UNIT – 1

Microbiology is the branch of science that deals with study of microorganisms. It deals with their form, structure,
physiology, classification, reproduction and metabolism. Microorganisms are the small living things that include
unicellular, multicellular or acellular.
Unicellular are single cell organisms like bacteria (Cocci, bacilli, vibrio and spirillae), unicellular fungi (yeast),
protozoa (Amoebazoa)
Multicellular are filaments and sheaths to form cell colonies like blue green algae (Cyanobacteria), fungi
Acellular are organisms without cells, like viruses, prions

HISTORY OF MICROBIOLOGY
History of microbiology is divided into three stages namely Discovery stage, Transition stage and Modern stage
Discovery Stage
• Aristotle (384 – 322) – Described living and non living organisms and their differentiation
• Roger Bacon (13th century) – Described that diseases are caused by living organisms
• Frascastorius (1546) – Communicable diseases were caused by living agents known as Germs
• Robert Hooke (1665) – First identified the cell structure
• Antony Van Leeuwenhoek (1632 – 1723) – First person used his own design microscope. In (1675) he
discovered microorganisms and named bacteria and protozoa as ‘Animalcules’
• Kircher (1659) – Reported minute worms in blood during plague attack to humans
Transition Stage
• Spontaneous generation – Many people in the 17th and 18th centuries believed that maggots are arised
from dead animals, meats
• Francesco Redii (1629 – 1679) – Disprove the theory of Spontaneous generation theory and described
that maggots are developed from flies.
• John Needham (1713 – 1781) – Supported the above theory and showed that tiny organisms are arised
from the mutton gravy.
• Finally Lazzaro spallanzani (1729 – 1799) demonstrated that air carried germs would give growth of
microorganism and which developed into maggots
• John Tyndall (1820 – 1893) – proved that on prolonged heating, heat stable and heat sensitive bacteria
both are killed
• Augustino Bassi (1835) – described that the silk worm disease was caused due to fungal infections
Modern Stage
• Actual development of microbiology came with Louis Pasteur, Robert koch, Lord Lister, Alexander
Fleming and Paul Elrich
Louis Pasteur
✓ Father of medical microbiology.
✓ Coined the term microbiology, Aerobic and Anaerobic
✓ In 1897, mild heating (63 °C for 30 min) was more effective than boiling to destroy microorganisms
without changing taste of the product. This method is known as Pasteurization.
Also invented fermentation process and developed live attenuated vaccines against Rabies and Anthrax
Robert Koch
✓ He demonstrated that how the diseases caused by bacteria and proposed the germ theory in 1876
✓ Also invented the isolation technique of bacteria from pure culture
✓ He prepared gelatin based solid media but it was failed to produce growth of bacteria since gelatin is
digested by bacteria.
✓ There are four Koch’s postulates viz
a. Every individual have disease causing microorganisms

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 b. The microorganisms could be isolated and grown in pure culture
c. When the pure culture is inoculated into any animal, it will surely cause disease
d. Again the causative organism must be re-isolated and re-identified from the animal
Lord Joseph Lister
✓ He is the father of antiseptic surgery.
✓ Also states that Wound infections were due to microorganisms
✓ Discovered the method of destroying microorganisms in the operation theatre by spraying a fine mist of
carbolic acid in the air
Paul Erlich
✓ He discovered the treatment of syphillis by using arsenic.
✓ Founder of biological standardization of toxins and antitoxins in quantitative manner
✓ Also invented the precursor technique to Gram staining bacteria
Alexander Flemming (1881 – 1955)
✓ First scientist who discovered Worlds first antibiotic called Penicillin from the mould of Penicillium
notatum in 1928 which destroy many pathogenic bacteria
✓ Received Nobel prize in 1945
Some important discoveries
• Emile Roux & Alexandre yersin – Diptheria toxin
• Joseph Hansen – Mycobacterium leprae
• Albert ludwig Neisser – Neisseria gonorrhoeae
• Louis pasteur – Rabbies vaccine
• Good pasteur – Cultivation of virus in chick embryos
• Alexander fleming – Penicillin
• James watson and Crick – Structure of DNA
• Robert Koch – Research on Tuberculosis
SCOPE AND IMPORTANCE
• Microbiology is a part of our daily life. It plays vital role to health care system.
• In Pharmaceutical microbiology – Microorganism is used in the production of antibiotics, enzymes,
vitamins, vaccines and other pharmaceutical products
• In Pharmaceutical industry: Many antimicrobial drugs are tested by assisting with microbiologist to
ensure the drug action against the certain microorganism
• In Medical microbiology – Used to study the disease pathology and immunology
• In Industrial microbiology – Utilization of microbes for use in industrial processes like fermentation and
waste water treatment
• In Microbial biotechnology – genetic and molecular level manipulation to generate useful products.
• In Agricultural microbiology - Used for the production of hybrid variety of plants and animals. It also
used as biofertilizer.
• In Environmental microbiology - Study of microorganism used as biopesticides , biodecomposers,
cleanup the pollutants.
• Medical device: Plays a significant role in medical devices. Immunofluroscence studies is applied to
detect pathogens in tissue samples

ASEPTIC TECHNIQUE AND ITS IMPORTANCE

• Aseptic techniques are used to prevent contamination from microbes, dust particles, pyrogens and bacterial
toxic products.
• An Aseptic area means room with clean area designed for preventing microbial contamination of the
products. Two major strategies of aseptic work are using a Bunsen burner and a laminar flow hood.

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 Bunsen burner:It will create a cone of hot air. It will heat things very quickly for sterilization of
inoculating loops, warming glass bottle necks.
Laminar air flow:It can deliver the air in vertical or horizontal. 99.97% pure air should be claimed in
laminar air flow. The air supplied to aseptic room is filtered is through HEPA filters. HEPA filter placed at
inlet for clean room. HEPA – High efficiency particulate air filter. Through various aseptic technique,
many materials are sterilized. They are,
1. Culture media
2. Fluids& Reagents used in the labs
3. Laboratory container and Laboratory equipments

CLASSIFICATION OF MICROORGANISM
Microbes are widely spread in environment in which some are responsible for serious harm and some are
beneficial to life. So microbes are classified into six major groups namely bacteria, archaea, fungi, protozoa,
algae and viruses.

Microorganisms

Acellular Cellular

Prokaryotic Eukaryotic

• Acellular organi[Link] cells or cell membrane. Eg: Virus

• Cellular organi[Link] definite cell structures. Based on cell structures and mode of reproduction they
are further divided into Prokaryotes and eukaryotes.

• Prokaryotes: These are cells without nucleus and have no membrane bound organelles. They are haploid
(Possess only one copy of genes in the cell) and reproduce asexually. Eg: Bacteria and archaea

• Eukaryotes: These are cells with true nucleus and well defined membrane. They are diploid (possess two
copies of genes) and reproduce sexually. Eg: Fungi, protozoa and algae.

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 Characteristic Eukaryotes Prokaryotes
Size Normally > 10 μm Typically 1-5 μm
Cell type Multicellular Unicellular
Contain
Cell wall composition Contains cellulose
peptidoglycan
Cell membrane
Sterols present Sterols absent
composition
Flagella Structurally complex Simple
Fimbriae Cilia Present
Pili Absent Present
ER, Mitochondria,
Lysosomes,
Present Absent
Microtubulesand Golgi
apparatus
Ribosomes Larger (80S) Smaller (70S)
Vacuoles large size Smaller size
Nuclear membrane Present Absent
Chromosomes More than one One (plasmid)
With in a true nucleus
In the cytoplasm,
Location of separated from the
usually attached to
chromosomes cytoplasm by nuclear
the cell membrane
membrane
Reproduction Asexual or sexual Asexual
Exhibit mitosis and Mitosis and meiosis
Nuclear division
meiosis are absent

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 BACTERIA
• Bacteria are prokaryotic and unicellular microorganisms that exist as independent or as parasites (dependent
for other organisms) in life. Bacteria are very small approximately 0.5 to 1.0 μm in dia.
• Their reproduction is by asexual method.
• They have rigid cell wall made of Peptidoglycon.
• They are in various shapes like Bacillus (Rod), Coccus (Spherical), Spirillum (Spiral), Vibrio (Comma).
MORPHOLOGY
ULTRA STRUCTURE OF BACTERIA:
EXTERNAL STRUCTURE:
Capsule:
• Capsule is the outer layer of the bacterial cell and it is well organized.
• This layer has different names called Glycocalyx (made of Carbohydrates), S- layer (made of glycoprotein),
Slime layer (not well organized).
• Functions: Prevent drying, protect from injury, temperature, helps in attachment on another surface.
Cell wall
• It is the rigid layer situated outside the plasma membrane.
• It provides shape to the bacteria and provides protection from osmotic lysis.
• Made of Peptidoglycan (Murein) is an enormous mesh like polymer made of glycoproteins. The polymer
contains two sugar derivatives – N- Acetyl glutamic acid and N – Acetyl muramic acid and 4 amino acids.
This peptidoglycan is very thick in gram positive bacteria and very thin in gram negative bacteria.
• The space between cell wall & plasma membrane is Periplasmic space filled by the substance Periplasm.

After Christian Gram developed the Gram stain in 1884, most bacteria could be divided into two major groups –
Gram positive and Gram negative.
Gram Positive Bacteria Gram Negative Bacteria
Cell wall is made of Glycoproteins called Peptidoglycan Cell wall is made of highly Lipoproteins and some
glycoproteins
Effect of dyes: Purple colour is formed Effect of dyes: Pink colour is formed
Effect of antibiotics: Easily susceptible Effect of antibiotics: Resistant
Flagella is made of two rings Flagella is made of four rings
Lipoproteins are absent Lipoprotein is present
Perisplasmic space is absent Perisplasmic space is present
Teichoic acid is present Teichoic acid is absent
Eg: Staphylococcus aureus, Streptococcus pneumonia Eg: E. coli, Salmonella typhi

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Pili and Fimbriae
• Bacteria contains short, fine made up of 100 - 1000 numbers of hair like structures called Fimbriae. They
are used for attachment to other and for twitching motility.
• Pili or Sex Pili are also hair like appendages which surrounds the cell about 1-10 numbers. These are
larger than fimbriae (9-10 nm in dia). It is non motile but has adhesive structure made up of Pilin
(Protein). It is used for attachment and helps in conjugation type of reproduction.

Flagella
Bacteria moves by use of flagella. It is thread like structure extending outward from the plasma
membrane and cell wall. Bacterial flagella are slender, rigid structures about 15 or 20 μm long. Flagella is
composed of three parts. Motile bacteria eg: E. coli, [Link]; Non motile bacteria: Staphylococcus aureus
1. Filament: Longest portion which is a hollow rigid cylinder extends from the cell surface.
2. Basal body: It is embedded into the cells. It has four rings (L, P, S and M Ring) connected to central rod.
3. Hook: These are short curved segment links the filament to the basal body and act as flexible coupling

Types of Flagella:
• Monotrichous bacteria: The bacteria have one flagellum, if it is located at an end it is known as Polar
flagellum Eg: Vibrio Cholerae
• Amphitrichous bacteria: The bacteria have a single flagellum at each pole. Eg: Spirillum minus
• Lophotrichous bacteria: The bacteria have a cluster of flagella at one or both ends Eg: Spirilla
• Peritrichous bacteria: Flagella are spread fairly evenly over the whole surface of peritrichous bacteria. Eg:
Salmonella typhi, [Link] etc.

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INTERNAL STRUCTURE:
Cell membrane or Plasma membrane or Cytoplasmic membrane:
• It is a thin structure that completely surrounds the cell components. It is composed of phospholipids (20-
30%) and proteins (60-70%).
• Main function is selective permeability and transport of solute and excretion of hydrolytic exoenzymes.

Cytoplasm:
• It is a homogenous aqueous solution bounded by cell membrane and is divided into three areas -
Cytoplasmic area, Chromatin area and Fluid portion.
• It does not contain ER, Golgi bodies, Mitochondria, and true nucleus.
Inclusion bodies:
• They are mainly used for storage of energy and reduce osmotic pressure.
Ribosomes:
• These are loosely attached to the plasma membrane (70S).
• They are made up of both protein and ribonucleic acid. They are the site of protein synthesis.
Mesosomes:
• They play a major role in cellular respiration.
Nucleoid:
• Nucleoid is the central area of the cell which contains chromosomes. It does not contain true nucleus. The
chromosome exists as a double stranded DNA.
Plasmid: Circular DNA used for transmission of gene characteristics to other organism

Endospores
• Bacteria – at unfavorable environmental condition (high temp or no food) produce spores – which is
highly resistant which are metabolically dormant. They are produced by the species like Bacillus
anthracis, Bacillus subtilis, Clostridium tetani, Clostridium botulinum

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 GROWTH OF BACTERIA
On the basis of Carbon and energy sources, bacteria are broadly divided into two groups
• Autotrophs – Able to synthesize their own food.
Phototrophs - Organism that uses energy from sunlight to synthesize organic compounds for its nutrition.
Eg: Cyanobacteria
Chemotrophs - Organisms that obtain energy by the oxidation of electron donors in their environments.
Rhodobacter species. These molecules can be organic (Organotrophs) or inorganic (Lithotrophs).
• Heterotrophs – Unable to synthesize their own food and depend on other sources.
Eg: Saprophytic bacteria (dead matter) - Clostridium acetobutylicum
Symbiotic bacteria (host organism) – Rhizobium species
Parasitic bacteria (animals and plants) – Staphylococcus aureus, Xanthomonas campestris
Nutritional Requirements for growth of bacteria –
Culture media
Culture medium: A culture media is used to grow different kinds of microorganisms.
• It is composed of different nutrients (Carbon, Nitrogen, vitamins, minerals, aminoacids, nucleotides etc)
that are essential for microbial growth.
• The Culture medium is a solid or liquid. The solid medium is made of brown jelly substance called agar.
Use or Importance of culture medium:
To identify the cause of infection from the clinical samples.
To study the characteristics and properties of microorganisms.
To prepare the biological products like vaccines, antigens, toxoids etc.
Classification of Culture medium:
• Media can be classified on the basis of several parameters,

Classification based on Physical state

• Solid media – Contains 1.5 – 2% agar in ingredients used for isolating the bacteria.
• Semisolid media – Contains 0.5% or less agar and is used for determination of bacterial motility.
• Liquid medium (Broth medium) – Does not contains gelling agents like agar or gelatin, used for
fermentation studies and various tests
Classification based on composition
• Simple media or basal media: It Contains peptone water and Beef extract (liquid medium). Addition of
2% agar to basal media gives Nutrient agar (solid medium)
• Complex media: It contains complex materials (blood or milk or yeast extract). Provides all growth
factors for the cultivation of bacteria
• Synthetic or defined media: It contains only pure chemical substances. Used for metabolic studies of
different microorganism.
Classification based on functional type
• Enriched media: Blood, serum and special nutrients are added to basal media to enhance the growth. Eg:
Blood agar.
• Enrichment media: Specific nutrients added to liquid medium to favor the growth of wanted bacteria Eg:
Tetrathionate broth.

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 • Selective media: Inhibit unwanted bacteria and provides growth of wanted bacteria Eg: EMB (Eosin
methylene blue) agar medium (allows only Gram -ve bacteria), MSA (Mannitol Salt Agar) medium – For
Gram +ve bacteria)
• Indicator media: Contains indicator which changes color when bacteria grows Eg: Wilson and Blair
medium. (Bismuth Sulphite agar produces metallic brown precipitate when [Link] grows)
• Differential media: Used to differentiate different types of bacteria Eg: MacConkeys agar medium
(Differentiate lactose fermenting and non fermenting bacteria)
• Sugar media: 1% sugar added in the base media. Microbes utilizing the sugar particularly will produce
gas to be identified.
• Transport media: To prevent contamination during transport Eg: Stuarts transport medium.
• Assay media: Used for assay (potency determination) of antibiotics, vitamins and aminoacids
• Storage media: Media help in preservation of bacteria for long period Eg: Cooked meat medium
Physical requirements for the growth of bacteria:
• Temperature
• pH
• Oxygen
• Osmotic pressure
• Light
Temperature: It will influence the growth of microorganism. Optimum growth temperature – Temperature at
which the bacteria grow rapidly. Based on temperature tolerance and its influence on growth, bacteria may be
classified into,
• Psychrophiles/ Cryophiles: Organisms that are capable of growth and reproduction in cold temperatures.
Temperature range: −20°C to +10°C. Eg: Chlamydomonas.
• Mesophiles: Grows best in moderate temperature. Temperature range: 20°C to 45°C. Eg: E. coli
• Psychrotrophs or Facultative psychrophiles: Cold‐tolerant bacteria. Temp. range: above 15 and 20°C,
respectively. Eg: Pseudomonas etc.
• Thermophiles: Heat-loving microorganisms. Grow at 50°C or higher. Eg: Thermus aquaticus.
• Hyperthermophiles: Lives in extremely hot environments. Temperature range: 80°C to 113°C. Eg:
Sulfolobus, Methanococcus jannaschii, Thermotoga, etc.

pH: Microbial growth is strongly affected by the pH of the medium. Drastic variations in cytoplasmic pH disrupt
the plasma membrane or inhibit the activity of enzymes and membrane transport proteins. Bacteria prefer media
of pH near neutrality, and usually cannot tolerate pH values much below 4-5.
• Acidophiles: Grow between pH 0 and 5.5. Eg: Ferroplasma
• Alkalophiles:Grow between pH range of 7.5 to 14. Eg: Thermococcus alcaliphilus, etc.
• Neutrophiles: Grow between pH 5.5 to 8.0. Eg: E. coli, Pseudomonas aerunginosa, etc.
Osmotic pressure: Osmotic pressure is the minimum pressure which needs to be applied to a solution to prevent
the inward flow of water across a plasma membrane.

