CONDUCT CLEAN-IN-PROCESS

Definition of clean in place

• Clean-In-Place (CIP) systems are automated systems used to clean the interior surfaces of
food and beverage process pipes, processing vessels, tanks, spiral freezers, mixers,
blenders, homogenizers, roasters and associated fittings, without disassembling the
process.
• Thorough, repeatable in-place cleaning is critical to the quality of your product, the safety
of consumers and your bottom line.
• Clean-In-Place (CIP) Systems are engineered to your specific plant application, layout
and utility requirements for effective and efficient sanitary process equipment cleaning.
• Our application-specific CIP design and sizing ensures sufficient flow.
• As a result, appropriate pressure is available to thoroughly remove residue, rinse
effectively, shorten cycle times and promote worker safety.
• CIP systems have a vital role in processing because of the need to keep processing
components free of bacteria.
• Cleaning occurs in process piping, tanks, heat exchangers, and other equipment to
prevent product contamination and maintain processing efficiency.
• CIP, or Cleaning in Place, is a method of cleaning
• Sanitary process lines
• Vessels
• Equipment commonly used in process plants, without having to remove or
disassemble piping or equipment to accommodate cleaning.
• CIP Systems pump cleaning, rinsing, and sanitizing solutions through the same piping
path as the product to eliminate product soil from all internal surfaces.
Advantages of a CIP system
• Minimizes Mistakes: Automating cleaning reduces the chance of human error that can
contribute to an unsafe product.
• Keeps Employees Safe: Reduces chemical exposure by containing cleaning solutions
within the system.
• More Production Time: As less production time is lost to cleaning, more time is spent
making product. Efficient cleaning through CIP systems ensures minimal downtime and
maximizes production output.
• Product Quality: Reliable and repeatable cleaning means sustainable product quality and
consistency. Less contamination means fewer product recalls and higher brand
confidence.
• Utility Savings: Water and energy usage is reduced through repeatable cycle control.
The CIP cleaning cycle
• CIP cycles are typically run either after a processing run that has produced normal soiling
or when changing over a processing line from one product to another, and these CIP
processes ensure thorough cleaning.
• Every CIP cleaning cycle has its own unique set of parameters, so there’s really no
such thing as a “typical” CIP cycle.
• The elements, sequence, and duration of the cleaning process can vary widely from one
system to another, but some common steps are included in most cleaning cycles:
Importance of keeping a factory clean
• Commercial factory cleaning is an indispensable aspect of maintaining a safe and
efficient industrial environment.
• It involves the removal of dirt, debris, and contaminants from the factory.
• Factory cleaning is an important process that helps ensure the production of quality
products.
• Dirty and dusty areas in a factory can lead to reduced efficiency, decreased product
quality, and higher manufacturing costs.
• By keeping your factory clean, you can improve productivity and reduce environmental
impact.
• Cleaning your factory is essential for both the health and safety of your employees and
customers.
• Dust, dirt, grease and other contaminants can cause serious illness if ingested or breathed
in, which is why it’s important to keep your facilities clean at all times.
• Additionally, dirty areas make it difficult to operate machinery correctly this can lead to
missed production deadlines and lost profits.
• A regular cleaning schedule also preserves your equipment’s lifespan
The importance of factory cleaning includes:
• Productivity: Clean factories operate at maximum efficiency.
• Safety: A clean environment reduces accidents.
• Hygiene: Cleanliness ensures a healthy workplace.
• Compliance: Meeting regulatory standards.
 • Appearance: A neat factory reflects professionalism.
• Pollution: Keeping the environment clean reduces environmental impact.

Describe the following types of CIP

Single use systems
• Single-tank, single-use systems operate on the basis of smaller volumes of solution
automatically adjusted to the required detergent concentration and temperature by using a
preparation loop.
• Singe-tank single-use systems (Fig. 10.6) are usually small packaged units (skids)
with one tank, pipes, centrifugal pumps, valves, a direct steam injection device
(direct heating of detergent solutions), a heating coil in the tank or an external heat
exchanger (indirect heating of detergent solutions), several dosing pumps to
automatically feed cleaning chemicals from the shipping containers or bulk storage, etc.
• These systems use the solution only once at the lowest possible strength, and discharge it
to the sewer at the end of each cycle.
• The tank must have a large enough capacity for the process equipment and pipes to be
cleaned.
• When located adjacent to the equipment to be cleaned and disinfected, the inlet and
outlet paths for the cleaning media are short, and losses from intermediate rinsing steps
and flush-outs are reduced.
• As such, consumption of cleaning solutions (and cleaning chemicals) can be
minimized and effluent rates reduced. Sometimes, an additional water tank is installed for
recovery of last rinse water, which can be used as pre-rinse in a next cleaning cycle.
• To further reduce the total amount of water, cleaning agents and energy required
for cleaning operations, several installations also have incorporated systems for
recovery of water from spent cleaning solutions.
• As an example, the dairy industry has attempted to recover water from spent
cleaning solution by concentration through ultrafiltration or through use of an
evaporator.
• Also that recovered water is then temporarily stored to be used as a pre-rinse for the
subsequent cleaning cycle.
• Single-use CIP systems are small in size, simple in design, low in initial investment, and
flexible in application.
• However, single-use CIP stations are seldom used in the agro-food industry (AFI).
• They are suitable for relatively small equipment that is heavily soiled, or for
processes where cross-contamination is strictly forbidden (e.g., process installations
with solids and chunks, process equipment containing allergens, membrane plants,
etc.).
• Single-use systems are especially used in the pharmaceutical industry due to the fear of
 cross contamination that could arise by recycling of cleaning solutions

• Re-use systems
• Preparation of cleaning solutions of required strength and at sufficient temperature A
typical reuse CIP system consists of a caustic tank(s), an acid tank, a water recovery tank
(e.g., to recover the last-rinse water of a previous cleaning cycle, which is re-used as
pre-rinse water for a next cleaning cycle), and one tank containing the water for the
final rinse.
• All tanks are interconnected by piping, provided with valves and manifold fitted with
CIP supply and return pumps.
• From containers, metering pumps feed metered amounts of concentrated caustic or
acid cleaning chemicals directly into the water-filled caustic and acid tank, or these
chemicals are injected in-line in a preparation loop.
• A preparation loop is a very efficient system, especially when the caustic and acid
tanks of the CIP station are tall.
• For big CIP stations, each tank (caustic, acid and water tanks) is equipped with its
own preparation loop.
• The content of each of the CIP tanks is mixed by recirculation over the corresponding
CIP tank through the CIP supply/recirculation pump.
• To bring and keep the cleaning solutions at adequate strength, conductivity sensors
are used because the conductivity is proportional to the detergent concentration.
• Detergent chemicals are generally fed directly on an “on-demand” basis of the
conductivity sensor signal.
• The recirculation loop is also fitted with a plate or tube heat exchanger to heat the
solutions to the desired temperature or to keep the required temperatures for CIP
solutions.
•
• Alternatively, in-tank heating by means of a heating coil or direct injection of steam in
the tank or preparation loop may be applied.
• If an external heat exchanger is used, the steam supply to this heat exchanger is
controlled by the temperature signal of the temperature sensor positioned in the
recirculation loop over respectively the acid or caustic CIP detergent tank.
• Recirculation goes on until the cleaning solution receives the adequate chemical strength
and temperature to start the CIP process.
CIP return
• The cleaning solutions can be routed back to the CIP system either by gravity (where
feasible) or via a low-speed CIP Return pump.
• The return pump should have a no-flow protection, to prevent premature failure of the
 pump.
• Sometimes, the return pump is aided by an eductor.
• An eductor generates suction in the return line, thus ensuring that the return pump
never air-locks.
• To generate that vacuum, the eductor requires a motive fluid that can be delivered by a
small motive pump.
• One of the CIP solutions (usually the same one as the returning solution) is sent from the
source tank through the eductor and back to the source tank. Thus the motive fluid is
different at different stages of CIP.
• The CIP return line may have a sample point and a sight glass, allowing validation
of a cleaning process (Seiberling, 1997; Christi, 1999).

Sorting and recovery

• Upon return to the CIP system, the solution can go into one of the CIP tanks or diverted
to drain.
• Re-use CIP systems are generally programmed to “waste” a small part of the solution at
the end of each cleaning cycle to continuously remove soiled solution from the system.
• This is followed by the addition of fresh water to bring the solution tank to the normal
operating level after which the conductivity-cell based chemical feed system will add
more cleaning chemical.
• A detergent solution that becomes less quickly polluted during its recirculation through
the process equipment being subjected to a caustic cleaning step can be re-used many
times.
• This is especially possible in process plants where parts of the process equipment are not
heavily soiled, and where the pre-rinse water succeeds to remove a high percentage of
soil during the preliminary rinse

CIP supply
• At that moment, the CIP tank recirculation valve closes and the CIP supply valve opens,
allowing the cleaning solution to pass a strainer, to finally flow in the CIP supply line.
• The strainer may be a self-cleaning type that discharges accumulated debris to drain
whenever the pressure drop across the device exceeds a pre-set value.
• The CIP supply line is connected to the spray devices located in a vessel or other
pieces of process equipment, and the piping that needs to be cleaned.
• Dry running of the supply pump which could damage the pump is prohibited by a no-
flow sensor.
 • Maximum recovery of caustic and/or acid detergent solutions is only possible after
adequate sorting of the cleaning solutions and rinse waters.
• Sorting/recycling of solutions is governed by a conductivity sensor which is installed
at the end of each CIP return line on the CIP station.
• When this sensor detects that the conductivity of a solution is higher than a pre-set target
value, the CIP solution is returned to the corresponding detergent tank.
• In a subsequent rinse step, the cleaning solution is flushed away by the rinse water,
with as result that the conductivity signal decreases and finally drops below a pre-set
value, triggering a changeover valve that routes the rinse water to drain instead of to the
relevant detergent tank.
• But once a pre-set minimum conductivity value has been reached, indicating
complete removal of acid or caustic from the system, the intermediate or final
rinse is stopped.
• Usually, the entire CIP sequence is automated, allowing the CIP system to stop regularly
at specific steps.
• Sorting of solutions is only efficient if intermixing between cleaning solutions and
water phases is minimal.
• Hence, the transition and boundary between two successive phases (in e.g., between
the caustic and rinsing sequence) must be sharp.
• The transition between two phases will be long if too much intermixing as the
consequence of poor hygienic design of the process equipment (e.g., due to dead legs)
occurs, or because different equipment units are cleaned in series.
• Sorting of solutions could also be governed by timers but is less appropriate than
sorting by means of conductivity sensors.
Additional tanks
• The water consumption in a re-use system can be further optimized by providing a
recirculation facility for the hot water.
• The unit could also be fitted with neutralisation tanks in which the alkali and/or acid
solutions are neutralized prior to their disposal into the effluent system.
• The capacity of the tanks is defined in advance by the circuit volume, temperature
requirements and desired cleaning.
• An ideal re-use CIP system has the ability to fill, empty, recirculate, heat and dispense
contents automatically.
CIP re-use systems versus single-use CIP systems
• Re-use systems are more complex than single-use systems, and hence the
additional investment costs are high.
• However, the payback period is very short because of the considerable savings in
water, detergent chemicals and energy.
Describe types of chemicals used in CIP
Outline the steps in CIP

