This document discusses the physiology of bacteria and the process of isolating a pure culture of aerobic bacteria. It covers bacteria metabolism and nutrition, including catabolism, anabolism, nutrient requirements, and mechanisms of nutrient transport. It also describes different types of bacteria based on their nutrient sources and how phototrophs and chemotrophs obtain energy. The document concludes by outlining the multi-stage process used to isolate a pure culture of aerobic bacteria, including seeding a sample and investigating cultural properties to obtain an isolated colony.

1. THEME: PHYSIOLOGY OF BACTERIA. TYPE AND MECHANISM OF
BACTERIA NUTRITION. ISOLATION OF PURE CULTURE OF AEROBIC
BACTERIA (FIRST STAGE)
I. STUDENTS’ INDEPENDENT STUDY PROGRAM
1. Bacteria metabolism. Catabolism and anabolism. To give definition of these terms.
2. Common nutrient requirements of bacteria.
3. Nutritional Types of Microorganisms. Classification of bacteria due their sources of
energy and carbon, nitrogen and oxygen.
4. To describe the mechanisms of nutrition: active and passive transport
5. Main growth factors of bacteria.
6. Isolation of the pure culture of aerobic bacteria, common principles and practical value.
7. First stage of the pure culture isolation:
a - main aim of the first stage
b - ways of seeding native material.
1. Metabolism is the total of all chemical reactions occurred in the cell. Metabolism may be
divided into major parts.
In catabolism larger and more complex molecules are broken down into smaller, simpler molecules
with the release of energy. The bacterial cell obtains the energy for biochemical reaction due catabolism
(energy-generating or energy-yielding process).
Anabolism is the synthesis of complex molecules from simpler ones with the input of energy
(energy-requiring process).
2. To obtain energy and construct new cellular components, organisms must have a
supply of raw materials or nutrients. Nutrients are substances used in biosynthesis and energy
production and therefore are required for microbial growth.
Analysis of microbial cell composition shows that over 95% of cell dry weight is made
up of a few major elements: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium,
calcium, magnesium, and iron. These are called macroelements or macronutrients because they
are required by microorganisms in relatively large amounts. The first six (C, O, H, N, S, and P)
are components of carbohydrates, lipids, proteins, and nucleic acids.
All microorganisms require several trace elements (also called microelements or
micronutrients). The trace elements—manganese, zinc, cobalt, molybdenum, nickel, and copper
—are needed by most cells. However, cells require such small amounts that contaminants in
water, glassware, and regular media components often are adequate for growth. Trace elements
are normally a part of enzymes and cofactors.
Elements can be categorized somewhat differently with respect to nutritional requirements.
The major elements (С, О, Н, N, S, P) are needed in gram quantities in a liter of culture medium.
The minor elements (K, Ca, Mg, Fe) often are required in milligram quantities. Trace elements
(Mn, Zn, Co, Mo, Ni, Cu) must be available in microgram amounts.
3. Classification of bacteria due their sources of energy and carbon, nitrogen and
oxygen.
The requirements for carbon, hydrogen, and oxygen often are satisfied together. Carbon is
required for the skeleton or backbone of all organic molecules, and molecules serving as carbon
sources usually also contribute both oxygen and hydrogen atoms. One carbon source for which
this is not true is carbon dioxide (CO2). Some microorganisms, called autotrophs, can use CO2
as their sole or principal source of carbon.
Organisms that use reduced, preformed organic molecules as carbon sources are
heterotrophs {these preformed molecules normally come from other organisms). Most
heterotrophs use organic nutrients as a source of both carbon and energy. This group may be
subdivided in two groups: saprophytes, which use organic compounds from died organisms, and

2. parasites, which use organic material from alive organism. Also parasites are divided in
facultative parasites and obligate ones.
All organisms also require sources of energy, hydrogen, and electrons for growth to take
place. Microorganisms can be grouped into nutritional classes based on how they satisfy these
requirements There are only two sources of energy available to organisms: (1) light energy
trapped during photosynthesis, and (2) the energy derived from oxidizing organic or inorganic
molecules. Phototrophs use light as their energy source; chemotrophs obtain energy from the
oxidation of chemical compounds (either organic or inorganic). Microorganisms also have only
two sources for hydrogen atoms or electrons. Lithotrophs (that is, "rock-eaters") use reduced
inorganic substances as their electron source, whereas organotrophs extract electrons or
hydrogen from organic compounds.
Table 1. Nutritional Diversity Exhibited Physiologycally Different Bacteria
Required Components for Bacterial Growth
Physiologic Type Carbon Sourece Nitrogen Sourcea
Sourceb
Energy Source Hydrogen Source
Heterotrophic
(chemoorganotrophic)
Organic Organic or
inorganic
Oxidation of
organic
compounds
–
Autotrophic
a
chemolithotrophic)
CO2 Inorganic Oxidation of
inorganic
compounds
–
Photosynthetic
Photolithotrophicb
(Bacteria)
CO2 Inorganic Sunlight H2Sor H2
Cyanobacteria CO2 Inorganic Sunlight Photolysis of H2Oc
Photoorganotrophic (Bacteria) CO2 Inorganic Sunlight Organic
compoundsd
a
Common inorganic nitrogen sources are NO3
-
or NH4
+
ions; nitrogen fixers can use N2;
b
Many prototrophs and chemotrophs are nitrogen-fixing organisms;
c
Results in O2 evolution (or oxygenic photosynthesis) as commonly occurs in plants;
d
Organic acids such as formate, acetate, and succinate can serve as hydrigen donors.
4. Since microorganisms often live in nutrient-poor habitats, they must be able to transport
nutrients from dilute solutions into the cell against a concentration gradient. Finally, nutrient
molecules must pass through a selectively permeable plasma membrane that will not permit the
free passage of most substances. In view of the enormous variety of nutrients and the complexity
of the task, it is not surprising that microorganisms make use of several different transport
mechanisms. The most important of these are:
1) passive diffusion; It is the process in which molecules move from a region of higher
concentration to one of lower concentration. Very small molecules such as Н2О, О2, and CO2
often move across membranes by passive diffusion.
2) facilitated diffusion; It is a diffusion, which are need in carrier proteins or permeases,
which are embedded in the plasma membrane. Because a carrier aids the diffusion process, it is
called facilitated diffusion;
Keep in mind that passive and facilitated diffusion do not require of energy. A concentra-
tion gradient spanning the membrane drives the movement of molecules.
3) active transport; It is the transport of solute molecules to higher concentrations, or
against a concentration gradient, with the use of metabolic energy input.
3) group translocation. It is a process in which a molecule is transported into the cell
while being chemically altered.
Note that active transport and group translocation with the use of metabolic energy input.
5. Growth factors are required in small amounts by cells because they fulfill specific roles
in biosynthesis.