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 • Osmotolerant: Able to tolerate high osmotic pressure. Eg: Staphylococcus spp, etc.
• Halophiles: Able to tolerate high salt concentration. Eg: Halobacterium halobium
Light: Except Phototrophs, Darkness usually favor the rapid growth of all microorganism. They are sensitive to
UV radiation, direct light and other radiations
Gaseous requirements for the growth of bacteria:
The principle gases that affect bacterial growth are Oxygen and Carbon dioxide. Depending on O2 requirement,
the bacteria can be classified as,
1. Aerobic bacteria: Require O2 for growth Eg: E. Coli, Staph. Aureus
2. Anaerobic bacteria: Do not utilize O2 for energy and growth.
Tolerant anaerobes – Tolerate small amount of O2.
Stringent anaerobes or obligate anaerobes – Cannot tolerate O2 Eg: Clostridium Species
3. Facultative aerobes: Anaerobic bacteria grow even in presence of O2.
3. Facultative anaerobes – Bacteria do not require O2 for growth but if O2 available, it will use it for energy
production. Eg: Pseudomonas aeruginosa.
4. Microaerophilic bacteria: Bacteria require low levels of O2 for growth but does not tolerate high level of O2
Eg: Lactobacillus plantarum

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 GROWTH CYCLE OF BACTERIA (GROWTH CURVE)
• When microorganisms are cultivated in culture medium, the growth of microorganisms reproduced by binary
fission which can be plotted as the logarithm of the number of viable cells versus the incubation time.
• The resulting curve has four distinct phases
Lag phase
Log phase or Exponential phase
Stationary phase
Decline or Death phase
Lag Phase:
• When microbes introduced into fresh culture medium, no immediate increase in cell number occurs. This
period is called the lag phase.
• Enzymes, intermediate metabolites and the cell components are synthesized in this phase, cell repairing
also done. This stage is known as initial stage or dormant stage.
• The average time of lag phase is 2 hrs but it varies from species to species and ranges from 1-4 hrs
Log Phase or Exponential phase:
• During the phase, microbes are growing and dividing at the maximal rate possible.
• Their rate of growth is constant and balanced growth.
• The average time of log phase is 8 hrs
Stationary Phase
• In this phase, Microorganism growth begin to stop and the growth curve becomes horizontal. Max.
growth achieved when 109 per ml.
• But all the microbes are viable that is they metabolically active.
• Reason of Stationary phase: Nutrients decreased, Accumulation of toxic waste products.
Decline Phase: (Senescence and Death)
• Death phase
• Nutrient loss and the increased toxic wastes caused loss of cell viability.
• That is, even when bacterial cells were transferred to fresh medium, no cellular growth was observed.
• And the cells are died.

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 QUANTITATIVE MEASUREMENT OF BACTERIAL GROWTH
Growth is determined on the basis of,
• Cell Count - Directly By Microscopy or by electronic particle counter or indirectly by colony count
• Cell mass - Directly By weighing or by cell nitrogen or Indirectly by turbidity
• Cell activity - Indirectly by relating the degree of biochemical activity
CELL COUNT
Direct Microscopic count:
• By Petroff Hausser Counting Chamber. Special Slide accurately ruled into squares (1/400 mm2 in area)
with Cover slip rest 1/50 mm above the slide. So the volume over a square - 1/20,000 mm3.
• Bacterial Suspension counted in chamber by Phase contrast microscope
• Adv: Very rapid & morphology can be studied
• Disadv: Bacteria at Log phase cannot be counted

Electronic Enumeration of Cell Numbers:

• Bacterial suspension placed inside an electronic particle counter.
• Bacteria passed thro tiny orifice 10 to 30 μm in dia. Orifice connected to counter contain electrically
conductive solution
• As Each Bacteria passes generates electric signal which is counted
• Disadv: Viable or inactive cells not identified.
The Plate count method: (Viable count method)
• Measured amount of bacterial suspension introduced into petridish
• Agar culture medium is added above. Mix well. If Agar get solidifies, microbes. entrapped into gel
• Each microbes. grows and developed into individual colonies.
• Colony count is performed in dark field microscope
• This method is used routine in bacterial estimation in milk, water, foods
• Disadv: If the cells have tendency to aggregate (staphylococci), the resulting count will be lower than
actual
Membrane filter count: (Viable Count method)
• This filter has uniform porosity of predetermined size which trap microbes
• Bacteria trapped in membrane is then placed in plate counter containing culture medium with dyes for
easy detection
• During incubation, the organism grow into colonies are counted
• Adv: Can determine small no. of viable cells
CELL MASS
Determination of the Dry weight of cells
• This is the most direct method. Apply at very dense suspensions
• The cells are washed off from the medium
• Disadv: Accumulation of metabolic products of some microbes will affect the result.
• But many organism give accurate and reliable result

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Turbidimetric method:
• Culture tube contains more than 107 to 108 cells per ml appears turbid to the naked eye.
• A spectrophotometer or calorimeter can be used for measurement of cell mass
• Disadv: Dead and living cells both are measured.
Determination of Nitrogen content
• Major constituent of cell is protein which contain nitrogen
• Bacteria contain 14% of nitrogen on dry weight basis
• Performed only on specimens free of all other sources of nitrogen
CELL ACTIVITY
Measurement of a specific chemical change produced:
• Some Bacteria produce organic acid by utilizing glucose fermentation
• The organic acid production is proportional to the bacterial population

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 CULTIVATION OF BACTERIA
Cultivation of Aerobic bacteria:
• Incubated under normal conditions
• Aeration ↑ by frequent shaking
• Can grow in test tubes, small flasks
bottles like Fernback and Roux bottles
Cultivation of Anaerobic bacteria:
Anaerobic bacteria do not require O2 for its growth. During cultivation, oxygen is removed by various methods,
1. By addition of reducing agents in the vacuum desiccator.
2. Anaerobic Chamber: The anaerobic chamber is made of air locked glove box and contains a gas mix of
5% hydrogen and 95% nitrogen. The palladium catalyst present in the chamber will made the reaction
between H2 and oxygen present inside the chamber to form H2O. The water is absorbed by Desiccant
silica gel.

3. By Candle jar method – Agar plates are placed inside the airtight container. Lighted candle is placed
inside the container which utilizes the O2 present in the container.

4. Addition of Pyrogallic acid or mixture of chromium & H2SO4 - Which absorbs O2.

5. McIntosh and Fildes anaerobic jar – It is a Glass or metal jar with lid. It has inlet and outlet. Inlet is
used for supply of gases (H2/N2) and the outlet is used as vacuum pump for evacuation of O2. McIntosh
and Fildes’ anaerobic jar works on the principle of evacuation and replacement. After O2 evacuation the
remaining air present in the jar is reacted with H2 in presence of palladium catalyst which is suspended in
the lid to form H2O.

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6. Gaspak system – Disposable envelope contains chemicals which combine with water to generate H2 &
CO2. Cold catalyst (Palladium crystals) present in the system will combine the H2 with O2 to produce
anaerobic environment

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 REPRODUCTION OF BACTERIA
1. Asexual Production: Bacteria mostly reproduce by 2. Sexual reproduction: Bacteria cannot reproduce
asexual reproduction. They are sexually but it transfer its genetic information to other
• Binary Fission bacteria in three ways and hence it is also called as
• Budding Sexual reproduction.
• Fragmentation • Conjugation, Transformation, Transduction
• Formation of Conidiospores or sporangiospores

Binary Fission:
In this process, two daughter cells formed are
identical to each other. The whole process of binary
fission involves two step - Genome replication and
Septum formation. Eg: Bacillus subtilis.

Budding:
Some bacteria produce protrusions, called buds. Eg:
Hyphomicrobium vulgare and Rhodo pseudomonas
acidophila are common examples of budding
bacteria

Fragmentation:
At unfavorable conditions, bacterial forms minute
bodies called gonidia. Under favorable conditions,
each gonidium grows to a new bacterium. Eg:
Nocardia species

Formation of spores: Sporulation is the formation of nearly dormant forms of bacteria at its unfavorable
condition. Spores can preserve the genetic material of the bacteria. At favorable conditions, each spores will
form into new bacteria. Types of spores are,
• Conidiospores - The terminal portions of bacterial filaments form into rounded structures in chains, called
conidia. These conidia on detachment form new individuals. Eg: Streptomyces sp
• Sporangiospores - sporangia-like structures may develop at the end of certain hyphae. On liberation of
the sporangiospores, it germinate to produce bacteria.
• Endospores: Some Gram +ve bacteria, especially bacilli, and certain blue-green bacteria (e.g.
Stichosiphon), Bacteria produce thick-walled, resistant structures around the entire cell at unfavorable
called endospores. Endospores can be formed in middle, sub-terminal or terminal of the cell. At favorable
conditions, This will breakup into new cell

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Sexual reproduction:
• It involves the transfer of gene factors, occurs in bacteria. But, there is no fertilization and meiosis. The
gene transfer in bacteria occurs by three methods—Conjugation, Transformation and Transduction.
• Conjugation involves transfer of DNA from a donor cell to a recipient cell through pili.
• Transduction involves transfer of bacterial genes from a donor cell to a recipient cell by a bacteriophage.
• Transformation involves the uptake of naked DNA molecules from one bacterium (the donor cell) by
another bacterium (the recipient cell).

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Synchronous growth or Culture: If all the cells in a culture were in same stage of division cycle, then it is
called synchronous growth. This is achieved by manipulating the growth of cultures by altering the physical
environment or nutrient concentration etc. These cells are separated out by filtration or centrifugation and used to
study the stages or cell division of the bacteria and their interrelationships.

Continuous Culture: The bacterial population growing at a particular rate in the log phase is maintained. This
condition is called steady state growth. The volume of culture medium and the cell concentration are kept
constant by allowing fresh sterile medium to enter the culture vessel and the spent culture containing cells are
removed. By this the growth in the culture vessel is balanced. This system is chemostat.

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 ISOLATION OF BACTERIA (or) PURE CULTURE
Individual Bacteria from various sources (Like soil) are isolated by various techniques. The techniques
commonly used for isolation of Individual bacteria from group of colonies,
1. Streak plate method
2. Pour plate method
3. Spread plate method
4. Roll tube method
5. Micromanipulator method
6. Serial dilution method
1. Streak plate method: Rapid qualitative isolation method. Loop of mixed culture streaked across the surface
of agar medium. More and more streaks will separate the microbes from each other.
Two methods of streaking are,
a. Continuous Streaking method: This involves inoculating the top half of the plate, rotating it 180 degrees, and
inoculating the other half of the plate without sterilizing the loop or dragging bacteria from the previous section.

b. Discontinuous streaking method: Streaking is discontinuous. It is done by T-Streaking and Quadrant method.
The portions of agar is streaked and on every streaking the loop should be sterilized.

2. Pour plate method: Agar medium is melted. The small amount of diluted mixture of microorganism sample
is mixed with melted agar at 40°C. Then it is poured into the Petridish and cooled. Allow to solidify. After
incubation, the plates are examined for individual colonies. Pure colonies are isolated and transferred into new
broth cultures.

Spread plate method:

• Small amount of mixed culture placed in centre of agar plate and it is spread by a sterile L shaped glass rod
called spreader by spinning the petriplate.
• Principle: As petriplate spins while spreading the microbes with spreader, the cells separated from each other
by a distance sufficient to allow the colonies

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Roll tube method:
• Used for the isolation of obligate anaerobes.
• Stoppered culture test tube wall is coated with O2 free reduced agar medium
• Stopper is removed, the tube is continuously flushed with N2 or CO2 from a gas cannula.
• Inoculation is done with transfer loop as the tube is rotated by a motor. Inoculation started at bottom first and
drawn up gradually. By this the inoculum get thinned and the isolated colonies are developed.

Micromanipulator Method:
• This device is used with microscopes.
• Single cell is sucked into a micropipette and transferred to drop of sterile medium.

Serial dilution method

• This is used for the microbial culture formed only in liquid media.
• Inoculum is subjected to serial dilution
• If the culture contains 10 ml of liquid medium and contains 1000 no. of organisms. Take 1 ml from this
and dilute to 9 ml of sterile liquid medium and again it is diluted to contain 1 m.o in 1 ml.
• This medium is incubated and the growth represents the pure culture.

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 PRESERVATION OF PURE CULTURES
After isolation of any microorganism, it is important to maintain the viability and purity of such microbes that are
free from contamination. By reducing the metabolism, the ageing and death of bacteria are slowed down and
maintaining inactive state, microbes are maintained for several years. Methods for preserving pure cultures are,
1. Refrigeration:
• Stored in refrigerators or cold rooms at 0-4° C.
• This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi).
• Here the metabolism are slowed down but not stopped.
2. Paraffin method: (storage in mineral oil)
• Simple and economic
• Sterilize liquid paraffin in oven for 1 hr at 180 °C.
• It is poured over the agar slope for 1 cm.
• Layer of paraffin ensures reduced O2 and prevents dehydration.
• Microbe’s growth is slowed down and remained in dormant state

3. Lyophilization or freeze-drying method:

• Most bacteria will die when it is dried but freeze drying will preserve the bacteria
• Dense cell suspension or Microbial culture is placed in small vials and frozen at -60 to -70 °C

Apparatus:
• Small cotton plugged vials containing frozen suspension of bacteria which are placed in the glass flask
attached to the condenser. The Condenser is connected to vacuum pump. Through condenser cold
condition is passed to the glass flask.
• Bacteria become dessicated (dry ice) as the ice in the suspension sublimes to water vapor
• After dessication, the vials are removed and placed in large tube.
• The inner vials are insulated with plug of glass wool pack. Then the outer tube is sealed under vacuum
4. Cryopreservation
• Suitable for long term preservation that donot survive in freeze drying
• The loss is reduced by adding Cryoprotectants like glycerol and DMSO(Dimethyl sulphoxide)
• Microbes are rapidly frozed in liquid N2 at -196°C in presence of Cryoprotectants.
• This remain the viability for 10-30 years without any change in characteristics
Procedure
• Grow the microbial culture in sterile Petridish.
• Add glycerol or DMSO. Pour 0.5 ml of suspension in cryotube
• Transfer the liquid N2 to cryotube and store it. For reviving, the culture is removed from liquid N2 and
keep it in room temp for 2-3 hrs and transfer to suitable growth media
5. Storage at -70 C
• Deep freezers are used
• 0.5 ml of suspension stored in small glass tube and stored at -70 °C

21
6. Storage on glass beads at -60°C to -70°C:
• Bacterial liquid culture medium is mixed with 15% glycerol (Cryoprotectants)
• Neutralized glass bead are transferred to the bacterial culture medium. The glass beads
will pick up approx 0.5 ml of suspension and these beads are transferred to vials.
• Freeze the vials at -60 to 70°C.
• It is revived by removing the beads by rubbing over the surface of solid culture
medium

7. Storage in gelatin discs:

• Bacterial suspension is suspended in molten nutrient gelatin.
• The molten gelatin is placed again in petridish and allow to solidify and dried through dessicator.
• Used to preserve Enterobacteriaceae, Staphylococcus bacteria.

8. Storage in Soil
• So, Bacteria converted into Spores and they are suspended in sterile water.
• Then it is seeded in bottle containing sterile nutrient soil.
• Allow the spore for growth in 10 days and kept in refrigerator.
• Fungi like Rhizopus, Aspergillus and some bacteria can be stored in sterile soil as spores for
longer period of time.

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 MICROSCOPY
A microscope is an instrument used for viewing objects that are too small to be seen by the naked eye. The
science of investigating small objects using microscope is called Microscopy
TYPES OF MICROSCOPE
LIGHT MICROSCOPE ELECTRON MICROSCOPE
Magnification is obtained by a system of optical lenses It is a microscope that uses a beam of accelerated
using light waves. They are classified into, electrons as a source of illumination. As the
wavelength of an electron can be up to 100,000 times
shorter than that of visible light photons, electron
microscopes have a higher resolving power than light
microscopes and can reveal the structure of smaller
objects.
1. Bright field Microscope 1. Scanning Electron Microscope
2. Dark field Microscope 2. Transmission Electron Microscope
3. Phase Contrast Microscope
4. Fluorescence Microscope

TERMINOLOGIES USED IN MICROSCOPY

• Magnification: Degree of enlargement ie., no. of times length, breadth or diameter of object has been
multiplied
• Resolving power: Ability of objective lens to differentiate two points of an object.
• Limit of Resolution: Minimum distance by which two objects can be separated
• Numerical aperture: It is the ratio of diameter of lens to its focal length → ability to gather light and
resolve fine detail of the specimen at a fixed distance.
It is derived by mathematical formula
NA = nsinθ/2
Where n is refractive index and θ is aperture angle

COMPOUND MICROSCOPE - LIGHT / BRIGHT FIELD MICROSCOPE

• Basic tool for education and research
• Helps in magnifying an image in two stages
• Uses objective lens → many different powers to magnify the image
• Uses eye piece → magnify the image formed from objective lens
• Its mechanical and optical parts are,
Mechanical parts
• Made of metals that include
• Foot/base - Horse shoe like solid part @ bottom of microscope
• Pillar - Small projection from foot, with inclination joint at the top
• Inclination joint - It is a hinge joint by which the upper part of microscope can be tilted in upward or
downward direction
• Stage - Metallic plate with two clips which hold the glass slide and hole in centre which allows the light
from the mirror
• Arm - Curved handle → carry microscope
• Body tube - Holds the optical parts like eyepiece and objective lens at proper distance

23
 • Nose piece - Fitted at the base of the body tube and holds different objective lens with different
magnifying powers (10X, 40X and 100X)
• Coarse adjustment knob - Used to move body tube up and down to focus the image
• Fine adjustment knob - Used to view fine details of objects
Optical parts:
Consists of glass lens →produce magnified image that includes
• Mirror - Fixed below the stage → to regulate the light to illuminate the object through the hole on stage
→has two faces; When natural light is available, plane mirror may be used and in artificial light concave
mirror is used
• Iris/ Diaphragm - Located bet. the mirror and object. Has an aperture which can be widened or narrowed
as required → regulates the entry of light to the specimen
• Condenser - Fixed between mirror and stage. It concentrates or focuses the light rays on the object
• Objective lens -
Low power objective - has magnification of 10X
High power objective - has magnification of 40X
Oil immersion objective - has magnification of 100X. Oil is used (Cedar wood oil) - prevents the
refraction of light outwards in ari and allows it to pass straight to the objective. The refractive index of oil
is 1.516 which equals the RI of glass. RI of air is 1.000.
• Eye piece/ Ocular lens - Consists of a lens placed at upper end of body tube and has magnification of
10X
Working principle:
• Used widely in clinical and educational laboratories. It uses combination of lenses that enhances both
magnifying and resolving power
• The specimen mounted on glass slide and placed between the condenser lens and objective lens. A beam
of visible light is focused onto the specimen through condenser
• It create a magnified image of the specimen called Primary image inside the body tube through objective
lens
• This image again magnified by the eye piece
• For higher magnification, the objective lens is rotated to bring high power lens (generally 40 or 45X).
• Fine adjustment is required to get clear image. More light is required
• For very high magnification (observing the bacterial cell), immersion objective lens (100X) is employed.
• A drop of immersion oil (Cedar oil, Olive oil) placed over the coverslip.
• The lens is contacted with oil for sharp observation. Clear the oil after observation.