Step 1: Pre-rinse
• The pre-rinse is a very important step in the CIP process because a well-monitored and
well-executed pre-rinse makes the rest of the wash cycle predictable and repeatable.
The pre-rinse cycle:
• Wets the interior surface of the lines and tanks
• Removes most of the remaining residue
• Dissolves sugars and partially melts fats
• Provides a non-chemical pressure test of the CIP flow path
• Meeting the cleaning requirements during the pre-rinse step is crucial for ensuring the
effectiveness of the subsequent cleaning stages.
• Use potable plant water, de-ionized water (DI), water that has been processed through
reverse osmosis (RO), or re-use the final rinse solution from the previous cleaning
sequence.
• A Turbidity Sensor may be used to verify that the pre-rinse effectively removes all solids.
Step 2: Caustic Wash – (140° – 185° F)
• Caustic washes soften fats, making them easier to remove.
• Also known as caustic soda, sodium hydroxide or NaOH, the alkali used in caustic
washes have a very high pH in a concentration range of 0.5-2.0%.
• Concentrations as high as 4% may be used for highly soiled surfaces.
• Caustic is typically used as the main detergent in most CIP wash cycles.
• The use of appropriate cleaning chemicals, such as caustic soda, is essential for breaking
down and removing residues effectively.
• A non-foaming formulation can help reduce pump cavitation and increase efficiency.
• It will also prevent tanks from overfilling with foam when the system starts to recirculate.
• Water Saving Tip: In many cases, the caustic wash can be returned to its tank and re-used
multiple times, which significantly reduces water, chemical, and energy costs over a
single tank system.
Step 3: Intermediate Rinse
• Fresh water flushes out residual traces of detergent remaining from the caustic wash.
• Use proper instrumentation during each step of the CIP Cycle, including rinsing, to
 ensure proper cleaning.
• Level Transmitters and Probes monitor tank levels of wash and rinse tanks.
• Flow Transmitters ensure optimum flow for spray devices to control wash and rinse steps
precisely.
• Conductivity Transmitters ensure chemical levels are hitting the predetermined set point.
Step 4: Final Rinse
• Rinse with either DI, RO, or city water to flush residual cleaning agents.
• In many systems, the final rinse water may be recovered and reused as the pre-rinse
solution for the next cleaning cycle.
• The residual heat and chemicals it retains from the final rinse will help make the next pre-
rinse more effective and economical.
Step 5. Sanitizing Rinse
• May be required to help kill microorganisms before starting the next production run.
• For many years, various hypochlorite solutions (potassium, sodium or calcium), also
known as “hypo,” have been used as sanitizers in many CIP cycles.
• The active ingredient in a sanitizing rinse is chlorine (bleach), which is:
• Relatively inexpensive to use.
• Very effective as a sanitizing rinse for soils that are prone to bacterial growth such as
dairy products.
• Potentially harmful to stainless steel, causing staining, corrosion and pitting.
• In recent years more sanitation managers have turned away from bleach-based sanitizers
in favor of peracetic acid (PAA) — a combination of hydrogen peroxide and acetic acid.

ORGANISATION AND STUDY OF LIVING ORGANISMS

Explain characteristics of living organisms

Biology is the study of living organisms. For something to be alive it needs to perform all seven
functions of living things.
MRS GREN
Movement, Respiration, Sensitivity, Growth, Reproduction, Excretion, Nutrition.
1. Movement
• Most organisms are able to move their whole body even plants can shift their stem
towards
 • the sunlight and their roots move towards healthy soil.
2. Respiration
• It is the breakdown of food inside a living organism it is vital for survival. 2 types
• Aerobic Respiration which involves O2 & glucose breaking down to form CO2 water &
• energy.
• Anaerobic Respiration which is the incomplete breakdown of food. Happens when there
is
• not enough oxygen. Equation, Glucose & oxygen (not enough) to form carbon dioxide
Lactic Acid or Alcohol (depending on the organism) & a little energy.
3. Sensitivity
• It is the ability to detect and respond to a stimulus.
4. Growth
• It is the permanent increase in size and quantity of cells using materials absorbed from
the
environment.
5. Reproduction
• It is forming new individuals of the same species either sexual (2 parents) or asexual (1
parent)
6. Excretion
• It is removal of harmful products of metabolism. Egestion is the removal of undigested
products which haven’t entered the cell.
7. Nutrition
• It is the intake of food material from the environment.
• Autotrophic nutrition: Organisms that make their own food such as plants.
• Heterotrophic nutrition: Organisms that need readymade food including herbivores,
carnivores &
• omnivores

Group living organisms

• A specie is a group of organisms that share the many similar appearances and can breed
with each other.
• Species are scientifically named by two names in Latin to avoid differences in languages.
• The first name is the name of the genus while the second name is the species name e.g.
 WOLF (Cannis Lupus) (must be italic and underlined)
• The main groups of living organisms are the 5 kingdoms. They don’t include virus since
it doesn’t obey some characteristics of life. The kingdoms are: Bacteria, Protoctista,
Fungi, Plants, and Animals.
• The prokaryotes are divided between the domains Bacteria (corresponding to domain
Eubacteria) and Archaea (corresponding to kingdom Archaebacteria), (many live in
extreme conditions); eukaryotes are placed into the domain Eukarya (corresponding to
kingdoms fungi, plantae, Animalia and kingdom Protista)
• The domain is a larger, more inclusive category than a kingdom. It is the highest
taxonomic rank in the hierarchical biological classification system, above the kingdom.
Domain Bacteria:
• Prokaryotic cells (no nucleus)
• DNA exists as circular ‘chromosome’ and does not have histone proteins associated with
it
• Plasmids (smaller circular molecules of DNA) are often present
• No membrane-bound organelles (e.g. mitochondria, E.R., Golgi body, chloroplasts) are
present
• Ribosomes (70 S) are smaller than in eukaryotic cells
• Cell wall is always present and contains peptidoglycans
• Cells divide in binary fission (not mitosis)
• Usually exist as single cells or small group of cells
Domain Archaea (archaeans):
• Prokaryotic cells (no nucleus)
• DNA exists as circular ‘chromosome’ and does not have histone proteins associated with
it
• Plasmids are often present
• No membrane-bound organelles are present
• Ribosomes (70 S) are smaller than in eukaryotic cells, but have features similar to those
of eukaryotic ribosomes
• Cell wall is always present, but does not contain peptidoglycans
• Cells divide in binary fission (not mitosis)
• Usually exist as single cells or small group of cells
• Metabolism similar to bacteria, but transcription common with eukaryotes

Domain Eukarya:
 • Cells with nuclei and membrane-bound organelles
• DNA in the nucleus arranged in linear chromosomes with histone proteins
• Ribosomes (80 S) in the cytosol larger than prokaryotes’; chloroplasts and mitochondrial
DNA have 70 S ribosomes
• Chloroplast and mitochondrial DNA is circular as in prokaryotes
• May be unicellular, colonial and multicellular organisms
• Cell division by mitosis
• Method of reproduction: asexual and sexual
Kingdom Fungi:
• Eukaryotic
• No chlorophyll (no photosynthesis)
• Heterotrophic nutrition – use organic compounds made by other organisms as their
source of energy and source of molecules for metabolism
• Reproduce by means of spores
• Simple body form (may be unicellular or made up of long threads called hyphae (with or
without cross walls))
• Have cell walls made up of chitin or other substances (not cellulose)
• Does not have cilia or flagella
Kingdom Plantae:
• Multicellular eukaryotes with cells that are differentiated to form tissues and organs
• Few types of specialised cells
• Some cells have chloroplasts and photosynthesize
• Cells have large, often permanent vacuoles for support
• Autotrophic nutrition
• Cell walls are always present (made of cellulose)
• Cells may have flagella
Kingdom Animalia:
• Multicellular eukaryotes with many different types of specialised cells
• Cells that are differentiated to form tissues and organs
• No chloroplasts (no photosynthesis)
• Small and temporary cell vacuoles (e.g. lysosomes and vacuoles)
• Heterotrophic nutrition
 • No cell walls
• Communication is by the nervous system
• Cells may have cilia or flagella
Kingdom Protoctista:
• Any eukaryote that is not fungus, plant or animal is a Protoctista
• Features:
• Eukaryotic
• Mostly single-celled, or exist as groups of similar cells
• Protozoa – having animal-like cells (no cell wall)
• Algae – having plant-like cells (cellulose cell walls and chloroplasts
Viruses:
• Acellular – no cellular structure like bacteria and fungi
• No features traditionally use for classification
• Infectious but has no metabolism
• Their taxonomic system is based on the type of nucleic acid they contain (DNA or RNA),
and whether the nucleic acid is single-stranded or double-stranded (DNA and RNA)

Use binomial systems

• The binomial naming system is the system used to name species.
• Each species is given a name that consists of two parts.
• The first part is the Genus to which the species belongs and the second part is the species
name.
• For example, Apis mellifera (the honey bee), Homo sapien (human).
• It was developed by Linnaeus.
• The levels of classification are; kingdom, phylum, class, order, family, genus, and
species.
• The name of the genus is always read first and must be capitalized upon.

Advantages of the system

• The organism can be easily categorized; it helps in making it easier to understand the
characteristics of a specific organism in an organized way
 • The names are unique with each creature having only one scientific name.
• Scientific names are standardized and accepted universally.

Describe prokaryotic and eukaryotic cells

• At one time it was common practice to try to classify all living organisms as either
animals or plants.
• With advances in knowledge of living things, it has become obvious that the living world
is not that simple.
• Fungi and bacteria, for example, are very different from animals and plants, and from
each other.
• Eventually it was discovered that there are two fundamentally different types of cell.
• The most obvious difference between these types is that one possesses a nucleus and the
other does not.
• Organisms that lack nuclei are called prokaryotes (‘pro’ means before; ‘karyon’ means
nucleus).
• They are, on average, about 1000 to 10 000 times smaller in volume than cells with
nuclei, and are much simpler in structure – for example, their DNA lies free in the
cytoplasm.
• Organisms whose cells possess nuclei are called eukaryotes (‘eu’ means true).
• Their DNA lies inside a nucleus.
• Eukaryotes include animals, plants, fungi and a group containing most of the unicellular
eukaryotes known as protoctists.
• Most biologists believe that eukaryotes evolved from prokaryotes, 1500 million years
after prokaryotes first appeared on Earth.
• All eukaryotic cells have certain features in common.

