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Chapter 4 Microbial Diversity.

Microbial diversity’ considers the vast array of microorganisms—the smallest forms of life—
which exist everywhere. The three primary groups of microorganisms are bacteria, archaea, and
eukaryotes. Bacteria and archaea are prokaryotes with their genetic material held in a single
chromosome. In eukaryotes, most of the genome is held in multiple chromosomes. Over 11,000 species
of bacteria have been identified using microscopic identification of cell shape and metabolic activity,
Gram-staining techniques, and genetic identification of RNA and DNA sequences. There are 500 named
species of archaea, divided into two phyla: the euryarchaeota and the crenarchaeota. There are eight
supergroupings of eukaryotes, all of them include single-celled organisms, and five are entirely
microbial.
 Microorganisms are very diverse and are found in all three domains of life: Archaea, Bacteria,
and Eukarya.

 Archaea and bacteria are classified as prokaryotes because they lack a cellular nucleus. Archaea
differ from bacteria in evolutionary history, genetics, metabolic pathways, and cell wall and
membrane composition.

 Archaea inhabit nearly every environment on earth, but no archaea have been identified as
human pathogens.

 Eukaryotes studied in microbiology include algae, protozoa, fungi, and helminths.

 Algae are plant-like organisms that can be either unicellular or multicellular, and derive energy
via photosynthesis.

 Protozoa are unicellular organisms with complex cell structures; most are motile.

 Microscopic fungi include molds and yeasts.

 Helminths are multicellular parasitic worms. They are included in the field of microbiology
because their eggs and larvae are often microscopic.

 Viruses are acellular microorganisms that require a host to reproduce.

 The field of microbiology is extremely broad. Microbiologists typically specialize in one of many
subfields, but all health professionals need a solid foundation in clinical microbiology.

Prokaryotic Microorganisms

Bacteria are found in nearly every


habitat on earth, including within and
on humans. Most bacteria are harmless
or helpful, but some are pathogens,
causing disease in humans and other
animals. Bacteria are prokaryotic
because their genetic material (DNA) is
not housed within a true nucleus.
Bacteria are often described in terms of
their general shape. Common shapes
include spherical (coccus), rod-shaped
(bacillus), or curved (spirillum,
spirochete, or vibrio). Figure 2 shows
examples of these shapes.

Archaea are also unicellular prokaryotic


organisms. Archaea and bacteria have
different evolutionary histories, as well as
significant differences in genetics, metabolic
pathways, and the composition of their cell
walls and membranes. Unlike most bacteria,
archaeal cell walls do not contain
peptidoglycan, but their cell walls are often
composed of a similar substance called
pseudopeptidoglycan. Like bacteria, archaea
are found in nearly every habitat on earth,
even extreme environments that are very cold,
very hot, very basic, or very acidic (Figure 3).
Some archaea live in the human body, but none
have been shown to be human pathogens.

Eukaryotic Microorganisms

The domain Eukarya contains all eukaryotes, including uni- or multicellular eukaryotes such as
protists, fungi, plants, and animals. The major defining characteristic of eukaryotes is that their cells
contain a nucleus.

Protists are unicellular eukaryotes that are not plants, animals, or fungi. Algae and protozoa
arexamples of protists.

Algae (singular: alga) are plant-like protists that can be


either unicellular or multicellular (Figure 4). Their cells
are surrounded by cell walls made of cellulose, a type of
carbohydrate. Algae are photosynthetic organisms that
extract energy from the sun and release oxygen and
carbohydrates into their environment. Because other
organisms can use their waste products for energy,
algae are important parts of many ecosystems. Many
consumer products contain ingredients derived from
algae, such as carrageenan or alginic acid, which are
found in some brands of ice cream, salad dressing,
beverages, lipstick, and toothpaste. Agar, a gel derived
from algae, can be mixed with various nutrients and
used to grow microorganisms in a Petri dish. Algae are
also being developed as a possible source for biofuels.
Protozoa (singular: protozoan) are protists that make up the
backbone of many food webs by providing nutrients for other
organisms. Protozoa are very diverse. Some protozoa move with
help from hair-like structures called cilia or whip-like structures
called flagella. Others extend part of their cell membrane and
cytoplasm to propel themselves forward. These cytoplasmic
extensions are called pseudopods (“false feet”). Some protozoa
are photosynthetic; others feed on organic material. Some are
free-living, whereas others are parasitic, only able to survive by
extracting nutrients from a host organism. Most protozoa are
harmless, but some are pathogens that can cause disease in
animals or humans (Figure 5).

Multicellular parasitic worms called helminths are not


technically microorganisms, as most are large enough to see
without a microscope. However, these worms fall within the
field of microbiology because diseases caused by helminths
involve microscopic eggs and larvae. One example of a helminth
is the guinea worm, or Dracunculus medinensis, which causes
dizziness, vomiting, diarrhea, and painful ulcers on the legs and
feet when the worm works its way out of the skin (Figure 8).
Infection typically occurs after a person drinks water containing
water fleas infected by guinea-worm larvae. In the mid-1980s,
there were an estimated 3.5 million cases of guinea-worm
disease, but the disease has been largely eradicated. In 2014,
there were only 126 cases reported, thanks to the coordinated
efforts of the World Health Organization (WHO) and other
groups committed to improvements in drinking water
sanitation.[

Viruses are acellular microorganisms, which means they are not


composed of cells. Essentially, a virus consists of proteins and
genetic material—either DNA or RNA, but never both—that are
inert outside of a host organism. However, by incorporating
themselves into a host cell, viruses are able to co-opt the host’s
cellular mechanisms to multiply and infect other hosts.

November 10, 2017


News Update: Archaea and the origin of eukaryotes

The Archaea was recognized as a third domain of life 40 years ago. In this Review, Eme et al.
outline a brief history of the changing shape of the tree of life and examine how the recent discovery of
diverse archaeal lineages has changed our understanding of the evolutionary relationships between the
three domains of life and the origin of the eukaryotic cell.

Microbes living in a toxic volcanic lake could hold clues to possible life on Mars

Date:

May 2, 2018

Source:

University of Colorado at Boulder

Summary:

Researchers have discovered microbes living in one of the harshest environments on Earth.
Their findings could guide scientists looking for signs of ancient life on Mars.
Chapter 5 Microbial Structures and Functions

Definition of eukaryotes and prokaryotes

Prokaryotes (pro-KAR-ee-ot-es) (from Old Greek


pro- before + karyon nut or kernel, referring to the cell
nucleus, + suffix -otos, pl. -otes; also spelled "procaryotes")
are organisms without a cell nucleus (= karyon), or any other
membrane-bound organelles. Most are unicellular, but some
prokaryotes are multicellular.

Eukaryotes (IPA: [juːˈkæɹɪɒt]) are organisms whose


cells are organized into complex structures by internal
membranes and a cytoskeleton. The most characteristic
membrane bound structure is the nucleus. This feature gives
them their name, (also spelled "eucaryote,") which comes
from the Greek ευ, meaning good/true, and κάρυον,
meaning nut, referring to the nucleus. Animals, plants, fungi,
and protists are eukaryotes.
Chapter 6 Microbial Growth and Reproduction

Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria. Multiplication
is the consequence of growth. Death of bacteria is the irreversible loss of ability to reproduce.

Factors Required for Bacterial Growth

The requirements for bacterial growth are:

(A)) Environmental factors affecting growth, and

(B) Sources of metabolic energy.

A. Environmental Factors affecting Growth

1. Nutrients. Nutrients in growth media must contain all the elements necessary for the synthesis of
new organisms. In general the following must be provided : (a) Hydrogen donors and acceptors, (b)
Carbon source, (c) Nitrogen source, (d) Minerals : sulphur and phosphorus, (e) Growth factors: amino
acids, purines, pyrimidines; vitamins, (f) Trace elements: Mg, Fe, Mn.

Growth Factors: A growth factor is an organic compound which a cell must contain in order to grow but
which it is unable to synthesize. These substances are essential for the organism and are to be supplied
as nutrients. Thiamine, nicotinic acid, folic acid and para-aminobenzoic acid are examples of growth
factors.

Essential Metabolites: These metabolites are essential for growth of bacterium. These must be
synthesized by the bacterium, or be provided in the medium. Mg, Fe and Mn are essential trace
elements.

Autotrophs live only on inorganic substances, i.e. do not require organic nutrients for growth. They are
not of medical importance.

Heterotrophs require organic materials for growth, e.g. proteins, carbohydrates, lipids as source of
energy. All bacteria of medical importance belong to heterotrophs. Parasites may depend on the host
for certain foods. Saprophytes grow, on dead organic matter.

