MIC 311 LECTURES

Thur: 11.40 – 12.25

Fri: 9.50 – 10.30

KEY RULES

1. ATTENDANCE AT LECTURES AND PRACTICAL COMPULSORY

2. CELL PHONES MUST BE SWITCHED OFF DURING LECTURES AND

PRACTICALS

3. PUNCTUALITY IN CLASSES IS NON NEGOTIABLE. LECTURES WILL

START 5 MIN LATE AND ONCE STARTED THE GATE CLOSES. DO
NOT ENTER THE CLASSROOM WHEN THE GATE IS CLOSED.

4. CLASS CAPTAIN WILL BE LIASON PERSON.

 CHAPTER 1

CELL STRUCTURE AND FUNCTIONS

1.1 Introduction

Living and Nonliving Things are composed of molecules made from chemical elements
such as carbon, hydrogen, oxygen, and nitrogen.

These molecules are organized into cells in living things thus distinguishing them from
non-living things.

The CELL is the smallest unit of life because it retains the properties of life such as
metabolism, reproduce-DNA, response to its environment.

1.2 Discovery of the cell and origin of the cell theory

1. Before the seventeenth century, no one knew that cells existed.

2. Most cells are too small to be seen with the unaided eye. Therefore, they were not
discovered until after the invention of the microscope in the early seventeenth century.

3. One of the First Microscopes was made by Anton von Leewenhoek. With his hand-
held microscope, Leewenhoek became the FIRST person to observe and describe
microscopic organisms and living cells.

4. In 1665, the English Scientist Robert Hooke used a microscope to examine a thin slice
of cork and originated the term “Cell”.

5. In 1820, Robert Brown founded the “Nucleus” in the cell.

6. In 1838, German Botanist Matthias Schleiden studied a variety of plants and

concluded that all plants "Are composed of cells".

7. The next year, German Zoologist Theodor Schwann reported that animals are also
made of cells and proposed a cellular basis for all life.

8. In 1855, German Physician Rudolf Virchow induced that "Every cell only comes
from already existing cells".

9. The COMBINE Work of Schleiden, Schwann, and Virchow make up what is now
known as the modern Cell theory.
The Cell Theory consists of three principles:
1. All living things are composed of one or more cells.
2. Cells are the smallest units of life (metabolism, DNA, response to its environments).
3. New cells come only from reproduction of existing cells.
1.3 Main structural organization of cell and their function

Plasma membrane – separate interior and anterior of cell, made up of two layers of
lipids (bilayer) with diverse proteins embedded of which roles are pumping substances
across membrane and play as a receptor to trigger changes in cell activities.

Nucleus – localizing DNA

Cytoplasm – everything between the cell membrane and the nucleus; ribosome and other
components (organelles).

1.3.1 CELL SIZE

Read up.
Assignment 1: Cells exist in different sizes. Discus in relation to their functions.

1.3.2 CELL SHAPE

1. Cells come in a variety of shapes.

2. This diversity of form reflects a diversity of function. For example, cells of the nervous
system are threadlike. And, blood cells are shaped like round disk that can squeeze
through tiny blood vessels.
Microscope – major contribution for modern biology

Light microscopes – different colors of light have different wavelengths (the size of
object can be observed as small as 200nm).

Electron microscopes – using electron particles to improve resolution of light microscope

(down to 0.5nm).

Scanning tunneling microscopes - using electric needle with a point detecting electron
tunneling (the density of the electron clouds) on the surface of specimen (mainly
molecules) - magnify 100 million times the real size of a object; can be used with living
things.

Eukaryotic cells

Features of eukaryotic cells

- Organelle: an internal, membrane-bound compartment that serves specialized

functions inside eukaryotic cells.

* Benefits of the compartmentalization

(1) allows many activities to occur simultaneously in very limited space; and
(2) allows compatible and interconnected reactions to proceed at different times.

- Major organelles and their function;

 Nucleus – localizing DNA

Endoplasmic reticulum (ER) – routing & modifying newly formed polypeptide

chains; synthesizing lipid.

Golgi bodies – peptide chains into proteins; sorting & shipping proteins & lipids

Mitochondria – efficiently producing ATP during aerobic respiration.

Various vesicles – transporting, storing, or digesting a variety of substances; other

Functions.

- Nonmembranous structures and their function;

Ribosomes - the site of protein synthesis (production or construction), most numerous

structures in almost all cells

Cytoskeleton – maintaining the cell structure, organizing and moving organelles.

* Comparisons of organelles between typical plant and animal cells

With regards to

Only plants – chloroplast, central vacuole, cell wall

Only animals – centrioles, lysosome,

1.4.2 Major Organelles in details

A. The Nucleus (plural, Nuclei)

- The Nucleus is the control center (like brain) of the cell and the most prominent
structure within a eukaryotic cell.

• Structure of nucleus:

Nuclear envelope – double membrane (two lipid bilayers) with many small pores
through which proteins and chemical mesages from the nucleus can pass. Golf
ball like dimples (pores).

Nucleoplasm – fluid interior portion of the nucleus.

Nucleolus – dense cluster of RNA and proteins that makes (synthesizes) subunits
of ribosomes. When a cell prepares to reproduce, the nucleolus disappears!

Chromosome – one DNA molecule and many proteins that are intimately
associated with it. Particularly during cell division, it appears.

Chromatin – total collection of all DNA molecules and their associated proteins

• Function of nucleus:
1. Separates and localizes all DNAs in a sac which makes easier for parent cell to
copy and sort out hereditary instructions before cell divides.
2. The nuclear membranes (pores on the nuclear envelope) help control the movement of
substances (proteins) and signals between the nucleus and the cytoplasm.

B. Endoplasmic Reticulum (ER)

- ER is an extensive network of membranes that connect the nuclear envelope and
extends through the cytoplasm. The ER is a system of membranous tubules and sacs.
- Two different types of ER:
a) Rough ER - studded with ribosomes and processes proteins to be exported from the
cell.
b) Smooth ER - free of ribosomes and curves through cytoplasm. Lipids are assembled.
The Smooth ER is involved in the synthesis of steroids in gland cells, the regulation of
calcium levels in muscle cells, and the breakdown of toxic substances by liver cells.

C. Golgi Bodies:
- The Golgi bodies are the processing, sorting of proteins and lipids and then packaging
and shipping them to specific location by forming vesicles.
- The Golgi body is a series of flattened, membrane bound sacs like a stack of pancakes.
- It works closely with the ER.

D. Other Vescicles:
Lysosomes:
- small spherical organelles that enclose hydrolytic enzymes within a single membrane
and the site of food digestion in the cell.
- formed from pieces of the golgi bodies that break off.
- common in the animal, fungi, and protist cells, but rare in plant cells.
Peroxisomes:
– Sacs of enzymes that break down fatty acids and amino acids. Detoxify the
product of reaction ‘hydrogen peroxide (H2O2)’ and other toxins.

E. Mitochondria
- found only in the eukaryotic cell and surrounded by two membranes. The smooth outer
membrane serves as a boundary between the mitochondria and the cytosol. The inner
membrane has many long folds, known as cristae, which greatly increases the surface
area of the inner membrane, providing more space for the chemical reactions to occur.
- the site of the most efficiently formed ATP (requires O2).
Therefore, mitochondria are usually more numerous in cells that have a high energy
requirement - muscle cells contain a large number of mitochondria.
- Mitochondria have their own DNA, and new mitochondria arise only when existing
ones grow and divide (may be evolved through ‘endosymbiosis’).

F. Specialized Plant Organelles

Includes three additional structures not found in animals cells - cell walls, vacuoles,
plastids that are extremely important to plant function.

(1) Plastids – specialize in photosynthesis or storage.

Three different types;

i. Chloroplasts – the main function is to convert sunlight, CO 2, and water into sugar
(called photosynthesis). These are composed of thylakoid membranes – disklike shape
and interconnected, produce ATP and NADPH (from sunlight); stroma – semifluid inner
membrane, produce sugar and starch using ATP and NADPH (from CO 2 and water);
granum - a stack of thylakoid membranes. Chloroplasts are green because they contain
a pigment called ‘chlorophyll’.

ii. Chromoplasts – store reddish-orange pigments ‘carotenoids’ that color fruits,

vegetables, flowers, and autumn leaves.

iii. Amyloplasts – lack pigments, often store starch, abundant in cells of stems, and seeds.

Central Vacuole – a large membrane-bound sac that takes up a large amount of space in
most Plant Cells and a storage for amino acids, sugars, ions, and toxin wastes.

II. Nonmembranous Structures

A. Ribosomes – tiny round particles (most are attached on rough ER). They are involved
in synthesizing proteins .

B. Cytoskeleton – Read up
1.4.3 How organelles and cells move?

A. Inside cell – Organelles (or cell compartments) can move along microtubules or
microfilaments using motor proteins (kinesin and dynein – microtubules, myosin –
microfilaments).

B. Outside cell - Beside moving internal compartment, many cells move through
environment using motile organelles (cilia, flagella) or false feet (pseudopods).

Cilia & Flagella – short hair (cilia) or long whip (flagella) like organelles that extend
from the surface of the cell, the bundles microtubules into structures.

Pseudopods – temporal and irregular lobes (elongated microfilaments with motor

proteins), used for locomotion and prey capture. Amoebas and macrophages form
pseudopods.

1.4.4 CELL WALL

- a structural component that wraps continuously around the plasma.

- Present in plant cells as well as fungi.
- The rigidity of cell walls helps support and project the plant.

Two types of cell walls:

1. primary cell wall

- just outside the cell membrane.
- When the cell reaches full size, a secondary cell wall may form.

2. secondary cell wall - forms between the primary cell wall and the plasma membrane.

- It is tough and woody

- Once 2o cell wall is formed plant cell can grow no further. The cells are dead.
- Cartilage and bone tissues in animals do similar role as cell wall in plants.

In multicelled organisms, diverse proteins at the plasma membrane function as physical

links, cytoplasmic bridges, and receptors for communication signals between cells.
1.5 Prokaryotic cells

Prokaryotes are unicellular organisms of relatively simple construction, especially if

compared to eukaryotes. Whereas eukaryotic cells have a preponderance of organelles
with separate cellular functions, prokaryotes carry out all cellular functions as individual
units.

A prokaryotic cell has five essential structural components:

 a genome (DNA),
 ribosomes,
 cell membrane,
 cell wall,
 and some sort of surface layer which may or may not be an inherent part of
the wall.

Diversity within the primary structure of these molecules accounts for the diversity that
exists among prokaryotes.

Structurally, a prokaryotic cell (Figure 1 below) has three architectural regions:

- appendages (attachments to the cell surface) in the form of flagella and pili (or
fimbriae);

- a cell envelope consisting of a capsule, cell wall and plasma membrane;

- and a cytoplasmic region that contains the cell genome (DNA) and ribosomes and
various sorts of inclusions.
 A typical prokaryotic cell.

Table 1. Summary: Characteristics of typical bacterial cell structures.

Predominant chemical
Structure Function(s)
composition
Flagella Swimming movement
Protein
Pili
Sex pilus Mediates DNA transfer during conjugation Protein
Common pili or Attachment to surfaces; protection against
Protein
fimbriae phagotrophic engulfment
Attachment to surfaces; protection against
Capsules (includes
phagocytic engulfment, occasionally killing Usually polysaccharide;
"slime layers" and
or digestion; reserve of nutrients or occasionally polypeptide
glycocalyx)
protection against desiccation
Cell wall
Gram-positive Prevents osmotic lysis of cell protoplast Peptidoglycan (murein)
bacteria and confers rigidity and shape on cells complexed with teichoic acids
Peptidoglycan prevents osmotic lysis and Peptidoglycan (murein)
Gram-negative confers rigidity and shape; outer membrane surrounded by phospholipid
bacteria is permeability barrier; associated LPS and protein-lipopolysaccharide
proteins have various functions "outer membrane"
Plasma membrane Permeability barrier; transport of solutes; Phospholipid and protein
 energy generation; location of numerous
enzyme systems
Ribosomes Sites of translation (protein synthesis) RNA and protein
Often reserves of nutrients; additional Highly variable; carbohydrate,
Inclusions
specialized functions lipid, protein or inorganic
Chromosome Genetic material of cell DNA
Plasmid Extrachromosomal genetic material DNA

1.5.1 Appendages

Figure 3. Salmonella enteritidis TEM about 10,000X. Salmonella is an enteric bacterium related
to E. coli. The enterics are motile by means of peritriochous flagella.

1.5.1.1 Flagella

Flagella are filamentous protein structures attached to the cell surface that provide the
swimming movement for most motile procaryotes. Prokaryotic flagella are much thinner
than eukaryotic flagella.

Read up details

Flagella may be variously distributed over the surface of bacterial cells in distinguishing
patterns, but basically flagella are either polar (one or more flagella arising from one or
both poles of the cell) or peritrichous (lateral flagella distributed over the entire cell
surface).

The arrangements of flagella in a cell are referred to as flagellation.

Flagella enables procaryotes to exhibit a variety of types of tactic behavior, i.e., the
ability to move (swim) in response to environmental stimuli. E.g

Chemotaxis a bacterium can sense the quality and quantity of certain chemicals in its
environment and swim towards them (if they are useful nutrients) or away from them (if
they are harmful substances).

Phototaxis, aerotaxis and magnetotaxis. The occurrence of tactic behavior provides

evidence for the ecological (survival) advantage of flagella in bacteria and other
procaryotes.

Detecting Bacterial Motility: Since motility is a primary criterion for the diagnosis and
identification of bacteria, several techniques have been developed to demonstrate
bacterial motility, directly or indirectly.

1. Flagellar stains outline flagella and show their pattern of distribution. If a bacterium
possesses flagella, it is presumed to be motile.