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 DARK FIELD MICROSCOPY
• It forms a bright image against dark background.
• Unstained samples are easily observed.
• The transparent and semi transparent objects are not easily visible in bright field microscope.
• Visibility is increased in Dark field microscopy.
Principle:
• A hollow cone of light is focused on the specimen
• By this unreflected and unrefracted rays donot enter the objective lens and pass outside the lens
• Only light that has been reflected or refracted by the specimen forms an image
• The field surrounding the specimen appears black but the specimen is brightly illuminated
• Three requirements are made for adapting ordinary microscope into dark background microscope,
- A dark background condenser which focuses rays of light only on the specimen
- A high intensity lamp
- A funnel stop or filter which reduces the numerical aperture of the objective to less than 1.0.
• The Abbe condenser, Paraboloid condenser and cardioid condenser are commonly used for dark field
illumination.
Abbe condenser
• It is more commonly used Suitable for objects require small magnification.
• Dark field element is inserted.
Paraboloid condenser
• To be used with oil immersion objectives
• Use intense source of light.
• Object mounted in the liquid and protected with coverslip.
• Numerical Aperture of the objective lens is smaller than condenser.
Cardioid condenser
• It uses Strong arc lamp.
• Due to high light, Fused quartz slides and cover slips are used.
• Designed for examining Colloidal suspensions and solutions.
Application of dark field Microscope:
• For examining unstained objects
• Microbes suspended in fluid (wet mount and hanging drop preparations)
• Eg: It is used to identify the microbes Treponema palladium in the diagnosis of syphilis

25
 PHASE CONTRAST MICROSCOPY
Principle:
- The phase contrast microscopy is based on the principle that small phase (Wavelength) changes in the
light rays, induced by differences in the thickness and refractive index of the different parts of an object,
can be transformed into differences in brightness or light intensity.
Construction
• It contains special type of condenser and phase plate.
• Condenser contains special diaphragm which consists of annular stop - allow hollow light to enter
• The phase plate is a optical disc made of special ring located to back to the objective lens which retards or
advance the direct light rays.
• The light passed through the specimen is altered to observe the various internal parts.
• This will differentiate the different parts of the cell or specimen
Applications:
• Visualization of internal cell structures.
• Unstained specimen is easily observed.
• Diagnosis of tumor cells.
• For examination of cell growth and cell division of bacteria, flagellar movements, spore formation.

FLUORESCENCE MICROSCOPY
Principle:
• Certain compounds absorb UV light and then they become excited and later it release energy in the form
of visible light. Such Substance are called fluorescent or fluochrome Eg: Uranium ores, Oil droplets,
Dyes
• This phenomenon is known as Fluorescence
• Application of this phenomenon in microscope to observe the image of an object is the basis of
fluorescence microscopy
Working procedure
• Mercury vapor lamp used as a source for the production of light beam that passes through the Exciter
filter.
• This filter transmits only the light having capacity of exciting the samples
• This excitation light is directed to the microscope by a special mirror called Dichromatic mirror
• This mirror reflects the (Excitation light) but allows light of Larger wavelength to pass through.
• The excitation light passes through the objective lens to the specimen.

26
 ELECTRON MICROSCOPE
• Was first developed by German engineers Max Knoll and Ernest Ruska (1931)
• Utilizes beam of electrons for illumination.
• The electron wavelength is 100000 times shorter that visible light and hence it produces more frequency
and energy and so it reveal even very small objects than light.
Principle:
• Beam of electrons are produced by Tungsten filament.
• Electron with high voltage, pass through electromagnetic lenses to produce thin beam of electrons.
• This electron scans the surface of the specimen which detected by detector.
Components of Electron microscope:
It is the form of tall column, which is vertically mounted contains the following components,
• Electron gun
• Electron Column
• Electromagnetic lens system
• Detectors
• Water chilling system
• Specimen/ Sample chamber
• Main Control panel and operational controls
• Image Capture
Electron Gun:
• Heated tungsten filament generates electron beam.
• Placed on the top of instrument column.

27
Electron column:
• It is filled with three set of electromagnetic lens, sample port and airlock and set of apertures.
• The inner contents are under vacuum
Electromagnetic lens system:
• It will shapes the electron beam.
• Lens is constructed with copper wire and there is hole in the center of the lens through which the electron
beam travels.
Specimen Holder
• Contains one or two wells at the end. The sample is loaded into it
• A grid present in it, which protects the sample from falling.
Detectors:
• X ray energy dispersive spectroscopy is used as detector.
• Double walled flask holds the liquid N2 for cooling
Main control panel and Operational controls
• It consists of button, toggles and knobs, foot operated tilt pedas to control the equipment
Image capture
• At the base of the column, a viewing chamber with binoculars
• This will focus the image. Then it is captured by CCD camera

• The electron beam of (100kv - 1000 kv) passes through the condenser lens and thin beam is entered to the
specimen. Electrons scattered depending upon the specimen thickness.
• These scattered electrons pass down the magnetic coils called objective lens which magnify the image.
• Then it will come out of ocular lens produces finalized image.
• Image is then projected on fluorescent screen which contains camera for recording the image.
• Since the electron microscope works in vacuum, the specimen should be completely dry.
• Air molecules present in the column will scatter the electrons causing flicker in the electron beams.
Preparation of specimen for Electron microscopy
Dehydration and fixation:
• The specimen contains water is replaced with organic solvents such as ethanol or acetone
• Or through filtration with resins
• Chemical fixation like glutaraldehyde which crosslink the proteins, Osmium tetroxide crosslink the lipids.
Shadow casting or metal shadowing
• Deposit thin layer of metal (platinum) at an slanting angle of organism.
• This will create the variations in brightness and contrast

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Ultra-thin sectioning
• Microbial cells are too thick does not allow to visualize the internal fine structure.
• So the specimens are cut into thin sections by using ultra microtome made of diamond or glass knife
Staining:
• Specimens are stained with heavy metals such as lead, uranium.
• This is used to visualize the image in dark background.
Freeze etching
• Special technique for revealing the fine details of internal and external surface of microbes.
• Cell suspension is frozen rapidly in Liquid N2 temp and then fractured by microtome.
• Fractured specimen is etched by increasing the temperature to 95°C within a few mins.
• This etching reveals the microscopic details
• This is used for SEM analysis.
• For TEM analysis the freeze etched specimen is exposed to the vapors of heavymetal at 45° angle to
produce shadow effect. Then it is rotated to 90° and exposed again to vapors of carbon for improving the
stability of image
Localization of cell constituents:
• Thin sections of cell is treated with ferritin - labelled antibody.
• Ferritin is an iron containing substance with high density, affects the passage of electron beam.
• The combination of antibody with antigen in the cell produce complex and it is particularly identified.
Autoradiography
• Used for the location of particular chemical constituent in the specimen.
• The cells are exposed to radioactive substance and stored in dark place.
• Radioactive subs complexed - part of the cell which emits the radiation which is observed.

29
 UNIT - II
IDENTIFICATION OF BACTERIA
Bacteria are identified by,
1. Staining techniques
2. Biochemical tests
STAINING TECHNIQUES FOR IDENTIFICATION OF BACTERIA
1. Simple Staining: Purpose - To demonstrate the cell size, shape and arrangement of bacteria cells. Single
staining reagent (Methylene Blue or Crystal Violet or Carbol fuchsin) is used. These are two types.
• Positive Staining: The stain is basic and has positive charge. This will attached to negative charges of
contents of Cytoplasm. Eg: Methylene Blue
• Negative Staining: The stain is acidic have -ve charge. Bacteria repells the stain and hence it stains the
background of the bacteria. Cells appears transparent. Eg: India ink, Nigrosin
2. Gram Staining: Used in differential staining. It determines the entire morphology of bacteria and
differentiate the bacteria into two major groups. They are Gram positive and Gram negative bacteria.
Reagents: Crystal violet, Grams iodine solution, Ethyl alcohol (95%) or alcohol - acetone (1:1) solution,
Safranin solution.
Procedure: Thin bacterial smear is prepared on the glass slide → Air dry → Heat fix → Add staining solution
(Crystal violet → wait for 1 min → wash → Add mordant (Grams Iodine) → wait for 1 min → wash → Add
Decolorizing agent (Ethanol 95%) drop by drop for 20-30 sec → wash →Add Counter stain (Saffranin) →
wash → Air dry the glass slide or dry with blotting paper → Observe in Microscope

Heat fixation

Gram Staining Procedure

30
 Results:
Blue or violet color for Gram positive bacteria
Red color for Gram negative bacteria

Principle:
Gram positive bacteria Gram negative bacteria
✓ Cellwall is thick, which is made of glycoproteins ✓ Cellwall is thin, which is majorly made of
(peptidoglycon layer). lipoproteins.
✓ On heat fixing the bacterial smear, it loosened the ✓ Lipoproteins are easily dissolved by alcohol results
cellwall. in breaking of pores in the cellwall.
✓ On addition of crystal violet, the bacterial inner ✓ This leads to leakage of Crystal violet - Iodine
layer is stained by blue color. Grams iodine is complex from the cell which results in
added which made complex with crystal violet (CV decoloration
– I Complex) to produce intense blue color. ✓ After addition of saffranin, the bacteria cell takes
✓ On application of ethanol, glycoprotein of cell wall the safranin color and appears red.
layer gets precipitated and so the pores of cellwall ✓ Eg: Salmonella typhi, [Link], Proteus vulgaris
are closed and not allow the blue color of CV-I
complex to exit the cellwall and also not allowed
the color of saffranin.
✓ Hence the gram positive bacteria appear blue or
purple in microscope.
✓ Eg: Streptococcus pyogens, Staphylococcus aureus

31
32
3. Acid fast staining:
It is also known as Ziehl Neelson Staining. It is used to identify the members of Mycobacterium group.
These organisms are difficult to stain by ordinary staining method due to presence of high lipid content
(Mycolic acid) in their cell wall.
Reagents:
- Carbol fuchsin solution. Carbolfuchsin is a lipid-soluble dye that can penetrate the waxy cell walls
of acid-fast bacteria. This red dye is used as the primary stain in the acid-fast procedure.
- Acid - Alcohol (3% Hcl in alcohol) or 20-25% H2SO4 in Water. Methylene blue Stain formed.
Procedure:
- Prepare thin smear of bacterial culture on glass slide. Air dry and Heat fix
- Stain with fresh carbol fuchsin. Heat gently in steam for 5 minutes. Donot allow the stain to dry. if
it gets dry add more stain from time to time
- Then cool the stain and wash with water.
- Add acid alcohol solution for 3 min and wash with water and drain it.
- Flood the slide with methylene blue and leave it for 2 min
- Wash with tap water, blot dry or air dry the slide and observe under oil immersion obj. lens
- Results: Acid fast org. will appear bright red on blue background and Non acid fast org. will
appear dark blue

33
BIOCHEMICAL TEST FOR THE IDENTIFICATION OF BACTERIA
Various bacteria are identified by various biochemical tests and gives valuable information in identifying and
differentiating microorganism. They are
1. Sugar or carbohydrate fermentation
2. Litmus milk reactions
3. IMVIC Test***
4. Nitrate reduction test
5. Hydrogen sulphide production
6. Potassium cyanide test
7. Catalase Production
8. Urease test
9. Oxidase test

1. Carbohydrate fermentation:
- Used to differentiate Carbohydrate fermenting and Carbohydrate non
fermenting bacteria
Procedure:
• The test microorganism is inoculated in sugar media containing pH indicator dye.
• Bacteria + sugar (Carbohydrate) in the medium → Production of acids → Decrease
pH of medium → Yellow color and gas production
Result:
• Yellow color and gas production → Carbohydrate fermenting bacteria is present.
([Link])

2. Litmus milk reactions:

- Used to identify the different species of bacteria. Litmus milk contains litmus dye,
lactose, casein, lactoalbumin & lactoglobulin.
Procedure:
• The test microorganism is inoculated in litmus milk media.
Result:
• Pinkish red & gas production → Lactose fermenting bacteria is present ([Link])
• Blue → Reduction occurs → Lactose non fermenting bacteria is present
(Salmonella)
• Clot or curd formation → Milk protein coagulating bacteria is present
(Lactobacillus)
• Clear solution & Brown ring formation→ Digestion of medium occurs →
Proteolytic bacteria is present (Bacillus)

3. IMViC TEST:

It incorporates four tests (I- Indole; M-Methyl red; V-Voges Proskauer; iC- in Citrate). IMViC is useful to
identify and differentiate the bacteria of Enterobacteriaceae family (i.e., Different types of Coliform
bacteria).
Coliform bacteria are defined as rod shaped, gram-negative, non-spore forming and motile or non-motile
bacteria. Eg: [Link], Enterobacter, Klebsiella, Shigella, Salmonella etc.

34
Indole production test Procedure:
• This test identifies the bacteria which are able to decompose tryptophan into indole
• The test microorganism is inoculated in tryptophan broth contains kovac’s reagent.
• Bacteria + tryptophan → Produces Indole react with kovac’s reagent → Cherry red.
Kovacs reagent is a biochemical reagent consisting of isoamyl alcohol, para-
dimethylaminobenzaldehyde (DMAB), and concentrated hydrochloric acid.
Result:
• Cherry red color → Indole Production test Positive – E. Coli;
• Yellow color → Indole Production test Negative – Klebsiella

Methyl red test Procedure:

The test microorganism is inoculated in Methyl red Voges Proskauer (MR-VP)
Medium (Glucose Phosphate Broth, Buffered Glucose Broth) containing methyl red.
• Bacteria + glucose in the medium → Produce Pyruvic acid → converted into lactic
acid & acetic acid → decreased pH which changes dye color from yellow to red.
Result:
• Red color → Methyl red test Positive – E. Coli
• Yellow color → Methyl red test Negative - Klebsiella
Voges Proskauer test Procedure:
• The test microorganism is inoculated in MRVP broth containing alpha-naphthol and
potassium hydroxide.
• Bacteria + glucose in the medium → Produces Acetoin → oxidized into diacetyl.
Result:
• Pinkish red color - Voges Proskauer test Positive – Klebsiella;
• No pink red color - Voges Proskauer test Negative – E. Coli
In – Citrate Utilization test Procedure:
• The test microorganism is inoculated in simmon’s citrate agar (Sodium Citrate
(dehydrate), Ammonium Dihydrogen Phosphate, Dipotassium Phosphate
Magnesium Sulfate (heptahydrate)) and contains bromothymol blue.
• Bacteria + Citrate in medium + inorganic ammonium salt → Broken down to
ammonia → increases alkalinity of medium which changes green color to blue.
Result:
- Intense blue color → Klebsiella
- Green color → [Link]

4. Nitrate reduction test

To determine the ability of an organism to reduce nitrate to nitrite. Many Anaerobic bacteria utilizes nitrate
as energy source on behalf of O2.
Procedure:
• The test microorganism is inoculated in the nitrate broth contains sulphanilic acid and α-naphthylamine.
• Bacteria + Nitrate → Converted into Nitrite →Again reduced to Nitric oxide → react with sulphanilic
acid and α-naphthylamine to form azo dye (red precipitate) → on addition of Zn powder → the red
color disappears.
Result:
• Red color disappears → Test positive → Neisseria, [Link]
• Red color not disappears on additionof Zn → Test negative → Aerobic bacteria
5. H2S gas production test: Used to identify the microorgansism which utilizes the medium containing
sulphur and aminoacids and produces H2S gas. Gas production is detected by filter paper made of lead
acetate which turn into black or brown when H2S gas is produced.
6. Potassium Cyanide test: Used to identify the microorganism which grows in presence of Potassium
Cyanide. Development of turbidity indicates the positive test.

35
7. Catalase test: When a small amount of test microorganism is added to hydrogen peroxide, if the bacteria
possess catalase enzyme, bubbles of oxygen are observed. Eg: Staphylococci, Bacillus species shows
catalase positive.
8. Urease test: This test is used to identify the bacteria (like H. pylori) which secrete urease enzyme. The
test microorganism is added in the urea medium, when the microorganism grows, it produces urease
enzyme which catalyzes the conversion of urea to ammonia and carbon dioxide.
9. Oxidase test: This test used in microbiology to determine if a bacterium produces certain cytochrome c
oxidases. The test microorganism is inoculated in nutrient broth contain α-naphthol and 1% p-
aminodimethylaniline oxalate. Result deep purple or blue color.

36
 STERILIZATION
Definition: Sterilization refers to any process that removes, kills, or inhibit the growth of all forms of
microorganisms such as fungi, bacteria, viruses, spores, plasmodium, etc. and other biological agents like
prions present in a specific surface, object or fluid. After sterilization an object is referred to as being sterile or
aseptic.
The growth of micro organism can be controlled by two ways
- By killing microorganism is known as ‘Cidal’ (eg: Bactericidal)
- By inhibiting growth of microorganism is known as ‘Static’ (eg: Bacteriostatic)
And this sterilization is achieved by Physical and Chemical agents
OFFICIAL STERILIZATION METHODS
1. PHYSICAL METHOD / PHYSICAL PROCESS
• Dry heat Sterilization
• Moist heat Sterilization
• Filtration
• Radiation
2. CHEMICAL METHODS
• Gaseous Sterilization – Formaldehyde, Ethylene oxide
• By using disinfectants – Cresol, Phenol
HEAT STERILIZATION
DRY HEAT STERILIZATION
Mechanism of action: Denature protein and Oxidation of cell constituents of microorganism
• Sunlight: It has bactericidal effect (due to IR and UV rays). It is one of the natural methods of sterilization
of water in rivers, lakes and tanks.
• Red Hot: Inoculating loop, forceps, spatula are sterilized by holding them on a flame of Bunsen burner
untill it turns red hot.
• Flaming: Glass slides, Mouth of culture tubes & bottles, Cover slips, blades & needles are sterilized by
passing them over the flame of bunsen burner for 3-4 times.
• Incineration: Efficient method for destroying infective materials (such as bedding, soiled dressing and
pathogenic materials like sputum, stool & carcasses) & converted to ashes in incinerator. About 500 °C is
used for incineration.
Instrumentation
HOT AIR OVEN:
- It is made of Insulated Stainless steel or aluminium double walled chamber.
- The outer wall contains electric heaters.
- A fan is fitted in the rear of the chamber to facilitate uniform air circulation to prevent the development of
cool spots inside the chamber.
- Temperature is monitored with thermocouples.
- Sterilization temperature & Holding time in Hot air oven is:
✓ 3 hrs at 140°C;
✓ 1 hrs at 160°C
✓ 30 mins at 180°C
Applications
- Ideal method for sterilizing glass wares and surgical instruments
- Spores are killed by heating at 160 °C for 2 hours

Merits
- It offers complete destruction of pyrogens. Easy removal of dust and rust.

37
 - Suitable for moisture sensitive materials. Easy storage.
Demerits
- Not suitable for surgical dressing sterilization.
- Volatile oils and aqueous liquids cannot be sterilized.
- Penetration power of dry heat is very slow

MOIST HEAT STERILIZATION

Mechanism of action: Denaturation and Coagulation of Proteins. Microorganism is killed by saturated steam or
hot water.
✓ Moist heat temperature range:
• Temp < 100 °C
• Temp at 100 °C
• Temp > 100 °C
TEMPERATURE < 100 °C
1. Vaccine bath – Bacterial vaccines are sterilized at 60 °C for 1 hr

2. Pasteurization – It is used for sterilization of Milk without altering its taste and nutritional value. It is done
by heating & cooling immediately. Pasteurization was first developed by Louis Pasteur in 1862. Two
methods are there,
Holder method – Milk is heated at temperature 63 °C for 30 mins. All non sporing organisms are killed.
Flash method – This method is done by raising the temperature to 72 °C for 15 Sec.
3. Inspissations – Heating at 80 °C for 30 mins on 3 consecutive days by inspissator. It is also the fractional
sterilization. Eg. Used for sterilization of egg and serum based culture medium like Lowestein – Jensens
medium.

38
TEMPERATURE AT 100 °C
1. Boiling at 100 °C for 10-30 mins: Glass syringes, rubber stoppers, surgical instrument are sterilized.
2. Boiling at 100 °C for 90 mins: Culture Media subject to decompose in autoclave are sterilized by this
method. Koch or Arnold Steamer is used. All non sporing organisms except thermophiles are killed.
3. Tyndallization – This method is developed by John Tyndall also called fractional or Intermittent
sterilization. It is carried out at 100°C for 30 mins on 3 successive days. It is performed in Koch or Arnold
steamer. On first exposure, vegetative cells are killed but not spores. On second next day, the spore is
converted into vegetative cells. While heating on the next day, these converted vegetative cells are killed.
But still anaerobic spores and thermophilic bacteria are not killed by this method. It is used for sterilization
of egg, serum, sugar based culture media which is not withstanding the high temp of autoclave.

TEMPERATURE ABOVE 100 °C:

AUTOCLAVE - Instrumentation
• Modified Pressure Cooker. Sterilization achieved by saturated steam.
• Available in Horizontal or vertical chambers.
Construction: Double walled or jacketed chamber made of Stainless steel or gun metal with supporting frame.
Steam Circulates within the jacket under high pressure to closed inner chamber where materials or products for
sterilization are kept
Principle - Water boils in closed or air tight container under increased pressure, temp of water rises above 100°C
(saturated steam) affect both vegetative forms (non sporing) and spores of bacteria. Quick, great action, penetrate
easily in materials.
Mechanism of action: Denaturation and coagulation of proteins.
Operation:
• One fifth of autoclave is filled with water.
• Materials to be sterilized are placed inside and the lid is closed.
• Heater is switch on & Safety valve is adjusted for required pressure.
• Boiling water gets steamed and it allowed to escape in small amount for maintaining the inner pressure.
• Discharge tap is closed & Pressure inside the chamber get raised and it is maintained for the fixed time
[Link]. Autoclaving conditions
Temperature (°C) Pressure (psi) Minimum holding time (mins)
1. 115 – 116 10 30
2. 121 – 123 15 15
3. 126 – 129 20 10
4. 134 – 138 32 3
Applications:
1. Sterilizing Bulk solutions, suspensions
2. Culture media vials and transfusion equipments are sterilized.
3. Closures made from plastic, rubber, glass wares, surgical dressings, cotton wool are sterilized.
Merits
1. Economic and simple.
2. Efficient and high penetrating power.
3. Both spores and vegetative forms of bacteria can be killed.

39
Demerits
1. Thermolabile and Moisture sensitive substance cannot be sterilized
2. Due to high temperatures and pressure, glass vessels can be damaged.
3. If air is not evacuated properly in autoclave, leads to decrease in temperature, leads to sterilization
inefficiency.

Vertical Autoclave Horizontal Autoclave

40
 FILTRATION STERILIZATION
Mechanism of Action: Rather than killing the microorganism, it removes the microorganism through physical
screening by using filters of various pore sizes. Filtration is an excellent way to reduce the microbial population
for heat sensitive material but it only applicable to liquids. Eg. Sera, Sugars, urea, enzyme & antibiotics.
Instrumentation or types of filters are,
1. Depth Filters
2. Membrane Filters
DEPTH FILTERS:
It is made of fibrous or granular materials that are bind into a thick layer filled with twisting channels. Solution
containing microorganism is drawn through filter by vacuum and microbial cells are removed by physical
screening and it adsorbed to the surface of the filter material. Eg. Asbestos filter, Sintered glass filter, Berkefield
filter etc
Asbestos Filter: (Seitz Filter)
- This filter is disposable, made of asbestos (Magnesium tri silicate).
- Supported on a perforated metal plate within a metal funnel. Pore size is 0.01 to 5 microns.
- Then the metal funnel is fit into sterile conical flask by silicone rubber bung.
- Flask is connected to vacuum pump & Fluid to be sterilized is transferred into the funnel.
Sintered glass filters (Fritted glass/ Mortan filters)
- Finely powdered borosilicate glass packed in disc and heated to stick the powdered glass to form a filter.
- Then this filter is supported on metal funnel.
- It is available in several diff. porosities.
- Brittle and expensive and have small area for filtration but easy to clean.
Filter candles (Ceramic/Berkefeld filter)
- Used for purification of water in industries.
- They are made of Porcelain or kieselghur.
- Cylindrical in shape with comparatively thick walls.
- Filter is fitted to the filter assembly and placed in a mantle.
- Liquid to be filtered is poured into the mantle where vacuum forces it through filter.
- Inexpensive but easily clogged and blocked and required high pressure for filtration.

MEMBRANE FILTER (MILLIPORE/ ULTRA FILTER)

- Filter is made of cellulose and cellulose esters, polycarbonate, polyvinylidene flouride or synthetic materials.
- Pore size of membrane is 0.45 µm or 0.22 µm (Millipore) particularly to filter even very small Bacteria
- Arrangement: Filters are supported on a rigid base of perforated metal or plastic or sintered glass container.
These membranes are preceded by depth filters to remove the larger particles & for preventing clogging.

41
- Eg. Culture media, Opthalmic solutions, Oils, antibiotics and other heat sensitive materials are filtered
through membrane filters.

HEPA FILTER: This is also a filter used for filtering the air free from microorganism. See in unit IV.

Applications of Filtration sterilization:

✓ Filtration is an excellent way to reduce the microbial population in solutions of heat sensitive material.
Eg. Sera, Sugars, urea, enzyme and antibiotics.
✓ Hepa filters are used in research labs and industries during handling of microorganisms, conducting
assays, preparing media, examining tissue cultures at sterile working surface
Merits
- Suitable for Heat sensitive substance (Opthalmic, Parenterals) and even sterilize the environmental air.
- All forms of bacteria & fungi (Spores & vegetative form) are filtered.
Demerits
- Suspensions cannot be filtered.
- Viruses, toxins, pyrogens are easily passed.
- Leakage of filter and Clogging of filter pores are the main problem.

RADIATION STERILIZATION
Mechanism of Action: Directly hit the DNA of the microorganisms and cause ionization of the cell components
leads to death of the microorganisms.
Radiation sterilization is also known as Cold sterilization because ionizing radiation produces little heat in
materials. So heat sensitive material also sterilized by this method. Based on wavelength and penetrating power,
radiations are divided into two types, 1. Non ionizing radiations; Ionizing radiations
Non-ioinizing radiations (Ultra Violet radiations):
• UV radiation in the region of 2537 A° have greatest activity in destroying microorganism
• Air borne contaminants & most vegetative forms of bacteria are mainly killed. Spores are not destroyed.
• UV lamp is the artificial source for creating UV radiation.
• Employed in aseptic areas and rooms and in pharmaceutical industry, in hospital operating rooms, food and
diary industries.
• Also used for sterilizing biological fluids such as blood, plasma and vaccines.
Ionizing radiations (Cold Sterilization)
• X-rays, gamma rays, Cathode rays are highly lethal to DNA and other cell constituents.
• Have very high penetration power and energy. Factors that affect ionizing radiation sterilization are oxygen,
protective compounds, sensitizing agents, pH of the culture, freezing, moisture and recovery conditions
X-rays:
✓ This has considerable energy and penetration ability to produce lethal effect to microorganism.

42
 ✓ But it is very expensive and difficult to use efficiently.
✓ These are widely used to produce mutation of microorganism.
Gamma rays:
✓ Gamma rays have shorter wavelength and higher energy.
✓ These can be obtained by using radioactive isotopes of 60Co (Cobalt). Gamma rays are emitted as a result of
breakdown of 60Co due to its unstable of atoms.
✓ Have high penetrating power and microbicidal effect, these are ideal for sterilization of bulk materials such
as packaged food, medical instruments etc.
Cathode rays (Electron beam radiation):
✓ When high voltage potential is established between cathode and anode in evacuated tube, Cathode emits
beam of electrons called Cathode rays.
✓ Special instruments required to produce Cathode rays Eg. Electron accelerator, which is used for sterilization
of drugs, surgical and other materials
✓ This is also used for sterilization of antibiotics (benzyl pencillin, streptomycin sulphate) and vitamins
✓ Also used for producing vaccines by inactivating the live viruses such as influenza, Rabies, Polio virus

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 CHEMICAL METHODS - GASEOUS STERILIZATION
It is defined as the destruction of microorganism with a chemical in gaseous or vapor state. Material which is not
sterilized by heat (dry and moist) sterilization is sterilized by this method. But these gases are very harmful to
humans and produce undesirable effects.
• Ethylene oxide, formaldehyde, Beta propiolactone are most used in gaseous sterilization.
• Various glycols, methyl bromide and alcohol are used for room sterilization
Formaldehyde (HCHO):
Mechanism of action: Formaldehyde combines readily with vital organic nitrogen compounds such as proteins
and nucleic acids of bacteria.
- Formaldehyde in aqueous solution called Formalin (37-40% of Formaldehyde)
- Vaporization of formalin (liquid form) or paraformaldehyde (Solid form) is used for sterilization
- Formaldehyde gas is generated by adding 150 g of KMnO4 to 280 ml formalin for every 1000 cubic feet
rooms which is used for room sterilization. This process of sterilization is known as Fumigation.
- After vapor generation, doors are sealed and left unopened for 48 hrs.
- Humidity to be maintained 70% and temperature is 22 °C
- It kills both vegetative and spore producing bacteria
- Employed in Hospitals, operation theatres, microbiological labs
Beta propiolactone (BPL):

• Heterocyclic ring compound is a colourless liquid with boiling point of 155 °C

• Capable of killing all microorganism and is very active against viruses.
• BPL Vapour is approx. 25 times active than formaldehyde and about 4000 times active than ethylene oxide
and 50000 times active than methyl bromide.
• Highly bactericidal and used in concentrations of 2 to 5 mg/litre.
• It has low power of penetration and also has high irritation and carcinogenic properties.
Ethylene oxide: (ETO)
Mechanism of Action: It will alkalize the amino, carboxyl, hydroxyl and sulfhydryl groups of microorganism and
reacts with DNA and RNA. Ring in the ETO molecule splits and attached to the hydrogen of the reactive
organism.
+ Microorganism– SH = Microorganism – S – CH2CH2OH

✓ Colourless liquid with boiling point of 10.8 °C.

✓ Highly inflammable and explosive when 3% conc mixed with air.
✓ So it is to be mixed with CO2 and flourinated hydrocarbons (freons) to make non inflammable.
✓ Effect depends on conc. of gas, temperature, moisture, time, conditions and microorganism.
✓ It is very powerful sterilizing agent for heat and moisture sensitive materials.
✓ Used for sterilization of medical and biological preparations, catgut, plastic equipments, antibiotics,
bandages, media, hospital bedding, food stuff, clothing etc.

ANTIMICROBIAL AGENTS
Chemical agents are used for sterilization such as Disinfectants and antiseptics Eg: Phenols, alcohols, halogens,
dyes, aldehydes etc.

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 STERILIZATION MONITORS
Sterile products are strictly monitored for its sterility and it should be free from microorganism. This is
controlled or validated by two types.
1. Controls on the Sterilization equipments i.e., Sterilization monitors / Sterilization indicators
2. Sterility testing of pharmaceutical products (done after sterilization)
Sterilization monitors or Sterilization indicators:
It is done by the Physical, Chemical and Biological indicators for monitoring the efficiency of sterilization
method used.
Physical Indicators:
1. Moist heat: A Master process record (MPR) is prepared as part of the validation procedure for a Autoclave
and for each product. This Master record is then used as reference for the batch process record (BPR). MPR
should be revised whenever any changes occur in the BPR and found that change occurred is permanent.
2. Dry heat: Temperature record Chart is made of each sterilization cycle and is compared against a master
temperature record (MTR)
3. Radiation: Plastic Dosimeter is placed for measurement of radiation
4. Gaseous: Temperature, Humidity and pressure are monitored and recorded. Concentration of gas is
measured independently of pressure rise by reference to the weight of the gas.
5. Filtration: Bubble point pressure test is used for determining the pore size of the filters and to check the
integrity of the filter after use. Principle is that filter is soaked in appropriate fluid and the pressure is
applied to the filter. The pressure difference when first bubble arises is equivalent to maximum pore size.
Chemical Indicators: Based on alteration of the characteristics of chemical substances by Heat, Steam, radiation
or Gas sterilization.
1. Browne’s tubes: The most commonly used chemical indicators for heat processes are Browne’s tube.
These are small sealed tubes containing chemical mixtures and indicator. At extreme temperature, the
chemical mixtures get reacted together and colour of the indicator change and confirm the particular
temperature.

2. Witness tubes: This tubes contains crystalline substances of known melting point eg., Sulphur (115 °C),
Succinic anhydride (120 °C), benzoic acid (121 °C) etc. A dye may be included to show clear identification.
3. Heat Sensitive tape: Bowie-Dick test is used for detection of complete air removal in the test pack. Used
for monitoring the sterilization of surgical dressings. Placed in centre of the Pack and sterilized in autoclave.
If the sterilization is efficient, moist steam enters the pack up to centre and change the colour of the
indicator from blue to pink.
4. Royce Sachet: It is the indicator for Ethylene oxide sterilization. It is a polythene sachet contains MgCl2,
HCl and bromophenol blue. Exposure of ETO gas results in formation of Ethylene chlorohydrin and colour
change from yellow to purple.
5. Chemical Dosimeters: To monitor the radiation sterilization. According to the amount of radiation
exposed, dosimeter absorb the radiation and indicated the change of color from yellow to red.
Biological Indicators: Suitable microorganism is deposited on the career and placed on the sterilizer. At the end
of sterilization, the career is recovered and cultured to determine the growth

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TERMS USED FOR EXPRESSIONS OF HEAT RESISTANCE BY MICROORGANISM
TDT - Thermal Death time: Minimum time it takes to kill a population of microbes at a specific temperature.

TDP – Thermal Death point: The lowest temperature at which all microbes are killed in a 10-minute exposure.

D – Value (Decimal reduction time): Time in minutes at any defined temperature to destroy 90 % of viable
microorganisms is called the D – Value.

Z – Value (Thermal destruction value): Number of degrees of change in temperature required to produce 10
fold changes in D – Value.

F – Value: In the food industry, a unit of lethality has been devised. Defined as that the equivalent in minutes
of 121 °C of all heat considered with respect to its capacity to destroy spores or vegetative cells of microbes It
is used to calculate the probable number of survivors remained in the load.

Q10 – Value or Temperature coefficient: It is the increase in the reaction or killing rate brought about by
arise in temperature of 10 °C

Bioburden: Initial number of microorganisms present in a given product before sterilization is known as
Bioburden or Bioload.

Minimum Inhibitory Concentration (MIC) is the lowest concentration of antimicrobial agents that inhibit the
growth of bacteria (i.e. bacteriostatic).
MIC= (Lowest conc. inhibit growth + Highest conc. allow growth)
2
Minimum Bactericidal Concentration (MBC) is the lowest concentration of antibacterial agent required to
kill ≥ 99.9% of bacteria (i.e. bactericidal).

Mean generation time: Rate of bacterial growth during their exponential phase, under standard nutritional
conditions is known as mean generation time. Mean Generation times for bacteria vary from about 12 minutes
to 24 hours or more.

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 UNIT- III
FUNGI
• Mycology is the branch of biology concerned with the study of fungi.
• Fungi are group of Eukaryotic and heterotrophic organism. They are called as Saprophytes which obtain their
food from dead matter and also called as Parasites which obtain their food from living matter. Fungi
reproduce by both sexually and asexually. It contains yeast and molds. Yeasts are usually unicellular. Molds
are Multicellular and filamentous. Eg: Yeast - Sacchromyces Cerevesiae; Mold - Penicillium notatum,
Fusarium graminerium.

MORPHOLOGY / STRUCTURE OF FUNGI

• Body of the fungus known as 'Thallus'. Thallus of yeast is made of single cell, which is oval in shape.
Thallus of mold is made of mycelium (Group of cells)
• The mycelium is a group of cells which is thread like filaments called 'Hyphae'. Mycelia can be
vegetative and reproductive. Vegetative mycelium provides nutrients absorbed through cell wall.
Reproductive mycelium is responsible for spore production.
• New hyphae is developed from spores during its germination. Young Hyphae divided into cells by inward
growth called 'Centripetal invagination'. It is separated by 'septum' that has central pore contains
protoplasm (cytoplasm and nuclei).
• Hyphae occur in three forms
- formed without septum called 'Coenocytic or aseptate Hyphae'.
- formed with septum and one nuclei for each septum called 'Septate Hyphae'.
- formed with septum and with multinuclei.

• Hyphae are composed of outer cellwall made of chitin and inner 'Lumen' filled with protoplasm.
• Between Protoplasm and lumen there is 'Plasmalemma' is made of phospholipid bilayered membrane,
and also made of lipids, proteins and especially Sterols.
• In protoplasm, the cell genes are present in 'Nucleus'.
• Nucleus is surrounded by 'Nuclear membrane' made of pores for communication with other cell
• The cell possesses well defined 'Mitochondria' which is Power house of cell.
• It also contains number of 'Ribosomes' which is used for Protein synthesis.
• The proteins are exported through 'Golgi apparatus' and 'Endoplasmic reticulum' for various cells and
nucleus
• Protoplasm also contains 'Vacuoles' for storage of nutrients and hydrolytic enzymes.

