Micr 221 - Environmental Microbiology

1. MOST PROBABLE NUMBER

Introduction

Most probable number (MPN) is a method used to estimate the number of viable microorganisms living in a test sample. It is
based upon the application of probability to the number of observed positive growth responses to a series of standard dilutions. It
offers an effective means to estimate the number of microorganims in a test sample without direct count by such techniques as
microscopy. MPN is commonly used in heterogeneous samples such as soil, water, and agricultural products in which exact cell
numbers of individual microorganims may be impossible to determine.

The methodology for MPN uses dilution and incubation of duplicate cultures across many serial dilution intervals. This particular
technique relies on various patterns of positive and negative growth aftter the inocculation of a desirable media in an appropriate
vessel such as a test tube or microwell plates (shown below). The results used to infer an estimate of the population size are
based on a rather complicated mathematical formula developed by Halvorson and Ziegler in 1933; though an MPN table or
calculator which show accurate estimates are commonly used today (Halvorson and Ziegler, 1933).

Materials and Methods

To begin this procedure it is best to know the dilution ratio (such as ten-fold dilutions), the number of replicates at each ratio as
well as the initial dilution volume and inocculation volume.

The example that will be used as to give a clearer understanding on how the MPN method works uses a hypothetical scenario
using a ten-fold dilution series consisitng of a water sample in which one ml of the dilution is inocculated into a separate test tube
with an appropriate medium. Although this is only a hypothetical example MPN can be used for just about any dilution series
depending on the durability of the microorganism in use.
The picture above shows a ten-fold serial dilution after prolonged period of incubation and can be replicated as follows:

1) An original undiluted sample which contains the microorganism(s) from which the dilution series can be made is needed.

2) Using a Beral pipette, alloquot one ml from the undiluted sample to a sanitized test tube containing an appropriate media of an
appropriate volume.

3) Shake or vortex the newly inocculated test tube for at least 10 seconds to be certain that all contents are thoroughly mixed.

4) Using a Beral pipette, alloquot one ml from the well mixed test tube to a sanitized test tube containing the same media and
volume as the first test tube before it was inocculated.

5) After the second test tube has been inocculated, shake or vortex the tube as in step 3.

6) Follow steps 2,3, and 4 for the rest of the tubes until the dilution series is finished.

7) Once the dilution series has been inocculated let the test tubes incubate until growth or no growth is observed (the amount of
time for growth to occur may depend on the organism).

8) Once growth is observed, then the amount of organisms contained in the original sample may be estimated.

The picture above shows that all dilutions up to 10^-3 showed signs of growth but dilutions starting at 10^-4 showed no signs of
growth whatsoever. Given this scenario it is possible to say that there are 1x10^3 organisms per ml in the orignal sample but less
then 1x10^4 organims per ml. To increase the statistical accuracy of MPN more then one test tube can be inocculated per each
dilution. MPN has historically used 3,5, or 10 test tubes per dilution with each tube being assigned a positive (+) or negative (-)
based on any presence of growth (Woomer et al., 1990).
To estimate the number of microorganisms with multiple test tubes growth must be recorded in the order the dilutions were made
(10^-1, 10^-2, 10^-3 etc). Once growth has been recorded for each succeding dilution it is then possible to compare the results
obtained with that of a MPN Table, or enter the results in a MPN Calculator to obtain the desired results ( A sample MPN Table is
shown below).

2. Ribosomal DNA (rDNA) is a DNA sequence that codes for ribosomal RNA. Ribosomes are
assemblies of proteins and rRNA molecules that translate mRNA molecules to produce proteins.

3. FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by biomedical researchers in the early
[1]
1980s that is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. FISH
uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of
sequence complementarity. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the
chromosomes.

4. epifluorescence:

5. This phrase in situ when used in laboratory science such as cell science can mean something intermediate between in
vivo and in vitro. For example, examining a cell within a whole organ intact and under perfusion may be in situ investigation. This
would not be in vivo as the donor is sacrificed by experimentation, but it would not be the same as working with the cell alone (a
common scenario for in vitro experiments).

6. DNA barcoding is a taxonomic method that uses a short genetic marker in an organism's DNA
to identify it as belonging to a particular species.[1] It differs from molecular phylogeny in that the
main goal is not to determine patterns of relationship but to identify an unknown sample in terms
of a preexisting classification.[2] Although barcodes are sometimes used in an effort to identify
unknown species or assess whether species should be combined or separated, [3] the utility of
DNA barcoding for these purposes is subject to debate. [4] The most commonly used barcode
region, for animals, at least, is a segment of approximately 600 base pairs of the mitochondrial
gene cytochrome oxidase I (COI).

Applications include, for example, identifying plant leaves even when flowers or fruit are not
available, identifying insect larvae (which may have fewer diagnostic characters than adults and
are frequently less well-known), identifying the diet of an animal, based on its stomach contents
or faeces[5] and identifying products in commerce (for example, herbal supplements or wood). [2]
7. Fatty acid methyl esters (FAME) are a type of fatty acid ester that can be produced by
an alkali-catalyzed reaction between fats or fatty acids and methanol. The molecules
in biodiesel are primarily FAMEs, usually obtained from vegetable oils by transesterification.