FUNCTIONS OF CELLS PARTS

Chloroplasts
• Chloroplasts tend to have an elongated shape and a diameter of about 3 to 10 μm
(compare 1μm diameter for mitochondria).
• The main function of chloroplasts is to carry out photosynthesis.
Lysosomes
• Lysosomes are spherical sacs, surrounded by a single membrane and having no internal
structure.
• They are commonly 0.1–0.5μm in diameter.
• They contain digestive (hydrolytic) enzymes which must be kept separate from the rest of
the cell to prevent damage from being done.
• Lysosomes are responsible for the breakdown (digestion) of unwanted structures such as
old organelles or even whole cells, as in mammary glands after lactation (breast feeding).
• In white blood cells, lysosomes are used to digest bacteria.
• Enzymes are sometimes released outside the cell for example, in the replacement of
cartilage with bone during development.
• The heads of sperm contain a special lysosome, the acrosome, for digesting a path to the
ovum (egg).

Golgi body
• The Golgi body is a stack of flattened sacs.
• More than one Golgi body may be present in a cell.
• The stack is constantly being formed at one end from vesicles which bud off from the ER,
and broken down again at the other end to form Golgi vesicles.
• The stack of sacs together with the associated vesicles is referred to as the Golgi
apparatus or Golgi complex.
• The Golgi body collects, processes and sorts molecules (particularly proteins from the
rough ER), ready for transport in Golgi vesicles either to other parts of the cell or out of
the cell (secretion).
• Two examples of protein processing in the Golgi body are the addition of sugars to
proteins to make molecules known as glycoproteins, and the removal of the first amino
acid, methionine, from newly formed proteins to make a functioning protein.
• In plants, enzymes in the Golgi body convert sugars into cell wall components. Golgi
vesicles are also used to make lysosomes.
Endoplasmic reticulum and ribosomes
• The ER is continuous with the outer membrane of the nuclear envelope.
• There are two types of ER: rough ER and smooth ER. Rough ER is so called because it is
covered with many tiny organelles called ribosomes.
• These are just visible as black dots.
• At very high magnifications they can be seen to consist of two subunits: a large and a
small subunit.
 • Ribosomes are the sites of protein synthesis.
• They can be found free in the cytoplasm as well as on the rough ER.
• They are very small, only about 25nm in diameter.
• They are made of RNA (ribonucleic acid) and protein.
• Proteins made by the ribosomes on the rough ER enter the sacs and move through them.
• The proteins are often modified in some way on their journey.
• Small sacs called vesicles can break off from the ER and these can join together to form
the Golgi body.
• They form part of the secretory pathway because the proteins can be exported from the
cell via the Golgi vesicles
• Smooth ER, so called because it lacks ribosomes, has a completely different function.
• It makes lipids and steroids, such as cholesterol and the reproductive hormones oestrogen
and testosterone
Bacterial structure

Viruses
• In 1852, a Russian scientist discovered that certain diseases could be transmitted by
agents that, unlike bacteria, could pass through the finest filters.
• This was the first evidence for the existence of viruses, tiny ‘organisms’ which are much
smaller than bacteria and are on the boundary between what we think of as living and
non-living.
• Unlike prokaryotes and eukaryotes, viruses do not have a cell structure.
• In other words, they are not surrounded by a partially permeable membrane containing
cytoplasm with ribosomes.
• They are much simpler in structure.
• Most consist only of:
• a self-replicating molecule of DNA or RNA which acts as its genetic code
• a protective coat of protein molecules
• It has a very symmetrical shape.
• Its protein coat (or capsid) is made up of separate protein molecules, each of which is
called a capsomere.
• Viruses range in size from about 20–300nm (about 50 times smaller on average than
bacteria).
 • All viruses are parasitic because they can only reproduce by infecting and taking over
living cells.
• The virus DNA or RNA takes over the protein synthesizing machinery of the host cell,
which then helps to make new virus particles.

The Mitochondria
Structure
• Mitochondria (singular: mitochondrion) are usually about 1 μm in diameter and can be
various shapes, often sausage-shaped as in the figure below.
• They are surrounded by two membranes (an envelope).
• The inner of these is folded to form finger-like cristae which project into the interior
solution, or matrix.
• The space between the two membranes is called the intermembrane space.
• The outer membrane contains a transport protein called porin, which forms wide aqueous
channels allowing easy access of small, water-soluble molecules from the surrounding
cytoplasm into the intermembrane space.
• The inner membrane is a far more selective barrier and controls precisely what ions and
molecules can enter the matrix.
• The number of mitochondria in a cell is very variable.
• As they are responsible for aerobic respiration, it is not surprising that cells with a high
demand for energy, such as liver and muscle cells, contain large numbers of
mitochondria.
• A liver cell may contain as many as 2000 mitochondria.
• If you exercise regularly, your muscles will make more mitochondria.
• Function of mitochondria and the role of ATP
• As we have seen, the main function of mitochondria is to carry out aerobic respiration,
although they do have other functions, such as the synthesis of lipids.
 • During respiration, a series of reactions takes place in which energy is released from
energy-rich molecules such as sugars and fats.
• Most of this energy is transferred to molecules of ATP. ATP (adenosine triphosphate) is
the energy-carrying molecule found in all living cells.
• It is known as the universal energy carrier.
• The reactions of respiration take place in solution in the matrix and in the inner
membrane (cristae).
• The matrix contains enzymes in solution, including those of the Krebs cycle and these
supply the hydrogen and electrons to the reactions that take place in the cristae.
• The flow of electrons along the precisely placed electron carriers in the membranes of the
cristae is what provides the power to generate ATP molecules
• The folding of the cristae increases the efficiency of respiration because it increases the
surface area available for these reactions to take place.
• Once made, ATP leaves the mitochondrion and, as it is a small, soluble molecule, it can
spread rapidly to all parts of the cell where energy is needed.
• Its energy is released by breaking the molecule down to ADP (adenosine diphosphate).
• This is a hydrolysis reaction.
• The ADP can then be recycled into a mitochondrion for conversion back to ATP during
aerobic respiration.

Discuss multicellularity and cell specialization

• A multicellular organism is an organism composed of many cells.

• The multicellular organisms are developed by cellular specialization and division of
labor.
• The four essential processes by which a multicellular organism is made: cell proliferation,
cell specialization, cell interaction and cell movement
Characteristics of multicellular organisms
• They possess distinct organs and organ systems
• They are eukaryotes i.e. they contain membrane bound organelles
• Their cells exhibit division of labor
• Their size increases with the number of cells in an organism.
• Cell specialization is the process by which a single cell after division becomes a distinct
 cell type according to its function.
• All cells start as stem cells, which are cells that can become many other types of cells.
• During cell specialization, cells undergo three stages: specification, determination and
differentiation.
MICROSCOPE TECHNIQUES

State functions of parts of microscope

Describe use and manipulation of microscope

MICROSCOPY

• When a ray of light passes from one medium to another, refraction occurs, that is, the
ray is bent at the interface.
• The refractive index is a measure of how greatly a substance slows the velocity of light,
and the direction and magnitude of bending is determined by the refractive indexes of the
two media forming the interface.
• When the light source is distant so that parallel rays of light strike the lens, a convex lens
will focus these rays at a specific point, the focal point.
• The distance between the center of the lens and the focal point is called the focal length.
Types of microscopes
• electron microscope: scanning electron microscope, transmission electron microscope
• light microscope: bright-field, dark-field, phase-contrast, and fluorescence microscopes

• The light microscope uses light as a source of radiation, while the electron microscope
uses electrons.
• The cell theory states that the basic unit of structure and function of all living organisms
is the cell.

LIGHT MICROSCOPY
• Modern microscopes are all compound microscopes. That is, the magnified image
formed by the objective lens is further enlarged by one or more additional lenses.
 • Cytology - microscope design and, equally important, preparation of material for
examination with microscopes.
• The ordinary microscope is called a bright-field microscope because it forms a dark
image against a brighter background.
• The microscope consists of a sturdy metal body or stand composed of a base and an arm
to which the remaining parts are attached.
• A light source, either a mirror or an electric illuminator, is located in the base.
• Two focusing knobs, the fine and coarse adjustment knobs, are located on the arm and
can move either the stage or the nosepiece to focus the image.
• The stage is positioned about halfway up the arm and holds microscope slides by either
simple slide clips or a mechanical stage clip.
• A mechanical stage allows the operator to move a slide around smoothly during viewing
by use of stage control knobs.
• The substage condenser is mounted within or beneath the stage and focuses a cone of
light on the slide.
• Its position often is fixed in simpler microscopes but can be adjusted vertically in more
advanced models.
• The curved upper part of the arm holds the body assembly, to which a nosepiece and one
or more eyepieces or oculars are attached.
• More advanced microscopes have eyepieces for both eyes and are called binocular
microscopes.
• The body assembly itself contains a series of mirrors and prisms so that the barrel holding
the eyepiece may be tilted for ease in viewing.
• The nosepiece holds three to five objectives with lenses of differing magnifying power
and can be rotated to position any objective beneath the body assembly.
• Ideally microscopes should be parfocal, that is, the image should remain in focus when
objectives are changed.
• The objective lens forms an enlarged real image within the microscope, and the eyepiece
lens further magnifies a primary image.
• When one looks into a microscope, the enlarged specimen image, called the virtual
image, appears to lie just beyond the stage about 25 cm away.
• The total magnification is calculated by multiplying the objective and eyepiece
magnifications together.
• For example, if a 45X objective is used with a 10X eyepiece, the overall magnification of
the specimen will be 450X.
MICROSCOPE RESOLUTION
• The most important part of the microscope is the objective, which must produce a clear
image, not just a magnified one.
 • Resolution is the ability of a lens to separate or distinguish between small objects that are
close together.
• The minimum distance (d) between two objects that reveals them as separate entities is
given by the Abbé equation, in which lambda (λ) is the wavelength of light used to
illuminate the specimen and n sin Ø is the numerical aperture (NA).
• d= 0.5 λ / n sin Ø
• As d becomes smaller, the resolution increases, and finer detail can be discerned in a
specimen.
• The preceding equation indicates that a major factor in resolution is the wavelength of
light used.
• The wavelength must be shorter than the distance between two objects or they will not be
seen clearly.
• Thus the greatest resolution is obtained with light of the shortest wavelength, light at the
blue end of the visible spectrum (in the range of 450 to 500nm).
• Numerical aperture n sin Ø.
• Theta is defined as 1/2 the angle of the cone of light entering an objective.
• Light that strikes the specimen after passing through a condenser is cone-shaped.
• When this cone has a narrow angle and tapers to a sharp point, it does not spread out
much after leaving the slide and therefore does not adequately separate images of closely
packed objects.
• The resolution is low.
• If the cone of light has a very wide angle and spreads out rapidly after passing through a
specimen, closely packed objects appear widely separated and are resolved.
• The angle of the cone of light that can enter a lens depends on the refractive index (n) of
the medium in which the lens works, as well as upon the objective itself.
• The refractive index for air is 1.00. Since sin Ø cannot be greater than 1 (the maximum Ø
is 90° and sin 90° is 1.00), no lens working in air can have a numerical aperture greater
than 1.00.
• The only practical way to raise the numerical aperture above 1.00, and therefore achieve
higher resolution, is to increase the refractive index with immersion oil, a colorless
liquid with the same refractive index as glass.
• If air is replaced with immersion oil, many light rays that did not enter the objective due
to reflection and refraction at the surfaces of the objective lens and slide will now do so.
• An increase in numerical aperture and resolution results.
MAGNIFICATION
• Magnification is the number of times larger an image is, than the real size of the object.
 • If you know two of these values, you can work out the third one. For example, if the
observed size of the image and the magnification are known, you can work out the actual
size: A = I /M.
MEASURING CELLS
• Cells and organelles can be measured with a microscope by means of an eyepiece
graticule.
• This is a transparent scale.
• It usually has 100 divisions.
• The eyepiece graticule is placed in the microscope eyepiece so that it can be seen at the
same time as the object to be measured.
• Figure 1.8b shows the scale over a human cheek epithelial cell. The cell lies between 40
and 60 on the scale.
• We therefore say it measures 20 eyepiece units in diameter (the difference between 60
and 40).
• We will not know the actual size of the eyepiece units until the eyepiece graticule scale is
calibrated.
• To calibrate the eyepiece graticule scale, a miniature transparent ruler called a stage
micrometer scale is placed on the microscope stage and is brought into focus.
• This scale may be etched onto a glass slide or printed on a transparent film.
• It commonly has subdivisions of 0.1 and 0.01mm.
• The images of the two scales can then be superimposed as shown in Figure 1.8c.

