Chapter 9
Microscopic Techniques
Timberley M. Roane, Ian L. Pepper, and Raina M. Maier
1. List four possible applications in environmental microbiology for each of the following
microscopic techniques: bright-field, fluorescence, electron, and in situ microscopy.
Bright-field microscopy can be used to observe the morphological diversities of microorganisms
in a sample. For example, bright-field microscopy is used to visualize the microorganisms
growing on a glass surface as in the buried slide technique. Often used in combination with
stains, bright-field microscopy is used to observe the general characteristics of an organism(s),
e.g., gram reaction, presence of spores or flagella.
Fluorescence microscopy is commonly used to enumerate the number of microorganisms in a
soil sample. For example, once the microorganisms are extracted from a soil, they are stained
with a fluorescent stain, such as acridine orange, for visualization with a fluorescent microscope.
Statistically significant number of organisms are determined through direct counting with the
fluorescence microscope.
There are two types of electron microscopies: transmission and scanning. Transmission electron
microscopy is used to determine internal cellular structure, for example, cellular accumulations
of heavy metals or in evaluating the effects of chemical exposure to cell wall integrity. Scanning
electron microscopy produces a 3-D image of cell interactions with each other and
environmental surfaces. In analyzing the structure of biofilms, scanning electron microscopy
provides information on the location, thickness and architectural complexities of biofilms.
In situ microscopy is based in fluorescence microscopy. As with the labeling of specific nucleic
acid sequences in fluorescent in situ hybridization, in situ microscopy allows for the concurrent
identification and visualization of cells in their environment. For example, in situ microscopy
can be used to determine bacterial diversity on a plant root or in a soil sample.
2. Direct microscopic counts using acridine orange (AODC) are often two or three orders
of magnitude greater than viable counts. Why?
Direct counts are greater than viable counts because of the presence of viable, dead, viable but
nonculturable (VBNC), and viable but difficult to culture (VBDC) organisms in a sample. Direct
counts with the fluorescence microscope does not distinguish amongst these groups, while viable
counts are a reflection of only those organisms easily grown under the conditions used. For
example, the use of nutrient agar, a non-selective heterotrophic bacterial growth medium, will
grow many types of organisms; however, if the plates are incubated aerobically then only
aerobic, heterotrophic organisms will grow. The numbers reflected on the plates will not include
fastidious, autotrophic, or anaerobic organisms, while direct microscopic counts will account for
all microorganisms in a sample.
3. If you wanted to determine whether or not a specific membrane protein was being
produced by a microorganism, which microscopic techniques might you use and why?
Atomic force microscopy allows for determination of surface topography, thus allowing for
visualization of membrane-associated structures. When performed in comparison with a control
organism, determination of the presence or absence of a particular protein could be performed.
Fluorescent immunolabeling with an antibody designed for the protein of interest will mark those
cells containing the protein in their membrane. Fluorescence microscopy can be used to
visualize the cells.
4. List at least four things about a microorganism you can learn from viewing it with
bright-field microscopy.
You can determine the (i) morphology, (ii) gram reaction (gram positive or gram negative), (iii)
cell size, and (iv) motility. Others include spore formation, presence/absence of chloroplasts,
etc.
5. Which is most important in microscopy: resolution, contrast, or magnification? Why?
Contrast is the most important factor in microscopy as contrast is the ability to see an object.
Without contrast, resolution, the ability to see two points on a specimen as two, and
magnification, the ability to increase the size of an image, cannot be visualized.
6. List two procaryotic structures that would stain in response to an acidic dye. List two
that would stain in response to a basic dye.
Acidic dyes have a negative charge and will be attracted to positively charged proteins on the
cell surface, for example, some transport proteins for nutrient uptake and flagellar-associated
proteins in motility. Basic dyes have a positive charge and will be attracted to negatively
charged components in the cell, such as nucleic acids and lipopolysaccharide on the cell surface.