will check on the viability of the cells through a dye exclusion technique.

The second procedure will be somewhat more complicated. Students will remove chicken embryos from the egg, trypsinize them to disaggregate the cells, and transfer the resulting cells to culture flasks. This procedure establishes a primary culture, or one that is a first generation growth from "in vivo" cells. Established culture lines are the result of long term selection for cells capable of growth under "in vitro" conditions. As such, they are more consistent clones, but often have genetic and structural alterations that differ significantly from the starting cell lines.

Table 12.1 Eukaryote and prokaryote culture media

As the cells grow in culture, we can observe three distinct phases. The first is a Lag Phase, usually no more than 1-2 days in length, and during which there is little or no increase in cell number. During this time, the cells are "conditioning" the media, undergoing internal cytoskeletal and enzyme changes and adjusting to the new media.

This is followed by a Log Phase. During this phase the cell number increases exponentially. This growth will continue as long as there is sufficient nutrient to support the increasing cell number. Eventually some critical nutrient will become limiting, however.

The final phase is the Plateau Phase. During this phase the number of cells remains constant (although not necessarily viable). Eventually, of course, the cells will die unless subcultured or fresh media is added. The final procedure in this laboratory exercise involves establishment of a primary culture from a chick embryo. Here, the cells are not established in culture, but must be disaggregated (detached from each other) and placed into a foreign environment (the culture media). Disaggregation in embryos is reasonably easy, but depends on the enzymatic dissolution of the cells glycocalyx, and the disruption of many plasma membrane structures and chemical elements. Consequently, the cells will take a longer time before they grow (that is, there is a long Lag Phase), and the selection process will favor cells which grow in contact with the culture vessel surface.

The cells will continue to grow in contact with the vessel and give rise to a "monolayer" culture. The cells will cease to divide when they reach confluency; they are said to demonstrate contact inhibition.

Consequently, growth curves are not measured by removing aliquots and determining cell concentration, but are measured by the density of cells growing on the vessel surface. This is accomplished through the use of an ocular grid inserted into an inverted phase contrast microscope. Cell density (cells/cm2) is then plotted on a log scale against time in culture.