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 CLASSIFICATION OF FUNGI
MORPHOLOGICAL CLASSIFICATION OF FUNGI:
Depend on cell morphology; it can be divided into 4 classes: Mould, Yeast, Yeast like fungi, Dimorphic fungi.
Moulds:
• Molds are Filamentous fungi which form mycelium made of numerous threads like structure called Hyphae.
• Hyphae are divided by cross walls called septae which contains uninucleus or Multinucleus in every septae.
• Hyphae which is not divided by septum and appear as long continuous cells with many nuclei. This is known
as Coenocytic Fungi.
• Eg: Aspergillus niger, Aspergillus fumigatus, Penicillium notatum, Microsporum gypsum

Yeasts:
• These are Round, oval or elongated unicellular fungi
• They are reproduced by asexual process like budding (cell develops protuberance called buds)
• Eg: Sacchromyces cerevisae, Cryptococcus neoformans

Yeast like Fungi:

• Some yeast produces buds which will not separated to form new cell during asexual reproduction and
form again new buds. This continues to form like pseudo hyphae.
• Eg: Candida albicans

Dimorphic Fungi: ****

• Fungi exist in two forms (as unicellular or multicellular).
• These are environment and nutrient dependents
• Eg: Mucor rouxii, Histoplasma capsulatum, Blastomyces dermitidis

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TAXONOMICAL CLASSIFICATION OF FUNGI
Kingdom - Fungi (Family: Myceteae)
Division
Gymnomycota (slime molds): Organism without cell wall.
Class -
• Acrasiomycetes – Unicellular, cell wall less, Free-living amoebae which aggregate to form spores Eg:
Dictyostelium discoideum.
• Myxomycetes - Multicellular, cell wall less plasmodium which transforms into spores Eg: Physarum
polycephalum.
Mastigomycota (Flagellated lower fungi): Aquatic fungi producing motile flagellated cells.
Class –
• Chytridiomycetes - Cells contain posterior whiplash type flagella Eg: Allomyces macrogynus
• Hyphochytridiomycetes - Cells contain tinsel type flagella Eg: Rhizidiomyces arbuscula
• Plasmodiophoromycetes - Cells contain two unequal anterior whiplash flagella Eg: Plasmodiophora
brassica
• Oomycetes - Cells with laterally inserted flagella one is anterior tinsel and another is posterior whiplash
Eg: Saprolegnia ferax.

Amastigomycota (Terrestrial fungi): Higher fungi with absorptive nutrition without motile cells contain
mycelium with septae or aseptae.
Class -
• Deuteromycetes or Fungi imperfecti - Sexual reproduction absent or not perfect. Vegetative reproduction
by conidiospores Eg: Molds and Mildews.
• Zygomycetes - Sexual reproduction by gametangial fusion, vegetative reproduction by mitospores Eg:
Mucor rouxii
• Ascomycetes - Sexual reproduction by ascus spores (Sac like cells). Vegetative reproduction by
conidiospores. Eg: Yeast
• Basidiomycetes - Sexual reproduction by basidiospores (Club like cells) Eg: Mushrooms, Puff balls

49
 REPRODUCTION OF FUNGI
Fungi reproduce by asexual and sexual reproduction.

Asexual reproduction: It is also called Somatic or vegetative reproduction. No involvement of nuclei/ sex cells/
sex organs. Done by
1) Fission of somatic or vegetative cells yields two daughter cells
2) Budding of somatic cells, each bud (a small outgrowth) develops into new cells
3) Fragmentation of hyphae or Disjoint of hyphae develops into new cells
4) Spore formation (Asexual spores)
- Sporanigospores – Many single cell spores formed inside the sacs called Sporangia at the end of hyphae.
When sporangia are motile, it is called Zoospores. When sporangia is nonmotile, it is called Aplanospores
- Conidiospores - Small single cell spores formed at the tip or side of hypha
- Oidia or Arthrospores - Formed by disjoint of hyphal cells
- Chlamydospores - Thick walled single celled spores which are highly resistant formed from cells of
hyphae
- Blastospores - Spores formed by budding

Sexual reproduction
According to fusion of nucleus of two cells
• Sexual reproduction is carried out by fusion of nuclei of two parent cells
• The process of sexual reproduction begins with joining of two cells and fusion of their protoplasts
(Plasmogamy)
• Thus enabling the two haploid nuclei fuse together to form diploid nuclei (Karyogamy)
• This is followed by meiosis, which again reduces to form 4 haploid cells
According to fusion of sex organs of fungi
• If sex organs present in fungi it is called Gametangia, they form gamates. Male gamatangia is called
Antheridium and Female gamatangia is called Oogonium. Various methods of sexual reproduction
according fusion of sex organs is summarized as
1. Gametic Copulation - Fusion of naked gamates
2. Gamete - Gametangial Copulation - Two gametangia come together but donot fuse. Male gamates come
out from male gametangia and fuse into Female gametangium
3. Gametangial Copulation - Two gametangia fuse and give rise to Zygote
4. Somatic Copulation - Fusion of Vegetative cells
5. Spermatization - Union of special male structure called Spermatium with female receptive structure

50
Types of sexual spores formed after sexual reproduction,
• Ascospores - Single celled spores are produced in a sac called Ascus. Usually eight spores present in
each ascus
• Basidiospores - Single cell spores formed on club shaped structure called basidium
• Zygospores - Large thick walled spores formed when two sexual gametangia fuse together
• Oospores - Fertilization of eggs or oospores formed within female structure called Oogonium by male
gametes formed by Antheridium

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 VIRUSES
• Viruses, are simple, acellular infectious agents can only be seen through Electron microscope. They
are 10-100 times smaller than bacteria with aproximate size range of 20-300 nm. They easily pass
through membrane filters. Viruses are called as obligate intracellular parasites.
• They can grow only in animal or plant or bacterial cells and lack metabolic machinery of their own to
generate energy or to synthesize proteins, so they depend on host cells to carry out these vital functions.
• Viruses have genetic material (Either DNA or RNA but not both)
• Study of virus is known as Virology
• Mostly they are insensitive to Antibiotics
MORPHOLOGY OF VIRUS
• Virus particles are composed of core of genetic material either DNA which is single stranded or double
stranded, or RNA which can be linear or circular.
• It is surrounded by protein coat called Capsid. Capsid with Nucleic acid is called Nucleocapsid.
• This protein coat will protect the viral genes from environmental factors. It also plays an important role in
attachment of virus to the receptor cells and also facilitates insertion of viral genome into the host cell.
• Capsid or protein coat is composed of large number of sub untis called Capsomeres
• This subunits are important for many aspects
- It contains the considerable quantity of genetic information
- It permits the construction of virus particles
- It involves only dissociation of subunits during intracellular release of viral genomes rather the
degradation of total protein sheath
• In addition to protein coat, Many animal virus are surrounded by Envelope, made of Lipoprotein.
• Envelope may or may not be covered by spikes made of carbohydrate protein complex called Peplomers.
This will help in attaching to host organism
• When capsid are not covered by envelope are known as Naked viruses or Non enveloped viruses

52
 CLASSIFICATION OF VIRUS
On the basis of morphology:
• Helical virus - Long rod shaped rigid or flexible Eg: Rabies virus, Tobacco mosaic virus.
• Icosahedral virus - Polyhedral or polygon shaped contains 20 triangular faces and 12 corners and 30 sides
Eg: Polio and Adeno virus.
• Complex virus - Bacterial viruses are very complex in structure Eg: T2, T4, T6 phages which infect [Link]
bacteria.
• Enveloped virus - They are spherical in shape. The viruses are enclosed in a lipoprotein envelope called
Enveloped virus Eg: Influenza for helical and Herpes simplex virus for polyhedral.
• Naked Virus – These virus contains only nucleic acid and Capsid. Eg: Parvo virus

On the basis of genetic material:

[Link]. DNA Virus RNA Virus
1 Pox Vaircella Corona Virus
2 Variola Virus AIDS (HIV)
3 Adeno Virus Measles (Rubeola Virus)
4 Herpes Virus Mumps (Paramyxo virus)
5 Cytomegalo virus Polio Virus (Picorna virus)
6 Epstein bar virus Rubella Virus
7 Papilloma Virus Toga virus
8 Hepatitis virus
On the basis of their host:
1. Animal virus – Viral Zoonoses/ Zoophage
2. Plant virus – Viral Phytophage
3. Bacterial virus – Bacteriophage – It is generally called as Phage. It is a type of virus that infects
bacteria. Eg: M13 Bacteriophage, Lambda bacteriophage.

53
 REPRODUCTION OF VIRUS OR MULTIPLICATION OF VIRUS OR REPLICATION OF VIRUS
Viruses multiply only intracellularly. The replication is divided into various stages.
1. Adsorption
2. Penetration
3. Uncoating
4. Biosynthesis
5. Virion Assembly
6. Release
Adsorption (Attachment):
• Virus contact with host cells by random collision and gets attached to the host cells
• Virus have special arrangement for this attachment called Spikes which easily attached to the host cell
Penetration:
• Second step is that attached virus to be penetrated into the cell done by engulfment of virus by the cell
• This process is called Viropexis
Uncoating:
• Physical Separation of nucleic acid from its outer structural components done by proteolytic enzymes
produced from lysosomes.
Biosynthesis (Viral compound synthesis)
• After uncoating, the viral genes (DNA or RNA) direct the biosynthetic machinery of host cell to shut
down the normal cell metabolism and to produce the viral components. Synthesis mostly done in Nucleus
of host cell.
• Viral DNA →enters nucleus of host cell → transcripted into early mRNA→ transported to ribosomes
→early mRNA converted to Regulatory viral proteins → formation of new viral DNA→ again goes to
nucleus →Act as templates to produce many copies →transcript late mRNA → transported to the
ribosomes→ late mRNA into late viral structural proteins (Capsid, Envelope, Spikes and enzymes)
which get back to nucleus for next stage of replication.
• For RNA viruses, Viral RNA stays in cytoplasm where it is replicated and translated into viral proteins.
Assembly (Virion assembly)
• Assembly of various viral components into complete virus particle is started just after replication of Viral
nucleic acid and may takes place in either nucleus or cytoplasm.
• The Capsomere subunits enclose the nucleic acid to form the viral nucleocapsid. This process is called
Encapsidation
Release:
• Finally the virus is replicated and released as mature virions from the host cell.
• Non enveloped virus released by cell lysis (Reverse phagocytosis). This process of releasing of virus
from the host cell by causing cell death is called Lytic cycle. Some viruses will not release from the host
cell and the multiplied Viral DNA will integrated or incorporated into the host DNA as mutation. This
process is called Lysogenic cycle. Enveloped virus, released through budding process.

54
 CULTIVATION OF VIRUS IN LABORATORY
Cultivation of virus in laboratory is done by three methods.
1. Animal inoculation
2. Embryonated eggs
3. Tissue culture method.

1. Animal Inoculation:
• White mice, Rabbits, Guinea pigs and monkeys are used
• Virus are inoculated in animals through intracerebral, intranasal, intraperitoneal or subcutaneous routes
• After inoculation, the animals are observed for signs and illness or death
• Then the animals are killed or the blood is collected from the animal
• Viruses are identified by microscopy or by neutralization and haemagglutination tests

2. Embryonated Eggs (Chick Embryo):

Procedure: Egg must be sterile and the shell should be intact and healthy. Embryos of 7-12 days old are used.
Virus are inoculated in the various parts of eggs. After inoculation, the eggs are incubated for 2-9 days and
examined for viral growth. The route of inoculation in egg is,
1. Chorioallantoic membrane (CAM): Pox virus and Herpes simplex virus are inoculated in CAM. The
replication of virus is indicated by visible lesions called pocks. Each pocks are derived from single virus.
So the Number of lesion indicates the number of virus
2. Allantoic cavity: Influenza and Paramyxo virus are inoculated in allantoic cavity. This area is inoculated
mainly for vaccine production. Rabbies vaccine, Yellow fever vaccines are also produced. Ducks egg are
used in vaccine production since it is larger than hens egg
3. Amniotic cavity: It is mainly inoculated for the primary isolation of influenza virus

55
 4. Yolk sac: Some virus and some bacterial like Chlamydia and Rickettsiae are inoculated in this area.

3. Tissue Culture:
There are three types
1. Organ culture: Small bit of organ maintained in vitro for days to weeks as like that of origical
morphology and Physiology. Formalin used for preservation. Eg: Tracheal ring organ culture used for
isolation of corono virus.
2. Explant culture: Fragments of minced tissue are used as culture. Virus is inoculated in plasma clots of
tissues. Eg: Adenovirus are isolated from Adenoid tissue explant culture
3. Cell culture: These are routinely employed nowadays. Cells of various tissues like from fibroblast,
epithelial cells are obtained and it used for cultivation and isolation of virus
Procedure:
Tissue is first removed. It is digested and converted into tissue fragments by physically (Homogenization) or
chemically by enzymes. These fragments are washed with saline solution or Hanks solution or Eagles solution.
Individual cells are obtained from these fragments by treating the tissue with trypsin called Trypsinization. It is
treated for about 18 hrs at 4 °C. During this period, the fragments of tissue gradually dispersed into individual
cells in presence of EDTA. Cells are then centrifuged and resuspended in saline medium. The suspended cells
are allowed to grow in suitable medium containing essential nutrients and antibiotics for preventing
contamination by bacteria. On incubation for 72 hrs at 36 °C, the cell layers are formed called monolayer.

56
 DISINFECTION
Definition: Disinfection is the process of destruction of microorganisms (or) reducing them to a low level that
are not harmful to the health. Disinfectants generally kill the sensitive vegetative cells (non sporing organism)
but not the heat resistant endospores. Disinfection is less effective than Sterilization process.

CLASSIFICATION OF DISINFECTANTS
Based on Consistency
1. Liquid (Alcohols and phenols)
2. Gaseous (Formaldehyde vapour)
Based on Spectrum of Activity
1. High level Disinfectants
2. Intermediate level Disinfectants
3. Low level
Based on Mode of Action
1. Action on plasma membrane
2. Denaturing cellular proteins
3. Oxidation of essential sulfahydral groups of enzymes (eg., H2O2, halogens)
4. Alkylation of amino-, carboxyl- and hydroxyl group (eg. Formaldehyde)
5. Damaging nucleic acid (eg. Formaldehyde)

The major antimicrobial agents or disinfectants can be grouped as shown below,

1. Acids and alkalies
2. Halogens
3. Heavy metals and their compounds
4. Phenol and Phenolic compounds
5. Alcohols
6. Aldehydes
7. Quarternary ammonium compounds
8. Dyes
9. Detergents and soaps

ACIDS AND ALKALIS:

Strong acid: The germicidal activity of acids is due to the strong dissociation of Hydrogen (H+) ions. Mode of
action: It will irreversibly denature the proteins of bacteria. It will take very short time to kill the bacteria. But
handling is very difficult. Eg., Most mineral acids ie., Hydrochloric acid, Sulphuric acid etc
Weak acid: Dissociation of H+ ions is less. But it can act differently against microorganism from strong acids.
Mode of action: Undissociated ions directly attack bacterial cell components. Eg. Benzoic acid, Citric acid.
Strong alkali: These are strong dissociation of (OH–) ions. These are very good detergents because they even
penetrate the spore producing bacteria. They are non toxic and have little odor when diluted. So they are used to
clean floors, in food industry etc, Eg: Sodium hydroxide
Weak alkali: Have weak dissociation of OH- ions Eg. Sodium bicarbonate, Quick lime (Anti infective agent in
agri) and Borax used for control of foot and mouth disease.

HALOGENS
Chlorine, Iodine, bromine and fluorine in the Free State as well as their compounds are strongly acted as
germicidal. Bromine and fluorine are irritants and are difficult to handle. So, Chlorine and Iodine are commonly
used for disinfectant action.

57
Mode of Action:
• Chlorine used especially in municipality water for sterilization
Cl2 + H2O = HCl + HClO (Hypochlorous acid)
HClO = 2HCl + O2
Cl2 – acts on the proteins of cell membrane
O2 – Oxidizing agent – acts on the cell organelles
• Chlorine compounds like Ca(OCl)2 and Na(OCl) – used in diary and eating equipments
• Chlorine compounds are used to disinfect the wounds, treat athletes foot and other infections
• I2- Sporicidal, fungicidal and active against many viruses. As a skin disinfectant and used for cold
sterilization of surgical sutures.
• I2 + PVP = Iodophores – Non irritating, non staining and odourless. Its a oxidizing agent which inactivates
essential proteins. I2 compounds used for disinfection of water, air and sanitization of food utensils.
HEAVY METALS
Mercury, Silver and Copper – Heavy metals
Mode of Action – combined with cellular proteins and coagulates it.
• Mercury compounds like, Mercuric chloride, Mercurous chloride, Mercuric oxide – are inorganic
compounds used as bactericidal and bacteriostatic in ointment preparations.
Merthiolate, Metaphen, Mercurochrome – are organic compounds used as bactericidal/static, fungicidal.
• Silver compounds – Silver nitrate – Bactericidal, Antiseptic.
• Copper compounds – Copper sulphate – Fungicide, prevent algal growth in lakes and pools.
PHENOL AND ITS DERIVATIVES
Mode of Action: Disruption & precipitation of cells, inactivation of enzymes, leakage of aminoacids from cells.
• Cresols are more germicidal than phenol when they are emulsified in soaps and alkalies.
• Lysol is a commercial preparation containing cresol
• Hexylresorcinol used as solution in glycerine and water and used as mouth gargles.