Figure 6. Flagellar stains of three bacteria a. Bacillus cereus b. Vibrio cholerae c. Bacillus brevis.
(CDC). Since the bacterial flagellum is below the resolving power of the light microscope,
although bacteria can be seen swimming in a microscope field, the organelles of movenent cannot
be detected. Staining techniques such as Leifson's method utilize dyes and other components
that precipitate along the protein filament and hence increase its effective diameter. Fkagellar
distribution is occasionally used to differentiate between morphologically related bacteria. For
example, among the Gram-negative motile rod-shaped bacteria, the enterics have peritrichous
flagella while the pseudomonads have polar flagella.
2. Motility test medium demonstrates if cells can swim in a semisolid medium. A
semisolid medium is inoculated with the bacteria in a straight-line stab with a needle.
After incubation, if turbidity (cloudiness) due to bacterial growth can be observed away
from the line of the stab, it is evidence that the bacteria were able to swim through the
medium.

3. Direct microscopic observation of living bacteria in a wet mount. One must look for
transient movement of swimming bacteria. Most uincellular bacteria, because of their
small size, will shake back and forth in a wet mount observed at 400X or 1000X. This is
Brownian movement, due to random collisions between water molecules and bacterial
cells. True motility is confirmed by observing the bacterium swim from one side of
the microscope field to the other side.

Figure 7. A Desulfovibrio species. TEM. About 15,000X. The bacterium is motile by means of a
single polar flagellum. Of course, one can determine the presence of flagella by means of
electron microscopy. Perhaps this is an alternative way to determine bacterial motility, if you
happen to have an electron microscope.

1.5.1.2 Fimbriae

Fimbriae and pili are interchangeable terms used to designate short, hair-like structures
on the surfaces of procaryotic cells. Like flagella, they are composed of protein. Fimbriae
are shorter and stiffer than flagella, and slightly smaller in diameter. Generally, fimbriae
have nothing to do with bacterial movement (there are exceptions, e.g twitching
movement on Pseudomonas) . Fimbriae are very common in Gram-negative bacteria, but
occur in some archaea and Gram-positive bacteria as well. Fimbriae are most often
involved in adherence of bacteria to surfaces, substrates and other cells or tissues in
nature. In E. coli, a specialized type of pilus, the F or sex pilus, mediates the transfer of
DNA between mating bacteria during the process of conjugation, but the function of the
smaller, more numerous common pili is quite different.

Common pili (almost always called fimbriae) are usually involved in specific
adherence (attachment) of procaryotes to surfaces in nature. In medical situations, they
are major determinants of bacterial virulence because they allow pathogens to attach
to (colonize) tissues and/or to resist attack by phagocytic white blood cells.
Fimbriae (common pili) and flagella on the surface of bacterial cells. Left: dividing Shigella
enclosed in fimbriae. The structures are probably involved in the bacterium's ability to adhere to
the intestinal surface. Right: dividing pair of Salmonella displaying both its peritrichous flagella
and its fimbriae. The fimbriae are much shorter and slightly smaller in diameter than flagella.
Both Shigella and Salmonella are enteric bacteria that cause different types of intestinal
diarrheas.The bacteria can be differentiated by a motility test. Salmonella is motile; Shigella is
nonmotile.

Table 2. Some properties of pili and fimbriae.

Bacterial species where Typical Distribution on cell

Function
observed number on cell surface
mediates DNA transfer during
Escherichia coli (F or sex pilus) 1-4 uniform
conjugation
Escherichia coli (common pili or surface adherence to epithelial cells
100-200 uniform
Type 1 fimbriae) of the GI tract
surface adherence to epithelial cells
Neisseria gonorrhoeae 100-200 uniform
of the urogenital tract
Streptococcus pyogenes (fimbriae adherence, resistance to
? uniform
plus the M-protein) phagocytosis; antigenic variability
Pseudomonas aeruginosa 10-20 polar surface adherence
Sulfolobus acidocaldarius
? ? attachment to sulfur particles
(an archean)
1.5.1.3 The Cell Envelope

The cell envelope is a descriptive term for the several layers of material that envelope or
enclose the protoplasm of the cell. The cell protoplasm (cytoplasm) is surrounded by the
plasma membrane, a cell wall and a capsule. The cell wall itself is a layered structure in
Gram-negative bacteria. All cells have a membrane, which is the essential and definitive
characteristic of a "cell". Almost all procaryotes have a cell wall to prevent damage to the
underlying protoplast. Outside the cell wall, foremost as a surface structure, may be a
polysaccharide capsule, or at least a glycocalyx.

Figure 9. Profiles of the cell envelope the Gram-positive and Gram-negative bacteria. The Gram-
positive wall is a uniformly thick layer external to the plasma membrane. It is composed mainly of
peptidoglycan (murein). The Gram-negative wall appears thin and multilayered. It consists of a
relatively thin peptidoglycan sheet between the plasma membrane and a phospholipid-
lipopolysaccharide outer membrane. The space between the inner (plasma) and outer membranes
(wherein the peptidoglycan resides) is called the periplasm.

Capsules

A true capsule is a discrete detectable layer of polysaccharides deposited outside the

cell wall.

A less discrete structure or matrix which embeds the cells is a called a slime layer or a
biofilm.

A type of capsule found in bacteria growing in nature is called a glycocalyx. Not found in
lab cultures.
Figure 11. Negative stain of Streptococcus pyogenes viewed by transmission electron microscopy
(28,000X). The halo around the chain of cells is the hyaluronic acid capsule that surrounds the
exterior of the bacteria. The septa between dividing pairs of cells may also be seen.

Capsules are generally composed of polysaccharide; rarely they contain amino sugars or
peptides (see Table 3).

Table 3. Chemical composition of some bacterial capsules.

Bacterium Capsule composition Structural subunits

Gram-positive Bacteria
polypeptide (polyglutamic
Bacillus anthracis D-glutamic acid
acid)
polypeptide and
Bacillus megaterium D-glutamic acid, amino sugars, sugars
polysaccharide
Streptococcus mutans Polysaccharide (dextran) glucose
Streptococcus pneumoniae Polysaccharides sugars, amino sugars, uronic acids
polysaccharide (hyaluronic N-acetyl-glucosamine and glucuronic
Streptococcus pyogenes
acid) acid
Gram-negative Bacteria
Acetobacter xylinum Polysaccharide (cellulose) glucose
glucose, galactose, fucose glucuronic
Escherichia coli polysaccharide (colonic acid)
acid
Pseudomonas aeruginosa Polysaccharide mannuronic acid
Azotobacter vinelandii Polysaccharide glucuronic acid
Agrobacterium
Polysaccharide (glucan) glucose
tumefaciens

Capsules have several functions e.g.

 often mediate adherence of cells to surfaces.

 protect bacterial cells from engulfment by predatory protozoa or white
blood cells (phagocytes), or from attack by antimicrobial agents of plant or
animal origin.

 protect cells from perennial effects of drying or desiccation in certain soil

bacteria.

 Could act as reserves of carbohydrate for subsequent metabolism.

Figure 12. Colonies of Bacillus anthracis. The slimy or mucoid appearance of a bacterial colony
is usually evidence of capsule production. In the case of B. anthracis, the capsule is composed of
poly-D-glutamate. The capsule is an essential determinant of virulence to the bacterium. In
the early stages of colonization and infection the capsule protects the bacteria from assaults by the
immune and phagocytic systems.

Read up details
Cell Wall

Most prokaryotes have a rigid cell wall.

An essential structure that protects the cell protoplast from mechanical

damage and from osmotic rupture or lysis.

The prokaryotic cell wall is made of porous, rigid material that has high tensile strength
called murein (Why do cell walls need murein?)

The cell walls of bacteria deserve special attention for several reasons:

 They are an essential structure for viability.

 They are composed of unique components found nowhere else in nature e.g
murein.

 They are one of the most important sites for attack by antibiotics.

 They provide ligands for adherence and receptor sites for drugs or viruses.

 They cause symptoms of disease in animals.

 They provide for immunological distinction and immunological variation

among strains of bacteria.

All Bacterial peptidoglycans contain N-acetylmuramic acid, which is the definitive

component of murein.

The cell walls of Archaea may be composed of protein, polysaccharides, or

peptidoglycan-like molecules, but never do they contain murein. This feature
distinguishes the Bacteria from the Archaea.

In the Gram-positive Bacteria (those that retain the purple crystal violet dye when
subjected to the Gram-staining procedure) the cell wall is thick (15-80 nanometers),
consisting of several layers of peptidoglycan.

In the Gram-negative Bacteria (which do not retain the crystal violet) the cell wall is
relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan
surrounded by a membranous structure called the outer membrane. The outer membrane
of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide
(LPS or endotoxin), which is toxic to animals. In Gram-negative bacteria the outer
membrane is usually thought of as part of the cell wall.
The structure of the muramic acid subunit of the peptidoglycan of Escherichia coli.
This is the type of murein found in most Gram-negative bacteria. The glycan backbone is
a repeat polymer of two amino sugars, N-acetylglucosamine (G) and N-
acetylmuramic acid (M). Attached to the N-acetylmuramic acid is a tetrapeptide
consisting of L-ala-D-glu-DAP-D-ala. b. Abbreviated structure of the muramic acid
subunit. c. Nearby tetrapeptide side chains may be linked to one another by an
interpeptide bond between DAP on one chain and D-ala on the other. d. The polymeric
form of the molecule.

The glycan backbone of the peptidoglycan molecule can be cleaved by an enzyme called
lysozyme that is present in animal serum, tissues and secretions, and in the phagocytic
lysosome.

Assignment: Describe the structural differences of the peptidoglycan molecules of a

Gram positive and Gram negative bacterium.
\

The Outer Membrane of Gram-negative Bacteria

Outer membrane, a discrete bilayered structure on the outside of the peptidoglycan

sheet present only in the Gm negative bacteria.

It is a permeability barrier, but primarily due to its lipopolysaccharide content, it

possesses many interesting and important characteristics of Gram-negative bacteria.

The outer membrane is a lipid bilayer intercalated with proteins, superficially resembling
the plasma membrane.

The inner face of the outer membrane is composed of phospholipids.

The outer face of the outer membrane may contain some phospholipid, but mainly it is
formed by a different type of amphiphilic molecule which is composed of
lipopolysaccharide (LPS).

Outer membrane proteins usually traverse the membrane and in one case, anchor the
outer membrane to the underlying peptidoglycan sheet.

Hydro- and Lipo-philic properties

Figure 15. Schematic illustration of the outer membrane, cell wall and plasma membrane of a
Gram-negative bacterium. Note the structure and arrangement of molecules that constitute the
outer membrane.
The LPS molecule that constitutes the outer face of the outer membrane is composed of a
hydrophobic region, called Lipid A that is attached to a hydrophilic linear
polysaccharide region, consisting of the core polysaccharide and the O-specific
polysaccharide.

Figure 16. Structure of LPS

Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts LPS
or endotoxin activates macrophages to produce pyrogens, activates the complement
cascade causing inflammation, and activates blood factors resulting in intravascular
coagulation and hemorrhage.

The toxic component of endotoxin (LPS) is Lipid A.

the O-specific polysaccharides contribute to virulence of Gram-negative bacteria.

The proteins in the outer membrane of Escherichia coli are well characterized (see Table
4). Read up.

Table 4. Functions of the outer membrane components of Escherichia coli.

Component Function
Lipopolysaccharide
Permeability barrier
(LPS)
Mg++ bridges Stabilizes LPS and is essential for its permeability characteristics
Braun lipoprotein Anchors the outer membrane to peptidoglycan (murein) sheet
Omp C and Omp F proteins that form pores or channels through outer membrane for passage
porins of hydrophilic molecules
provides receptor for some viruses and bacteriocins; stabilizes mating
Omp A protein
cells during conjugation

Table 5. Correlation of Grams stain with other properties of Bacteria.

Property Gram-positive Gram-negative
Thickness of wall thick (20-80 nm) thin (10 nm)
Number of layers 1 2
Peptidoglycan (murein) content >50% 10-20%
Teichoic acids in wall Present absent
Lipid and lipoprotein content 0-3% 58%
Protein content 0 9%
Lipopolysaccharide content 0 13%
Sensitivity to Penicillin G Yes no (1)
Sensitivity to lysozyme Yes no (2)

Cell Wall-less Forms

A few bacteria are able to live or exist without a cell wall.

The mycoplasmas are a group of bacteria that lack a cell wall. Mycoplasma pneumoniae
is the cause of primary atypical bacterial pneumonia, known in the vernacular as
"walking pneumonia". For obvious reasons, penicillin is ineffective in treatment of
this type of pneumonia.

Assignment: Why is penicillin not effective for the treatment of Mycoplasma

pneumoniae infection?

Sometimes, under the pressure of antibiotic therapy, pathogenic streptococci can revert to
cell wall-less forms (called spheroplasts) and persist or survive in osmotically-protected
tissues. When the antibiotic is withdrawn from therapy the organisms may regrow their
cell walls and reinfect unprotected tissues.
The Plasma Membrane

Also called the cytoplasmic membrane, is the most dynamic structure of a procaryotic
cell.

Its main function is as a selective permeability barrier that regulates the passage of
substances into and out of the cell.

It allows passage of water and uncharged molecules up to mw of about 100 daltons, but
does not allow passage of larger molecules or any charged substances except by means
special membrane transport processes and systems.

It has a variety of functions in energy generation, and biosynthesis in prokaryotes. For

example, the electron transport system that couples aerobic respiration and ATP
synthesis is found in the prokaryotic membrane. The photosynthetic chromophores that
harvest light energy for conversion into chemical energy are located in the membrane.

Hence, the plasma membrane is the site of oxidative phosphorylation and

photophosphorylation in prokaryotes, analogous to the functions of mitochondria and
chloroplasts in eukaryotic cells.

It is also locations of transport proteins, sensing proteins and binding proteins.

Membranes also contain enzymes involved in many metabolic processes such as cell
wall synthesis, septum formation, membrane synthesis, DNA replication, CO 2
fixation and ammonia oxidation. The predominant functions of bacterial membranes
are listed in the table below.

Functions of the prokaryotic plasma membrane.

1. Osmotic or permeability barrier

2. Location of transport systems for specific solutes (nutrients and ions)

3. Energy generating functions, involving respiratory and photosynthetic electron

transport systems, establishment of proton motive force, and transmembranous, ATP-
synthesizing ATPase
 4. Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells)

5. Synthesis of murein (cell wall peptidoglycan)

6. Assembly and secretion of extracytoplasmic proteins

7. Coordination of DNA replication and segregation with septum formation and cell
division

8. Chemotaxis (both motility per se and sensing functions)

9. Location of specialized enzyme system

Transport Processes

The proteins that mediate the passage of solutes through membranes are referred to
variously as transport systems, carrier proteins, porters, and permeases.

Transport systems operate by one of three transport processes as described below. In a

uniport process, a solute passes through the membrane unidirectionally. In symport
processes (also called cotransport) two solutes must be transported in the same direction
at the same time; in antiport processes (also called exchange diffusion), one solute is
transported in one direction simultaneously as a second solute is transported in the
opposite direction.

Transport processes in bacterial cells. Solutes enter or exit from bacterial cells by means of one of
three processes: uniport, symport (also called cotransport) and antiport (also called exchange
diffusion). Transport systems operate by one or another of these processes.
Types of Transport Systems

Passive diffusion is the net movement of gases or small uncharged polar

molecules across a phospholipid bilayer membrane from an area of higher
concentration to an area of lower concentration. Examples of gases that
cross membranes by passive diffusion include N2, O2, and CO2; examples of
small polar molecules include ethanol, H2O, and urea.

However, bacteria may need to concentrate substances inside the cytoplasm against a
concentration gradient. Concentration of solutes in the cytoplasm requires the operation
of an active transport system.

The definitive feature of an active transport system is the accumulation of the solute in
the cytoplasm at concentrations far in excess of the environment.

Active transport system requires energy and are operated by transport proteins (sometimes
called carriers, porters or permeases) in the plasma membrane.

There are four types of carrier-mediated transport systems in procaryotes.

1. Facilitated diffusion systems (FD)

2. Ion driven transport systems (IDT) and

3. Binding-protein dependent transport systems (BPDT)

4. Group translocation systems (GT)

 Operation of bacterial transport systems.

Facilitated diffusion is a carrier-mediated system that does not require energy and does not
concentrate solutes against a gradient.

Active transport systems such as Ion-driven transport and Binding protein-dependent

transport, use energy and concentrate molecules against a concentration gradient.

Group translocation systems, such as the phosphotransferase (pts) system in Escherichia coli,
use energy during transport and modify the solute during its passage across the membrane. The
PTS derives energy from the metabolic intermediate phosphoenol pyruvate (PEP). PEP is
hydrolyzed to pyruvate and glucose is phosphorylated to form glucose-phosphate during
the process. Thus, by the expenditure of a single molecule of high energy phosphate,
glucose is transported and changed to glucose-phosphate.
The Cytoplasm

The cytoplasmic constituents of procaryotic cells invariably include the procaryotic

chromosome and ribosomes.

Procaryotes sometimes possess smaller extrachromosomal pieces of DNA called

plasmids. The total DNA content of a procaryote is referred to as the cell genome.

The distinct granular appearance of procaryotic cytoplasm is due to the presence and
distribution of ribosomes.

Procaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits unlike
the 80S ribosomes of eukaryotes are made up of 40S and 60S subunits.

Protein synthesis using 70S ribosomes occurs in eukaryotic mitochondria and

chloroplasts, and this is taken as a major line of evidence that these organelles are
descended from prokaryotes (endosymbiosis theory).

Table 9. Molecular composition of E. coli under conditions of balanced growth.

Molecule Percentage of dry weight

Protein 55

Total RNA 20.5

DNA 3.1

Phospholipid 9.1

Lipopolysaccharide 3.4

Murein 2.5

Glycogen 2.5

Small molecules: precursors, metabolites, vitamins, etc. 2.9

Inorganic ions 1.0

Total dry weight 100.0

Table 10. Small molecules present in a growing bacterial cell.

Approximate number of
Molecule
kinds
Amino acids, their precursors and derivatives
120
Nucleotides, their precursors and derivatives
100
Fatty acids and their precursors
50
Sugars, carbohydrates and their precursors or derivatives
250
quinones, porphyrins, vitamins, coenzymes and prosthetic groups and
300
their precursors

Table 11. Inorganic ions present in a growing bacterial cell.

Ion Function
+
K Maintenance of ionic strength; cofactor for certain enzymes
+
NH4 Principal form of inorganic N for assimilation
Ca++ Cofactor for certain enzymes
++
Fe Present in cytochromes and other metalloenzymes
++
Mg Cofactor for many enzymes; stabilization of outer membrane of Gram-negative bacteria
Mn++ Present in certain metalloenzymes
Trace element constituent of vitamin B12 and its coenzyme derivatives and found in certain
Co++
metalloenzymes
Cu++ Trace element present in certain metalloenzymes
Mo++ Trace element present in certain metalloenzymes
Ni++ Trace element present in certain metalloenzymes
++
Zn Trace element present in certain metalloenzymes
--
SO 4 Principal form of inorganic S for assimilation
PO4--- Principal form of P for assimilation and a participant in many metabolic reactions
Inclusions

Inclusions are distinct granules that may occupy a substantial part of the cytoplasm.

They are usually reserve materials of some sort e.g. glycogen (a polymer of glucose) or
as polybetahydroxybutyric acid (a type of fat) granules.

Polyphosphate inclusions are reserves of PO4 and possibly energy;

Elemental sulfur (sulfur globules) are stored by some phototrophic (gains energy from
light) and some lithotrophic (synthesizes all organic molecules from inorganic sources)
procaryotes as reserves of energy or electrons.

Table 12. Some inclusions in bacterial cells.

Cytoplasmic inclusions Where found Composition Function

reserve carbon and
Glycogen many bacteria e.g. E. coli Polyglucose
energy source
polybetahydroxyutyric many bacteria e.g. polymerized hydroxy reserve carbon and
acid (PHB) Pseudomonas butyrate energy source
reserve phosphate;
polyphosphate (volutin many bacteria e.g. linear or cyclical
possibly a reserve of
granules) Corynebacterium polymers of PO4
high energy phosphate
reserve of electrons
phototrophic purple and
(reducing source) in
green sulfur bacteria and
sulfur globules elemental sulfur phototrophs; reserve
lithotrophic colorless
energy source in
sulfur bacteria
lithotrophs
buoyancy (floatation)
aquatic bacteria especially protein hulls or shells
gas vesicles in the vertical water
cyanobacteria inflated with gases
column
endospore-forming bacilli unknown but toxic to
parasporal crystals Protein
(genus Bacillus) certain insects
orienting and
magnetite (iron oxide)
Magnetosomes certain aquatic bacteria migrating along geo-
Fe3O4
magnetic field lines
enzymes for
Carboxysomes many autotrophic bacteria autotrophic CO2 site of CO2 fixation
fixation
light-harvesting
Phycobilisomes Cyanobacteria Phycobiliproteins
pigments
 lipid and protein and light-harvesting
Chlorosomes Green bacteria
bacteriochlorophyll pigments and antennae

Endospores

A bacterial structure sometimes observed as an inclusion is actually a type of dormant

cell called an endospore.

Endospores exhibit no signs of life, being described as cryptobiotic.

They are highly resistant to environmental stresses.

Although cryptobiotic, they retain viability indefinitely such that under appropriate
environmental conditions, they germinate back into vegetative cells.

Endospores are formed by vegetative cells in response to environmental signals that

indicate a limiting factor for vegetative growth, such as exhaustion of an essential
nutrient.

Hence, endospore-formation is a mechanism of survival rather than a mechanism of

reproduction.
Table 13. Differences between endospores and vegetative cells.

Property Vegetative cells Endospores

Typical Gram-positive murein Thick spore coat, cortex, and
Surface coats
cell wall polymer peptidoglycan core wall
Microscopic appearance Nonrefractile Refractile
Calcium dipicolinic acid Absent Present in core
Cytoplasmic water activity High Very low
Enzymatic activity Present Absent
Macromolecular synthesis Present Absent
Heat resistance Low High
Resistance to chemicals
Low High
and acids
Radiation resistance Low High
Sensitivity to lysozyme Sensitive Resistant
Sensitivity to dyes and
Sensitive Resistant
staining

Figure 23. Bacterial endospores. Phase microscopy of sporulating bacteria demonstrates the
refractility of endospores, as well as characteristic spore shapes and locations within the mother
cell.
Variations in endospore morphology. (1, 4) Central endospore, (2, 3, 5)
terminal endospore, (6) lateral endospore

Figure 24. Electron micrograph of a bacterial endospore. The spore has a core wall of unique
peptidoglycan surrounded by several layers, including the cortex, the spore coat and the
exosporium. The dehydrated core contains the bacterial chromosome and a few ribosomes and
enzymes to jump-start protein synthesis and metabolism during germination.
 CHAPTER II

MICROBIAL GROWTH AND ITS REGULATION

2.1 Nutritional Requirements of Cells

Every organism must find in its environment all of the substances required for energy
generation and cellular biosynthesis. The chemicals and elements of this environment
that are utilized for bacterial growth are referred to as nutrients or nutritional
requirements. In the laboratory, bacteria are grown in culture media which are designed
to provide all the essential nutrients in solution for bacterial growth.

2.1.1 The Major Elements

At an elemental level, the nutritional requirements of a bacterium such as E. coli are

revealed by the cell's elemental composition, which consists of C, H, O, N, S. P, K,
Mg, Fe, Ca, Mn, and traces of Zn, Co, Cu, and Mo. These elements are found in the
form of water, inorganic ions, small molecules, and macromolecules which serve
either a structural or functional role in the cells. The general physiological functions of
the elements are outlined in Table 1 below.

Table 1. Major elements, their sources and functions in bacterial cells.

% of dry
Element Source Function
weight
organic compounds
Carbon 50 Main constituent of cellular material
or CO2
H2O, organic
Constituent of cell material and cell water;
Oxygen 20 compounds, CO2,
O2 is electron acceptor in aerobic respiration
and O2
NH3, NO3, organic Constituent of amino acids, nucleic acids
Nitrogen 14
compounds, N2 nucleotides, and coenzymes
H2O, organic Main constituent of organic compounds and
Hydrogen 8
compounds, H2 cell water
inorganic phosphates Constituent of nucleic acids, nucleotides,
Phosphorus 3
(PO4) phospholipids, LPS, teichoic acids
o
SO4, H2S, S , organic Constituent of cysteine, methionine,
Sulfur 1
sulfur compounds glutathione, several coenzymes
Main cellular inorganic cation and cofactor
Potassium 1 Potassium salts
for certain enzymes
Inorganic cellular cation, cofactor for certain
Magnesium 0.5 Magnesium salts
enzymatic reactions
Inorganic cellular cation, cofactor for certain
Calcium 0.5 Calcium salts
enzymes and a component of endospores
 Component of cytochromes and certain
Iron 0.2 Iron salts nonheme iron-proteins and a cofactor for
some enzymatic reactions

2.1.2 Trace Elements

Trace elements are metal ions required by certain cells in such small amounts that it is
difficult to detect (measure) them, and it is not necessary to add them to culture media
as nutrients. Includes Mn, Co, Zn, Cu, and Mo.

2.1.3 Carbon and Energy Sources for Bacterial Growth

In order to grow in nature or in the laboratory, a bacterium must have an energy source,
a source of carbon and other required nutrients, and a permissive range of physical
conditions such as O2 concentration, temperature, and pH.

All living organisms require a source of energy. Organisms that use radiant energy (light)
are called phototrophs. Organisms that use (oxidize) an organic form of carbon are
called heterotrophs or chemo(hetero)trophs. Organisms that oxidize inorganic
compounds are called lithotrophs.

In terms of organic carbon demand Organisms that use organic carbon are heterotrophs
and organisms that use CO2 as a sole source of carbon for growth are called autotrophs.

Thus, on the basis of carbon and energy sources for growth four major nutritional
types of procaryotes may be defined (Table 2).

Table 2. Major nutritional types of prokaryotes.

 Carbon
Nutritional Type Energy Source Examples
Source
Cyanobacteria, some
Photoautotrophs Light CO2 Purple and Green
Bacteria
Organic Some Purple and Green
Photoheterotrophs Light
compounds Bacteria
Chemoautotrophs or Inorganic
A few Bacteria and
Lithotrophs compounds, e.g. H2, CO2
many Archaea
(Lithoautotrophs) NH3, NO2, H2S
Chemoheterotrophs or Organic Most Bacteria, some
Organic compounds
Heterotrophs compounds Archaea

Almost all eukaryotes are either photoautotrophic (e.g. plants and algae) or heterotrophic
(e.g. animals, protozoa, fungi). Lithotrophy is unique to procaryotes and
photoheterotrophy, common in the Purple and Green Bacteria, occurs only in a very few
eukaryotic algae. Phototrophy has not been found in the Archaea, except for
nonphotosynthetic light-driven ATP synthesis in the extreme halophiles.

2.1.4 Growth Factors

Small amounts of certain organic compounds which are essential substances that the
organism is unable to synthesize from available nutrients are called growth factors.

Growth factors are required in small amounts by cells because they fulfill specific roles
in biosynthesis. 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

Table 3. Common vitamins required in the nutrition of certain bacteria.

Vitamin Coenzyme form Function
p-Aminobenzoic Precursor for the biosynthesis of folic
-
acid (PABA) acid
Transfer of one-carbon units and
required for synthesis of thymine,
Folic acid Tetrahydrofolate
purine bases, serine, methionine and
pantothenate
Biosynthetic reactions that require CO2
Biotin Biotin
fixation
Transfer of acyl groups in oxidation of
Lipoic acid Lipoamide
keto acids
Mercaptoethane-
Coenzyme M CH4 production by methanogens
sulfonic acid
NAD (nicotinamide adenine Electron carrier in dehydrogenation
Nicotinic acid
dinucleotide) and NADP reactions
Coenzyme A and the Acyl Oxidation of keto acids and acyl group
Pantothenic acid
Carrier Protein (ACP) carriers in metabolism
Transamination, deamination,
Pyridoxine (B6) Pyridoxal phosphate decarboxylation and racemation of
amino acids
FMN (flavin
Riboflavin (B2) mononucleotide) and FAD Oxidoreduction reactions
(flavin adenine dinucleotide)
Thiamine pyrophosphate Decarboxylation of keto acids and
Thiamine (B1)
(TPP) transaminase reactions
Cobalamine coupled to
Vitamin B12 Transfer of methyl groups
adenine nucleoside
Quinones and
Vitamin K Electron transport processes
napthoquinones

2.2 Culture Media for the Growth of Bacteria

For any bacterium to be propagated for any purpose it is necessary to provide the
appropriate biochemical and biophysical environment. The biochemical (nutritional)
environment is made available as a culture medium.

* a large variety and types of culture media have been developed with different purposes
and uses.

* Culture media are employed

- in the isolation and maintenance of pure cultures of bacteria

- and are also used for identification of bacteria according to their biochemical and
physiological properties.

Liquid media are used for growth of pure batch cultures,

Solidified media are used widely for the

- isolation of pure cultures,

- for estimating viable bacterial populations,

- and a variety of other purposes.

Gelling agent for solid or semisolid medium is agar, a hydrocolloid derived from red
algae. Agar melts at 100 oC and remains liquid until cooled to 40 oC, the temperature at
which it gels and because it cannot be metabolized by most bacteria. Hence as a medium
component it is relatively inert; it simply holds (gels) nutrients that are in aqueous
solution.

2.2.1 Types of Culture Media

Culture media may be classified into several categories depending on their composition
or use.

* A chemically-defined (synthetic) medium is one in which the exact chemical

composition is known.

* A complex (undefined) medium is one in which the exact chemical constitution of

the medium is not known such as blood or milk or yeast extract or beef extract, the exact
chemical composition of which is obviously undetermined.

* A defined medium is a minimal medium if it provides only the exact nutrients

(including any growth factors) needed by the organism for growth.

Complex media usually provide the full range of growth factors that may be required by
an organism so they may be more handily used to cultivate unknown bacteria or bacteria
whose nutritional requirement are complex (i.e., organisms that require a lot of growth
factors, known or unknown).

* Most pathogenic bacteria of animals, which have adapted themselves to growth in

animal tissues, require complex media for their growth. Blood, serum and tissue extracts
are frequently added to culture media for the cultivation of pathogens.

Other concepts employed in the construction of culture media are the principles of
selection and enrichment.

A selective medium is one which has a component(s) added to it which will inhibit or
prevent the growth of certain types or species of bacteria and/or promote the growth of
desired species. One can also adjust the physical conditions of a culture medium, such as
pH and temperature, to render it selective for organisms that are able to grow under these
certain conditions.

A culture medium may also be a differential medium if allows the investigator to

distinguish between different types of bacteria based on some observable trait in their
pattern of growth on the medium.

Typical example of a selective, differential medium is the mannitol salt agar for the
isolation of Staphylococcus aureus.

EMB (Eosin Methylene Blue) Agar

- selective for: gram-negative
bacteria
o growth of gram-positive bacteria (e.g.: Staphylococcus aureus in the image below) is
inhibited by the eosin & methylene blue dyes in the media
- differential for: lactose
fermentation
o gram-negative Enterobacteria Escherichia coli and Enterobacter aerogenes ferment
lactose
o E. coli produces colonies with a characteristic green metallic sheen on EMB agar
o E. aerogenes produces pink colonies often with a central dark purple dot (fish eye
colonies) on EMB agar
o gram-negative bacteria Proteus vulgaris and Salmonella typhimurium grow on EMB
agar, but do not ferment lactose

MSA (Mannitol Salt Agar)

- selective for: gram-positive Staphylococci
bacteria
o 7% salt in the medium inhibits the growth of most gram-positive and gram-negative
bacteria
- differential for: mannitol
fermentation
o phenol red pH indicator turns yellow in the presence of acid by-products of mannitol
fermentation
o Staphylococcus aureus ferments mannitol
o S. aureus changes the color of the medium from pink to yellow due to acid by-
products of mannitol fermentation
o Staphylococcus epidermidis grows on MSA, but does not ferment mannitol (media
remains light pink in color & colonies are colorless

* An enrichment medium contains some component that permits the growth of specific
types or species of bacteria, usually because they alone can utilize the component from
their environment. However, an enrichment medium may have selective features.
Table 4a. Minimal medium for the growth of Bacillus megaterium. An example of a chemically-
defined medium for growth of a heterotrophic bacterium.

Component Amount Function of component

sucrose 10.0 g C and energy source
K2HPO4 2.5 g pH buffer; P and K source
KH2PO4 2.5 g pH buffer; P and K source
(NH4)2HPO4 1.0 g pH buffer; N and P source
MgSO4 7H2O 0.20 g S and Mg++ source
FeSO4 7H2O 0.01 g Fe++ source
MnSO4 7H2O 0.007 g Mn++ Source
water 985 ml
pH 7.0

Table 4b. Defined medium (also an enrichment medium) for the growth of Thiobacillus
thiooxidans, a lithoautotrophic bacterium.

Component Amount Function of component

NH4Cl 0.52 g N source
KH2PO4 0.28 g P and K source
MgSO4 7H2O 0.25 g S and Mg++ source
CaCl2 2H2O 0.07 g Ca++ source
Elemental Sulfur 1.56 g Energy source
CO2 5%* C source
water 1000 ml
pH 3.0
* Aerate medium intermittently with air containing 5% CO2.

Table 5a. Complex medium for the growth of fastidious bacteria.

Component Amount Function of component
Beef extract 1.5 g Source of vitamins and other growth factors
Yeast extract 3.0 g Source of vitamins and other growth factors
Peptone 6.0 g Source of amino acids, N, S, and P
Glucose 1.0 g C and energy source
Agar 15.0 g Inert solidifying agent
water 1000 ml
pH 6.6

Table 5b. Selective enrichment medium for growth of extreme halophiles.

Component Amount Function of component

Casamino acids 7.5 g Source of amino acids, N, S and P
Yeast extract 10.0 g Source of growth factors
Trisodium citrate 3.0 g C and energy source
KCl 2.0 g K+ source
MgSO4 7 H2O 20.0 g S and Mg++ source
FeCl2 0.023 g Fe++ source
NaCl 250 g Na+ source for halophiles and inhibitory to nonhalophiles
water 1000 ml
pH 7.4

2.3 Physical and Environmental Requirements for Microbial Growth

Microorganisms exist in nature under an enormous range of physical conditions such as

O2 concentration,

Hydrogen ion concentration (pH) and

temperature.

A thermophile grows at high temperatures,

an acidophile grows at low pH; alkalinophile grows at high pH

an osmophile grows at high solute concentration, and so on.

2.3.1 The Effect of Oxygen

Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in
aerobic respiration.

Obligate anaerobes (occasionally called aerophobes) do not need or use O2 as a

nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth.
Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial
photosynthesis, or the novel process of methanogenesis.

Facultative anaerobes (or facultative aerobes) are organisms that can switch between
aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O 2) they
grow by fermentation or anaerobic respiration, but in the presence of O 2 they switch to
aerobic respiration.

Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type

of metabolism but they are insensitive to the presence of O 2. They live by fermentation
alone whether or not O2 is present in their environment.

Table 6. Terms used to describe O2 Relations of Microorganisms.

Environment
Group Aerobic Anaerobic O2 Effect
Required (utilized for
Obligate Aerobe Growth No growth
aerobic respiration)
Growth if level Required but at levels
Microaerophile No growth
not too high below 0.2 atm
Obligate Anaerobe No growth Growth Toxic
Not required for growth
Facultative Anaerobe
Growth Growth but utilized when
(Facultative Aerobe)
available
Not required and not
Aerotolerant Anaerobe Growth Growth
utilized

All cells contain enzymes capable of reacting with O2.

Toxic radicals that could be produced as a result of the enzymatic interaction with oxygen
include superoxide or O2.- and singlet oxygen.

In aerobes and aerotolerant anaerobes the potential for lethal accumulation of superoxide
is prevented by the enzyme superoxide dismutase (Figure 1).

Figure 3. The action of superoxide dismutase, catalase and peroxidase. These enzymes detoxify
oxygen radicals that are inevitably generated by living systems in the presence of O 2. The
distribution of these enzymes in cells determines their ability to exist in the presence of O 2

All organisms which can live in the presence of O 2 (whether or not they utilize it in
their metabolism) contain superoxide dismutase. Nearly all organisms contain the
enzyme catalase, which decomposes H2O2. Even though certain aerotolerant bacteria such
as the lactic acid bacteria lack catalase, they decompose H 2O2 by means of peroxidase
enzymes which derive electrons from NADH 2 to reduce peroxide to H 2O. Obligate
anaerobes lack superoxide dismutase and catalase and/or peroxidase, and therefore
undergo lethal oxidations by various oxygen radicals when they are exposed to O 2. See
Figure 3 below.

All photosynthetic (and some nonphotosynthetic):

Singlet oxygen (toxic) caronenoid pigments Triplet oxygen (nontoxic)

Table 7. Distribution of superoxide dismutase, catalase and peroxidase in procaryotes with

different O2 tolerances.

Superoxide
Group Catalase Peroxidase
dismutase
Obligate aerobes and most facultative
+ + -
anaerobes (e.g. Enterics)
Most aerotolerant anaerobes (e.g. Streptococci) + - +
Obligate anaerobes (e.g. Clostridia,
- - -
Methanogens, Bacteroides)

2.3.2 The Effect of pH on Growth

The pH, or hydrogen ion concentration, [H +], of natural environments varies from about
0.5 in the most acidic soils to about 10.5 in the most alkaline lakes. Appreciating that pH
is measured on a logarithmic scale, the [H +] of natural environments varies over a billion-
fold and some microorganisms are living at the extremes, as well as every point between
the extremes! Most free-living procaryotes can grow over a range of 3 pH units, about a
thousand fold change in [H+]. The range of pH over which an organism grows is defined
by three cardinal points: the minimum pH, below which the organism cannot grow, the
maximum pH, above which the organism cannot grow, and the optimum pH, at which
the organism grows best. For most bacteria there is an orderly increase in growth rate
between the minimum and the optimum and a corresponding orderly decrease in growth
rate between the optimum and the maximum pH, reflecting the general effect of changing
[H+] on the rates of enzymatic reaction (Figure 4).

Microorganisms which grow at an optimum pH well below neutrality (7.0) are called
acidophiles. Those which grow best at neutral pH are called neutrophiles and those that
grow best under alkaline conditions are called alkaliphiles. Obligate acidophiles, such as
some Thiobacillus species, actually require a low pH for growth since their membranes
dissolve and the cells lyse at neutrality. Several genera of Archaea, including Sulfolobus
and Thermoplasma, are obligate acidophiles. Among eukaryotes, many fungi are
acidophiles, but the champion of growth at low pH is the eukaryotic alga Cyanidium
which can grow at a pH of 0.

In the construction and use of culture media, one must always consider the optimum pH
for growth of a desired organism and incorporate buffers in order to maintain the pH of
the medium in the changing milieu of bacterial waste products that accumulate during
growth. Many pathogenic bacteria exhibit a relatively narrow range of pH over which
they will grow. Most diagnostic media for the growth and identification of human
pathogens have a pH near 7.

Figure 4. Growth rate vs pH for three environmental classes of procaryotes. Most free-living
bacteria grow over a pH range of about three units. Note the symmetry of the curves below and
above the optimum pH for growth.

Table 8. Minimum, maximum and optimum pH for growth of certain procaryotes.

Organism Minimum pH Optimum pH Maximum pH

Thiobacillus thiooxidans 0.5 2.0-2.8 4.0-6.0
Sulfolobus acidocaldarius 1.0 2.0-3.0 5.0
Bacillus acidocaldarius 2.0 4.0 6.0
Zymomonas lindneri 3.5 5.5-6.0 7.5
Lactobacillus acidophilus 4.0-4.6 5.8-6.6 6.8
Staphylococcus aureus 4.2 7.0-7.5 9.3
Escherichia coli 4.4 6.0-7.0 9.0
Clostridium sporogenes 5.0-5.8 6.0-7.6 8.5-9.0
Erwinia caratovora 5.6 7.1 9.3
Pseudomonas aeruginosa 5.6 6.6-7.0 8.0
Thiobacillus novellus 5.7 7.0 9.0
Streptococcus pneumoniae 6.5 7.8 8.3
Nitrobacter sp 6.6 7.6-8.6 10.0

2.3.3 The Effect of Temperature on Growth

Microorganisms have been found growing in virtually all environments where there is
liquid water, regardless of its temperature. In 1966, Professor Thomas D. Brock, then at
Indiana University, made the amazing discovery in boiling hot springs of Yellowstone
National Park that bacteria were not just surviving there, they were growing and
flourishing. Brock's discovery of thermophilic bacteria, archaea and other
"extremophiles" in Yellowstone is summarized for the general public in an article at this
web site. See Life at High Temperatures.

Subsequently, procaryotes have been detected growing around black smokers and
hydrothermal vents in the deep sea at temperatures at least as high as 120 degrees.
Microorganisms have been found growing at very low temperatures as well. In
supercooled solutions of H2O as low as -20 degrees, certain organisms can extract water
for growth, and many forms of life flourish in the icy waters of the Antarctic, as well as
household refrigerators, near 0 degrees.

A particular microorganism will exhibit a range of temperature over which it can grow,
defined by three cardinal points in the same manner as pH (Figure 6, cf. Figure 4).
Considering the total span of temperature where liquid water exists, the procaryotes may
be subdivided into several subclasses on the basis of one or another of their cardinal
points for growth. For example, organisms with an optimum temperature near 37 degrees
(the body temperature of warm-blooded animals) are called mesophiles. Organisms with
an optimum T between about 45 degrees and 70 degrees are thermophiles. Some
Archaea with an optimum T of 80 degrees or higher and a maximum T as high as 115
degrees, are now referred to as extreme thermophiles or hyperthermophiles. The cold-
loving organisms are psychrophiles defined by their ability to grow at 0 degrees. A
variant of a psychrophile (which usually has an optimum T of 10-15 degrees) is a
psychrotroph, which grows at 0 degrees but displays an optimum T in the mesophile
range, nearer room temperature. Psychrotrophs are the scourge of food storage in
refrigerators since they are invariably brought in from their mesophilic habitats and
continue to grow in the refrigerated environment where they spoil the food. Of course,
they grow slower at 2 degrees than at 25 degrees. Think how fast milk spoils on the
counter top versus in the refrigerator.

Psychrophilic bacteria are adapted to their cool environment by having largely

unsaturated fatty acids in their plasma membranes. Some psychrophiles, particularly
those from the Antarctic have been found to contain polyunsaturated fatty acids, which
generally do not occur in procaryotes. The degree of unsaturation of a fatty acid
correlates with its solidification T or thermal transition stage (i.e., the temperature at
which the lipid melts or solidifies); unsaturated fatty acids remain liquid at low T but are
also denatured at moderate T; saturated fatty acids, as in the membranes of thermophilic
bacteria, are stable at high temperatures, but they also solidify at relatively high T. Thus,
saturated fatty acids (like butter) are solid at room temperature while unsaturated fatty
acids (like safflower oil) remain liquid in the refrigerator. Whether fatty acids in a
membrane are in a liquid or a solid phase affects the fluidity of the membrane, which
directly affects its ability to function. Psychrophiles also have enzymes that continue to
function, albeit at a reduced rate, at temperatures at or near 0 degrees. Usually,
psychrophile proteins and/or membranes, which adapt them to low temperatures, do not
function at the body temperatures of warm-blooded animals (37 degrees) so that they are
unable to grow at even moderate temperatures.

Thermophiles are adapted to temperatures above 60 degrees in a variety of ways. Often

thermophiles have a high G + C content in their DNA such that the melting point of the
DNA (the temperature at which the strands of the double helix separate) is at least as high
as the organism's maximum T for growth. But this is not always the case, and the
correlation is far from perfect, so thermophile DNA must be stabilized in these cells by
other means. The membrane fatty acids of thermophilic bacteria are highly saturated
allowing their membranes to remain stable and functional at high temperatures. The
membranes of hyperthermophiles, virtually all of which are Archaea, are not composed
of fatty acids but of repeating subunits of the C5 compound, phytane, a branched,
saturated, "isoprenoid" substance, which contributes heavily to the ability of these
bacteria to live in superheated environments. The structural proteins (e.g. ribosomal
proteins, transport proteins (permeases) and enzymes of thermophiles and
hyperthermophiles are very heat stable compared with their mesophilic counterparts. The
proteins are modified in a number of ways including dehydration and through slight
changes in their primary structure, which accounts for their thermal stability.

Figure 5 (above).SEM of a thermophilic Bacillus species isolated from a compost pile at 55 o C. ©

Frederick C. Michel. The Ohio State University -OARDC, Wooster, Ohio. Licensed for use by
ASM Microbe Library http://www.microbelibrary.org. The rods are 3-5 microns in length and 0.5
to 1 micron in width with terminal endospores in a slightly-swollen sporangium.

Figure 6 (below). Growth rate vs temperature for five environmental classes of procaryotes. Most
procaryotes will grow over a temperature range of about 30 degrees. The curves exhibit three
cardinal points: minimum, optimum and maximum temperatures for growth. There is a steady
increase in growth rate between the minimum and optimum temperatures, but slightly past the
optimum a critical thermolabile cellular event occurs, and the growth rates plunge rapidly as the
maximum T is approached. As expected and as predicted by T.D. Brock, life on earth, with
regard to temperature, exists wherever water remains in a liquid state. Thus, psychrophiles grow
in solution wherever water is supercooled below 0 degrees; and extreme thermophilic archaea
(hyperthermophiles) have been identified growing near deep-sea thermal vents at temperatures up
to 120 degrees. Theoretically, the bar can be pushed to even higher temperatures.

Terms used to describe microorganisms in relation to temperature requirements for growth .

Temperature for growth (degrees C)

Group Minimum Optimum Maximum Comments
Psychrophile Below 0 10-15 Below 20 Grow best at relatively low T
Able to grow at low T but prefer
Psychrotroph 0 15-30 Above 25
moderate T
Most bacteria esp. those living in
Mesophile 10-15 30-40 Below 45 association with warm-blooded
animals
Among all thermophiles is wide
Above 100
Thermophile* 45 50-85 variation in optimum and maximum
(boiling)
T

Minimum, maximum and optimum temperature for growth of certain bacteria and archaea.

Temperature for growth (degrees C)

Bacterium Minimum Optimum Maximum
Listeria monocytogenes 1 30-37 45
Vibrio marinus 4 15 30
Pseudomonas maltophilia 4 35 41
Thiobacillus novellus 5 25-30 42
Staphylococcus aureus 10 30-37 45
Escherichia coli 10 37 45
Clostridium kluyveri 19 35 37
Streptococcus pyogenes 20 37 40
Streptococcus pneumoniae 25 37 42
Bacillus flavothermus 30 60 72
Thermus aquaticus 40 70-72 79
Methanococcus jannaschii 60 85 90
Sulfolobus acidocaldarius 70 75-85 90
Pyrobacterium brockii 80 102-105 115

Optimum growth temperature of some procaryotes.

Genus and species Optimal growth temp (degrees C)
Vibrio cholerae 18-37
Photobacterium phosphoreum 20
Rhizobium leguminosarum 20
Streptomyces griseus 25
Rhodobacter sphaeroides 25-30
Pseudomonas fluorescens 25-30
Erwinia amylovora 27-30
Staphylococcus aureus 30-37
Escherichia coli 37
Mycobacterium tuberculosis 37
Pseudomonas aeruginosa 37
Streptococcus pyogenes 37
Treponema pallidum 37
Thermoplasma acidophilum 59
Thermus aquaticus 70
Bacillus caldolyticus 72
Pyrococcus furiosus 100

Hyperthermophilic Archaea.

Temperature for growth(degrees C)

Genus Minimum Optimum Maximum Optimum pH

Sulfolobus 55 75-85 87 2-3
Desulfurococcus 60 85 93 6
Methanothermus 60 83 88 6-7
Pyrodictium 82 105 113 6
Methanopyrus 85 100 110 7
2.3.4 Water Availability

Water is the solvent in which the molecules of life are dissolved, and the availability
of water is therefore a critical factor that affects the growth of all cells.

The availability of water for a cell depends upon its presence in the atmosphere
(relative humidity) or its presence in solution or a substance (water activity).

The water activity (Aw) of pure H2O is 1.0 (100% water). Water activity is affected by
the presence of solutes such as salts or sugars, that are dissolved in the water.

The higher the solute concentration of a substance, the lower is the water activity and
vice-versa. Microorganisms live over a range of A w from 1.0 to 0.7. The Aw of human
blood is 0.99; seawater = 0.98; maple syrup = 0.90; Great Salt Lake = 0.75. Water
activities in agricultural soils range between 0.9 and 1.0.

The only common solute in nature that occurs over a wide concentration range is
salt [NaCl], and some microorganisms are named based on their growth response to salt.

Microorganisms that require some NaCl for growth are halophiles.

Mild halophiles require 1-6% salt,

moderate halophiles require 6-15% salt;

extreme halophiles that require 15-30% NaCl for growth are found among the archaea.

Bacteria that are able to grow at moderate salt concentrations, even though they grow
best in the absence of NaCl, are called halotolerant.
Although halophiles are "osmophiles" (and halotolerant organisms are "osmotolerant")
the term osmophiles is usually reserved for organisms that are able to live in
environments high in sugar.

Organisms which live in dry environments (made dry by lack of water) are called
xerophiles.

The concept of lowering water activity in order to prevent bacterial growth is the basis
for preservation of foods by drying (in sunlight or by evaporation) or by addition of high
concentrations of salt or sugar.

Growth rate vs osmolarity for different classes of procaryotes. Osmolarity is determined by solute
concentration in the environment. Osmolarity is inversely related to water activity (A w), which is
more like a measure of the concentration of water (H 2O) in a solution. Increased solute
concentration means increased osmolarity and decreased A w. From left to right the graph shows
the growth rate of a normal (nonhalophile) such as E. coli or Pseudomonas, the growth rate of a
halotolerant bacterium such as Staphylococcus aureus,and the growth rate of an extreme
halophile such as the archaean Halococcus. Note that a true halophile grows best at salt
concentrations where most bacteria are inhibited.

Limiting water activities (Aw) for growth of certain procaryotes.

Organism Minimum Aw for growth

Caulobacter 1.00
Spirillum 1.00
Pseudomonas .91
Salmonella/E. coli .91
Lactobacillus .90
Bacillus .90
Staphylococcus .85
Halococcus .75

CHAPTER III

GROWTH OF BACTERIAL POPULATIONS

3.1 Measurement of Bacterial Growth

Growth is an orderly increase in the quantity of cellular constituents. It depends upon the
ability of the cell to form new protoplasm from nutrients available in the environment. In
most bacteria, growth involves increase in cell mass and number of ribosomes,
duplication of the bacterial chromosome, synthesis of new cell wall and plasma
membrane, partitioning of the two chromosomes, septum formation, and cell
division. This asexual process of reproduction is called binary fission.

Figure 1. Bacterial growth by binary fission.

For unicellular organisms such as the bacteria, growth can be measured in terms of two
different parameters:

(i) changes in cell mass

(ii) and changes in cell numbers.

3.1.1 Methods for Measurement of Cell Mass

Methods for measurement of the cell mass involve both direct and indirect techniques.

1. Direct physical measurement of dry weight, wet weight, or volume of cells after
centrifugation.

2. Direct chemical measurement of some chemical component of the cells such as total
N, total protein, or total DNA content.

3. Indirect measurement of chemical activity such as rate of O2 production or

consumption, CO2 production or consumption, etc.

4. Turbidity measurements employ a variety of instruments to determine the amount of

light scattered by a suspension of cells. Particulate objects such as bacteria scatter light
in proportion to their numbers. The turbidity or optical density of a suspension of cells is
directly related to cell mass or cell number, after construction and calibration of a
standard curve. The method is simple and nondestructive, but the sensitivity is limited to
about 107 cells per ml for most bacteria.
3.1.2 Methods for Measurement of Cell Numbers

Measuring techniques involve direct counts, visually or instrumentally, and indirect

viable cell counts.

1. Direct microscopic counts are possible using special slides known as counting
chambers. Dead cells cannot be distinguished from living ones. Only dense suspensions
can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or
filtration to increase sensitivity.

2. Electronic counting chambers count numbers and measure size distribution of cells.
For cells the size of bacteria the suspending medium must be very clean. Such electronic
devices are more often used to count eukaryotic cells such as blood cells.

3. Indirect viable cell counts, also called plate counts, involve plating out (spreading) a
sample of a culture on a nutrient agar surface. The sample or cell suspension can be
diluted in a nontoxic diluent (e.g. water or saline) before plating. If plated on a suitable
medium, each viable unit grows and forms a colony. Each colony that can be counted is
called a colony forming unit (cfu) and the number of cfu's is related to the viable
number of bacteria in the sample.

Advantages of the technique are its sensitivity (theoretically, a single cell can be
detected), and it allows for inspection and positive identification of the organism counted.
Disadvantages are

(1) only living cells develop colonies that are counted;

(2) clumps or chains of cells develop into a single colony;

(3) colonies develop only from those organisms for which the cultural conditions are
suitable for growth. The latter makes the technique virtually useless to characterize or
count the total number of bacteria in complex microbial ecosystems such as soil or the
animal rumen or gastrointestinal tract. Genetic probes can be used to demonstrate the
diversity and relative abundance of procaryotes in such an environment, but many species
identified by genetic techniques have so far proven unculturable.
Table 1. Some Methods used to measure bacterial growth

Method Application Comments

Enumeration of bacteria in Cannot distinguish living
Direct microscopic count
milk or cellular vaccines from nonliving cells
Enumeration of bacteria in
Viable cell count (colony Very sensitive if plating
milk, foods, soil, water,
counts) conditions are optimal
laboratory cultures, etc.
Estimations of large Fast and nondestructive, but
Turbidity measurement numbers of bacteria in clear cannot detect cell densities
liquid media and broths less than 107 cells per ml
Measurement of total cell
Measurement of total N or only practical application is
yield from very dense
protein in the research laboratory
cultures
Measurement of Biochemical
Requires a fixed standard to
activity e.g. O2 uptake CO2
Microbiological assays relate chemical activity to
production, ATP production,
cell mass and/or cell numbers
etc.
Measurement of dry weight or probably more sensitive than
Measurement of total cell
wet weight of cells or volume total N or total protein
yield in cultures
of cells after centrifugation measurements
Figure 2. Bacterial colonies growing on a plate of nutrient agar.
Hans Knoll Institute. Jena, Germany.

3.2 The Bacterial Growth Curve

In the laboratory, under favorable conditions, a growing bacterial population doubles at

regular intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 2 0, 21, 22,
23.........2n (where n = the number of generations). This is called exponential growth. In
reality, exponential growth is only part of the bacterial life cycle, and not representative
of the normal pattern of growth of bacteria in Nature.

When a fresh medium is inoculated with a given number of cells, and the population
growth is monitored over a period of time, plotting the data will yield a typical bacterial
growth curve (Figure 3 below).
Figure 3. The typical bacterial growth curve.

Four characteristic phases of the growth cycle are recognized.

1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the
population remains temporarily unchanged. Although there is no apparent cell division
occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins,
RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including
the size of the inoculum; time necessary to recover from physical damage or shock in the
transfer; time required for synthesis of essential coenzymes or division factors; and time
required for synthesis of new (inducible) enzymes that are necessary to metabolize the
substrates present in the medium.

2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced

growth wherein all the cells are dividing regularly by binary fission, and are growing by
geometric progression. The cells divide at a constant rate depending upon the
composition of the growth medium and the conditions of incubation. The rate of
exponential growth of a bacterial culture is expressed as generation time, also the
doubling time of the bacterial population. Generation time (G) is defined as the time (t)
per generation (n = number of generations). Hence, G=t/n is the equation from which
calculations of generation time (below) derive.

3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture

(e.g. a closed system such as a test tube or flask). Population growth is limited by one of
three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory
metabolites or end products; 3. exhaustion of space, in this case called a lack of
"biological space".
During the stationary phase, if viable cells are being counted, it cannot be determined
whether some cells are dying and an equal number of cells are dividing, or the population
of cells has simply stopped growing and dividing. The stationary phase, like the lag
phase, is not necessarily a period of quiescence. Bacteria that produce secondary
metabolites, such as antibiotics, do so during the stationary phase of the growth cycle
(Secondary metabolites are defined as metabolites produced after the active stage of
growth). It is during the stationary phase that spore-forming bacteria have to induce or
unmask the activity of dozens of genes that may be involved in sporulation process.

4. Death Phase. If incubation continues after the population reaches stationary phase, a
death phase follows, in which the viable cell population declines. (Note, if counting by
turbidimetric measurements or microscopic counts, the death phase cannot be observed.).
During the death phase, the number of viable cells decreases geometrically
(exponentially), essentially the reverse of growth during the log phase.

3.3 Growth Rate and Generation Time

As mentioned above, bacterial growth rates during the phase of exponential growth,
under standard nutritional conditions (culture medium, temperature, pH, etc.), define the
bacterium's generation time. Generation times for a few bacteria are shown in Table 2.

Table 2. Generation times for some common bacteria under optimal conditions of growth.

Bacterium Medium Generation Time (minutes)

Escherichia coli Glucose-salts 17
Bacillus megaterium Sucrose-salts 25
Streptococcus lactis Milk 26
Streptococcus lactis Lactose broth 48
Staphylococcus aureus Heart infusion broth 27-30
Lactobacillus acidophilus Milk 66-87
Rhizobium japonicum Mannitol-salts-yeast extract 344-461
Mycobacterium tuberculosis Synthetic 792-932
Treponema pallidum Rabbit testes 1980

Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial population is by

geometric progression. If we start with one cell, when it divides, there are 2 cells in the
first generation, 4 cells in the second generation, 8 cells in the third generation, and so on.
The generation time is the time interval required for the cells (or population) to divide.

G (generation time) = t(time, in minutes or hours)/n(number of generations)

G = t/n

If

G = generation time (time for the cells to divide)

t = time interval in hours or minutes

B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

n = number of generations (number of times the cell population doubles during the time
interval)

b = B x 2n (This equation is an expression of growth by binary fission)

Hence,

logb = logB + nlog2

n = logb - logB
log2
n = logb - logB
0.301

n = 3.3 logb/B

G = t/n

Solve for G

G= t
3.3 log b/B

Example: What is the generation time of a bacterial population that increases from 10,000 cells to
10,000,000 cells in four hours of growth?

G= t
3.3 log b/B

G= 240 minutes
3.3 log 107/104

G = 240 minutes
3.3 x 3

G = 24 minutes

Class assignment

A bacterium with an initial cell population of 103 cells was

cultivated in a closed system and has a generation time of 15
minutes. The bacterium was estimated to attain a final cell
population of X cells in 6 hours. Estimate the final cell population
(X).

3.4 Continuous Culture of Bacteria

The cultures so far discussed for growth of bacterial populations are called batch
cultures. Since the nutrients are not renewed, exponential growth is limited to a few
generations. Bacterial cultures can be maintained in a state of exponential growth over
long periods of time using a system of continuous culture, designed to relieve the
conditions that stop exponential growth in batch cultures. Continuous culture, in a device
called a chemostat, can be used to maintain a bacterial population at a constant density, a
situation that is, in many ways, more similar to bacterial growth in natural environments.

In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once

growth is initiated, fresh medium is continuously supplied from the reservoir. The
volume of fluid in the growth chamber is maintained at a constant level by some sort of
overflow drain. Fresh medium is allowed to enter into the growth chamber at a rate that
limits the growth of the bacteria. The bacteria grow (cells are formed) at the same rate
that bacterial cells (and spent medium) are removed by the overflow. The rate of
addition of the fresh medium determines the rate of growth because the fresh
medium always contains a limiting amount of an essential nutrient. Thus, the
chemostat relieves the insufficiency of nutrients, the accumulation of toxic
substances, and the accumulation of excess cells in the culture, which are the
parameters that initiate the stationary phase of the growth cycle. The bacterial
culture can be grown and maintained at relatively constant conditions, depending on the
flow rate of the nutrients.

Figure 4. Schematic diagram of a chemostat a device for the continuous culture of bacteria.
The chemostat relieves the environmental conditions that restrict growth by continuously
 supplying nutrients to cells and removing waste substances and spent cells from the culture
medium.

Synchronous Growth of Bacteria

Studying the growth of bacterial populations in batch or continuous cultures does not
permit any conclusions about the growth behavior of individual cells, because the
distribution of cell size (and hence cell age) among the members of the population is
completely random. Information about the growth behavior of individual bacteria can,
however, be obtained by the study of synchronous cultures. Synchronized cultures must
be composed of cells which are all at the same stage of the bacterial cell cycle.
Measurements made on synchronized cultures are equivalent to measurements made on
individual cells.

A number of clever techniques have been devised to obtain bacterial populations at the
same stage in the cell cycle. Some techniques involve manipulation of environmental
parameters which induces the population to start or stop growth at the same point in the
cell cycle, while others are physical methods for selection of cells that have just
completed the process of binary fission. Theoretically, the smallest cells in a bacterial
population are those that have just completed the process of cell division.
Synchronous growth of a population of bacterial cells is illustrated in Figure 5.
Synchronous cultures rapidly lose synchrony because not all cells in the population
divide at exactly the same size, age or time.

Figure 5. The synchronous growth of a bacterial population. By careful selection of cells that
have just divided, a bacterial population can be synchronized in the bacterial cell division cycle.
Synchrony can be maintained for only a few generations.
 CHAPTER IV

CONTROL OF MICROBIAL GROWTH

4.1 Introduction

The control of microbial growth is necessary in many practical situations, and significant
advances in agriculture, medicine, and food science have been made through study of this
area of microbiology.

"Control of growth", as used here, means to prevent growth of microorganisms. This

control is effected in two basic ways: (1) by killing microorganisms or (2) by inhibiting
the growth of microorganisms. Control of growth usually involves the use of physical or
chemical agents which either kill or prevent the growth of microorganisms. Agents which
kill cells are called cidal agents; agents which inhibit the growth of cells (without killing
them) are referred to as static agents. Thus the term bactericidal refers to killing bacteria
and bacteriostatic refers to inhibiting the growth of bacterial cells. A bactericide kills
bacteria, a fungicide kills fungi, and so on.
Sterilization is the complete destruction or elimination of all viable organisms (in or on
an object being sterilized). There are no degrees of sterilization: an object is either sterile
or not. Sterilization procedures involve the use of heat, radiation or chemicals, or physical
removal of cells.

4.2 Methods of Sterilization

Heat: most important and widely used. For sterilization always consider type of heat,
time of application and temperature to ensure destruction of all microorganisms.
Endospores of bacteria are considered the most thermoduric of all cells so their
destruction guarantees sterility.

Incineration: burns organisms and physically destroys them. Used for needles ,
inoculating wires, glassware, etc. and objects not destroyed in the incineration process.

Boiling: 100o for 30 minutes. Kills everything except some endospores (Actually, for the
purposes of purifying drinking water 100o for five minutes is probably adequate though
there have been some reports that Giardia cysts can survive this process). To kill
endospores, and therefore sterilize the solution, very long or intermittent boiling is
required.

Autoclaving (steam under pressure or pressure cooker): 121o for 15 minutes (15#/in2
pressure). Good for sterilizing almost anything, but heat-labile substances will be
denatured or destroyed.

Dry heat (hot air oven): 160o/2hours or 170o/1hour. Used for glassware, metal, and
objects that won't melt.
The protocol and recommendations for the use of heat to control microbial growth are
given in Table 1.

Table 1. Recommended use of heat to control bacterial growth

Treatment Temperature Effectiveness

Vaporizes organic material on nonflammable
Incineration >500o surfaces but may destroy many substances in
the process
30 minutes of boiling kills microbial pathogens
Boiling 100o and vegetative forms of bacteria but may not
kill bacterial endospores
Three 30-minute intervals of boiling, followed
Intermittent boiling 100o
by periods of cooling kills bacterial endospores
kills all forms of life including bacterial
Autoclave and pressure
121o/15 minutes endospores. The substance being sterilized
cooker (steam under
at 15# pressure must be maintained at the effective T for the
pressure)
full time
 For materials that must remain dry and which
are not destroyed at T between 121o and 170o
Dry heat (hot air oven) 160o/2 hours
Good for glassware, metal, not plastic or
rubber items
Same as above. Note increasing T by 10
Dry heat (hot air oven) 170o/1 hour degrees shortens the sterilizing time by 50
percent
kills most vegetative bacterial cells including
Pasteurization (batch
63o/30 minutes pathogens such as streptococci, staphylococci
method)
and Mycobacterium tuberculosis
Effect on bacterial cells similar to batch
Pasteurization (flash method; for milk, this method is more
72o/15 seconds
method) conducive to industry and has fewer
undesirable effects on quality or taste

Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is usually used
(commonly used to sterilize the surfaces of objects), although x-rays and microwaves are
possibly useful.

Filtration: involves the physical removal (exclusion) of all cells in a liquid or gas,
especially important to sterilize solutions which would be denatured by heat (e.g.
antibiotics, injectable drugs, amino acids, vitamins, etc.)

Chemical and gas: (formaldehyde, glutaraldehyde, ethylene oxide) toxic chemicals kill
all forms of life in a specialized gas chamber.

4.3 Control of Microbial Growth by Physical Agents

Applications of Heat The lethal temperature varies in microorganisms. The time

required to kill depends on the number of organisms, species, nature of the product being
heated, pH, and temperature. Whenever heat is used to control microbial growth
inevitably both time and temperature are considered.

Sterilization (boiling, autoclaving, hot air oven) kills all microorganisms with heat;
commonly employed in canning, bottling, and other sterile packaging procedures.

Pasteurization is the use of mild heat to reduce the number of microorganisms in a

product or food. In the case of pasteurization of milk the time and temperature depend on
killing potential pathogens that are transmitted in milk, i.e., staphylococci, streptococci,
Brucella abortus and Mycobacterium tuberculosis. For pasteurzation of milk: batch
nethod: 63o/30minutes; flash method: 71o/15 seconds.
Low temperature (refrigeration and freezing): Most organisms grow very little or not
at all at 0 oC. Store perishable foods at low temperatues to slow rate of growth and
consequent spoilage (e.g. milk). Low temperatures are not bactericidal. Psychrotrophs,
rather than true psychrophiles, are the usual cause of food spoilage in refrigerated foods.

Drying (removal of H2O): Most microorganisms cannot grow at reduced water activity
(Aw < 0.90). Often used to preserve foods (e.g. fruits, grains, etc.). Methods involve
removal of water from product by heat, evaporation, freeze-drying, addition of salt or
sugar.

Irradiation (microwave, UV, x-ray): destroys microorganisms as described under

"sterilization". Many spoilage organisms are easily killed by irradiation. In some parts of
Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.
The practice has not been accepted in the U.S.

4.4 Control of microbial growth by chemical agents

Antimicrobial agents are chemicals that kill or inhibit the growth microorganisms.
Antimicrobial agents include chemical preservatives and antiseptics, as well as drugs
used in the treatment of infectious diseases of plants and animals. Antimicrobial agents
may be of natural or synthetic origin, and they may have a static or cidal effect on
microorganisms.

Types of antimicrobial agents

Antiseptics: microbicidal agents harmless enough to be applied to the skin and mucous
membrane; should not be taken internally. Examples: mercurials, silver nitrate, iodine
solution, alcohols, detergents.

Disinfectants: Agents that kill microorganisms, but not necessarily their spores, not safe
for application to living tissues; they are used on inanimate objects such as tables, floors,
utensils, etc. Examples: chlorine, hypochlorites, chlorine compounds, lye, copper sulfate,
quaternary ammonium compounds.

Note: disinfectants and antiseptics are distinguished on the basis of whether they are safe
for application to mucous membranes. Often, safety depends on the concentration of the
compound. For example, sodium hypochlorite (chlorine), as added to water is safe for
drinking, but "chlorox" (5% hypochlorite), an excellent disinfectant, is hardly safe to
drink.
Common antiseptics and disinfectants and their uses are summarized in Table 2.

Table 2. Common antiseptics and disinfectants.

Chemical Action Uses

Denatures proteins and
Ethanol (50-70%) Antiseptic used on skin
solubilizes lipids
Denatures proteins and
Isopropanol (50-70%) Antiseptic used on skin
solubilizes lipids
Reacts with NH2, SH
Formaldehyde (8%) Disinfectant, kills endospores
and COOH groups
Tincture of Iodine (2% I2 in 70%
Inactivates proteins Antiseptic used on skin
alcohol)
Forms hypochlorous
Disinfect drinking water;
Chlorine (Cl2) gas acid (HClO), a strong
general disinfectant
oxidizing agent
General antiseptic and used in
Silver nitrate (AgNO3) Precipitates proteins
the eyes of newborns
Inactivates proteins by Disinfectant, although
Mercuric chloride reacting with sulfide occasionally used as an
groups antiseptic on skin
Detergents (e.g. quaternary Skin antiseptics and
Disrupts cell membranes
ammonium compounds) disinfectants
Phenolic compounds (e.g.
Antiseptics at low
carboloic acid, lysol, Denature proteins and
concentrations; disinfectants at
hexylresorcinol, disrupt cell membranes
high concentrations
hexachlorophene)
Disinfectant used to sterilize
Ethylene oxide gas Alkylating agent heat-sensitive objects such as
rubber and plastics

Preservatives: static agents used to inhibit the growth of microorganisms, most often in
foods. If eaten they should be nontoxic. Examples; calcium propionate, sodium benzoate,
formaldehyde, nitrate, sulfur dioxide. Table 3 is a list of common preservative and their
uses.

Table 3. Common food preservatives and their uses

Effective
Preservative Uses
Concentration
Propionic acid and Antifungal agent in breads, cake, Swiss
0.32%
propionates cheeses
Sorbic acid and Antifungal agent in cheeses, jellies, syrups,
0.2%
sorbates cakes
Benzoic acid and Antifungal agent in margarine, cider,
0.1%
benzoates relishes, soft drinks
Sodium diacetate 0.32% Antifungal agent in breads
Antimicrobial agent in cheeses, buttermilk,
Lactic acid unknown
yogurt and pickled foods
Antimicrobial agent in dried fruits, grapes,
Sulfur dioxide, sulfites 200-300 ppm
molasses
Sodium nitrite 200 ppm Antibacterial agent in cured meats, fish
Prevents microbial spoilage of meats, fish,
Sodium chloride unknown
etc.
Prevents microbial spoilage of preserves,
Sugar unknown
jams, syrups, jellies, etc.
Prevents microbial spoilage of meats, fish,
Wood smoke unknown
etc.

Chemotherapeutic agents: antimicrobial agents of synthetic origin useful in the

treatment of microbial or viral disease. Examples: sulfonilamides, isoniazid, ethambutol,
AZT, chloramphenicol. Note that the microbiologist's definition of a chemotherapeutic
agent requires that the agent be used for antimicrobial purposes and so excludes synthetic
agents used for therapy against diseases that are not of microbial origin.

Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other

microorganisms. This is the microbiologist's definition. A more broadened definition of
an antibiotic includes any chemical of natural origin (from any type of cell) which has the
effect to kill or inhibit the growth of other types cells. Since most clinically-useful
antibiotics are produced by microorganisms and are used to kill or inhibit infectious
Bacteria, we will follow the classic definition.

Antibiotics are low molecular-weight (non-protein) molecules produced as secondary

metabolites, mainly by microorganisms that live in the soil. Among the molds, the
notable antibiotic producers are Penicillium and Cephalosporium , which are the main
source of the beta-lactam antibiotics (penicillin and its relatives). In the Bacteria, the
Actinomycetes, notably Streptomyces species, produce a variety of types of antibiotics
including the aminoglycosides (e.g. streptomycin), macrolides (e.g. erythromycin), and
the tetracyclines. Endospore-forming Bacillus species produce polypeptide antibiotics
such as polymyxin and bacitracin. The table below (Table 4) is a summary of the classes
of antibiotics and their properties including their biological sources.

Table 4. Classes of antibiotics and their properties.

Chemical class Examples Biological Spectrum Mode of action

 (effective
source
against)
Inhibits steps in
Penicillium cell wall
Beta-lactams
Penicillin G, notatum and Gram-positive (peptidoglycan)
(penicillins and
Cephalothin Cephalosporium bacteria synthesis and
cephalosporins)
species murein
assembly
Inhibits steps in
Gram-positive cell wall
Semisynthetic Ampicillin, and Gram- (peptidoglycan)
penicillin Amoxycillin negative synthesis and
bacteria murein
assembly
Gram-positive
Clavamox is Suicide inhibitor
Streptomyces and Gram-
Clavulanic Acid clavulanic acid of beta-
clavuligerus negative
plus amoxycillin lactamases
bacteria
Inhibits steps in
Gram-positive cell wall
Chromobacter and Gram- (peptidoglycan)
Monobactams Aztreonam
violaceum negative synthesis and
bacteria murein
assembly
Inhibits steps in
Gram-positive cell wall
Streptomyces and Gram- (peptidoglycan)
Carboxypenems Imipenem
cattleya negative synthesis and
bacteria murein
assembly
Gram-positive Inhibit
Streptomyces and Gram- translation
Aminoglycosides Streptomycin
griseus negative (protein
bacteria synthesis)
Gram-positive
Inhibit
and Gram-
Micromonospora translation
Gentamicin negative
species (protein
bacteria esp.
synthesis)
Pseudomonas
Glycopeptides Vancomycin Streptomyces Gram-positive Inhibits steps in
orientales bacteria, esp. murein
Staphylococcus (peptidoglycan)
aureus biosynthesis and
 assembly
Gram-positive
and Gram- Inhibits
Streptomyces negative translation
Lincomycins Clindamycin
lincolnensis bacteria esp. (protein
anaerobic synthesis)
Bacteroides
Gram-positive
bacteria, Gram-
negative Inhibits
Streptomyces bacteria not translation
Macrolides Erythromycin
erythreus enterics, (protein
Neisseria, synthesis)
Legionella,
Mycoplasma
Damages
Bacillus Gram-negative
Polypeptides Polymyxin cytoplasmic
polymyxa bacteria
membranes
Inhibits steps in
murein
Gram-positive
Bacitracin Bacillus subtilis (peptidoglycan)
bacteria
biosynthesis and
assembly
Inactivate
Streptomyces membranes
Polyenes Amphotericin Fungi
nodosus containing
sterols
Inactivate
Streptomyces membranes
Nystatin Fungi (Candida)
noursei containing
sterols
Gram-positive
Inhibits
and Gram-
transcription
Streptomyces negative
Rifamycins Rifampicin (eubacterial
mediterranei bacteria,
RNA
Mycobacterium
polymerase)
tuberculosis
Gram-positive
Inhibit
and Gram-
Streptomyces translation
Tetracyclines Tetracycline negative
species (protein
bacteria,
synthesis)
Rickettsias
Semisynthetic Doxycycline Gram-positive Inhibit
 and Gram-
negative
translation
bacteria,
tetracycline (protein
Rickettsias
synthesis)
Ehrlichia,
Borellia
Gram-positive Inhibits
Streptomyces and Gram- translation
Chloramphenicol Chloramphenicol
venezuelae negative (protein
bacteria synthesis)

CHAPTER V

MICROBIAL MORPHOGENESIS AND DIFFERENTIATION

5.1 Microbial Morphogenesis
5.1.1 Colony morphology

A. Colony surface: smooth; rough; dull; glistening; wrinkled; contoured;

granular
B. Optical characteristics: opaque; translucent; dull; glossy; iridescent;
opalescent
C. Consistency: butyrous; membranous; viscid; brittle
D. Pigmentation: any color; soluble or non-soluble
E. Forms: circular; irregular; spindle; filamentous; rhizoid

F. Elevation: raised; flat; convex; umbonate; pulvinate

 G. Margins: entire; undulate; lobate; filamentous; curled; spreading

5.1.2 Cell morphology:

H. Bacterial morphologies: Straight rod; club-shaped rod; branching rod;

comma forms; spore forming rod; spiral; coccus
I. Colony arrangements (for cocci): diplococci; chain; tetrad; cluster
J. Flagella arrangements: monotrichous; lophotrichous; amphitrichous;
peritrichous
 CHAPTER VII

Carbohydrate metabolism and energy production etc

Summary of glycolysis

a. Phase I: Input of 2 ATP for 1 glucose

b. Phase II: Output of 4 ATP and 2 NADH for 1 glucose
c. Overall reaction:

Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 pyruvate + 2 NADH + 2 H+ + 2 ATP

d. The ATP produced represents only a small portion (about 5%) of the energy
available from complete oxidation of glucose.

Energy yield from glucose

(a) Under anaerobic conditions:

(i) Phase I of glycolysis requires input of 2 ATP per glucose
(ii) Phase II of glycolysis produces 4 ATP, 2 NADH and 2 pyruvate per glucose.
(iii) Electrons from NADH donated to another molecule (e.g pyruvate) so net
energy yield is 2 ATP.
(iv) Very small (<5%) of the energy potential available if glucose was completely
oxidized.

(b) Under aerobic conditions:

(i) Able to complete the oxidation
(ii) Convert pyruvate to acetyl-CoA
(iii) Acetyl-CoA will be oxidized via the citric acid cycle.
(iv) The electrons will be carried to the electron transport system and ultimately
be given to oxygen.
(v) These processes will result in the production of an additional 34-36 ATP.
Test 2: 17 May 2012 @ 5 pm
Lipid Metabolism

Triglyceride is the major form of lipids. Lipids are a group of chemicals, usually fats that
do not dissolve in water, but dissolve in ether.

Triglycerides serve as prime energy source in many organisms.

(a) Carbon in fatty acids is highly reduced state.

(b) More highly reduced than carbonhydrate.

(c) Burning one gram of fat yield approx. 9.5 kcal and one gram of CHO yields
approx. 4.2 kcal.

(d) Difference even greater in stored state due to the attraction of hydroxyl groups of
CHO for water.

(e) Total effect is stored lipid has about six times the energy value of CHO.
Stages in Fatty acid oxidation reaction

(a) Activation – conversion of FAs to an energy-rich molecule.

(b) Transport into mitochondrion

(c) First oxidation (insertion of double bond)

(d) Hydration – addition of water across the double bond

(e) Second oxidation

(f) Cleavage resulting in the production of acetyl-CoA and fatty acyl-CoA that is two
carbon less.
Fatty Acid Synthesis

 Occurs under conditions of excess fuel from carbohydrate or protein and is

characteristic of all living organisms – animal cells and yeasts, prokaryotes and
plants alike.
 Takes place in the liver of animal – in cytoplasm of cells
 Does not take place while F/A oxidation is occurring
 Reaction sequences not just reverse of oxidation. A lot of differences exist.

Reaction sequence: begins with acetyl-CoA (from CHO or protein) and involved the
following:

(a) Transport of acetyl group from inside mitochondria

(b) Activation of the acetyl group

The regulated enzyme is activated by insulin and inhibited by glucagon and

epinephrine.

(c) Condensation reaction

 Acetyl group from (acetyl-CoA) and malonyl group (from malonyl-CoA)

transferred to acyl carrier protein (ACP) on multifunctional protein (fatty acid
synthase).
 Acetyl group and malonyl group condense with loss of CO2

(d) First reduction reaction

(e) Dehydration

(f) Second reduction reaction

(g) Reaction continues with butyryl-ACP combining with another malonyl-ACP and
loss of CO2 – add two more carbons.
(h) Cycle repeated until reaches palmityl-ACP and then palmitic acid is released.
Nitrogen and Amino Acid Metabolism
 Amino acid (Nitrogen) metabolism

Introduction I General: digestion, absorption transamination and urea.

Q01. In which form atmospheric nitrogen is used by all organisms?

A01.

 Nitrogen ranks fourth important element after carbon, hydrogen and

oxygen, of the mass of living cells.
 Though atmospheric nitrogen N2 is most abundant but is too inert for use in
most biochemical processes.
 Only few microorganisms can convert N2 to biological useful form such as
NH3, amino groups are used with great economy in biological system.
 Atmospheric nitrogen needs to be converted by some organisms into forms
acceptable to the all the organisms.
 Nitrogen fixation is carried out by bacterial nitrogensases forming reduced
nitrogen, NH4+ that can then be used by all organisms to form amino acids.

Q02. How nitrogen enters the human body?

A02. Reduced nitrogen enters the human body as dietary free amino acids, protein,
and the ammonia produced by intestinal tract bacteria. Amino acids derived from
dietary proteins are the main source of amino groups.

Q03. In which metabolic circumstances, amino acids in body can undergo oxidative
degradation?

A03. In body, amino acids can undergo oxidative degradation in three different
metabolic conditions:

1. During the normal synthesis and degradation of cellular proteins (protein

turnover). Some of amino acids released during protein breakdown will
undergo oxidative degradation if they are not needed for new protein
synthesis.

2. When a diet is rich in protein, the surplus amino acids may be

catabolized when they are in excess. Amino acids cannot be stored.

3. During starvation or in diabetes mellitus, when carbohydrates are either

unavailable or not properly utilized, body proteins are used as fuel.

Under these different circumstances amino acids lose their amino groups and the
alpha-keto acids so formed may undergo oxidation to CO2 and H2O. In addition,
the carbon skeleton of amino acids provides three and four carbon units that can be
converted to glucose to be used by body.
Q04. Does metabolic energy derived from amino acids varies greatly with the type of
organism and with metabolic situation?

A04.

 Carnivores immediately following a meal may obtain up to 90% of their

energy requirements from amino acid oxidation.
 Herbivores may obtain a small fraction of their energy needs from amino
acids.
 Most microorganisms can scavenge amino acids form their environment and
can use for their metabolic need.
 Photosynthetic plants rarely oxidize amino acids for energy purpose. Instead
they convert CO2 and H2O into carbohydrates that are mainly used as
energy source.
 The amount of amino acids in plant tissues are carefully used for
biosynthesis of proteins, nucleic acids and other molecules needed to support
the growth.
 Amino acids catabolism does occur in plants and their metabolites are used
for other biosynthetic pathways.

Q05. What is gastrin and what is its role in protein digestion?

A05. Entry of protein in to stomach stimulates gastric mucosa to secreate the

hormone gastrin. Which stimulates the secretion of hydrochloric acid by the parietal
cells of gastric glands and pepsinogen by the chief cells.

Q06. How protein digestion takes place?

A06. Protein digestion begins in the stomach, where a proenzyme called pepsinogen
is secreted, autocatalytically converted to Pepsin A, and used for the first step of
proteolysis. However, most proteolysis takes place in the duodenum as a
consequence of enzyme activities secreted by the pancreas. All of the serine
proteases and the zinc peptidases of pancreatic secretions are produced in the form
of their respective proenzymes. These proteases are both endopeptidase and
exopeptidase, and their combined action in the intestine leads to the production of
amino acids, dipeptides, and tripeptides, all of which are taken up by enterocytes of
the mucosal wall.

Q07. How preoteolytic enzymes are regulated?

A07. A circuitous regulatory pathway leading to the secretion of proenzymes into

the intestine is triggered by the appearance of food in the intestinal lumen.

- Special mucosal endocrine cells secret the peptide hormones cholecystokinin

(CCK) and secretin into the circulatory system.
 - Together, CCK and secretin cause contraction of the gall bladder and the
exocrine secretion of a bicarbonate-rich, alkaline fluid, containing protease
proenzymes from the pancreas into the intestine.

- A second, paracrine role of CCK is to stimulate adjacent intestinal cells to

secrete enteropeptidase, a protease that cleaves trypsinogen to produce trypsin.

- Trypsin also activates trypsinogen as well as all the other proenzymes in the
pancreatic secretion, producing the active proteases and peptidases that
hydrolyze dietary polypeptides.

Q08. What is celiac disease?

A08. Celiac disease is a condition in which the intestinal enzymes are unable to
digest certain water insoluble proteins of wheat, particularly gliadin, which is
injurious to the cells lining the small intestine. Wheat products must be avoided in
this condition.

Q09. What is acute pancreatitis?

A09. In this condition the normal pathway of secretion of pancreatic juice into the
intestine is obstructed. Thus the zymogens of the proteolytic enzymes are converted
to the active forms inside the pancreatic cells, prematurely. This active proteolytic
enzymes act on the pancreatic tissue itself, causing serious destruction of pancreas,
which is very painful and can be fatal.

Q10. How intestinal bacterial activity contributes to nitrogen metabolism?

A10. Many other nitrogenous compounds are formed in the intestine as a result of
intestinal bacterial activity. Some have powerful pharmacological (vasopressor)
effects. Intestinal bacteria convert lysine, arginine, tyrosine, ornithine and histidine
to their vasopressor amines such as cadaverene, agmatine, tyramine, putrescine and
histamine respectively.

Q11. What are essential amino acids?

A11.

Prokaryotes such as E. coli can make the carbon skeletons of all 20 amino acids and
transaminate those carbon skeletons with nitrogen from glutamine or glutamate to
complete the amino acid structures.

Humans cannot synthesize the branched carbon chains found in branched chain
amino acids or the ring systems found in phenylalanine and the aromatic amino
acids; nor can we incorporate sulfur into covalently bonded structures.
Therefore, the 9 so-called essential amino acids must be supplied from the diet.
They are:

Branched chain amino acids: leucine, Isoleucine, valine,

Aromatic amino acids: Phenylalanine, tryptophan,

Sulphur containing amino acid: methionine

Basic amino acids: Histidine and lysine.

And threonine.

Q12. What are semi-essential amino acids?

A12.

 Depending on the composition of the diet and physiological state of an

individual, one or another of the non- essential amino acids may also become
a required dietary component forming a group of semi-essential amino acids.
 For example, arginine is not usually considered to be essential, because
enough for adult needs is made by the urea cycle. However, the urea cycle
generally does not provide sufficient arginine for the needs of a growing
child.
 To take a different type of example, cysteine and tyrosine are considered
non-essential but are formed from the essential amino acids methionine and
phenylalanine, respectively.
 If sufficient cysteine and tyrosine are present in the diet, the requirements
for methionine and phenylalanine are markedly reduced; conversely, if
methionine and phenylalanine are present in only limited quantities, cysteine
and tyrosine can become essential dietary components.

Finally, it should be recognized that if the α-keto acids corresponding to the carbon
skeleton of the essential amino acids are supplied in the diet, aminotransferases in
the body will convert the keto acids to their respective amino acids, largely
supplying the basic needs.

Q13. What happens during the degradation of amino acids?

A13. Amino acids can undergo oxidative degradation as a consequence of protein

turnover; when the diet is particularly rich in protein or when carbohydrates are
not available like in starvation or in diabetes mellitus.

The degradative pathway of every amino acid requires the separation of the amino
group from the carbon skeleton. The carbon skeletons enter the Krebs cycle or are
channeled into gluconeogenesis. Part of the ammonia is reused for biosynthetic
purpose; part is excreted directly and the rest is excreted as urea.

Q14. How removal of nitrogen from amino acids takes place?

A14. Most of the amino acids are metabolized in liver. Some of the ammonia that is
generated is recycled and used in a variety of biosynthetic processes. The excess
ammonia is either excreted directly or converted to uric acid or urea for excretion
depending on the organism. Excess ammonia generated in extrahepatic tissues is
transported to the liver for excretion after converting to a proper form. Nitrogen
elimination begins intracellularly with protein degradation. There are two main
routes for converting intracellular proteins to free amino acids: a lysosomal
pathway, by which extracellular and some intracellular proteins are degraded, and
cytosolic pathways that are important in degrading proteins of intracellular origin.

Q15. What happens in cystosolic pathway?

Q15. In one cytosolic pathway:

- A protein known as ubiquitin is activated by conversion to an AMP

derivative.

- And cytosolic proteins that are damaged or otherwise destined for

degradation are enzymically tagged with the activated ubiquitin.

- Ubiquitin-tagged proteins are then attacked by cytosolic ATP-dependent

proteases that hydrolyze the targeted protein, releasing the ubiquitin for
further rounds of protein targeting.

Q16. How amino acid nitrogen is removed?

A16. The dominant reactions involved in removing amino acid nitrogen from the
body are known as transaminations. This class of reactions funnels nitrogen from all
free amino acids into a small number of compounds; then, either they are
oxidatively deaminated, producing ammonia, or their amine groups are converted
to urea by the urea cycle.

Q17. How transaminations take place?

A17. Transaminations involve moving a α-amino group from a donor α-amino acid
to the keto carbon of an acceptor α-keto acid. These reversible reactions are
catalyzed by a group of intracellular enzymes known as transaminases
(aminotransferases), which employ covalently bound pyridoxal phosphate as a
cofactor.

Transaminases exist for all amino acids except threonine and lysine.
Q18. Which compounds are most commonly involved in transamination?

A18. The most common compounds involved as a donor/acceptor pair in

transamination reactions are glutamic acid and α-ketoglutaric acid, which
participate in reactions with many different aminotransferases.

Q19. Which aminotransferase is clinically important?

A19. Serum aminotransferases such as serum glutamate-oxaloacetate-

aminotransferase (SGOT) have been used as clinical markers of tissue damage, with
increasing serum levels indicating an increased extent of damage.

Q20. How creatinine is formed and what is its clinical significance?

A20.

 The first reaction in creatinine formation is the transfer of the amido (or
amidine) group of arginine to glycine, forming guanidinoacetate.
 Subsequently, a methyl group is transferred from the ubiquitous 1-carbon-
donor S-adenosylmethionine to guanidinoacetate to produce creatine (from
which phosphocreatine is formed), some of which spontaneously cyclizes to
creatinine, and is eliminated in the urine.
 The quantity of urine creatinine is generally constant for an individual and
approximately proportional to muscle mass.
 In individuals with damaged muscle cells, creatine leaks out of the damaged
tissue and is rapidly cyclized, greatly increasing the quantity of circulating
and urinary creatinine.
 A small but clinically important amount of creatinine is excreted in the urine
daily, and the creatinine clearance rate is often used as an indicator of kidney
function.

Q21. How glutamete is a prominent intermediate in nitrogen elimination?

A21.

 Because of the participation of α-ketoglutarate in numerous transaminations,

glutamate is a prominent intermediate in nitrogen elimination as well as in
anabolic pathways.
 Glutamate formed in the course of nitrogen elimination is either oxidatively
deaminated by liver glutamate dehydrogenase, forming ammonia.
 The ammonia thus formed is converted to glutamine by glutamine synthase
and transported to kidney tubule cells.
 There the glutamine is sequentially deamidated by glutaminase and
deaminated by kidney glutamate dehydrogenase.
 The ammonia produced in the latter two reactions is excreted as NH4+ in the
urine, where it helps maintain urine pH in the normal range of pH 4 to pH 8.
  The extensive production of ammonia by peripheral or liver glutamate
dehydrogenase is not feasible because of the highly toxic effects of circulating
ammonia.
 Normal serum ammonium concentrations are in the range of 20-40 mmol,
and an increase in circulating ammonia to about 400 mmol causes alkalosis
and neurotoxicity.

Q22. Which amino acid related reaction is therapeutically significant?

A22.

 A therapeutically useful amino acid-related reaction is the amidation of

aspartic acid to produce asparagine.
 The enzyme asparagine synthase catalyzes the ATP, requiring the
transamidation reaction shown below:
aspartate + glutamine + ATP --> glutamate + asparagine + AMP + PPi
 Most cells perform this reaction well enough to produce all the asparagine
they need.
 However, some leukemia cells require exogenous asparagine, which they
obtain from the plasma. Chemotherapy using the enzyme asparaginase takes
advantage of this property of leukemic cells by hydrolyzing serum
asparagine to ammonia and aspartic acid, thus depriving the neoplastic cells
of the asparagine that is essential for their characteristic rapid growth.

Q23. How clinically important are sterospecific amino acid oxidases?

A23.

 In the peroxisomes of mammalian tissues, especially liver, there are 2

stereospecific amino acid oxidases involved in elimination of amino acid
nitrogen.
 D-amino acid oxidase is an FAD-linked enzyme, and while there are few D-
amino acids that enter the human body the activity of this enzyme in liver is
quite high. L-amino acid oxidase is FMN-linked and has broad specificity for
the L amino acids.
 A number of substances, including oxygen, can act as electron acceptors
from the flavoproteins. If oxygen is the acceptor the product is hydrogen
peroxide, which is then rapidly degraded by the catalases found in liver and
other tissues.
 Missing or defective biogenesis of peroxisomes or L-amino acid oxidase
causes generalized hyper-aminoacidemia and hyper-aminoaciduria,
generally leading to neurotoxicity and early death.

Q24. Discribe the role and significance of glutamate dehydrogenase?

A24.

The reaction catalyzed by glutamate dehydrogenase is:

NH4+ + α -ketoglutarate + NAD (P) H + H+ <----> glutamate + NAD (P)+ + H2O

Glutamate dehydrogenase can utilize either NAD orNADP as cofactor.

 In the forward reaction as shown above glutamate dehydrogenase is

important in converting free ammonia and α-ketoglutarate (α-KG) to
glutamate, forming one of the 20 amino acids required for protein synthesis.
 However, it should be recognized that the reverse reaction is a key
anapleurotic process linking amino acid metabolism with TCA cycle activity.
 In the backward reaction, glutamate dehydrogenase provides an oxidizable
carbon source used for the production of energy.
 As expected for a branch point enzyme with an important link to energy
metabolism, glutamate dehydrogenase is regulated by the cell energy charge.
 ATP and GTP are negative effectors, whereas ADP and GDP are positive
allosteric effectors. Thus, when the level of ATP is high, conversion of
glutamate to α-KG and other TCA cycle intermediates is limited; when the
cellular energy charge is low, glutamate is converted to ammonia and
oxidizable TCA cycle intermediates.

Q25. Describe the role and significance of glutamine synthase.

A25.

The reaction catalyzed by glutamine synthase is:

glutamate + NH4+ + ATP -------> glutamine + ADP + Pi + H+

The glutamine synthatase reaction is also important in several respects. First it

produces glutamine, one of the 20 major amino acids. Second, in animals, glutamine
is the major amino acid found in the circulatory system.

Its role there is to carry ammonia to and from various tissues but principally from
peripheral tissues to the kidney, where the amide nitrogen is hydrolyzed by the
enzyme glutaminase (reaction below); this process regenerates glutamate and free
ammonium ion, which is excreted in the urine.

glutamine + H2O -------> glutamate + NH3

Note that, in this function, ammonia arising in peripheral tissue is carried in a

nonionizable form, which has none of the neurotoxic or alkalosis-generating
properties of free ammonia.
 

Q26. What is Urea Cycle?

A26. About 80% of the excreted nitrogen is in the form of urea, which is also largely
made in the liver, in a series of reactions that are distributed between the
mitochondrial matrix and the cytosol. The series of reactions that form urea is
known as the Urea Cycle or the Krebs-Henseleit Cycle.
Q27. What are essential features of the urea cycle?
A27. The essential features of the urea cycle reactions and their metabolic
regulation are as follows:
1. Arginine from the diet or from protein breakdown is cleaved by the cytosolic
enzyme arginase, generating urea and ornithine.
2. Ornithine arising in the cytosol is transported to the mitochondrial matrix,
where ornithine transcabamoylase catalyzes the condensation of ornithine
with carbamoyl phosphate, producing citrulline. The energy for the reaction
is provided by the high-energy anhydride of carbamoyl phosphate.
3. The product, citrulline, is then transported to the cytosol, where the
remaining reactions of the cycle take place.
4. In a 2-step reaction, catalyzed by cytosolic argininosuccinate synthetase,
citrulline is converted to argininosuccinate. The reaction involves the
addition of AMP (from ATP) to the amido carbonyl of citrulline, forming an
activated intermediate on the enzyme surface (AMP-citrulline), and the
subsequent addition of aspartate to form argininosuccinate.
5. Arginine and fumarate are produced from argininosuccinate by the cytosolic
enzyme argininosuccinate lyase. In the final step of the cycle arginase cleaves
urea from aspartate, regenerating cytosolic ornithine, which can be
transported to the mitochondrial matrix for another round of urea synthesis.

Beginning and ending with ornithine, the reactions of the cycle consumes 3
equivalents of ATP and a total of 4 high-energy nucleotide phosphates. Urea is the
only new compound generated by the cycle; all other intermediates and reactants
are recycled.

Q28. How the regulation of the Urea Cycle takes place in the body?

A28.

The urea cycle operates only to eliminate excess nitrogen.

On high-protein diets the carbon skeletons of the amino acids are oxidized for
energy or stored as fat and glycogen, but the amino nitrogen must be excreted.

To facilitate this process, enzymes of the urea cycle are controlled at the gene level.

When dietary proteins increase significantly, enzyme concentrations rise. On return

to a balanced diet, enzyme levels decline. Under conditions of starvation, enzyme
levels rise as proteins are degraded and amino acid carbon skeletons are used to
provide energy, thus increasing the quantity of nitrogen that must be excreted.

Q29. What happens when excretion of ammonia is deranged?

A29. Built up of ammonia is neurotoxic. Marked brain damage is seen in cases of

failure to make urea via the urea cycle or to eliminate urea through the kidneys. The
result of either of these events is a buildup of circulating levels of ammonium ion.
Aside from its effect on blood pH, ammonia readily traverses the brain blood
barrier and in the brain is converted to glutamate via glutamate dehydrogenase,
depleting the brain of α-ketoglutarate. As the α-ketoglutarate is depleted
oxaloacetate falls correspondingly, and ultimately TCA cycle activity comes to a
halt. In the absence of aerobic oxidative phosphorylation and TCA cycle activity,
irreparable cell damage and neural cell death ensue.

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