||Middle school science, high school biology, introductory undergraduate microbiology|
|Revision Date||May 7, 2008|
The human body represents an enormous and extraordinarily diverse collection of surface habitats and micro-niches potentially available for colonization by biofilm associated micro flora (Denyer et al. 1993). At birth the body is essentially sterile but soon becomes colonized by a variety of microorganisms. With continuous exposure to environmental surfaces and to human contact the biofilms become increasingly complex and gradually approach those typical of adults for a given geographical and perhaps even ethnic population. This accumulation of microbial cells eventually out numbers the human cells in the body by as much as tenfold. Despite this huge population of microbes, much of the body remains sterile in healthy individuals. The blood, and most body fluids, the heart, skeleton and skeletal muscles and the tissues of most organs, brain, liver, kidneys, the lymphatic system and the body cavities remain sterile until exposed to serious illness or injury. Other body regions including the skin, the gastrointestinal tract, the urogenital tract and the oropharynx develop a rich and diverse array of biofilms.
The flora of the mouth is one of the most intensively studied human microbial communities. Over 500 different species of bacteria have been identified in the oral cavity. Microbiologists have concentrated on the microbes that form plaque on the teeth because this flora is responsible for dental caries and periodontal disease (Kolenbrander and Palmer, 2004). Relatively few investigations of the buccal mucosa have been done and those pertain mostly to children (Wilson 2005).
Permission pending, J. Plochocki
The major colonizers of oral squamous epithelial cells are facultatively anaerobic Streptococci of the viridans group, including S. oralis, S. mitis, S. salivarius and S. sanguis. Other organisms present in abundance are Neisseria spp., Haemophilus spp., Veillonella spp., Lactobacilli, Actinomyces spp. and Propionibacterium spp. (Wilson 2005). In healthy individuals, these are all considered normal flora and not associated with any disease process.
Figure 1 shows a squamous epithelial cell covered with a large number of bacterial cells, most which appear to be diplococci. Wilson (2005) reports a typical number of bacteria per cell between 5 and 25 with this low number being due to the high rate of shedding (desquamation) of epithelial cells. The cell pictured, however, is estimated to have more than 1300 adherent bacterial cells.
Subjects appropriate for the microscopic examination of human biofilms on human tissue are not common. Although biofilms exist on many human tissues, most are not easy to observe. Most are either inconvenient or inappropriate to sample in the undergraduate laboratory (gut), they are not amenable to the techniques available in the undergraduate laboratory (teeth), or do not show the biofilm in its natural state (nasal or throat swabs). The examination of squamous epithelial cells with their adherent bacteria circumvents all of these difficulties. The tissue is accessible, easily and safely gathered and shows a representative biofilm in situ.
Students should see large angular cheek epithelial cells some of which will exhibit adherent bacterial cells, either as single cells or as micro colonies.
Students can use this exercise to estimate the difference in cell volume between prokaryotic and eukaryotic cells (see extensions).
It is estimated that prokaryotic cells outnumber eukaryotic cells in the human body by at least 10 to 1. This observational exercise can be used to make that point. Not all squamous epithelial cells will exhibit bacterial cells but in those that do the ratio of prokaryotic to eukaryotic cells can be spectacular as in the figure.
The detailed instructions are given in the student attachment. Briefly, buccal cells are harvested from the cheek with a tongue depressor and are placed into a drop of nigrosine dye on a slide. This drop is then spread with a technique similar to making a blood smear. The slide is not heat fixed. Students search for squamous epithelial cells bearing surface attached bacteria.
Beyond simply observing the bacteria, there are two significant points that students may consider. 1. One is the enormous difference in size between a typical prokaryotic cell and a typical eukaryotic cell. Some may argue that a squamous epithelial cell is not typical but it is within an order of magnitude of the average size for mammalian cells.
|As Necessary||tongue depressors
|As Necessary||1 x 3 inch glass microscope slides
|As Necessary||nigrosine dye
|1||microscope, oil immersion equipped immersion oil