Examine prepared slides

Preparation and Staining of Specimens
• Although living microorganisms can be directly examined with the light microscope, they
often must be fixed and stained to increase visibility, accentuate specific morphological
features, and preserve them for future study.
Fixation
• The stained cells seen in a microscope should resemble living cells as closely as possible.
• Fixation is the process by which the internal and external structures of cells and
microorganisms are preserved and fixed in position.
• It inactivates enzymes that might disrupt cell morphology and toughens cell structures so
that they do not change during staining and observation.
• A microorganism usually is killed and attached firmly to the microscope slide during
fixation.
• There are two fundamentally different types of fixation.
 (1) Bacteriologists heat-fix bacterial smears by gently flame heating an air-dried film of
bacteria. This adequately preserves overall morphology but not structures within cells.
(2) Chemical fixation must be used to protect fine cellular substructure and the
morphology of larger, more delicate microorganisms. Chemical fixatives penetrate cells
and react with cellular components, usually proteins and lipids, to render them inactive,
insoluble, and immobile. Common fixative mixtures contain such components as ethanol,
acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.
Staining
• In the first step of the Gram-staining procedure, the smear is stained with the basic dye
crystal violet, the primary stain.
• It is followed by treatment with an iodine solution functioning as a mordant.
• That is, the iodine increases the interaction between the cell and the dye so that the cell is
stained more strongly.
• The smear is next decolorized by washing with ethanol or acetone.
• This step generates the differential aspect of the Gram stain; gram-positive bacteria retain
the crystal violet, whereas gram-negative bacteria lose their crystal violet and become
colorless.
• Finally, the smear is counterstained with a simple, basic dye different in color from
crystal violet.
• Safranin, the most common counterstain, colors gram-negative bacteria pink to red and
leaves gram-positive bacteria dark purple

• When starting to examine a slide, select the low powered (X4) objective lens first. Your
eye level should be just above the eye pieces. Then look down the eye pieces and gently
slide them together until you see a single image.

Discuss the hanging drop technique

The Hanging-Drop Preparation
• The simplest method for examining living microorganisms is to suspend them in a fluid
(water, saline, or broth) and prepare a ''hanging drop,'' using a cover glass and a hollow
ground slide.
• The slide is ground with a concave well in the center; the cover glass holds a drop of the
suspension.
• When the cover glass is inverted over the well of the slide, the drop hangs from the glass
in the hollow concavity of the slide.
• Microscopic study of such a wet preparation can provide very useful information.
• This method is used to determine whether an organism is motile or not.
 • Such a slide can be observed for a fairly long time period, because the drop does not dry
up quickly.
• The slide for a hanging drop is ground with a concave well in the centre; the cover glass
holds a drop of the suspension.
• When the cover glass is inverted over the well of the slide, the drop hangs from the glass
in the hollow concavity of the slide.
• Since the drop lies within an enclosed glass chamber, drying out occurs very slowly.
• A ring of Vaseline around the edge of the cover slip keeps the slide from drying out.

Procedures
1. Take a cover glass and clean it thoroughly, making certain it is free of grease (the drop to be
placed on it will not hang from a greasy surface). It may be dipped in alcohol and polished dry
with tissue; or washed in soap and water, rinsed completely, and wiped dry.
2. Take one hollow-ground slide and clean the well with a piece of dry tissue. Place a film of
petroleum jelly around the rim of the well.
3. Gently shake the broth culture of Proteus until it is evenly suspended. Using the wire
inoculating loop, sterilize on the Bunsen flame, remove a loopful of culture. Close, and return the
tube to the rack.
4. Place the loopful of culture in the center of the cover glass (do not spread it around). Flame the
loop and put it down.
5. Hold the hollow-ground slide inverted, well down, over the cover glass; then press it down
lightly so that the petroleum jelly adheres to the four edges of the cover glass. Now turn the slide
over. You should have a sealed wet mount, the drop of culture hanging in the well.
6. Place the slide on the microscope stage, cover glass up. Start your examination with the
low-power objective to find the focus. It is helpful to focus first on one edge of the drop,
which will appear as a dark line.
7. The light should be reduced with the iris diaphragm, and, if necessary, by lowering the
condenser. If you have trouble with the focus, ask the instructor for help.
8. Continue your examination with the high-dry and oil immersion objectives (be very careful
not to break the cover slip with the latter).
9. Make a hanging-drop preparation of the staphylococcus culture, following the same
procedure described above.
10. Record your observation of the shape, cell groupings, and motility of the organisms.
11. Discard your slides in a container with disinfectant solution.

Why?
This method is used to determine whether an organism is motile or not.
Hanging drop microscopy offers several advantages compared to other microscopy techniques. It
allows for the culturing of 3D micro tissues under varying perfusion conditions, with precise
positioning and stability over time.
The platform is simple and robust, requiring no dedicated equipment

MICROBIOLOGY
Microbiology is the study of organisms that are usually too small to be seen by the unaided eye;
it employs techniques—such as sterilization and the use of culture media—that are required to
isolate and grow these microorganisms.

Classify bacteria according to shape using Bergey’s manual of bacteriology

• Publication of the second edition of Bergey’s Manual of Systematic Bacteriology was

begun in 2001.
• The 2nd edition gives the most up-to-date phylogenic classification of prokaryotic
organisms, including both eubacteria and archaea.
• When it is completed, it will consist of 5 volumes.
• The classification in Bergey’s Manual is accepted by most microbiologists as the best
consensus for prokaryotic taxonomy.
Microbial phylogeny
Domains
• Based on the research of Woese and others in the 1980s and 1990s, most biologists
divide all living organisms into 3 domains:
 Domain Archaea
• odd bacteria that live in extreme environments, high salt, heat, etc. (usually called
extremophiles)
Domain Bacteria/ Eubacteria
• true bacteria, peptidoglycan
Domain Eucarya/ Eukarya
• have a nucleus & organelles (humans, animals, plants)
rRNA sequence data suggests that Archaea & Eucarya may share a more recent common
ancestor with each other than with Bacteria

Phylogeny of domain Archaea

• Based primarily on rRNA sequence data, domain Archaea is divided into two
phyla:
• Phylum Crenarchaeota
• Originally containing thermophylic and hyperthermophilic sulfur-
metabolizing archaea
• Recently discovered Crenarchaeota are inhibited by sulfur & grow
at lower temperatures
• Phylum Euryarchaeota
• Contains primarily methanogenic archaea, halophilic archaea, and
thermophilic, sulfur-reducing archaea
Phylogeny of domain Bacteria
• The 2nd edition of Bergey’s Manual of Systematic Bacteriology divides domain Bacteria
into 23 phyla. Nine of the more notable phyla are described here.
• Phylum Aquiflexa
• The earliest “deepest” branch of the Bacteria
• Contains genera Aquiflex and Hydrogenobacter that can obtain energy from hydrogen via
chemolithotrophic pathways.
• Phylum Cyanobacteria
• Oxygenic photosynthetic bacteria
• Phylum Chlorobi
 • The “green sulfur bacteria”
• Anoxygenic photosynthesis
• Includes genus Chlorobium
Phylum Proteobacteria
• The largest group of gram-negative bacteria
• Extremely complex group, with over 400 genera and 1300 named species
• All major nutritional types are represented: phototrophy, heterotrophy, and several types
of chemolithotrophy
• Sometimes called the “purple bacteria,” although very few are purple; the term refers to a
hypothetical purple photosynthetic bacterium from which the group is believed to have
evolved
Phylum Proteobacteria (cont.)
Divided into 5 classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,
Deltaproteobacteria, Epsilonproteobacteria
Significant groups and genera include:
• Photosynthetic genera such as Rhodospirillum (a purple non-sulfur bacterium) and
Chromatium (a purple sulfur bacterium)
• Sulfur chemolithotrophs, genera Thiobacillus and Beggiatoa
• Nitrogen chemolithotrophs (nitrifying bacteria), genera Nitrobacter and Nitrosomonas
• Other chemolithotrophs, genera Alcaligenes, Methylobacilllus, Burkholderia
• The family Enterobacteriaceae, the “gram-negative enteric bacteria,” which includes
genera Escherichia, Proteus, Enterobacter, Klebsiella, Salmonella, Shigella, Serratia, and
others
• The family Pseudomonadaceae, which includes genus Pseudomonas and related genera
• Other medically important Proteobacteria include genera Haemophilus, Vibrio,
Camphylobacter, Helicobacter, Rickessia, Brucella

Phylum Firmicutes
• “Low G + C gram-positive” bacteria
• Divided into 3 classes
• Class I – Clostridia; includes genera Clostridium and Desulfotomaculatum, and
others
• Class II – Mollicutes; bacteria in this class cannot make peptidoglycan and lack
cell walls; includes genera Mycoplasma, Ureaplasma, and others
• Class III – Bacilli; includes genera Bacillus, Lactobacillus, Streptococcus,
 Lactococcus, Geobacillus, Enterococcus, Listeria, Staphylococcus, and others
Phylum Actinobacteria
• “High G + C gram-positive” bacteria
• Includes genera Actinomyces, Streptomyces, Corynebacterium, Micrococcus,
Mycobacterium, Propionibacterium
• Phylum Chlamidiae
• Small phylum containing the genus Chlamydia
Phylum Spirochaetes
• The spirochaetes
• Characterized by flexible, helical cells with a modified outer membrane (the outer
sheath) and modified flagella (axial filaments) located within the outer sheath
• Important pathogenic genera include Treponema, Borrelia, and Leptospira
Phylum Bacteroidetes
• Includes genera Bacteroides, Flavobacterium, Flexibacter, and Cytophyga;
Flexibacter and Cytophyga are motile by means of “gliding motility”