Phenol o- Cresol m- Cresol p- Cresol

Chloro cresol Chloroxylenol o- Phenylphenol Hexachlorophane

ALCOHOLS
Mode of Action: Denaturation of the proteins. Damage the lipid complexes in the cell membrane. Also act as
dehydrating agents.
• These are having rapid bactericidal action against vegetative bacteria when diluted to 60 to 70% with water
• Ethanol (60-70%) and isopropanol (50-60%) used as skin disinfectants
• Methanol vapour used as fungicide.
• The higher alcohols (Propyl, butyl etc) are more germicidal than ethanol

58
 • Alcohols are used as preservatives in some vaccines. Are used prior to hypodermic injections and for the
disinfection of clinical oral thermometer. Alcohol conc above 60% are effective against virus.
Eg., Methanol – CH3OH, Ethanol – CH3CH2OH, Isopropanol – (CH3)2CHOH, n-Butanol – CH3(CH2)3OH, n-
Amyl alcohol - CH3(CH2)4OH, n-Octyl alcohol - CH3(CH2)7OH

2- Phenoxy ethanol 2- Phenylethyl alcohol Chlorobutanol

ALDEHYDES
• Formaldehyde (HCHO) used as a gaseous sterilization. Formalin solution BP contains 34 to 38% w/w of
formaldehyde in water.
• Glutaraldehyde CHOCH2CH2CH2CHO have rapid sporicidal and tuberculocidal action. It is used as 2%
aqueous solution, buffered with sodium bicarbonate to pH 7.5 to 8.5. It is less toxic and less irritant to skin
than formaldehyde. In medical field, it is used for sterilizing urological, lensed and other instruments.
QUATERNARY AMMONIUM COMPOUNDS
Mode of Action: Disruption of cell walls and membranes. Also inactivate enzymes and denature proteins
• Also called as Cationic detergents
• Highly active against G+ve bacteria and some against G-ve bacteria. Also active against fungi and some
pathogenic protozoa
• They are mainly used for control of microbes on floors, walls, nursing homes and other public places.
• Also used as skin antiseptics and sanitizing agents in diary, egg and fish industries

Cetrimide Cetyl Pyridinium Chloride

DYES
Mode of Action: Impair the DNA complex and destroy the reproduction
Basic dyes are more effective bactericidal than acidic dyes.
• Acridine and Triphenylmethane dyes are used as antimicrobial agents
• Acridine dyes are acriflavine, proflavine, aminacrine and enflavine. They are effective against G+ve
bacteria. Used for treatment of burns, opthalmic application and bladder irrigation
• Triphenylmethane or aniline dyes are brilliant green, malachite green and crystal violet. These dyes are
added to selective culture media to inhibit the growth of G+ve bacteria and helps in isolaton of G-ve
bacteria. Crystal violet also has fungicidal action
DETERGENTS
• They are used as surface active agents, wetting agents and emulsifiers.
• They are classified into four main groups such as anionic, cationic, nonionic and amphoteric.
• Anionic surfactants are Soaps, Sodium lauryl sulfate.
• Cationic surface active agents are the most antibacterial agents eg. Cetrimide, benzalkonium chloride.
• Non ionic detergents - does not possess much antimicrobial activity
• Amphoteric compounds have properties of both anionic and cationic detergents EG: Phospholipids

59
 FACTORS AFFECTING DISINFECTANT ACTION
The rate and extent of antimicrobial action of chemical disinfectants are influenced by a number of factors,
1. Concentration of disinfectant
2. Temperature
3. Time of contact
4. pH of the environment
5. Surface tension
6. Formulation of the disinfectant
7. Chemical structure of disinfectant
8. Type and number of microorganisms present
9. Interfering substances in the environment
10. Potentiation, synergism and antagonism of disinfectants
(1) Concentration of disinfectant
• Rate of killing of microorganism is directly proportional to concentration of the disinfectant.
• The effectiveness is generally related to the concentration exponentially and not linear.
• The dilution coefficient can be calculated from the following equation,

• Where n is the concentration exponent or dilution coeffecient for the disinfectant, t1 is the death time
with disinfectant concentration C1 and t2 is the death time with disinfectant concentration C2.
• Concentration exponent or dilution coeffecient is an important characteristic of each disinfectant and is
useful in determining the effect of dilution on the disinfectant.
(2) Temperature
• Rate of disinfection increases with the temperature.
• The effect of temperature on bactericidal activity is mentioned as temperature coeffecient. The
temperature coeffecient per degree rise in temperature is denoted by Ɵ. Where 10º C rise in temperature
is expressed by Ɵ 10 or Q10 values.
Ɵ 10 or Q10 = Time required to kill at Tº
Time required to kill at (T+10)º
Ɵ (T2-T1) = t1/t2
Where T2 and T1 are two temperatures differing by 10ºC; t2 and t1 are the corresponding lethal times.
(3) Time of contact
• Sufficient time of contact is must to exert the disinfectant action
• Principles of first order kinetics is implied for the time of contact,
K = 1/t log N0/Nt
• Where, t = time for the viable count to fall from N0 to Nt ; N0= initial number of microorganism; Nt=
final number of microorganisms
(4) pH of the environment
• A change of pH can affect the rate of growth of micro organism
• pH of 6-8 is optimal for growth of many bacteria
• Rate of growth decreases on either side of this range.
• Phenolic and acidic compounds have greatest activity in acidic conditions
• Acridine dyes and quartenary ammonium compounds have more activity in alkaline conditions.
• Amphoteric surface active agents have optimum activities at widely differing pH values, depending on
the number of nitrogen groups

60
(5) Surface tension:
• The contact between aqueous solutions of disinfectants and structure of microorganism is facilitated by
surfactant properties.
• This helps in adsorption of disinfectant on the surface of the cells as well as helps in wetting and
spreading properties in solutions
• A combination of soap with crude phenol (Carbolic acid) has excellent disinfectant properties
• Presence of soap reduces the extinction time of bacteria since it increases the contact time of
disinfectant with bacteria by lowering the surface tension of the solution.
(6) Formulation of the disinfectant:
• Formulation may be important for the effectiveness of the disinfectants.
• Chlorhexidine and quaternary ammonium compounds have greater activity when mixed with 70%
alcohol than in aqueous solution.
• Iodine is insoluble in water and so dissolved in alcohol or pot. Iodide solution or with solutions of
surfactants. Presence of surfactants in iodine solution will reduce the staining and corrosive properties
and increase the stability of preparation.
• For convenient and economic purpose, the formulation of disinfectant always be concentrated and it is
diluted with water at the time of use.
(7) Chemical structure of the disinfectant:
• Chemical structure may change the disinfectant activity.
• Substitution of alkyl chain in para position to phenolic OH group increases the activity but greater than
6 carbon chains addition will decreases water solubility and obviously disinfectant action also decreased
• Halogenation increases the activity of phenol but nitration decreases activity and systematic toxicity
also.
(8) Type and number of microorganism present:
• Disinfectant efficiency depends on the nature and number of contaminating microbes and presence or
absence of spores.
• Most vegetative bacteria except Acid fast bacilli are easily killed by most chemical disinfectants.
• Bacterial spores are difficult to destroy but aldehydes are sporicidal.
• Aldehydes with Halogens and Beta propiolactones are virucidal.
• Acid fast bacilli are resistant to many disinfectants but are susceptible to iodine, formaldehyde, alcohol
and phenolic compounds.
(9) Interfering substances in the environment:
• Material such as blood, body fluids, pus, milk, food residues or colloidal proteins may reduce the
effectiveness of disinfectant.
• This is due to adsorption or shielding of micro organism or interaction with disinfectants.
• Presence of oils and fats reduce the activity of phenolics.
(10) Potentiation, synergism and antagonism of disinfectants:
• Potentiation – enhanced antimicrobial activity by addition of any excipients. Eg., Polysorbate 80
addition with disinfectants
• Synergism – enhanced antimicrobial activity by addition of two antimicrobial or disinfectants Eg.,
Different esters of p-hydroxybenzoate are available, when combined together exhibit synergistic activity
• Antagonism – Decreased antimicrobial activity by addition of any excipients Eg., Sodium thiosulphate,
lecithin etc.

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 EVALUATION OF ANTIMICROBIAL AGENTS OR DISINFECTANTS
1. TUBE DILUTION AND AGAR PLATE METHOD:
• The anti-microbial agent is added into nutrient broth/ agar medium.
• Test microorganism is inoculated. Incubated at 30-35 ºC for 2 to 3 days.
• Turbidity or colonies are observed and the results are identified.

2. KIRBY- BAUER TEST (OR) CUP-PLATE & CYLINDER PLATE METHOD:/ DISK METHOD
• The Nutrient agar is melted and cooled to 45°C and add certain quantity of test microorganism.
• This solution (agar with microorganism) is poured in the Petridish. Allow to solidify.
• Make holes about 9 mm in dia made by a sterile cork borer. / Place the antibiotic disk at various conc.
• Different types or different concentration of antimicrobial agent is poured in the holes.
• The zone of inhibition is observed after incubation at 30-35 ºC for 2 to 3 days. The diameter of zone of
inhibition gives an indication of action of different antimicrobial agent.

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3. DITCH –PLATE METHOD:
• A solution of antimicrobial substance is carefully run into the ditch which is prepared in an agar plate.
• A loopful of test microbes is then streaked outwards from the ditch on the agar surface.
• Microbes resistant to the antimicrobial grow right up to the ditch whereas sensitive microbes show a
zone of inhibition adjacent to the ditch or centre of plate.
• Width of inhibition indicates the Antimicrobial action.

4. PHENOL COEFFICIENT METHOD: *****

Various disinfectants are tested for its antimicrobial property and they are compared with phenol activity as
reference. The same quantities of microorganisms are added to the different dilutions of phenol and disinfectants.
In UK, Salmonella typhi and in USA, Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa are
used as test microorganisms. The phenol coefficient test includes,
1. Rideal – Walker test (RW test)
2. Chick – Martin test (CM test)
3. USFDA test
4. The US Association of Official Agricultural Chemists test (AOAC test)
This test is only suitable for testing water soluble disinfectants and exerts the antimicrobial action which is
compared with phenol activity.
Rideal – Walker test procedure:
1. Nutrient Medium: Rideal – Walker broth (Meat extract, Peptone, Sodium chloride and Water) is used
2. Microorganism used: Salmonella typhi as sensitive microorganism.
3. Fresh Culture preparation: Microorganism is inoculated in Rideal walker broth before 1 day of testing.
4. Dilutions: Different dilutions of test disinfectants and phenol are prepared.
5. Reaction Mixture: 0.5ml of fresh culture ([Link] in Rideal walker broth) is mixed in the test tubes
containing 5 ml of each dilution of disinfectants and all tubes are placed in water bath (20 ºC) for specific
period of time.
6. After 2.5, 5, 7.5 and 10 min, 1ml of above solution is taken from the reaction mixture and transferred to 5 ml
nutrient medium (Rideal walker broth) in the fresh test tubes.
7. The test tubes are incubated at 37 ºC for 2-3 days and examined for the presence or absence of growth. The
Rideal – Walker coefficient of the test disinfectant is then calculated as follows,

63
 Determination of Rideal – Walker Coeffecient
Disinfectant Dilution Time interval for Sub-
Culture (min)
2.5 5 7.5 10
Test 1: 1000 + - - -
disinfectant 1: 2000 + + - -
1: 3000 + + + -
1: 4000 + + + +
Phenol 1: 80 + - - -
1:100 + + - -
1: 120 + + + -
1: 140 + + + +
(+ = growth; - = No growth)

If a R.W. Coeffecient is 1, then the test disinfectant has the same effectiveness as phenol
If it is less than 1 it means less effective and
if it is more than 1 it means more effective than phenol.
Differences between Phenol Coeffecient tests
Points of Difference RW Test FDA Test CM Test AOAC Test
Culture Medium Rideal Rideal Rideal Walker Rideal Walker
Walker Walker broth broth
broth broth
Culture Medium pH to be maintained 7.4 6.5 7.4 6.5
Test Microorganisms S. typhi S. typhi S. typhi S. typhi,
Staph. Aureus
Dilution of disinfectants using Water Water Yeast Water
Suspension
Volume of Reaction mixture (ml) 0.5 + 5.0 0.5 + 10.0 0.5 + 5.0 0.5 + 5.0
(Microbial culture qty + Disinfectants qty)
Reaction Temperature (°C) 17/18 20 30 20/37
Sampling times (min) 2.5, 5, 7.5, 5, 10, 15 30 5, 10
10
Volume of Rideal Walker broth (ml) to be
mixed with Reaction mixture during 5.0 10.0 10.0 10.0
incubation
Calculation of Phenol Coeffecient Dilution Dilution Mean of highest Greatest
test killing test killing phenol dilution of test
in 7.5 but in 10 but concentration killing in 10
not in 5 not in 5 inhibiting and min divided
min min lowest permitting by same for
divided by divided by growth divided phenol
same for same for by same for test
phenol phenol

64
Advantages of Phenol Coeffecient test:
• Inexpensive and can be performed quickly and have reproducible results
• They are helpful to eliminate to useless products
Disadvantages of Phenol Coeffecient test:
• Have only limited information (only one microbe. – [Link])
• Activity of bactericides done at only one concentration with fixed death time and reaction temperatures
• In presence of organic matter, the test has no indication of activity of disinfectants
• No information about toxicity and Sampling errors are large
5. KELSEY – SYKES METHOD:
• In this method, several test bacteria are used. They are, Staph. Aureus, Proteus vulgaris, E. coli and Pseud.
aeruginosa. Basic procedure is outlined in below table.
Steps Time (min) Procedure
Step 1 0 Inoculate - 3 ml Disinfectant + 1 ml bacterial suspension and shake gently.
Step 2 8 At 8th min,
→ Transfer each one drop from step 1, to 5 test tubes contains nutrient broth (or)
→ Transfer each one drop from step 1, to 5 places of a petridish contains nutrient agar
Step 3 10 At 10th min, add again 1 ml of bacterial suspension to the step 1
Step 4 18 At 18th min,
→ Transfer each one drop from step 1, to 5 test tubes contains nutrient broth (or)
→ Transfer each one drop from step 1, to 5 places of a petridish contains nutrient agar
Step 4 20 At 20th min, Add again 1 ml of bacterial suspension to step 1
Step 5 28 At 28th min,
→ Transfer each one drop from step 1, to 5 test tubes contains nutrient broth (or)
→ Transfer each one drop from step 1, to 5 places of a petridish contains nutrient agar

• The samples taken at 8th min, 18th min and 28th minutes are then incubated at 37 °C and the number of tubes
showing growth or the number of colonies from surface plate culture is recorded.

• Result: No growth occurs in 2 or more of 5 tubes of 18 min samples ie., subcultures taken after second
incremental addition of bacteria or there are not more than 5 colonies from the 5 drops on the agar plate.

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 STERILITY TESTING OF PRODUCTS****
Sterility Testing – The test which detects the presence of any viable microorganism in the pharmaceutical
preparations like Parenteral preparations, Ophthalmic preparations, Surgical dressings and devices, Solid dosage
form (Tablet, capsule), Antibiotic solids, powders, Liquids.
Principle: The pharmaceutical product is placed in the suitable culture medium, and then the medium is
incubated. If there is no growth, the sterility testing is passed. If there is turbidity in the culture medium, it shows
the presence of microorganism in the pharmaceutical product and the sterility testing is failed.
Culture media for sterility testing:
Media must show the growth of even small numbers of microorganism present in the sample.
1. For detection of Aerobic & Anaerobic bacteria – Fluid Thioglycolate media (FTM) , Alternate Thioglycolate
media (ATM)
2. For detection of Fungi – Soyabean casein digest medium (SCDM)
Fluid Thioglycollate Medium (FTM) – Intended for the culture of Aerobic & Anaerobic bacteria
Ingredients Qty Function
L – Cystine 0.5 g Antioxidant
NaCl 2.5 g Isotonicity
Dextrose 2.5 g Reducing agent, Carbon source
Agar 0.75 g Viscosity enhancer
Yeast extract 5.0 g Growth promoter
Pancreatic digest of Casein extract 15.0 g Nitrogen source
Sodium thioglycollate 0.5 g Reducing agent
Thioglycollic acid 0.3 ml -
Resazurin (0.1%) 1.0 ml Oxidation – reduction indicator
Distilled water 1000
ml
Alternative Thioglycollate medium (ATM) - Same as FTM (But Agar is removed)

Soyabean Casein Digest Medium (SCDM) – Intended for culture of fungi

Ingredients Qty Function
Pancreatic digest of Casein 17 g Source of C, N and essential amino acids
Pancreatic digest of Soyabean meal 3g Source of C, N and essential amino acids
Sodium Chloride 5g Isotonicity
Dibasic Potassium Phosphate 2.5 g Reducing agent
Distilled Water 1000 ml Growth promoter
Media sterility or suitability test:
Prior to test, make sure that the media is sterile and media supports growth of micro organisms. For that two tests
are performed,
Media Sterility test:
• It is Negative control test. Incubate portions of FTM/ATM at 30 -35 °C and SCDM at 20-25 °C for NLT 14
days and observe the growth.
Growth promotion test:
• It is Positive control test. Media is inoculated with < 100 cfu (colony forming unit) of microorganisms and
incubated. Growth of microorganism is observed through viable plate counts method.
• Bacterial growth observed within 3 days and Fungal growth observed in 5 days
Dilution fluids for diluting the pharmaceutical products:
Three types of diluting fluids used

66
 1. FLUID – A: 1 g of peptic digest of animal tissue + water → Make up to 1 litre
2. FLUID – B: Fluid A+1 ml of Polysorbate 80.
3. FLUID – K: 5g of peptic digest of animal tissue + 3g of beef extract + 10g of polysorbate 80 in water →
make up to 1 litre.
Methods for sterility testing:
Sterility testing can be carried out by using the following two methods,
1. Membrane filtration method
2. Direct inoculation method
MEMBRANE FILTRATION METHOD:
Apparatus:
• Consists of Closed reservoir and a container to collect the filtrate.
• Membrane of porosity 0.45 µm diameter is placed between reservoir and container, which allows flow
rate of 55-75 ml of water/ min at a pressure of 70 mm of mercury.
• Membrane is made of Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions.
• Membrane is made of Cellulose acetate filters are used for strong alcoholic solutions.
• The whole complete unit should be sterile and the operation should carry out aseptically.

General procedure:
1. The specific quantity of test product is taken and diluted with suitable dilution fluids and is made to pass
through a membrane filter.
2. Then the membrane is then collected & cut into two pieces
3. One half of membrane is placed in 100 ml of FTM incubated at 30 – 35 °C for NLT 14 days
4. Another half of membrane is placed in 100 ml of SCDM incubated at 20-25 °C for NLT 14 days.
5. Then observe the growth of organisms.
Advantages of Membrane Filtration method:
• Wide applications for mostly all kind of products. A very large volume can be tested
• Smaller volume of culture medium is required.
Disadvantages of Membrane Filteration:
• Possibility of adsorption of sufficient medicament to spoil the test cannot be discounted entirely
• Highly skilled staff and exceptionally good aseptic techniques are necessary
DIRECT INOCULATION METHOD
Procedure:
It is the most traditional testing method. It involves,
1. The specific quantity of test product is taken and diluted with suitable dilution fluids
2. Using sterile syringe and needle, withdraw the required volume of sample.
3. Inject one half of the sample into a test tube containing the FTM and the other half into a second test tube
containing SCDM and incubate both test tubes.

67
4. Observe the growth for turbidity

68
 TEST FOR PYROGEN
Greisman and Hornick (1969) first identified this test. Rabbits are used as test animal. But the recent test uses
horseshoe crab for testing the toxins
RABBIT EAR VEIN TEST:
Principle: This test is based on rise in temperature of rabbits when the preparation is injected intravenously.
Test animal:
Healthy adult rabbits of either sex; Weight NLT 1.5 kg; placed in uniform room temperature
Use the fresh rabbits for testing or 2 weeks elapsed rabbits for testing.
Materials:
• Sterile needles, syringes and glass wares. Retaining boxes for rabbits.
• Thermometer to monitor the temperature of rabbit. Thermometer measuring should be quick and read even
the change in 0.1 °C. No water and food is supplied throughout the test.
Preliminary test (Sham test):
• Performed one to three days before the main test
• Isotonic pyrogen-free saline solution injected 10ml/kg of body wt
• Record the temp of animals initially before the test begins atleast 90 mins
• Rabbits showing more than 0.6 °C are excluded from pyrogen tests
Main test:
• Performed by using a group of three rabbits.
• Dissolve test substances in saline solution.
• Administer slowly into the marginal ear vein within 4 mins (NLT 0.5ml/kg &NMT 10 ml/kg of body wt)
• Record the temp of each rabbit at beginning 90 mins before injection.
• After injection, record initial temp and continue recording at every 30 mins and it is continued for 3 hrs.
• The difference between maximum temp and initial temp is taken to the response.
• When the difference is negative, the result is counted as a zero response.
Interpretation of results:
• If the sum of responses of three rabbits not exceeds 1.4 °C and if the response of individual rabbit is Less
than 0.6 °C, the test sample passes the pyrogen test. If the individual rabbit shows 0.6 °C or more and the
sum of response of group of three rabbits exceeds 1.4 °C, continue the test with 5 other rabbits.
• If NMT 3 of 8 rabbits show individual response of 0.6 °C or more and the sum of response shows NMT
3.7 °C, the test sample passes the test. When pyrogen test is positive, the test sample should be rejected
LIMULUS AMEBOCYTE LYSATE TEST (LAL TEST)
Principle: The reaction of LAL is based on blood clotting mechanism that protects horseshoe crab from the
hostile sea. Serine protease zymogens in the LAL produce coagulin gel clot in presence of endotoxin
Test reagent: (Amebocyte lysate) obtained from lysing the amebocyte blood cells of Atlantic horse shoe crab
(Limulus polyphemus).
Procedure:
LAL + bacterial endotoxin (lipopolysaccharide, component of gram –ve bacteria) produces turbidity. If
chromogenic substance is added with LAL it produces color. Also known as BET (Bacterial Endotoxin test)
test. The rate of reaction is depend upon activity of endotoxins, pH and temperature
Advantages:
• LAL Reagents easily and commercially available
• LAL test is 100 times more sensitive than Rabbit test and is alternative to animal tests. Have wide similarity
bet Limulus and mammalian that response to endotoxin. ET produce proinflammatory cytokines enzymes
like clotting enzymes produced in Limulus.

69
 UNIT - IV
DESIGN, CONSTRUCTION AND PRODUCTION FACILITIES OF ASEPTIC AREA
Aseptic area: An Aseptic area is a room with clean area designed for preventing microbial contamination of the
products. Requirements for Design of Aseptic area:
1. Site of premises
2. Size of premises
3. Windows
4. Doors
5. Floor, walls and Bench tops
Site of premises:
• Aseptic area should be away from stairs, lift, corridors and general manufacturing area.
• Washing and change rooms should be in front of aseptic area.
• Each stage of production should be carried out in separate rooms of aseptic area.
• Storerooms should be adjacent to aseptic area.
Size of premises:
• Large and spacious
• Ceiling is in low height for easy cleaning
Windows:
• Large windows with transparent glass are suitable.
• Windows remain closed and ventilation is provided through HEPA filter system.
• Windows are double gazed to prevent the heat loss from glass material.
Doors:
• Entrance should have double doors with an air lock system.
• Positive pressure is maintained inside the room to avoid entry of outside air into the aseptic area.
• Sliding and Swing doors can be used.
Floor, walls and bench tops
• Floor should be made of materials like terrazzo (mixture of cement and marble), Linoleum (heavy grade
material) or Plastics (PVC)
• Surface of walls and ceilings is made of tiles, glass paint or plastic laminate.
• Lights are fitted within the ceiling – to avoid disturbance of airflow pattern.
• For reduction of fungal growth, 1% of 8-hydroxyquinoline are added to the paints.
• Epoxy resin and polyurethane paints used to avoid cracking.
• Tops of working benches are made of stainless steel or plastic laminate.
Flow diagram of building design of Aseptic area

70
CONDITIONS MAINTAINED IN ASEPTIC AREA TO PREVENT CONTAMINATION
Personnel and protective clothing:
• Persons working in aseptic area should be neat & clean with good health & free from skin infections.
• Number of persons working in aseptic area should be limited.
• They should wear protective clothing in the aseptic area and it should cover all the body parts except eye.
• Clothing should be sterilized before and after use. Eyes are covered with sterilized goggles.
• All persons working in the aseptic area should be trained for GMP and aseptic techniques.
Environmental control:
• Stringent environmental control is maintained before and during the processing of parenterals in aseptic area.
• It should be free from contamination and there is no accumulation of dust particles, lint and viable
microorganism.
Traffic control:
• Entry and exit of supply of materials, workers should be separate.
• Entry of unauthorized persons are not allowed.
• Once personnel enter the aseptic area, they cannot leave the manufacturing area until the work completed.
Cleaning and disinfection:
• Suitable disinfecting agents should be used for animate and inanimate things.
• Different disinfectants are used in rotation to prevent development of resistant strains.
• Area Sanitization is carried out using disinfectants or UV radiation before and completing the production.
Laminar air flow system:
• The greatest source of contamination is through air as it contains most form of microorganisms. Thus, it
important to clean the air before it enters the aseptic area to avoid contamination.
• This system supplies air to the aseptic room/ clean room through HEPA filters which claims 99.97 %
microbial free air The air flows at a uniform velocity of 100 ± 20 ft/ min.
• Laminar air flow pattern is of three types,
Uni-directional air flow:
✓ Air enters through HEPA filter present in the top of the ceilings and exits through outlets present
in the floor of the aseptic area. Air flow is highly efficient but highly expensive to construct.
✓ Used only in Clean rooms of Class 10 and Class 100.
Non – unidirectional air flow:
✓ Air enters through HEPA filter present in the top of the ceilings and exits thru outlet ducts placed
on the sides of the floor.
✓ Used in cleanrooms of class 1000 and 10000.
Combined air flow:
• In many pharmaceutical clean rooms, the background area is ventilated by non- unidirectional
airflow and critical aseptic area is supplied with unidirectional airflow.
Uni-directional airflow Non - Uni-directional airflow Combined airflow

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• HEPA FILTER
High efficiency particulate air filter. (HEPA). These are used for the sterilization of air. This can
remove 99.97% of particles of size greater than 0.3µm. These are fitted in laminar air flow cabinets,
walls, ceiling panels and ducts where the aseptic condition is required
Construction:
• It consists of bunches of filter medium and spacers.
• It is made up of pleated sheets of glass microfibers separated by spacers made up of aluminium.
• The pleated sheets of glass microfibers are placed parallel to each other.
• The whole filter setup is sealed with aluminium frame.

Working:
Laminar air flow cabinet is placed in an aseptic area. It is made of initial pre-filter and then with
HEPA filter. The cabinet setup is of two models – Vertical and horizontal laminar air flow cabinet. It
is started 15 mins prior to actual work. The fan forces the air through the HEPA filters which releases
pure air into the area. The velocity of air is delivered at 230±90 cm/min. The cabinet can deliver the
air in vertical, horizontal or curvilinear direction.
Maintenance: The quality is monitored every 6 to 12 months. Evaluated by DOP test, settle plates,
microbial air samplers and particle counters tests.
DOP Test: (Dioctyl phthalate test)
• DOP is a volatile liquid. Under pressure, it converts to vapor or smoke having a size range of 0.4
µm. DOP smoke is introduced through HEPA filter setup.
• Photometer probe present outside the filter will scan the surface of smoke particles. HEPA filter
does not allow the particles more than 0.3µm.
• If any leakage in filter will cause entry of DOP particles into the filter which is detected in
photometer. Sensors are fitted to indicate the pressure difference across the filter.
• Alarm system is attached with sensor indicated the filter blockage or failure of air supply.

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 Vertical laminar air flow cabinet Horizontal laminar air flow cabinet

• Laminar air directed vertically downwards in the • Laminar air comes from above prefilter and then it
working area changed the directions into horizontal flow
• The air can leave the working area via holes • It provides material and product protection ie.,
present in the base prevent from contamination by environment or
• This provide better operator protection. through personnel.

ULPA FILTER (Ultra low penetration air)

These are replaceable dry filters that have a minimum particle collection efficiency of 99.9997% efficient for
particles greater than or equal to 0.12 micron in size. Better than HEPA filter but very expensive.

HVAC SYSTEM (Heating, Ventilation, and Air Conditioning)

Advanced method of applying air conditioning system in the pharmaceutical industries and aseptic areas is
known as HVAC system. It is termed as Heating Ventilation and Air conditioning System. It is used to control
the temperature and moisture of the air. And there by it reduce the contamination of air borne particles in the
pharmaceutical facility.
.

73
 CLEAN AREA OR CLEAN ROOM CLASSIFICATION
Clean area is essential for the production of sterile products. The standard of cleanliness and purity of the
environment is designed by US and European countries and the international society of pharmaceutical
engineers. This specification standard is based on maximum number of air borne particles per m3 of area
Maximum number Maximum number United states European grade International society
of particles/m , ≥0.5 of particles/m , ≥5 classification
3 3
of pharmaceutical
µm µm engineers
35 0 Class 1 - -
352 0 Class 10 - -
3520 29 Class 100 Grade A Critical
35,200 293 Class 1000 Grade B Clean
3,52,000 2930 Class 10,000 Grade C Controlled
35,20,000 29,300 Class 100000 Grade D Pharmaceutical

Class 100 or Grade A area – Filling of products at particularly having microbiological risk.
Class 1000 or Grade B area - Aseptic products preparation and it is background environment for Class 100 zone.
Class 10000 or Grade C area – Manufacturing of sterile products.
Class 100000 or Grade D area - Wash area, change over area, corridors of aseptic building.

DIFFERENT SOURCES OF CONTAMINATION IN AN ASEPTIC AREA

1. Atmosphere: (Atmospheric air will contaminate the area)
- Outside air: Dust particles in air may carry soil microorganisms eg. Bacillus sp., yeasts, moulds, etc.
- Inside air: Sneezing, Coughing and talking
2. Operator:
- Through Skin, hair and clothing
- Poor personal hygiene and open wounds
- Eating, drinking or smoking in storage or processing area
- Inadequate training
3. Raw materials: (Highest proportion in industry)
- RM from natural sources (Bacterial, yeast and mould) and Water
- Improper storage and handling
- Incorrect sampling and testing
4. Equipment:
- Sedimentation of particles in the internal and external surfaces of the equipments
- Poor sanitization and cleaning
5. Buildings and facilities:
- Inadequate size and space of the organization
- Poor pest control
- Absence of HVAC system and HEPA filters
- Poor lighting and drainage system
6. Manufacturing process:
- Improper cleaning between the batches
- Open manufacturing system for exposing the product to environment
- Lack of cleaning status and manufacturing procedure.

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 QUANTITATIVE EVALUATION OF MICROBIAL CONTAMINATION:
The quantity or number of microorganism present in the aseptic area is evaluated by various methods. They are
divided into,
1. General methods; 2. Air sampling methods; 3. Surface sampling methods
General methods: These are used to validate HEPA filter, detect particulate contamination and monitor
environmental contamination. These methods includes,
✓ Filter efficiency test is done using scanning probes, particle counter.
✓ Induction leak test is done using scanning probes, photometer.
✓ Particulate contamination control test is done using particle counter.
✓ Air pressure test is done using Manometer.
✓ Air flow test is done using Anemometer or Velometer.
✓ Noise level test is done using Sound level meter or Noise dosimeter.
✓ Lighting test is done using Goniophotometer or Luxmeter.
✓ Temperature and humidity test (20-22 °C & 35-50% RH) is done using thermocouples and ERH meter.
Air sampling methods: These methods includes,
✓ Electronic air particle counters: Determine the number of particles/microbes count per cubic feet. But it
cannot differentiate count of viable and non viable microorganisms.

✓ Settle plates: It is also called as Sedimentation method. It is based on the principle of rate at which the
bacteria get settle down. Open plates of nutrient agar is placed in specific areas for 30 to 60 minutes. Then
the medium is incubated at 30-37 °C for 48 hrs. Growth is observed.

✓ Slit – air sampler:(Better than settle plate method). It is used in parenterals and opthalmic manufacturing
areas. A known volume of air (one cubic foot of air) is directed onto a plate of culture medium through a slit
of 0.25 mm width. The plate is mechanically rotated to ensure uniform distribution of organisms all over the
plate. Likewise 10 cubic foot of air is tested. Medium is then incubated and colonies counted

75
✓ Liquid impinger: The air sample drawn into a measured volume of nutrient broth in an impinger, then it is
incubated and counted.

✓ Centrifugal air sample: Known amount of air are drawn towards the centrifugal impeller blades from 16
inches above the sterile drum housing. By applying centrifugal forces (4000 – 4200 rpm), the microbial
particles are impacted at a high velocity onto the incubation; colonies on the strips are counted.

Surface sampling methods: These methods includes,

✓ Rodac plates: These are used to take a sample from any surface to determine the microbial load on the surface. It is
an alternate method for swab sampling.

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✓ Swab – rinse test: Employ sterile cotton swab tips to sample location. The swab then placed into tubes of culture
media.

MICROBIOLOGICAL ASSAY
Definition: Microbiological assay is the one type of bio-assay, which is defined as the qualitative or quantitative
determination of any chemical compound using microorganism.
Principle:
This assay is based upon inhibition or growth of micro organism by using standard antibiotics or vitamins or
amino acids of known concentration of standards is compared with unknown concentration of test chemicals.
Application
• For determination of potency
• For determining the pharmacokinetics of a drug in animals or man
• For monitoring and controlling antimicrobial chemotherapy

MICROBIOLOGICAL ASSAY OF ANTIBIOTICS

Definition: Inhibition of growth of microorganism is measured by comparing known concentration of standard

antibiotic preparation having known activity with unknown concentration of test antibiotic preparation

Principle:
Method A: Cylinder plate method or cup plate method: In that the diffusion of known conc. of antibiotic from a
vertical cavity through a solidified agar layer in the petridish and determine the zone of inhibition of
microorganism around the cavity.
Method B: Tube assay method or turbidimetric method: In this method, inhibition of growth of microorganism
in the fluid culture medium containing standard antibiotics is compared with fluid culture medium containing test
antibiotics. The turbidity indicates the growth and is measured through turbidimeter.

Requirements:
1. Culture medium:
The ingredients used for preparing the Culture media are listed below. Different types of media are used are
classified from A to J with different conc. and different types of ingredients used.

77
2. Preparation of standard antibiotic solution:
Required quantity of standard antibiotic sample is dissolved in suitable solvent and considered it as Stock
Soln. On the day of assay, the stock solution is diluted to achieve the different concentration.
3. Preparation of sample antibiotic solution:
From information available for the test sample, dilute it with suitable solvent.
4. Test microorganisms:
The test microorganism for each antibiotic is listed given below with its id. number in American Type
Culture Collection (ATCC). Maintain the microorganism in a culture medium of agar slants under the
incubation condition. Transfer weekly to fresh slants.

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5. Preparation of inoculum:
Test microorganism suspension is prepared by anyone of the following methods
Method 1:
Microorganism transferred to Culture medium A→ incubate →Wash with 3 ml Saline →Transfer to
Roux bottle contains 250 ml nutrient agar → Incubate for 1day → Wash with 50 ml Saline → Dilute with
nutrient broth → store in refrigerator.
Method 2:
Follow the procedure as per Method 1. But incubate the Roux bottle for 5 days. Wash with 50 ml saline
for three times and dilute with nutrient broth. Store in refrigerator.
Method 3:
Follow the procedure as per Method 1. But instead of Culture medium A, use Culture medium G.
Method 4:
Follow the procedure as per Method 1 using Culture medium A but without agar.

PROCEDURE:
ASSAY METHOD A
CUP AND PLATE TECHNIQUE
1. The nutrient agar is melted and poured into petri dish and Allowed to solidify.
2. Spread 0.2 ml of inoculum on the surface of the solidified agar (Spread Plate Technique).
3. Hole or cup or cavity is created in the solidified agar by using a sterile borer.
4. Now various concentrations of diluted standard antibiotics (0.2, 0.4, 0.6, 0.8, 1.0 ml) and unknown
concentration of antibiotic is poured into the cups of agar plate and then incubated at 37 °C for 24 hr.
5. If the antibiotic has any anti-bacterial effect it will show the zone of inhibition.

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Disc diffusion method:
• Follow the above procedure up to step 2.
• Place the prefilled disc of test antibiotics and standard antibiotics of various concentrations.
• Incubate it for 24 hrs at 37°C and measure and compare the zone of inhibition of test antibiotics with
standard antibiotic sample.

ASSAY METHOD B: TURBIDIMETRIC METHOD OR TUBE ASSAY METHOD

▪ Five different concentration of standard antibiotic solution is prepared.
▪ The test antibiotic solution is diluted to five different concentration.
▪ One ml of each concentration of standard and sample are added to the test tubes. To each tube, 9 ml of
inoculums is added
▪ At the same time, 3 control tubes are prepared, one contains inoculated culture medium without
antibiotic solution (Culture control), second one contains the inoculated culture medium with
formaldehyde solution (Blank), a third one contains culture medium without antibiotic solution &
without microorganism.
▪ All the test tubes are placed in incubator at specified temperature for 4 to 5 hrs.
▪ The growth of microorganism is measured by determining the absorbance at about 530 nm of each of the
solution in the test tubes against the blank.

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 MICROBIOLOGICAL ASSAY OF VITAMINS
This is applicable to only B vitamins.
Principe: The rate of growth of test microorganism is measured in culture media that contain standard vitamins
of known potency is compared with test vitamin sample of unknown concentration.
Requirements
1. Culture media or Basal Stock medium: It contains

2. Test organism:

Vitamins Microorganism Incubation temp Assay pH

Vitamin B12 or Cyanocobalamin Lactobacillus leichamanii 37 6.1
Vitamin B6 or Pyridoxine Sacchromyces uvarum 30 4.5
Vitamin B2 or Riboflavin Lactobacillus caesi 37 6.8
Vitamin B1 or Thiamine Lactobacillus viridescens 30 6.0
Vitamin B7 or Biotin Lactobacillus plantarum 37 6.8
Vitamin B3 or Niacin Lactobacillus plantarum 37 6.8
Vitamin B5 or Pantothenic acid Lactobacillus plantarum 37 6.8

3. Preparation of inoculums:
– Transfer Lactobacillus leichmannii into test tubes contains 10 ml nutrient broth.
– Incubate for 18 to 24 hrs at 37 °C. Centrifuge the culture till the cells settle to the bottom of the tube.
– Decant the supernatant fluid. Suspend the cells in 10 ml of Basal stock medium and with 25 ml of Water.
– Centrifuge again & decant off the supernatant fluid. Repeat the procedure if necessary.
– Dilute 1 ml of above suspension again to 10 ml of basal stock soln and mix. The resulting cell suspension is
inoculum.