2. pH of the medium. Most pathogenic bacteria grow best in pH 7.2-7.4. Vibno cholerae can grow in pH
8.2-9.0.

3. Gaseous Requirement

(a) Role of Oxygen. Bacteria may be classified into four groups on oxygen requirement :

(i) Aerobes. They cannot grow without oxygen, e.g. Mycobacterium tuberculosis.
(ii) Facultative anaerobes. These grow under both aerobic and anaerobic conditions. Most bacteria are
facultative anaerobes, e.g. Enterobacteriaceae.

(iii)Anaerobes. They only grow in absence of free oxygen, e.g. Clostridium, Bacteroides.

(iv) Microaerophils grow best in oxygen less than that present in the air, e.g. Campylobacter.

Aerobes and facultative anaerobes have the metabolic systems: (1) cytochrome systems for the
metabolism of oxygen, (2) Superoxide dismutase, (3) catalase.

Anaerobic bacteria do not grow in the presence of oxygen. They do not use oxygen for growth and
metabolism but obtain their energy from fermentation reactions. Anaerobic bacteria are killed by
oxygen or toxic oxygen radicals. Multiple mechanisms play role for oxygen toxicity : (1) They do not have
cytochrome systems for oxygen metabolism, (2) They may have low levels of superoxide dismutase, and
(3) They may or may not have catalase.

(b) Carbon dioxide. All bacteria require CO2 for their growth. Most bacteria produce CO2. N.
gonorrhoeae and N. meningitides and Br abortus grow better in presence of 5 per cent CO2.

4. Temperature. Most bacteria are mesophilic. Mesophilic bacteria grow best at 30-37°C. Optimum
temperature for growth of common pathogenic bacteria is 37°C. Bacteria of a species will not grow but
may remain alive at a maximum and a minimum temperature.

5. Ionic strength and osmotic pressure.

6. Light. Optimum condition for growth is darkness.

B.Sources of Metabolic Energy

Mainly three mechanisms generate metabolic energy. These are fermentation, respiration and
photosynthesis. An organism to grow, at least one of these mechanisms must be used.

REPRODUCTION

Bacteria reproduce by binary fission. Multiplication takes place in geometric progression. The
nucleus (chromosome) undergoes duplication prior to cell division. When the cell grows about twice its
size, the nuclear material divides, and a transverse septum originates from plasma membrane and cell
wall and divides the cell into two parts. The two daughter cells receive an identical set of chromosomes.
The daughter cells separate and may be arranged singly, in pairs, clumps, or chains.
GROWTH CURVE

The growth curve indicates multiplication and death of bacteria. When a bacterium is inoculated in a
medium, it passes through four growth phases which will be evident in a growth curve drawn by plotting
the logarithm of the number of bacteria against time. Number of bacteria in the culture at different
periods may be : (1) Total count. It includes both living and dead bacteria, or (2) Viable count. It includes
only the living bacteria. Microbial concentration can be measured in terms of cell concentration, i.e. the
number of viable cells per unit volume of culture, or of biomass concentration, i.e. dry weight of cells
per unit volume of culture.

Growth Phases
Methods of Measuring Microbial Growth :

There are different methods of counting microbial growth. These are based on different parameters of
cells such as dry-weight and wet-weight measurement, absorbance, cell plate, density, turbidity, ATP
measurement, viable count, ATPase activity and use of Coulter counter.

(a) Wet Weight Measurement:

Measuring cell mass is an easy step of cell growth measurement. A known volume of culture sample
from the ferment or is withdrawn and centrifuged, Wet weight of pellets is measured by using pre-
weighed filter paper. A pre-weighed filter paper of similar size is used to subtract the weight of wet filter
paper. Thus wet-weight of cells is calculated.

(b) Dry Weight Measurement:

Dry weight measurement of cell material is similar to that of wet weight. Here dry weight of pre-
weighed filter paper containing pellets of microbial cells is measured. Dry weight of filter paper is
nullified by subtracting the dry weight of only filter paper of similar size.

Thus dry weight of microbial cells can be obtained. For example dry weight of about one million cells of
E. coli is equal to 150 mg. Dry weight of bacterial cells is usually 10-20% of then- wet weight.

(c) Absorbance:

Absorbance is measured by using a spectrophotometer. Scattering of light increases with increase in cell
number. When light is passed through bacterial cell suspension, light is scattered by the cells.

Therefore, transmission of light declines. At a particular wavelength absorbance of light is proportional


to the cell concentration of bacteria present in the suspension.

Thus cell growth of any bacterial suspension at a particular wavelength at different intervals can be
measured in terms of absorbance and a standard graph (between absorbance and cell concentration)
can be prepared.

(d) Total Cell Count:

Cell growth is also measured by counting total cell number of the microbes present in that sample. Total
cells (both live and dead) of liquid sample are counted by using a special microscope glass slide called
Petroff-Hausser Counting Chamber.

In this chamber a grid is marked on the surface of the glass slide with squares of known area. The whole
grid has 25 large squares, a total area of 1 mm2 and a total volume of 0.02 mm3 (1/50 mm).

All cells are counted in large square and total number per ml sample is measured. If 1 square contains 12
cells, the total number of cells per ml sample will be: 12 cells x 25 square x 50x103 = 1.5xl07 cells.
If there is dilute culture, direct cell counting can be done. However, the cell culture of high density can
be diluted. Otherwise clumps of cells would be formed which would create problem in exact counting of
bacterial cells.

(e) Viable Count:

A viable cell is defined as a cell which is able to divide and increase cell numbers. The normal way to
perform a viable count is to determine the number of cells in the sample which is capable of forming
colonies on a suitable medium.

Here it is assumed that each viable cell will form one colony. Therefore, viable count is often called plate
count or colony count. There are two ways of forming plate count.

(i) Spread Count Method:

A volume of culture (0.1 ml) is spread over the surface of an agar plate by using a sterile glass spreader.
The plate is incubated to develop colonies. Then the number of colonies is counted (Fig. 6.6A).

(ii) Pour Plate Method:

In this method a known volume (0.1-1.0 ml) of the culture is poured into the sterile Petri dishes. Then
melted agar medium is poured and mixed gently. The plate is incubated. Colonies growing on the
surface of agar are counted.

(f) Coulter Counter:

It is an electronic device. The microbial culture is directly used to count cells present in the suspension.

Common Methods of isolation of pure culture

 Isolation of pure culture in liquid media

Pure culture can be cultivated in solid and liquid medium. When a large number of cells of a single
species are required for study, it is necessary to grow them in a liquid medium called broth.

A flask containing the medium is sterilized and few cells taken from a colony are inolculated in the
medium. The transfer of cells from the test tube or petri dish is done under a perfectly aseptic condition
to preclude contamination during inoculation. The flask is incubated at the temperature optimum for
the micro organism for its growth , the flask is mechanically shaken by a shaker for aerating the culture.

 Isolation of pure culture by plating method

Pure culture of microorganisms that form discrete colonies on solid media, e.g., yeasts, most bacteria,
many other microfungi, and unicellular microalgae, may be most commonly obtained by plating
methods such as streak plate method, pour plate method and spread plate method.
1. Streak Plate Method

This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed
culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar
medium. The successive streaks “thin out” the inoculums sufficiently and the microorganisms are
separated from each other. It is usually advisable to streak out a second plate by the same loop/needle
without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of
this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as
bacterial cells are deposited on the agar surface. Because of this dilution gradient, confluent growth
does not take place on that part of the medium where few bacterial cells are deposited.

2. Pour plate Method

This method involves plating of diluted samples mixed with melted agar medium. The main
principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a
thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted
directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-
45°C (agar solidifies below 42°C).

The bacteria and the melted medium are mixed well. The contents of each tube are poured into
separate petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one
finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the
medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked
onto another petri plate to insure purity.
Pour plate method has certain disadvantages as follows:

(i) The picking up of subsurface colonies needs digging them out of the agar medium thus interfering
with other colonies.

(ii) The microbes being isolated must be able to withstand temporary exposure to the 42-45°
temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of
psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is also used for
determining the number of viable bacterial cells present in a culture.

3. Spread Plate Method

In this method the mixed culture of microorganisms is not diluted in the melted agar medium
(unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually,
water or physiological saline. A drop of so diluted liquid from each tube is placed on the centre of an
agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod. The medium is
now incubated. When the colonies develop on the agar medium plates, it is found that there are some
plates in which well-isolated colonies grow. This happens as a result of separation of individual
microorganisms by spreading over the drop of diluted liquid on the medium of the plate.

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