Phylogeny of domain Eucarya

• The domain Eucarya is divided into four kingdoms by most biologists:
• Kingdom Protista, including the protozoa and algae
• Kingdom Fungi, the fungi (molds, yeast, and fleshy fungi)
• Kingdom Animalia, the multicellular animals
• Kingdom Plantae, the multicellular plants

Species and subspecies

• Species
• collection of bacterial cells which share an overall similar pattern of traits in
contrast to other bacteria whose pattern differs significantly
• Strain or variety
• culture derived from a single parent that differs in structure or metabolism from
other cultures of that species (biovars, morphovars)
• Type
 • subspecies that can show differences in antigenic makeup (serotype or serovar),
susceptibility to bacterial viruses (phage type) and in pathogenicity (pathotype)
Naming microorganisms
• Binomial (scientific) nomenclature
• Gives each microbe 2 names:
• Genus - noun, always capitalized
• species - adjective, lowercase
• Both italicized or underlined
• Staphylococcus aureus (S. aureus)
• Bacillus subtilis (B. subtilis)
• Escherichia coli (E. coli)
Describe aseptic techniques
• Aseptic technique is a set of practices that protects patients from healthcare
associated infections and protects healthcare workers from contact with blood,
body fluid and body tissue.
Methods of aseptic techniques
• Sterilization is the process by which all living cells, viable spores, viruses, and viroids
are either destroyed or removed from an object or habitat. A sterile object is totally free of
viable microorganisms, spores, and other infectious agents
• Sanitization is when the microbial population is reduced to levels that are considered
safe by public health standards.
• Germicide or biocide is a chemical agent that kills microbes e.g. disinfectants,
antiseptics.
• Disinfection is the killing, inhibition, or removal of microorganisms that may cause
disease. The primary goal is to destroy potential pathogens, but disinfection also
substantially reduces the total microbial population.
• Disinfectant is a chemical agent that kills harmful microbes, but is too harsh to be used
on human skin
• Antisepsis is the prevention of infection or sepsis and is accomplished with antiseptics.
• Antiseptic is a chemical agent that kills harmful microbes and is selectively toxic enough
that it can be used on human skin

How to select an antimicrobial procedure?

There is no ideal multipurpose non-toxic method:
• Consider the type of microbe,
 • Extent of contamination,
• Environ conditions &
• Potential risk of infection associated with use of the item

Action of antimicrobial agents

• Alteration of membrane permeability
- Lipids and membrane proteins damage
• Damage to proteins and nucleic acids
- Enzymes and cellular protein damage, DNA and RNA damage
Physical methods of control
Heat
• Cheap, fast, inexpensive and no potential toxic substances
• Can totally eliminate or simply reduce microbial load
• Heat resistance varies among different microbes; these differences can be expressed
through the concept of thermal death point.
Moist heat
• Boiling or pressurised steam
• Coagulates proteins irreversibly (100 degrees for 10mins) except endospores eg C.
botulinum or C. perfringens, can survive many hrs of boiling
• Pressurised steam that reaches 121 for 15 mins at 15 psi reliably kills endospores
(Autoclaving)
• Pressure cookers and autoclaves
• Pressure increases temp to 121 in less time but plays no part in actually killing the
microbes
Pasteurisation
• Used in wine, vinegar, chibuku for spoilage organisms
• Used to reduce disease causing orgs in milk & juices
• Increases shelf life and protects consumers (tuberculosis, salmonellosis, typhoid fever)
without altering food quality
• Use high temp short time approach (HTST)
• Milk is heated for 15secs at 72℃
• Why does ice-cream require 20 secs at 82℃?
• Chibuku is held at 80 ℃for 15mins
Ultra High temperature (UTH) Treatment
• UHT eliminates all microbes that can grow under normal storage conditions.
• Heat to 140-150℃, hold for several secs then rapidly cool, aseptically package eg milk,
boxed juices
Canning
• Use pressurised steam an autoclave- Retort
• Kills even the spore of C. botulinum
• Cans are commercially sterile, WHY?
Dry heat
• Heating in the absence of steam
• In the lab- flaming, glass Petri dishes and pipets are sterilised in the oven at 160-170℃
for 2-3hrs
• Dry heat can be use on powders and other anhydrous material where moist heat is N/A
Filtration
• Used on heat sensitive fluids
• Used to sterilize sugar solutions, air entering special hospital rooms, unpasteurised beer
production, enzymes, antibiotics, vaccines…
• Depth and membrane filters
• Depth filter: trap microbes by charges; allow passage of fluid but retain microbe
• Filters don't distinguish bacteria and protein, don't remove viruses, too much pressure
affect filtration
• Composed of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidine fluoride
• They absorb very little of enzymes or the suspending fluid
• Membrane filtered beer is free of spoilage organisms
• AIR filtration: high efficiency particulate air (HEPA) remove all microbes with a
diameter more than micrometres
• Laminar flow cabinets and hospitals

Radiation
• X-rays, gamma rays, UV and visible light
• Short wavelength radiation i.e. gamma, have more killing effect than long wavelength
radiation i.e. visible light
• Microwaves generate heat which then kill microbes
• Gamma radiation: ionizing radiation that cause biological damage by producing
reactive molecules such as superoxide (o2- ) and hydroxyl free radicals (OH•) when the
rays transfer their energy to microorganisms
• Generation of free radicals, damage macromolecules, especially DNA, blocking
microbes' ability to replicate
• Gamma radiation for cobalt 60 is effective against salmonella, pseudomonas among
others
• Effective for many substances (plastics, cloth, etc.) because these high energy rays
penetrate well
• Good means of sterilization
• UV: damages DNA using wavelengths of 220-300nm
• Absorption of these waves causes the formation of covalent bonds between adjacent
thymine molecules forming thymine dimmers
• Effective only on thin films or surfaces because UV rays (low energy) do not penetrate
well
• Used as a means of disinfection, not sterilization
• Spores are resistant while actively growing microbes are the most susceptible
Preservation
• Slow or halt the growth of microorganisms to delay spoilage
• Benzoic, sorbic and proprionic acids are organic acids used as preservatives
• Nitrate and nitrite are added to some foods to inhibit germination and subsequent growth
of C. botulinum endospores
• The later give colour to meat
• May pose danger if nitrosamines are formed-carcinogenic
• Low temp storage: enzymatic reactions are slow or non-existent at low temp
• Some psychrotrophic and psychrophilic bacteria can grow at normal refrigeration temp
• Freezing may kill microbes by forming ice crystals
• Reducing available water: salting or sugaring reduce water activity(aw) to levels below
the requirements of growth
• The high solute conc. causes plasmolysis
• Drying: removing water or desiccating; usually supplemented by adding salt
 • Lyophilization (freeze drying): the food is frozen then dried in a vacuum. When water is
added, it reconstitutes.

Chemical methods of disinfection

Describe culturing of bacteria

• Microbiological study depends on the ability to grow and maintain microorganisms in the
laboratory, (culture media availability).
• A culture medium is a solid or liquid preparation used to grow, transport, and store
microorganisms.
• Medium must contain all the nutrients the microorganism requires for growth for
effectiveness.
• Specialized media are essential in the isolation and identification of microorganisms, the
testing of antibiotic sensitivities, water and food analysis, industrial microbiology, and
other activities.
• The precise composition of a satisfactory medium will depend on the species one is
trying to cultivate (variation in nutritional requirements)
• Knowledge of a microorganism’s normal habitat often is useful (nutrient requirements
should reflect its natural surroundings).
• In some cases, the function of the medium also will determine its composition. (medium
to select and grow or identify specific microorganisms)

Types of culture media

• General purpose media is media that supports growth of many microorganisms e.g.
stryptic soy broth and tryptic soy agar
• Enriched media is one that has been specially fortified (e.g., blood agar) to allow for
growth of other fastidious microbes
 • Blood and other special nutrients may be added to general purpose media to encourage
the growth of fastidious heterotrophs
• Selective media favor the growth of particular microorganisms.
• E.g. Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-
negative bacteria by inhibiting the growth of gram-positive bacteria without affecting
gram-negative organisms.
• Examples include Endo agar, eosin methylene blue agar, and MacConkey agar.
• Bacteria also may be selected by incubation with nutrients that they specifically can use.
• E.g. A medium containing only cellulose as a carbon and energy source is quite effective
in the isolation of cellulose-digesting bacteria
• Differential media are media that distinguish between different groups of bacteria and
even permit tentative identification of microorganisms based on their biological
characteristics.
• For example: Blood agar is both a differential medium and an enriched one. It
distinguishes between hemolytic and non-hemolytic bacteria.
• Hemolytic bacteria (e.g., many streptococci and staphylococci isolated from throats)
produce clear zones around their colonies because of red blood cell destruction.
• MacConkey agar is both differential and selective. Since it contains lactose and neutral
red dye, lactose-fermenting colonies appear pink to red in color and are easily
distinguished from colonies of non-fermenters.

Methods in microbiology for identification and characterization of microorganisms

• Microscopy is a technique prominent in microbiology
• Chemical, physical and biological methods of selecting microorganisms.

Physical methods
• Heat Selection (select for endospore forming bacteria, 80°C for 10 mins, kill vegetative)
• pH (acid tolerance study) e.g. selecting lactobacilli in cheese production @ pH 5.35
• Cell size and motility (use of membranes)
Chemical methods
• Use of carbon and nitrogen sources e.g. selecting for nitrogen fixing bacteria using N₂
• Use of inhibitory chemicals e.g. certain dyes are inhibitory for most gram positive
bacteria
Isolation of pure cultures
 • A pure culture, a population of cells arising from a single cell, to characterize an
individual species.
• Pure colonies arise when a mixture of cells is spread out on an agar surface with each cell
growing into a completely separate colony, representing a pure culture.
• Pure culture techniques introduced by Robert Koch
• Several methods available:
The spread plate and streak plate
• The spread plate is an easy, direct way of achieving pure cultures.
• A small volume of dilute microbial mixture containing around 30 to 300 cells is
transferred to the center of an agar plate
• This is then spread evenly over the surface with a sterile bent-glass rod
• Dispersed cells develop into isolated colonies.
• Spread plates can be used to count the microbial population.

Spread plate technique

• Pure colonies also can be obtained from streak plates.

• Microbial mixture is transferred to the edge of an agar plate with an inoculating loop or
swab
• It is then streaked out over the surface in one of several patterns
• Single cells drop from the loop as it is rubbed along the
• agar surface and develop into separate colonies).
• In both spread-plate and streak-plate techniques, successful isolation depends on spatial
separation of single cells.
Streak plate
The pour plate
• Pour plate also can yield isolated colonies and is extensively used with bacteria and
fungi
• The original sample is diluted several times to reduce the microbial population
sufficiently to obtain separate colonies when plating.
• Then small volumes of several diluted samples are mixed with liquid agar that has been
cooled to about 45°C,
• Mixtures are poured immediately into sterile culture dishes.
• Most bacteria and fungi are not killed by a brief exposure to the warm agar.
• After the agar has hardened, each cell is fixed in place and forms an individual colony
• Total number of colonies equals the number of viable microorganisms in the diluted
sample.

Morphological features
• shape (circular-irregular)
• size (small- medium- large)
• color
• margin (entire- irregular)
• Texture (soft- hard- mucoid)
• optical characters (transparent- opaque- translucent)
• elevation (flat- convex- raised)
• surface: (smooth, rough, shiny)
Bacterial colony morphology

Maintenance and preservation of pure cultures

• Maintenance is mainly characterized by sub-culturing pure culture samples on fresh
media
• Preservation is mainly through refrigeration at low temperatures (which will not freeze
the samples)
• Reviving samples is another major aspect.
What is the secret behind storing samples in a refrigerator?
Biochemical tests (IMViC)
Indole Formation - Utilisation of Tryptophan
• Culture is done on peptone water, a liquid medium containing tryptophan,
• certain bacteria will produce indole.
• The presence of this indole is readily revealed through addition of Kovak's reagent,
producing a pink colour.
• This reagent contains the organic solvent amyl alcohol that extracts the coloured (pink)
substance.
The Methyl Red (MR) Test - Mixed Acid Fermentation Pathway
• The mixed acid fermentation pathway results in the formation of a number of organic
acids such as lactic and acetic acid.
• If this is a primary fermentation pathway of a bacterium, a noticeable drop in pH will
occur with incubation on MRVP media.
• This decrease in pH can be revealed by a methyl red solution which is yellow under
neutral conditions and red at a pH less than 5.
The Voges-Proskauer (VP) Test - The Butanediol Fermentation Pathway
• An alternate fermentation pathway performed by some other bacteria results in the
formation of a non-acidic product, Butanediol
• The occurrence of the pathway may be determined by a biochemical test for an
intermediate compound in the pathway, acetoin (acetylmethyl carbinol), which is detected
by the Voges-Proskauer test.
Citrate Utilisation - Growth Using a Single Carbon Source
• In Simmon's citrate agar, citrate, in the form of sodium citrate, is the sole carbon source.
• Organisms able to utilise the citrate grow on the surface of the medium
• Due to oxidative formation of sodium carbonate, raises the pH of the medium changing it
from green to blue (bromothymol blue is the indicator).
Urea Hydrolysis
• Some bacteria can produce urease, an enzyme which hydrolyses urea into ammonium and
carbon dioxide.
• The presence of this enzyme is detected by growing the bacteria in a medium containing
urea and a pH indicator, phenol red.
 • If ammonium is produced as a result of urea hydrolysis, the increase in pH will turn the
medium to a violet-red colour

Describe industrial uses of microorganisms

Industrial microbes
• Originally isolated from nature, but increasingly "improved" by genetic manipulation
via mutagenesis and selection or recombinant DNA technology or protoplast fusion
(fungi)
• To be useful in industrial microbiology, an organism must:
• produce usable substance(s) or effect(s)
• be available in pure culture
• be genetically stable, but amenable to genetic manipulation
• produce spores or other reproductive structures to allow easy inoculation
• grow rapidly and produce product quickly in large-scale culture
• grow in such a way that the cells are easily separated from the product
• not be harmful to humans or agricultural plants and animals, etc.

Agricultural uses
• Rhizobium - facilitates nitrogen fixation in symbiotic legumes
• Agrobacterium tumefaciens – Ti. plasmid is used as a gene vector for transferring genes
coding for important functions to plants
• Bacillus thuringiensis spores contain protein crystals toxic for tomato worms
• Polyhedrosis virus is used to control pine caterpillars and cotton bollworms
Food and beverage industry
• dairy products - fermentation of milk generates lactic acid, which precipitates
milk proteins and prevents other microbial growth
• buttermilk (from skim milk) - Streptococcus diacetylactis
• Yorghut- Streptococcus thermophilus and Lactobacillus bulgaricus
• sour cream (from cream) - Streptococcus diacetylactis
• acidophilus milk - Lactobacillus acidophilus
• cheeses (lactic acid fermentations; 2000 varieties, 20 types)
 • soft - cottage (Streptococcus lactis; Leuconostoc cremoris), cream
(S. cremoris, S. diacetylactis, S. thermophilus, Lactobacillus
bulgaricus, Brie (S. lactis, S. cremoris; Penicillium camemberti, P.
candidum, Brevibacterium linens), mozzarella (S. thermophilus, L.
bulgaricus)
• semi-soft - monterey jack (S. lactis, S. cremoris), Muenster (S.
lactis, S. cremoris; Brevibacterium linens), bleu (Roquefort - S.
lactis, S. cremoris; Penicillium roqueforti)
• hard - cheddar (Streptococcus lactis, S. cremoris, S. durans;
Lactobacillus casei, L. plantarum), edam (S. lactis, S. cremoris),
gouda (Streptococcus lactis, S. cremoris, S. diacetylactis), swiss (S.
lactis, L. helveticus, S. thermophilus; Propionibacterium
shermanii, P. freudenreichii)
• very hard - parmesan (goat milk fermented and flavored by
Streptococcus lactis, S. cremoris, S. thermophilus; Lactobacillus
bulgaricus), romano (cow milk fermented and flavored by
Streptococcus lactis, S. cremoris, S. thermophilus; L. bulgaricus

Brewing industry
• Alcoholic beverages
• brews (beer, ale)
• germinate grains (barley, wheat, rice) to allow amylase activity to release
fermentable sugars (malting)
• dry and crush malted grains, then rehydrate and allow further enzymatic
activity (mashing)
• add hops (dried flowers of the female vine Humulus lupulis), heat in brew
kettle
• remove hops, add Saccharomyces carlsbergensis for beer or S. cerevisiae
for ale (pitching), then ferment 7-12 days (---> 2-5% ethanol)
• "age" (lagering)
• pasteurize or filter, then package
• distilled alcoholic beverages (whiskey, brandy, rum, etc.)
• generate brew (Saccharomyces spp.)
Food production
• meat products (salami, summer sausage) - Pediococcus cerevisiae and
Lactobacillus plantarum
• baked goods - Baker's yeast (Saccharomyces cerevisiae) aerobically generates
carbon dioxide for breads and pastries
 • "regular" bread - S. cerevisiae provides flavor and carbon dioxide for
"holes" to give light texture
• sourdough bread - S. exiguus (provides flavor and carbon dioxide ) and
Lactobacillus (provides flavor)
• miscellaneous foods
• Chinese cabbage- Lactobacillus
green olives) - Leuconostoc mesenteroides together with Lactobacillus plantarum
• pickles (cucumbers) - Leuconostoc mesenteroides together with
Lactobacillus plantarum
• sauerkraut(cabbage) - Leuconostoc mesenteroides together with
Lactobacillus plantarum
• soy sauce (soybeans) - Aspergillus oryzae or A. soyae, Streptococcus
rouxii together with Lactobacillus delbrueckii
• tempeh (tofu = soybean curd) - Rhizopus oligosporus or Rhizopus oryzae
• vinegar (apple juice, wine) - Acetobacter or Gluconobacter
Microbes as direct food source
• single-cell protein - Candida grown on sulfite waste "liquors" from paper
manufacturing
• approximately 50% protein, but also 16% nucleic acid
• taste is objectionable to many
• may cause kidney stones or gout (high nucleic acid content) when
consumed in large quantities over long periods of time
• Spirulina (cyanobacterium - photosynthetic)
• mushrooms - Agaricus campestris bisporus

Food additives and supplements

• amino acids - used as food additives or starting materials in the chemical
industry
• microbes must be modified to overproduce their products by
eliminating feedback inhibition and repression mechanisms as well
as inducing secretion of product
• examples - glutamic acid (MSG), phenylalanine and aspartic acid
(aspartame = Nutrasweet), lysine, tryptophan
• vitamins - food supplements for humans and animals
 • microbial production is feasible due to complexity and expense of
chemical synthesis
• more than $700 million in market value produced annually
• examples - vitamin B1, riboflavin (Saccharomyces); ascorbic acid
(Acetobacter)
• brewer's yeast (Saccharomyces carlsbergensis) - used as vitamin B-rich
food additive (relatively high in nucleic acids, though)
• citric acid (Aspergillus niger) - food and beverage additive
Pharmaceutical industry
• products of genetically engineered microbes include:
• insulin - treatment of diabetes
• growth hormones - human (treat dwarfism), epidermal (promotes wound
healing), bone (treat osteoporosis), animal (promotes livestock growth)
• tissue plasminogen activator (TPA) - dissolves blood clots
• blood clotting factors (VII, VIII, IX) - restore clotting mechanisms in
hemophiliacs without chance of transmitting AIDS or hepatitis
• erythropoietin - treatment of certain forms of anaemia
• cytokines - interferons (IFN), interleukins (IL), and other cytokines that
act as anticancer agents or immune modulators
• IFN-gamma stimulates cancer cells to produce tumor-associated
antigens so they can be detected and eliminated by the immune
system
• IL-2 stimulates T cells to promote immune responses
tumor necrosis factor alpha (TNFa) and granulocyte-macrophage colony stimulating factor
(GM-CSF) work together with IL-2 in cancer therapy
• vaccine antigens - prevention of bacterial, fungal, metazoan, viral
diseases (e.g., recombinant Hepatitis B vaccine now in use)
• monoclonal antibodies (mAb)
• diagnostic applications - determine ovulation, pregnancy;
identification of infectious agents
• therapeutic applications - specific drug delivery in cancer
therapy; destruction of platelet-catalyzed blood clots in heart
disease therapy
• antibiotics are secondary metabolites produced by bacteria (Bacillus,
Nocardia, Streptomyces) or fungi (Aspergillus, Cephalosporium,
Penicillium)
 • cheaper to produce by fermentation than by chemical synthesis, but their
structures (and thus their activities) may be modified by subsequent
chemical steps (semisynthetic antibiotics)
• steps toward commercial production include:
• isolation - usually by screening (cross-streak method)
• testing for toxicity and efficacy
• optimization and purity of yield - gene amplification, other
genetic engineering of microbes
• developing extraction and purification steps - organic chemistry
applications
Environmental remediation
• Bioremediation is also called Biodegradation Enhancement and includes any
purposeful use of microbes to degrade unwanted substances in the environment
• natural products
• petroleum - certain bacteria (some cyanobacteria, pseudomonads, corynebacteria,
mycobacteria), green algae and fungi (several molds and yeasts) oxidize
hydrocarbons at aerobic water/oil interfaces (with optimal conditions, up to 80%
removal within 1 year after a spill)
• biodegradable plastics
• photobiodegradable - structure of polymer altered by UV light in
sunlight so that it is now amenable to biodegradation
• biochemically biodegradable starch-linked polymers - starch-digesting
bacteria in soil attack the starch, releasing polymer fragments which are
degraded by other microbes
• xenobiotics - chemically synthesized compounds not found in nature (pesticides,
synthetic polymers, etc.) and thus would seem unlikely to be degradable by naturally
existing microorganisms; persistent in nature
• microbes that can degrade xenobiotics are rather diverse and typically include both
bacteria and fungi
• PCBs - certain Pseudomonas species have been engineered to accelerate
breakdown of polychlorinated biphenyls (formerly used by electric industry as
transformer insulation)
• PAHs (poly aromatic hydrocarbons) can be difficult to degrade, but there are
microbes in the environment that can accomplish this task, especially when
working together
• pesticides - herbicides, insecticides and fungicides
• these are typically rather complex molecules
• some xenobiotics are good carbon sources and electron donors for soil
 microbes, so they are more readily degraded than others
• other xenobiotics, such as chlorinated insecticides, are recalcitrant to
degradation; thus they have rather long persistence times in the
environment
• lindane - 3 years for 75-100% disappearance
• DDT - 4 years for 75-100% disappearance
• chlordane - 5 years for 75-100% disappearance
• to degrade these xenobiotics, microbes may employ co-metabolism, in
which an organic material other than the xenobiotic is used as the primary
energy source and the xenobiotic is degraded as a secondary process
• Genetically engineered microbes in bioremediation
• Microbes can be "engineered" to carry out the biochemical processes needed
for bioremediation
• concerns about long-term effects of genetically engineered microbes on
the environment are manifold
• fate of genetically engineered microbes is similar to that of other
allochthonous organisms, but even more extreme because they are
typically engineered to require nutrients, etc. not naturally present in the
environments into which they may be introduced ... so they will die out
when the material they were engineered to degrade has been removed
from the area
• Naturally-occurring microbes bio remediate just as well as engineered
microbes in many cases ... it is important to adjust environmental conditions to
favor their growth, however
Microorganisms that can degrade plastics are:-
Aliphatic Polyesters :
• Aliphatic polyesters are derived from naturally occurring compounds, such as lactide
(LA), glycolide (GL), and ε-caprolactone (CL). These represent an important class of
biocompatible and biodegradable materials. Initially, the main application of these
materials was for reabsorbable sutures.
• PolyEthylene Adipate (PEA)- lipases from R. arrizus, R. delemar, Achromobacter sp.
and Candida cylindracea ; Poly (β-Propiolactone) PPL - estereases from Acidovorax sp.,
Variovorax paradoxus, Sphingomonas paucimobilis.
Aromatic Polyesters :
• Aromatic Polyester is resistant to high temperatures. Aromatic Polyester is slightly
hydrophilic, so it is preferable to use the material in dry environments. Aromatic
polyester-filled PTFE materials exhibit excellent wear resistance, and their non-abrasive
nature prevents the galling of soft mating surfaces.
• Poly-3-Hydroxybutyrate (PHB) – estereases from Pseudomonas lemoigne, Comamonas
 sp. Acidovorax faecalis, Aspergillus fumigatus ;Poly Lactic Acid (PLA) - proteinase K
from Tritirachium album, Amycolatopsis sp Strains of Actinimycetes has been reported to
degrade polyamide (nylon), polystyrene, polyethylen
Microbes for organic pollutants
• Phenolic compounds: Achromobacter, Alcaligenes, Acinetobacter, Arthobacter,
Azobacter, Flavobacterium, Pseudomonas putida, Candida tropicalis, Trichosporon
cutaneoum, Aspergillus, Penicillium
• Benzoate and related compound: Arthobacter, Bacillus spp, Micrococcus, P. putida
• Hydrocarbons: E. coli, P. putida, P. Aeuruginosa, Candida
• Surfactants: Bacillus, Alcaligenes, Achromobacter, Flavobacterium, Pseudomonas,
Candida
• Pesticides: P. Aeruginosa (DDT), B. sphaericu (Linurin), Arthrobacter and P. cepacia
(2,4-D), P. cepacia (2,4,5-T and Parathion )
Other products
• Commodity chemicals - organic compounds
• acetic acid and other organic acids
• acetone, butanol (Clostridium acetobutylicum), ethanol - solvents
• ethanol - energy production (fuel component)
• Enzymes
• secondary metabolites produced by bacteria or fungi during idiophase
• useful (due to specificity of reaction) in food processing (especially dairy
products), pharmaceutical, and textile industries
• examples: proteases (detergent additives); amylase, glucoamylase and glucose
isomerase (polysaccharide digestion to help start fermentations)
• Biopolymers
• exopolysaccharides can be used as stabilizers, etc.
• microbial plastics:
• poly-ß-hydroxybutyrate (PHB), which is commonly used by some bacteria
as a lipid storage material, can be used as a raw material for plastic based
packaging materials
• the nature of the raw material (and thus the plastic that can be synthesized
from it) can be selected by varying the carbon source used to grow the
bacteria - using acetate (C2) and butyrate (C4) yields PHB; caproate (C6)
yields poly-ß-hydroxycaproate PHC; valerate (C5) yields poly-ß-
hydroxyvalerate PHV; mixtures yield co-polymers
• Biosensors - bioelectronics utilize the abilities of microbes to measure pollutants and
contaminants
BIOCHEMISTRY
• the science of the atoms and molecules in living organisms
• the study of chemical processes within and relating to living organisms
• the study of the structure, composition and chemical reactions of substances in living
organisms.
Importance of biochemistry
• Contributes important information to biology, medicine, nutrition, agriculture,
physiology, genetics, and immunology:
• practically all of the primary specialties on the life sciences.
• helps one understand the actual chemical concepts of biology.
• In medicine: biochemists investigate the causes and cures of diseases.
• In nutrition: they study how to maintain health wellness and study the effects of
nutritional deficiencies.
• In agriculture: biochemists investigate soil and fertilizers, and try to discover ways to
improve crop cultivation, crop storage and pest control.
Define enzymes
• Enzymes are biological catalysts synthesized by living cells.
• Majority of enzymes are protein in nature.
Special characteristics
• High catalytic power
• Enhances the rate of reaction by as much as fold
• remains unchanged at the end of the reaction
• High specificity
• Ability to catalyze reactions under normal temperature and pressure
• Ability to get regulated by a variety of metabolites and environmental conditions
• Sometimes enzymes require additional organic or inorganic molecules called cofactors
for their activity.
• An enzyme complexed with its cofactor is called holoenzyme
• An enzyme lacking its cofactor is termed apoenzyme
• Apoenzyme (inactive) + cofactor = holoenzyme
Specificity of enzymes
• Enzymes are highly specific
 • Each enzyme generally catalyzes only one type of reaction
• This is called reaction specificity.
• Emil Fischer in 1884 proposed his famous ‘lock and key’ hypothesis after observing the
specificity of glycolytic enzymes toward their carbohydrate substrates.
Describe lock and key hypothesis
• Aka Fischer’s template Theory
• Active site is a rigid and pre-shaped template where only a specific substrate can bind
• Just like a key fits into a lock
• It does not give any scope for flexible nature of enzyme
• It does not explain some key facts about enzyme catalysis

Active site
• The active site is the special place, cavity, crevice, chasm, cleft, or hole that binds and
then magically transforms the substrate to the product.
• The kinetic behavior of enzymes is a direct consequence of the protein’s having a limited
number (often 1) of specific active sites.

Induced fit theory

• Aka Koshland’s model

• Proposed in 1958
• Considered a more realistic and acceptable model
• Active site is not rigid or pre-shaped
• Only upon binding of a proper substrate, a conformational change is induced in
the enzyme molecule which results in the formation of a strong binding site
• Due to the induced fit, appropriate amino acids are repositioned to form the active
site and bring about catalysis
• Induced fit hypothesis showed that:
• Enzyme molecules are not very rigid
• Enzyme structure is quite flexible
• Enzyme structure can acquire different conformational states with distinct properties
under different conditions.
Describe structure of carbohydrates and proteins
Amino acids
• Amino acids are biologically important organic compounds containing:
• amino (-NH2) functional group - amine
• carboxyl (-COOH) functional group - acidic
• a side-chain (R group) specific to each amino acid.
• Both the carboxyl and amino groups are attached at the same alpha carbon.
• At pH 7, both the -carboxyl and - amino groups are ionized.

Side Chains
• The R-group (side chain) is what makes each amino acid unique.
• Each of the 20 amino acids has a different side chain structure.
• Side chains contain mainly hydrogen, carbon, and oxygen atoms.
• Some amino acids have sulfur or nitrogen atoms in their R-groups.

Amino acids roles:

• building blocks of proteins
• Intermediates in metabolism
• Building blocks for a number of compounds with specific biological activity e.g.
enzymes, hormones, antibiotics, etc.
• All proteins whether from bacteria or animal origins consist of 20 amino acids
• These amino acids are covalently linked in a characteristic manner:
short chains – peptides
long chains – polypeptides or proteins
The 20 amino acids
• that make up proteins
• each have assigned to them both:
 • three-letter (can be upper or lower case)
• one-letter codes (upper case)
• alanine - ala - A
• arginine - arg - R
• asparagine - asn - N
• aspartic acid - asp - D
• cysteine - cys - C
• glutamine - gln - Q
• glutamic acid - glu - E
• glycine - gly -G
• histidine - his - H
• isoleucine - ile - I
• leucine - leu - L
• Lysine - lys – K
• methionine - met - M
• phenylalanine - phe – F
• proline - pro - P
• serine - ser - S
• Threonine - thr - T
• tryptophan - trp – W
• tyrosine - tyr - Y
• valine - val – V
• All standard amino acids except glycine have an asymmetric carbon atom, that is the -
carbon is attached to 4 different groups, therefore the α-carbon is a chiral centre.
• All amino acids exist in two stereoisomers, D and L configuration.
• But all proteins except glycine are present in L-stereoisomers.

Amino acid classification

• The most useful way to classify the 20 standard amino acids is by the polarities of their
side chains.
• The amino acids may be categorized in 4 classes:
• non-polar or hydrophobic
 • Polar but uncharged (neutral)
• Negatively charged
• Positively charged
Structure of proteins
• Proteins are linear polymers of amino acids.
• Proteins are the workhorses of biochemistry WHY?
• Because they participate in essentially all cellular processes.
• Proteins Are Built from a library of 20 Amino Acids
• Four levels of protein structure are:
• The primary structure: refers to the amino acid sequence.
• The secondary structure refers to the conformation adopted by local regions of the
polypeptide chain.
• Tertiary structure describes the overall folding of the polypeptide chain.
• Quaternary structure refers to the specific association of multiple polypeptide
chains to form multi-subunit complexes.

• Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains

Primary structure
• amide bonds formed between the carboxyl group of one amino acid and the amino group
of the next.
• Properties of the peptide bond
• it is resistant to hydrolysis so that proteins are remarkably stable kinetically.
• the peptide group is planar because the C-N bond has considerable double-bond
character.
• Each peptide bond has both a hydrogen-bond donor (the NH group) and a hydrogen-bond
acceptor (the CO group).
• hydrogen bonding between these backbone groups is a distinctive feature of
protein structure.
• The peptide bond is uncharged
 • This allows proteins to form tightly packed globular structures having significant
amounts of the backbone buried within the protein interior.

Secondary structure
• Polypeptide Chains Can Fold into Regular Structures Such as:
• the Alpha Helix
• the Beta Sheet
• Turns
• Loops
• Two major elements of secondary structure are the α helix and the β strand.
• In the α helix:
• the polypeptide chain twists into a tightly packed rod.
• Within the helix, the CO group of each amino acid is hydrogen bonded to the NH
group of the amino acid four residues along the polypeptide chain.
-Helix
• It is a spiral structure
• Each turn of an α-helix contains 3.6 amino acids.
• consisting of a tightly packed, coiled polypeptide backbone core
• with the side chains of the component amino acids extending outward from the central
axis to avoid interfering sterically with each other

In the β strand:
• the polypeptide chain is nearly fully extended.
• Two or more β strands connected by NH-to-CO hydrogen bonds come together to form β
sheets.
• Secondary structure exists to provide a way to form hydrogen bonds in the interior of a
protein.
• These structures (helix, sheet, turn) provide ways to form regular hydrogen bonds.
• These hydrogen bonds are just replacing those originally made with water. As a protein
folds, many hydrogen bonds to water must be broken.
• If these broken hydrogen bonds are replaced by hydrogen bonds within, the protein, there
is no net change in the number of hydrogen bonds.
 • Because the actual number of hydrogen bonds does not change as the secondary structure
is formed, it is often argued that hydrogen bonds don’t contribute much to the stability of
a protein.
• However, hydrogen bonds that form after the protein is already organized into the correct
structure may form more stable hydrogen bonds than the ones to water.
• Hydrogen bonding does contribute somewhat to the overall stability of a protein;
however, the hydrophobic interaction usually dominates the overall stability.
• The stability of secondary structure is also influenced by surrounding structures.
• Secondary structure may be stabilized by interactions between the side chains and by
interactions of the side chains with other structures in the protein.
Structure of carbohydrates
Monomers, polymers and macromolecules
• The term macromolecule means giant molecule.
• There are three types of macromolecule in living organisms, namely polysaccharides,
proteins (polypeptides) and nucleic acids (polynucleotides).
• The prefix ‘poly’ means many, and these molecules are polymers, meaning that they are
made up of many repeating subunits that are similar or identical to each other.
• These subunits are referred to as monomers.
• They are joined together like beads on a string.
• Making such molecules is relatively simple because the same reaction is repeated many
times.
• The monomers from which polysaccharides, proteins and nucleic acids are made are
monosaccharides, amino acids and nucleotides respectively.
Carbohydrates
• All carbohydrates contain the elements carbon, hydrogen and oxygen.
• The ‘hydrate’ part of the name comes from the fact that hydrogen and oxygen atoms are
present in the ratio of 2:1, as they are in water (‘hydrate’ refers to water).
• The general formula for a carbohydrate can therefore be written as (.
• Carbohydrates are divided into three main groups, namely monosaccharides,
disaccharides and polysaccharides.
• The word ‘saccharide’ refers to a sugar or sweet substance.
Monosaccharides
• Monosaccharides are sugars.
• Sugars dissolve easily in water to form sweet-tasting solutions.
 • Monosaccharides have the general formula (C and consist of a single sugar molecule
(‘mono’ means one).
• The main types of monosaccharides, if they are classified according to the number of
carbon atoms in each molecule, are trioses (3C), pentoses (5C) and hexoses (6C).
• The names of all sugars end with -ose.
• Common hexoses are glucose, fructose and galactose.
• Two common pentose are ribose and deoxyribose.
Molecular and structural formulae
• The formula for a hexose can be written as .
• This is known as the molecular formula.
• It is also useful to show the arrangements of the atoms, which can be done using a
diagram known as the structural formula.
• The structural formula of glucose, a hexose, which is the most common monosaccharide.

Disaccharides and the glycosidic bond

• Disaccharides, like monosaccharides, are sugars.
• They are formed by two monosaccharides joining together.
• The three most common disaccharides are maltose (glucose + glucose), sucrose (glucose
+ fructose) and lactose (glucose + galactose).
• Sucrose is the transport sugar in plants and the sugar commonly bought in shops.
• Lactose is the sugar found in milk and is therefore an important constituent of the diet of
young mammals.
• The joining of two monosaccharides takes place by a process known as condensation.
• For each condensation reaction, two hydroxyl (–OH) groups line up alongside each other.
• One combines with a hydrogen atom from the other to form a water molecule.
• This allows an oxygen ‘bridge’ to form between the two molecules, holding them
together and forming a disaccharide (‘di’ means two).
• The bridge is called a glycosidic bond.
• In theory any two –OH groups can line up and, since monosaccharides have many –OH
groups, there are a large number of possible disaccharides.
• The shape of the enzyme controlling the reaction determines which –OH groups come
alongside each other. Only a few of the possible disaccharides are common in nature.
• The reverse of condensation is the addition of water, which is known as hydrolysis.
 • This takes place during the digestion of disaccharides and polysaccharides, when they are
broken down to monosaccharides.

Testing for the presence of sugars Page 32

Classify sugars

Polysaccharides
• Polysaccharides are polymers whose subunits (monomers) are monosaccharides.
• They are made by joining many monosaccharide molecules by condensation.
• Each successive monosaccharide is added by means of a glycosidic bond, as in
disaccharides.
• The final molecule may be several thousand monosaccharide units long, forming a
macromolecule.
• The most important polysaccharides are starch, glycogen and cellulose, all of which are
polymers of glucose.
• Polysaccharides are not sugars. Since glucose is the main source of energy for cells, it is
important for living organisms to store it in an appropriate form.
• If glucose itself accumulated in cells, it would dissolve and make the contents of the cell
too concentrated, which would seriously affect its osmotic properties.
• Glucose is also a reactive molecule and would interfere with normal cell chemistry.
• These problems are avoided by converting glucose, by condensation reactions, to a
storage polysaccharide, which is a convenient, compact, inert (unreactive) and insoluble
molecule.
• The storage polysaccharide formed is starch in plants and glycogen in animals.
• Glucose can be made available again quickly by an enzyme-controlled reaction.
Starch and glycogen
• Starch is a mixture of two substances – amylose and amylopectin.
• Amylose is made by condensations between α-glucose molecules.
• In this way, a long, unbranching chain of several thousand 1,4 linked glucose
molecules is built up. (‘1,4 linked’ means they are linked between carbon atoms 1 and
4 of successive glucose units.)
• The chains are curved and coil up into helical structures like springs, making the final
molecule more compact.
• Amylopectin is also made of many 1,4 linked α-glucose molecules, but the chains are
shorter than in amylose, and branch out to the sides.
• The branches are formed by 1,6 linkages.
• Mixtures of amylose and amylopectin molecules build up into relatively large starch
grains, which are commonly found in chloroplasts and in storage organs such as
potato tubers and the seeds of cereals and legumes.
• Starch grains are easily seen with a light microscope, especially if stained; rubbing a
freshly cut potato tuber on a glass slide and staining with iodine–potassium iodide
solution is a quick method of preparing a specimen for viewing. Starch is never found
in animal cells.
• Instead, a substance with molecules very like those of amylopectin is used as the
storage carbohydrate.
• This is called glycogen.
• Glycogen, like amylopectin, is made of chains of 1,4 linked α-glucose with 1,6
linkages forming branches.
• Glycogen molecules tend to be even more branched than amylopectin molecules.
• Glycogen molecules clump together to form granules, which are visible in liver cells
and muscle cells, where they form an energy reserve.
Cellulose
• Cellulose is the most abundant organic molecule on the planet, due to its presence in
plant cell walls and its slow rate of breakdown in nature.
• It has a structural role, being a mechanically strong molecule, unlike starch and
glycogen.
• However, the only difference between cellulose and starch and glycogen is that cellulose
is a polymer of β-glucose, not α-glucose.
• Remember that in the β-isomer, the –OH group on carbon atom 1 projects above the ring.
 • In order to form a glycosidic bond with carbon atom 4, where the –OH group is below
the ring, one glucose molecule must be upside down (rotated 180°) relative to the other.
• Thus successive glucose units are linked at 180° to each other.
• This arrangement of β-glucose molecules results in a strong molecule because the
hydrogen atoms of –OH groups are weakly attracted to oxygen atoms in the same
cellulose molecule (the oxygen of the glucose ring) and also to oxygen atoms of –OH
groups in neighboring molecules.
• These hydrogen bonds are individually weak, but so many can form, due to the large
number of –OH groups, that collectively they provide enormous strength.
• Between 60 and 70 cellulose molecules become tightly cross-linked to form bundles
called microfibrils.
• Microfibrils are in turn held together in bundles called fibers by hydrogen bonding.

Classify vitamins
• Vitamins are chemical compounds that are required in small amounts with our regular
diet in order to carry out certain biological functions and for the maintenance of our
growth.
• Fat-soluble vitamins
Vitamin A, D, E and K are fat-soluble.
These are stored in adipose tissues and hence are called fat-soluble vitamins.
• Water- soluble vitamins
Vitamins in B-group and vitamin C are water-soluble and cannot be stored in our bodies
as they pass with the water in urine.
These vitamins must be supplied to our bodies with regular diets.

Functions of vitamins
• Vitamin A- hardening of the cornea in the eye, night blindness
• Vitamin B1- deficiency may cause beriberi and dwarfism
• Vitamin B2- deficiency can cause disorders in the digestive system, skin burning
sensations
• Vitamin B6- deficiency of B6 causes convulsions, conjunctivitis, and sometimes
neurological disorders.
• Vitamin B12- its deficiency can cause pernicious anaemia and a decrease in red blood
cells in haemoglobin
• Vitamin C- it is a water-soluble vitamin, its deficiency causes bleeding in gums and
scurvy
• Vitamin D- it is obtained by our body when exposed to sunlight. Its deficiency causes
improper growth of bones, soft bones in kids, and rickets.
• Vitamin E- deficiency of vitamin E leads to weakness in muscles and increases the
fragility of red blood cells.
• Vitamin K- it plays an important role in blood clotting. The deficiency of vitamin K
increases the time taken by the blood to clot. Severe deficiency may cause death due
to excessive blood loss of a cut or an injury.