This approach consists
of measuring the number of viable cells capable of forming colonies on a
suitable growth medium. Plate count is determined by using the pour plate
method (0.1–1 mL of microbial suspension is mixed with molten agar medium in a
petri dish), or the spread plate method (0.1 mL of bacterial suspension is
spread on the surface of an agar plate). The results of plate counts are
expressed as colony
forming units (CFU).
The number of CFU per plate should be between 30 and 300. Membrane filters can
also be used to determine microbial numbers in dilute samples. The sample is
filtered and the filter is placed directly on a suitable growth medium.
Friday, February 28, 2014
Measurement of the Number of Viable Microbes on Solid Growth Media
Thursday, February 27, 2014
Total Number of Microbial Cells
Total
number of cells (live and dead cells) can be measured by using special counting
chambers such as the Petroff–Hauser chamber for bacterial counts or the
Sedgewick–Rafter chamber for algal counts. The use of a phase-contrast
microscope is required when nonphotosynthetic microorganisms are under
consideration. Presently, the most popular method consists of retaining the cells
on a membrane filter treated to suppress autofluorescence (use of polycarbonate filters
treated with Irgalan Black) and staining the cells with fluorochromes such as
acridine orange (AO) or 40,6-diamidino-2-phenylindol
(DAPI). The microorganisms are subsequently counted using an epifluorescence
microscope (Kepner and Pratt, 1994).
An
advantage of DAPI is its stable fluorescence. A wide range of other
fluorochromes are available for many applications in environmental microbiology
studies. These include, among others, PicoGreen, SYBR-Green 1 and 2, Hoechst
33342, YOYO-1, and SYTO dyes (green, red, and blue) (Neu and Lawrence, 2002).
Scanning
electron microscopy (SEM) has also been considered for measuring total microbial
numbers. Electronic particle counters are also used for determining the total
number of microorganisms in a sample. These instruments do not differentiate,
however, between live and dead microorganisms, and very small cells may be
missed. Flow cytometers are fluorescence-activated cell sorters and include a
light source (argon laser or a mercury lamp) and a photodetector, which
measures fluorescence (use correct excitation wavelength) and scattering of the
cells. They sort and collect cells with predefined optical parameters. They are
often used in the biomedical and aquatic microbiology fields (Paul, 1993). They
have been used to sort algal cells and to distinguish between cyanobacteria
from other algae, based on phycoerythrin (orange) and chlorophyll (red)
fluorescence. They can help identify microorganisms when combined with
fluorescent antibodies.
Wednesday, February 26, 2014
Plasmids
A plasmid
is a circular extrachromosomal circular DNA containing from 1000 to 200,000
bp, and reproducing independent from the chromosomal DNA. Plasmids are inherited
by daughter cells after cell division. Plasmid replication can be inhibited by curing
the cells with compounds such as ethidium bromide. Some of the
plasmids may exist in a limited number (1–3) of copies (stringent plasmids) or
relatively large number (10–220) of copies (relaxed plasmids).
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