Since every microorganism has its specific FAME profile (microbial fingerprinting), it can be
used as a tool for microbial source tracking (MST). The types and proportions of fatty acids
present in cytoplasm membrane and outer membrance (gram negative) lipids of cells are
major phenotypic traits.

Clinical analysis can determine the lengths, bonds, rings and branches of the FAME. To
perform this analysis, a bacterial culture is taken, and the fatty acids extracted and used to
form methylesters. The volatile derivatives are then introduced into a gas chromatagraph,
and the patterns of the peaks help identify the organism. This is widely used in characterizing
new species of bacteria, and is useful for identifying pathogenic strains.

8. Gas chromatography (GC), is a common type of chromatography used in analytical chemistry for separating and analyzing
compounds that can bevaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or
separating the different components of a mixture (the relative amounts of such components can also be determined). In some
situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds
from a mixture

2
9. Sulfide (systematically named sulfanediide, and sulfide(2)) is an inorganic anion of sulfur with the chemical of formula S .
It contributes no color to sulfide salts. As it is classified as a strong base, even dilute solutions of salts such as sodium sulfide are
corrosive and attack the skin. Sulfide is the simplest sulfur anion.

10. Rhizobia are soil bacteria that fix nitrogen (diazotrophs) after becoming established inside root nodules of legumes
(Fabaceae). Rhizobia require a planthost; they cannot independently fix nitrogen. In general, they are Gram-negative, motile, non-
sporulating rods.

11. An epiphyte is a plant that grows non-parasitically upon another plant (such as a tree), and derives its moisture and nutrients
from the air, rain, and sometimes from debris accumulating around it instead of the structure it is fastened to. An epiphytic
[1]
organism that is not a plant is not called an epiphyte. Epiphytes are usually found in the temperate zone (e.g.,
[2]
many mosses, liverworts, lichens, and algae) or in the tropics (e.g., many ferns, cacti,orchids, and bromeliads). Epiphytes
provide a rich and diverse habitat for other organisms including animals, fungi, bacteria, and myxomycetes ()

12. Pseudomonas syringae is a rod-shaped, Gram-negative bacterium with polar flagella. As a plant pathogen, it can infect a
wide range of species, and exists as over 50 different pathovars, all of which are available to researchers from international
culture collections such as the NCPPB, ICMP, and others. It is unclear whether these pathovars represent a single species.

P. syringae pathogenesis is dependent on effector proteins secreted into the plant cell by the bacterial type III secretion system.
[7]
Nearly 60 different type III effector families encoded by hop genes have been identified in P. syringae. Type III effectors
contribute to pathogenesis chiefly through their role in suppressing plant defense. Owing to early availability of the genome
sequence for three P. syringae strains and the ability of selected strains to cause disease on well-characterized host plants,
including Arabidopsis thaliana, Nicotiana benthemiana, and tomato, P. syringae has come to represent an important model system
for experimental characterization of the molecular dynamics of plant-pathogen interactions.

13. Clathrate hydrates (or gas clathrates, gas hydrates, clathrates, hydrates, etc.) are crystalline water-based solids physically
resembling ice, in which small non-polar molecules (typically gases) or polar molecules with large hydrophobic moieties are
trapped inside "cages" of hydrogen bonded water molecules. In other words, clathrate hydrates are clathrate compounds in which
the host molecule is water and the guest molecule is typically a gas or liquid. Without the support of the trapped molecules,
the lattice structure of hydrate clathrates would collapse into conventional ice crystal structure or liquid water. Most low molecular
weight gases, including O2, H2, N2, CO2, CH4, H2S, Ar, Kr, and Xe, as well as some higher hydrocarbons and freons, will
form hydrates at suitable temperatures and pressures.

13. Lake Vostok (Russian: , Ozero Vostok, lit. "Lake East") is the largest of Antarctica's almost 400
known subglacial lakes. Lake Vostok is located at the southern Pole of Cold, beneath Russia's Vostok Station under the surface of
the central East Antarctic Ice Sheet, which is at 3,488 m (11,444 ft) above mean sea level. The surface of this fresh water lake is
approximately 4,000 m (13,100 ft) under the surface of the ice, which places it at approximately 500 m (1,600 ft) below sea level.

14. Panspermia (Greek: from / (pas/pan) "all" and (sperma) "seed")

is the hypothesis that life exists throughout the Universe, distributed
by meteoroids, asteroids, comets[1][2] and planetoids.[3]

15.
Amensalism refers to a relationship between two different organisms in different species in which one organism is
damaged or destroyed while the other remains unaffected. In competition, a stronger organism may keep a weaker
organism out of a living space or deprive it of food. In antibiosis, one of the organisms is damaged or killed by a chemical
secretion from the other.