3. Growth factors are organized into three categories:
1. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA);
2. Amino acids: required for the synthesis of proteins;
3. Vitamins: needed as coenzymes and functional groups of certain enzymes.
A microorganism requiring the same nutrients as most naturally occurring members of its
species is a prototroph. A microorganism that lacks the ability to synthesize an essential
nutrient due mutation and therefore must obtain it or a precursor from the surroundings is an
auxotroph.
Some vitamins that are frequently required by certain bacteria as growth factors are PABA,
folic acid (B9), biotin, nicotinic acid, pantothenic acid, pyridoxine (B6), riboflavin (B2),
thiamine (B1) etc.
6. Isolation of Microorganisms in Pure Culture
Pure culture is a population of cells arising from a single cell, to characterize an
individual species. In order to study the properties of a given organism, it is necessary to handle
it in pure culture. To do this, a single cell must be isolated from all other cells and cultivated in
such a manner that its collective progeny also remain isolated. Several methods are available.
A. Plating:
a. Spread Plate. A small volume of dilute microbial mixture containing is
transferred to the center of an agar plate and spread evenly over the surface with
a sterile bent-glass rod. The dispersed cells develop into isolated colonies. .
b. Streak Plate. The original suspension can be streaked on an agar plate with a
wire loop. As the streaking continues, fewer and fewer cells are left on the loop,
and finally the loop may deposit single cells on the agar.
c. Pour plate. In the pour-plate method, a suspension of cells is mixed with
melted agar at 50 °C and poured into a Petri dish. When the agar solidifies, the
cells are immobilized in the agar and grow into colonies.
B. Dilution: A much less reliable method is that of extinction dilution. The suspension is
serially diluted and samples of each dilution are plated. If only a few samples of a particular
dilution exhibit growth, it is presumed that some of these cultures started from single cells. This
method is not used unless plating is for some reason impossible. An undesirable feature of this
method is that it can only be used to isolate the predominant type of organism in a mixed
population.
Isolation of a pure culture allows the determination of morphological, cultural,
biochemical, and other properties of the tested microorganism, which in turn makes it
possible to identify it as a species.
Diagrammatically, the stages of isolation and identification of pure cultures of aerobic and
facultative anaerobic bacteria may be presented in the following way.
Isolation and identification of a pure culture
First day (material is seeded to obtain isolate colony)
1. Microscopic examination of the tested material.
2. Streaking of the material tested onto nutrient media (solid, liquid).
Second day (isolation and enrichment of pure culture)
1. Investigation of the cultural properties.
2. Sub-inoculation of colonies onto solid media to enrich for a pure culture.
Third day (identification of pure culture)
1. Checking of the purity of the isolated culture.
2. Investigation of biochemical properties: (a) sugarlytic, (b) proteolytic.
3. Determination of antigenic properties.
4. Study of phagosensitivity, phagotyping, colicinogensitivity, colicinogenotyping,
sensitivity to antibiotics, and other properties.

4. First day. Prepare smears of the tested material and study them under the microscope. Then, using
a spatula or a bacteriological loop, streak the material onto a solid medium in a Petri dish. This ensures
mechanical separation of microorganisms on the surface of the nutrient medium, which allows for their
growth in isolated colonies. In individual cases the material to be studied is streaked onto the liquid
enrichment medium and then transferred to Petri dishes with a solid nutrient medium. Place these dishes
in a 37 0
C incubator for 18-24 hrs.
ІI. Students` practical activities:
1. To prepare the smear from pathologic material (pus or wound exudation). To stain its
using Gram`s method. Study the morphology and tinctorial properties of the
microorganisms. Sketch it..
2. To seed the material using streak plate method. The seed has been done by
bacteriological loop. For obtaining microorganisms in pure culture tested material
inoculate onto surface media in Petri dish by bacteriological loop and after that
streak plating evenly for mechanical divorce. With that purpose in upper part of
Petri dish has been made dense streaking, set free bacteriological loop from
superfluous material. After that are made parallel streaks at the last part of the
agar.
3. The seeded plates are placed in the incubator for a 24 hours.
RESUME: