SECOND SEMESTER

SKILL ENHANCEMENT COURSE

TOOLS & TECHNIQUES IN BIOCHEMISTRY

Unit 3: Introduction to microbial world

Basic introduction to different microbes, growth medium for microbes- differential and
specific medium. Sterilization and Disinfection

Exercises:
a) Isolation of bacteria from soil, water or air, counting CFU (Colony forming units).
b) Differential staining techniques.
c) Preparation of hand sanitizers.

Basic introduction to different microbes

Microscopic organisms, commonly known as microorganisms or microbes, are found all
around us and even inside our bodies. The category ‘microbes’ includes a massive range of
organisms including acellular organisms like viruses and cellular organisms including bacteria,
fungi, algae, archaea and protozoa.
Microorganisms, as their name implies, are so small that they usually require a
microscope to see. The vast majority of microbes on the earth pose no real threat to humans,
plants or animals; in fact they actually work alongside humans to make the world go round,
aiding decomposition, decay and even helping us to digest our food. However, there are some
microorganisms which negatively impact our lives, causing illness, bad odours and damaging
products and surfaces. Some of the names we regularly hear in the media are Salmonella, E. coli,
MRSA, malaria and bird flu (avian influenza).

Types of Microbes
1. Bacteria, perhaps the most well-known microorganism, are a member of the prokaryotes;
they have no nucleus within the cell and contain no organelles (specialised cellular
‘organs’). Within bacteria there are two classes; Gram-positive bacteria, which possess a
thick peptidoglycan layer and no outer lipid membrane, and Gram-negative bacteria that
have a very thin peptidoglycan layer with an outer lipid membrane composed of
lipopolysaccharides.
Bacteria are extremely diverse and in terms of number are by far the most
successful organism on Earth. Many microflora bacteria can live harmlessly within the
human body, often aiding bodily functions such as digestion. In fact there are more
bacterial cells within the human body than humans cells, although they are much smaller
in size. Bacteria, of all the ‘living’ microorganisms, cause the most problems in terms of
disease in humans, despite only relatively few bacteria being dangerous.
2. Fungi are eukaryotes which means they have a defined nucleus and organelles. The cells
are larger than prokaryotes such as bacteria. Fungal colonies can be visible to the human
eye once they have achieved a certain level of growth, for example mould on bread.
Fungi can be split into three main groups,
 i. moulds which display thread-like (filamentous) growth and multicellular
structures
ii. yeasts which are typically non-filamentous and can be single celled
iii. mushrooms which possess a fruiting body for production of spores.
Fungal Infections (Mycosis) Fungal infections are any disease or condition you
get from a fungus. They usually affect your skin, hair, nails or mucous membranes but
they can also infect your lungs or other parts of your body. Fungi also contain significant
pathogens which can cause disease in plants. However, we also recognise organisms
from this group from widespread use in the food industry, for production of beer and
other foodstuffs, and some fungi can be used for the production of antibiotics.
3. Viruses are microscopic particles that exist almost everywhere on Earth. They are
biological entities that can only thrive and multiply in a host. The host range of a virus is
the spectrum of host cells the virus can infect. There are viruses that infect invertebrates,
vertebrates, plants, protists, fungi, and bacteria. Viruses are considered by many experts
to not be living organisms although this is still a controversial topic amongst scientists.
They essentially consist of nucleic acid (DNA or RNA,) and a protein coat. A virion (a
virus particle) requires a host cell in order to replicate. In many cases the immune system
detects the presence of the virus and takes action leaving us with the symptoms of a
common cold or influenza. Some viruses can cause permanent and irreversible damage to
cells, for example HIV.
4. Algae are a more difficult to define group of organisms, containing both prokaryotes and
eukaryotes by some definitions. Unlike other microorganisms, algae are typically
photosynthesisers and are generally found in marine environments. Confusingly,
cyanobacteria are most commonly referred to as ‘blue-green algae’, however they are in
fact prokaryotic bacteria and not algae. They are an ancient group of photosynthetic
bacteria, and the name blue-green algae is due to their relation to the chloroplast only in
eukaryotic algae. There are other groups such as Archaea and Protozoa which are
generally less well known.
5. Archaea are single-celled prokaryotic organisms. They have distinct molecular
characteristics separating them from bacteria as well as from eukaryotes. Archaea is
derived from the Greek word archaios, meaning “ancient” or “primitive.”
Archaeans include inhabitants of some of the most extreme environments on the
planet. Some live near rift vents in the deep sea at temperatures well over 100 degrees
Centigrade. Others live in hot springs, or in extremely alkaline or acid waters. They have
been found thriving inside the digestive tracts of cows, termites, and marine life where
they produce methane. They live in the anoxic muds of marshes and at the bottom of the
ocean, and even thrive in petroleum deposits deep underground. Some archaeans can
survive the in extremely saline waters, while some live in thermal vents.
6. Protists are simple eukaryotic organisms that are neither plants nor animals or fungi.
Protists are unicellular in nature but can also be found as a colony of cells. Most protists
live in water, damp terrestrial environments or even as parasites. The term ‘Protista’ is
derived from the Greek word “protistos”, meaning “the very first”. Scientists speculate
that protists form a link between plants, animals and fungi as these three kingdoms
 diverged from a common protist-like ancestor, billions of years ago. Though this
“protists-like” ancestor is a hypothetical organism, we can trace some genes found in
modern animals and plants to these ancient organisms. Therefore, these organisms are
traditionally considered the first eukaryotic forms of life and a predecessor to plants,
animals and fungi.

GROWTH MEDIUM FOR MICROBES- DIFFERENTIAL AND SPECIFIC MEDIUM.

Microbiological Culture Media
The survival and growth of microorganisms depend on available nutrients and a favorable
growth environment. In the laboratory, the nutrient preparations that are used for culturing
microorganisms are called media (singular, medium). Three physical forms are used: liquid, or
broth, media; semisolid media; and solid media. The major difference among these media is that
solid and semisolid media contain a solidifying agent (usually agar), whereas a liquid medium
does not.
i. Complex Media
Complex media, provide a variety of nutrients in unspecific quantities because they are
composed of extracts and enzymatic digests of living organisms (e.g., peptones from animal,
plant, or yeast). Therefore, they are suitable for most everyday needs for easy-to-grow microbes
except for metabolic or physiological studies due to the undefined levels of the components.
ii. Differential media contain compounds that allow groups of microorganisms to be visually
distinguished by the appearance of the colony or the surrounding media, usually on the basis of
some biochemical difference between the two groups.
Eg: Blood agar is one type of differential medium, allowing bacteria to be distinguished by the
type of hemolysis produced.
Hemolysis Observation of the hemolytic reactions on blood agar is a very useful tool in
the identification of bacteria, particularly streptococci. The types of hemolysis are defined as
follows:
 alpha (α) hemolysis: An indistinct zone of partial destruction of red blood cells (RBCs)
appears around the colony, often accompanied by a greenish to brownish discoloration of
the medium. Streptococcus pneumoniae and many oral streptococci are α hemolytic.
 beta (β) hemolysis: A clear, colorless zone appears around the colonies, in which the
RBCs have undergone complete lysis. S. pyogenes, S. agalactiae, and several other
species of streptococci are β hemolytic. Many other bacteria besides streptococci can be β
hemolytic, including Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria
monocytogenes, and hemolytic reactions can also be a useful diagnostic tool for these
organisms.
 no (γ) hemolysis: No apparent hemolytic activity or discoloration is produced by the
colony (also called gamma hemolysis).
Sterilization and Disinfection
Sterilization is a process to eradicate all forms of live microorganisms from a substance.
Microbiologically sterile material is one that contains no living organisms at all and the term
sterile is therefore an absolute one. Sterilization is done to preserve the substance for a long time
without decay. Secondly, if a substance is not sterilized, any microbes in it may cause infections
to the people who consume it. So sterilization is essential.
The microbes are invisible to naked eye and even those like bacteria have a protective
sheath on their surface making them resistant to sterilization. For this effective sterilization
techniques are designed and studied in microbiology.

I. Physical sterilization: This class includes heat sterilization and radiation sterilization as well.
Heat sterilization procedure is the one in which the destruction of microorganisms mainly
occurs due to the high temperatures employed. This process includes

(a) Moist heat sterilization.

Moist heat sterilization is otherwise referred as steam sterilization under pressure. Steam
sterilization under pressure is carried out in an autoclave, which is an airtight, jacketed chamber
designed to maintain a high pressure of saturated hot steam. Sterilization of aqueous solutions,
glassware, and rubber articles is best done by steam under pressure. This process is not suitable
or sterilizing solutions of drugs that are thermolabile in nature. Conditions to be followed for the
moist heat sterilization according to The USP XXI and BP 1988 are given below:
* Pressure: 15 lb / square inch (psi)
* Temperature: 121 C
* Time: 15 minutes
(b) Dry Heat Sterilization
Dry heat is the simplest and most economical method of sterilization. This method requires
higher temperatures and longer exposure times to achieve the same microbial-killing efficiency
as compared with that of steam sterilization. A major problem associated with dry heat
sterilization is non uniform distribution of temperature. A fan is usually installed in the oven to
overcome this problem, but unless the fan is properly baffled it may blow around powders that
are to be sterilized. Furthermore, dry heat cannot be used with materials that are heat sensitive. It
is mainly used for sterilization of glass and metal objects. It can also be used to sterilize
thermostable powders and fatty substances.
Conditions to achieve complete sterilization by dry heat sterilization are as follows:
 Cycles recommended as per BP 1988 are:
* A minimum of 180°C for not less than 30 minutes.
* A minimum of 170°C for not less than 1 hour.
* A minimum of 160°C for not less than 2 hours.

(c)Radiation Sterilization
This process is accomplished by exposure to ultraviolet (UV) light or high-energy ionizing
radiation such as gamma rays and accelerated electrons i.e particulate radiation as well. The
radiation employed for the sterilization may be ionizing or non ionizing radiation. Gamma and
X-rays, having energies more than about 10eV, are called ionizing radiations because they have
enough energy to knock electrons away from molecules and ionize them. These radiations create
free hydrogen radicals, hydroxyl radicals and some peroxides which cause different kinds of
intracellular damage. The less energetic ultraviolet radiation cannot ionize and it is absorbed and
excites the electrons and rises them to higher energy states, thus finally destroying the cellular
structure of the microorganism. When properly operated, the UV radiation technique can reduce
the level of airborne bacterial contamination in a room by 90% within 30 min.

II. Chemical sterilization

Chemical sterilization is the elimination of all viable microorganisms and their spores using
liquid or gaseous compounds. The method you use for chemical sterilization varies with the form
of chemical you use.
(a) Gaseous sterilization
Gaseous sterilization is accomplished by exposure to a gas that kills microorganisms. The most
commonly used gas for sterilization is ethylene oxide. Ethylene oxide acts by alkylation.
Ethylene oxide is a cyclic ether that has flammable and explosive properties, especially when
confined and mixed with oxygen. It is therefore often used in combination with inert gases such
as carbon dioxide to avoid hazard.
The effectiveness of ethylene oxide in the sterilization process depends on relative
humidity, gas concentration, temperature, exposure time, and the extent of contamination. The
normal working ranges for the relative humidity and ethylene oxide concentration are 30 to 60%
and 500 to 1000 mg/l, respectively. At these ranges, the times required for complete sterilization
are 2h to 5h at 55°C.
(b) Liquid sterilizers
Liquid sterilizers include Phenol and phenolic compounds which produces a variety of effects on
the microorganisms. Based on their concentration they exert various effects like disruption of
cells, precipitation of cell protein, inactivation of enzymes and leakage of aminoacids from the
cells. Other chemical agents include alcohols, halogens, heavy metals and their compounds,
dyes, detergents, quaternary ammonium compounds and aldehydes etc.