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PROCEDURE
TITRIMETRIC METHOD
1. Add various concentration of standard vitamin B12 solution (0 ml, 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, 3 ml, 4
ml, 4.5 ml and 5 ml) to the ten test tubes. To each test tube add 5 ml Basal medium stock solution. Adjust the
final volume (10 ml) with water.
2. In other four test tubes add 1ml, 2ml, 3ml, and 4ml of test vitamin B12 solution. To each test tube add 5ml
Basal medium stock solution and adjust the volume (10ml) with water.
3. Sterilize all the test tubes in autoclave (121 °C for 5 min) and cool to room temp.
4. Inoculate one drop of inoculum (Lactobacillus leichmannii). Incubate the tubes for 72 hrs at temperature 37
°C.
5. Titrate the contents of each tube with 0.05N NaOH or using 0.1% w/v bromothymol blue indicator
6. Determine the average value of standard and test sample.
7. Plot the graph by considering the titration values of 0.05 N NaOH (in ml) obtained for the corresponding
concentration of Standard vitamin B12 solution added.
8. Draw a curve and determine the concentration as activity per ml of test vitamin B12 solution by interpolation

TURBIDIMETRIC METHOD:
1. Apparatus, reagents and procedures are same as titrimetric method but this test has two more test tubes
without Vitamin B12 solution and inoculum is added.
2. Incubate all tubes at 30-37 °C for 16-24 hrs. Turbid produced in the test tubes due to growth of
microorganism.
3. By using un-inoculated blank tube adjust the transmittance at 640 nm in turbidometer.
4. Thoroughly mix the contents of each tube and record the transmittance reading.
5. Plot the graph by considering transmittance value against the corresponding level of standard Vitamin B12
solution.
6. Draw the curve and calculate the concentration of the test solution of Vitamin B12.

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 MICROBIAL ASSAY OF AMINO ACIDS

Amino acids are the growth factors which are essential for the growth and multiplication of microorganisms.
When the essential amino acids are absent in the culture medium, certain microorganism are fail to grow.

Principle:
The principle involved in the microbial assay of amino acid is based on the determination of quantity of
microbial growth in presence of standard amino acids and compared with test amino acids.

Test Microorganism used for microbial assay of amino acids:

➢ Streptococcus faecalis is used for analysis for valine, leucine.
➢ E. Coli is used for analysis of L – glutamic acid.
➢ Leuconostoc mesenteroides is used for analysis of Methionine, Histidine, Lysine
➢ Lactobacilli are used for analysis of other amino acids.

Procedure:
• Test organism is inoculated in culture medium without amino acids act as control. This shows no
growth or minimal growth. Since there is no essential amino acids present in the culture medium.
• In another test tube, the test organism is inoculated in culture medium containing known concentration
of amino acids which act as the standard. This shows normal growth of bacteria with known amount of
multiplication shows certain amount of turbidity in the culture medium.
• In another test tubes, the test organism is inoculated in culture medium containing unknown
concentration of amino acids (for which the assay is to be done) which act as the test. In this the growth
of bacteria and its quantity of multiplication is determined by the turbidity of test culture medium is
compared with turbidity of standard culture medium.
• Turbidity is measured by turbidimeter where the amount of light is reflected according to turbidity of the
culture medium due to microbial growth is calculated.

Test

Control

Standard

Microbiological assay of amino acids by turbidimetric method

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ASSESMENT OF NEW ANTIBIOTIC (OR) ASSAY DESIGN IN CUP PLATE METHOD
ONE LEVEL ASSAY WITH STANDARD CURVE:
Standard Solution:
Accurately weighed quantity of standard antibiotic sample is taken and diluted with suitable solvent to give five
different dilutions (S1, S2, S3, S4, S5), Where S3 is taken as Median and stored in refrigerator.
Sample Solution:
Accurately weighed quantity of known concentration of test antibiotic is taken approximately in the mid-range
concentration of antibiotic and diluted with suitable solvent and stored in refrigerator.
Procedure:
– Prepare12 petri dishes with solidified agar
– These 12 petri dishes are divided into 4 sets (Each set contain 3 petri dish)
– 6 holes are made in agar media of each petri dish, so there is total 72 holes in 12 petri dishes.
– On each of the three set of plates, fill alternative holes with dilution S3.
– Remaining holes of first set filled with dilution S1, second set filled with dilution S2, third set is filled
with dilution S4 and the fourth set is filled with dilution S5.
– All sets are incubated for 18 hrs.
– Measure the zone of inhibition
Calculation for Standard curve preparation

➢ L – Calculated Zone diameter of lowest concentration of Standard curve response line

➢ H – Calculated Zone diameter of highest concentration of Standard curve response line
➢ C – Average zone diameter of dilution S3.
➢ a, b, d, e = average zone diameter of dilution S1, S2, S4 and S5

Determination of unknown conc of antibiotic (one dilution)

Prepare a set of 3 petridishes with solidified agar. In each petridish, standard solution S3 is filled in alternate 3
holes and the remaining 3 holes of each Petridish is filled with the unknown antibiotic solution. The zone of
inhibition is measured. From the standard curve response line, extrapolate the readings obtained from unknown
concentration. By which the potency of the sample is calculated.

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TWO LEVEL FACTORIAL ASSAYS:
Approximately equal dilution of both standard and test solutions are prepared (S1S2 vs U1U2) in high and low
conc. Four petridish having four cavities are alternatively filled with standard and test antibiotics. Diameter of
Zone of inhibition is measured.
Estimation of Potency:
• Sum the diameters of the zones of each dilution of each Petridish and calculate the percentage potency of the
test antibiotic sample from the following equation.
• Percent Potency = Antilog 2.0 + a log I,
• I – ratio of dilution

• U1 and U2 are the sum of diameters of zone of inhibition by unknown samples high and low levels.
• S1 and S2 are the sum of diameters of zone of inhibition by standard of high and low level.

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 UNIT - V
Microbial Spoilage is the contamination of pharmaceutical products with the microbes which lead to spoilage of
the product affecting drug safety and quality and is not intended for use. Shortly Microbial Spoilage is defined as
deterioration of pharmaceutical products by the contaminant microbe.
TYPES OF MICROBIAL SPOILAGE
The types of microbial spoilage occur in the pharmaceutical products are,
1. Chemical spoilage
2. Physiochemical spoilage
3. Biological spoilage
1. Chemical Spoilage - Chemical spoilage means deterioration of chemical nature of drugs and excipients.
Molecular structure of the ingredients may change. This leads to decrease of potency and activity of the drug.
Chemical degradation of surfactants, organic polymers may damage preservative of the formulation and
hence the microbial growth occurs in the formulation.
2. Physicochemical spoilage - This type of spoilage may change physico-chemical properties of product.
Following types of changes may occur.
1. Viable growth - Viable growth of microbes may occur inside the container. This growth may be visible
in the form of floating layers, turbidity, hemps, etc. Contamination of products by fungus species like
aspergillus sp. may cause this type of spoilage. Some bacterial species may also show viable growth.
2. Gas production - Some microbes may produce gas inside the containers. These gases may be visible in
the form of bubbles, floccules etc. Contamination of products with bacteria like E. coli may produce gas
if it contains sugars.
3. Coloration / Discoloration - Some microbes may decolorize formulation, or it may produce unique
colour which is different from the normal color of the product.
4. Odor formation - Microbial growth in the finished product may produce bad odor or characteristic odor.
It may produce a characteristic rotten smell.
5. Taste change - Microbial spoilage may change the taste of the oral formulations. It may impart bitter or
obnoxious taste to the oral formulation.
3. Biological Spoilage - Spoilage of pharmaceuticals may produce some undesirable and dangerous molecule
which has undesirable biological effects. Some microbes may produce toxins, pyrogens or other harmful
metabolites.

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 FACTORS AFFECTING MICROBIAL SPOILAGE
1. Size of inoculums: Low level of contaminants in formulation leads to low or slow rate of detoriaration.
High level of contamination leads to high deterioration. But all time this will not work. Sometimes an
exceptionally low level of pseudomonas in formulation will produce greater risk.
2. Nutritional Factors: Microbial spoilage of a product occur mainly by growth of microorganism. Mostly
ingredients used in formulation which provide all nutrients, carbon, and nitrogen source to the growth of
m.o. Mainly use of vegetable or animal products which act as source of nutrients for the many m.o.
3. Moisture content: If the formulation has high moisture content or more water activity, the product is
subjected easily to microbial attack. So less water activity to be achieved by drying, addition of Sodium
chloride etc. Moisture films on the tablets leads to contamination of m.o. Storage condition also to be
maintained.
4. Temperature: Microbial spoilage can occur at the range of -10 °C to 60 °C, usually at 37C. Syrups,
multidose preparation are mentioned by ‘store in a cool place’ to avoid the risk of in-use contamination.
5. pH: Microorganism grow well at optimized pH (6-8). Below are above the pH will affect the growth of
microorganism
6. Redox potential:It is defined as measure of potential difference . Heating, Penetration of oxygen,
presence of reducing substance will cause change in redox potential of the products. This will affect the
microbial growth
7. Protective components like surfactants, enzymes will interfere in the microbial growth
SOURCES AND TYPES OF MICROBIAL CONTAMINATION
1. Atmosphere: (Atmospheric air will contaminate the area)
- Outside air: Dust particles in air may carry soil microorganisms eg. Bacillus sp., yeasts, moulds, etc.
- Inside air: Sneezing, Coughing and talking
2. Operator:
- Through Skin, hair and clothing
- Poor personal hygiene and open wounds
- Eating, drinking or smoking in storage or processing area
- Inadequate training
3. Raw materials: (Highest proportion in industry)
- RM from natural sources (Bacterial, yeast and mould) and Water
- Improper storage and handling
- Incorrect sampling and testing
4. Equipment:
- Sedimentation of particles in the internal and external surfaces of the equipments
- Poor sanitization and cleaning
5. Buildings and facilities:
- Inadequate size and space of the organization
- Poor pest control
- Absence of HVAC system and HEPA filters
- Poor lighting and drainage system
6. Manufacturing process:
- Improper cleaning between the batches
- Open manufacturing system for exposing the product to environment
- Lack of cleaning status and manufacturing procedure.

87
 ASSESMENT or EVALAUATION OF MICROBIAL CONTAMINATION AND SPOILAGE
1. Physical and chemical Change – Viscosity, pH, emulsion stability and loss of surface activity.
2. Measurement of O2 consumption
3. Sterility tests – Injectables and ophthalmic products of sterile products
4. Microbial limit test – Assessment of viable microorganism in non-sterile products
5. LAL and Rabbit test – Estimation of Pyrogen

MICROBIAL LIMIT TEST (MLT)

Microbial Limit test is the estimation of total number of viable aerobic microorganism in given sample ([Link],
Staph aureus, Pseudomonas aeruginosa, salmonella, shigella, clostridia and candida albicans) as per IP.

Preliminary procedure:
Before doing the microbial limit test, the preservatives present in the test sample (formulations) are identified and
neutralized or inactivated with suitable inactivators. Because it will interfere in the results of MLT test.
Antimicrobial agents
Inactivators
present in the formulation
Phenolics, Parabens Polysorbate 80
Iodine, Quaternary ammonium compound Lecithins
Alcohols, Aldehydes, Sorbates SLS

Mercurial Halogens Sodium Thiosulphate

Main test or Total aerobic microbial count test:

Total Aerobic microbial count present in the test subs is determined by the following methods,
1. Membrane filtration:
▪ 10ml of diluted sample → passed through membrane filter of 0.45µm pore size.
▪ Cut the membrane filter in to two halves.
▪ Transfer one half to FTM (Fluid Thioglycollate medium for measurement of bacteria) and another half
to SCDM (soyabean casein digest medium for measurement of fungi) plate.
▪ Incubate 30 - 40oC /48hrs for bacteria and 20-25oC /5 days for fungi.
▪ Observe the growth of microorganism.

88
2. Plate Count Method:
▪ 1 ml of diluted test sample mixed with each FTM (Fluid Thioglycollate medium for measurement of
bacteria) & SCDM (soyabean casein digest medium for measurement of fungi) and poured into the
plates at 45 °C.
▪ Incubate plates at 30 - 40oC /3- 5 days for aerobic bacteria and 20-25oC / 5-7 days for fungi.
▪ Observe the growth of microorganism.

3. Most Probable Number (MPN)

▪ Also known as Multiple tube or Serial Dilution method.
▪ 10 test tubes taken, add 9 ml of FTM (Fluid Thioglycollate medium for measurement of bacteria)
▪ Arrange 9 test tubes in 3 sets & one as control (Without sample)
▪ Dilute the test sample in to three concentrations (100 mg, 10 mg and 1 mg per ml).
▪ Transfer 1 ml of each concentration into three tubes of every set.
▪ Incubate all tubes and examine the growth of bacteria in each test tubes.
▪ The same procedure is repeated with SCDM (soyabean casein digest medium for measurement of fungi)

89
 PRESERVATION OF PHARMACEUTICAL PRODUCTS
To minimize the microbial spoilage and to kill any contaminant in drug, preservatives are included in the
formulation.
Ideal Properties of preservatives:
1. Non-irritant.
2. Inert.
3. Non-toxic.
4. Stable and effective throughout the expiry.
Development of Preservative system:
➢ Single preservative is not efficient.
➢ Choice of two or more preservatives is suggested depends upon the literatures, nature of formulation and
microbiological studies. This will produce synergistic effect and used as broad spectrum.
➢ In eye drops, benzalkonium chloride often used with Phenyl ethyl alcohol for preservative action.
➢ In oral formulation, MPS and PPS are used often in the conc. of 10:1 ratio.
Chemical Preservatives:
Classified into Four groups such as Acidic, Neutral, Mercurial and Quaternary ammonium compounds.

Factors affecting Preservative efficacy:

1. Excipients: Hydrocolloids (MC, PVP, alginates, tracaganth), Emulsifying agents, Tablet additives
(suspending agents - Kaolin, Mag trisilicate) interact with Preservative efficacy.
2. Properties of Preservatives: Should have both lipid and water soluble. pH independent. Should be inert.
3. Effect of Containers: Environmental moisture may penetrate through plastic containers which increase
the chance of microbial growth in the product, so prefer glass containers. Rubber also reacts with many
preservatives. Screw capped container and corks are common source of mold formation.
4. Type of Microorganism: Preservatives does not have antimicrobial activity against all microorganism.
5. Influence of pH: Many preservatives are pH sensitive. Have action against at certain pH only. Sodium
benzoate and Benzoic acid are active in the pH of 3 to 5. Whereas Methyl paraben sodium act at the pH
of 3 to 5 and the Propyl paraben sodium act at the pH of 6 to 7.

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 PRESERVATIVE EFFICACY TEST (OR) ANTIMICROBIAL EFFICACY TEST:

This test is applied to the packaged container after formulation, is used for determining the efficacy of
preservatives used in the formulation. Concentration and efficacy of preservatives should be mentioned in the
label of the product.

Procedure:
Medium – SCDM (Soybean Casein Digest Medium) for measuring both bacteria and fungi.

Test microorganism: Staph aureus, Pseud. aeruginosa, E. coli, Candida albicans and Aspergillus brosilliensis

Inoculum preparation: Microorganisms (Bacteria/ Fungi) transferred to the SCDM solution. Incubate bacteria
(30-35°C/18-24 hrs) and fungi (20-25°C/ 48 hrs). After incubation, Dilute with saline solution to obtain the
concentration (10-8) CFU/ml. (CFU means Colony forming unit)

Method: Mix the inoculum with the products and then it is transferred to petri dish containing culture medium.
Incubate at room temp. Determine the viable microorganism by plate count method at 7, 14, 21 and 28 days.
Calculate the percentage of reduction in CFU/ml

Interpretation of results

According to USP,

Category 1. Injections, otics, sterile nasals,

ophthalmic with aqueous bases

Category 2. Topicals, nonsterile nasals

Category 3. Orals other than antacids

(aqueous bases)

Category 4. Antacids (aqueous base)

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 CELL CULTURE

Cell Culture is defined as Process of cultivating the human or animal or insect cells and tissues in invitro
conditions (outside the body) in an artificial environment. It will mimic the in vivo conditions (inside the body)
such as temperature, nutrition.

➢ First animal cell culture done by Scientist Ross Harrison in 1907.

➢ Scientist Roux in 1885 first time maintained embryonic chick cells in animal cell culture successfully.
➢ Used to
➢ study cell growth and differentiation,
➢ to perform genetic manipulation and
➢ to understand the gene structure and function.
➢ Even under optimal conditions, animal cells division time itself takes 20 hrs ten times longer than the
yeast.
➢ Growth of visible colony takes a week or more (Yeast and bacteria seen in overnight)
➢ For animal cells, cells are taken from the organ of animal which is removed directly or by mechanical or
by enzymatic action.
➢ Example of animal Cells used to culture are fibroblast, lymphocytes, cells from cardiac and skeletal
tissue, cells from liver, breast, skin and kidney and different types of tumor cells.

CULTURE MEDIA FOR ANIMAL CELL CULTURE******

Culture media is more complex. It may be natural or artificial.

Natural media:
1. Body fluids: Plasma, Serum, lymph, amniotic fluid, ascetic and pleural fluids, aqueous humour from eyes
and insect hemolymph.
2. Tissue extracts: Chick embryo extract, Extracts of liver, spleen, bone marrow and leucocytes.
3. Blood Clots: Coagulants, Plasma clots.
Artificial media:
Balanced Salt Solutions (BSS):
• BSS are primarily composed of Inorganic salts and various nutrients like amino acids, vitamins, salts,
glucose other organic compound, growth factors, hormones, and antibiotics with HEPES (Hydroxyethyl
piperazine 2 sulfonic acid), Sodium bicarbonate (both act as buffer). It is used for providing essential
inorganic ions, maintain pH, desired osmolality, and supply energy to the animal cell growth.
• Commercially available media are,
EBSS – Earle’s balanced salt solution
HBSS – Hank’s balanced salt solution
EMEM – Eagle’s minimal essential medium
DMEM – Dulbecco’s modification of Eagles medium
GMEM – Glasgow’s modification of Eagles medium
RPMI – Media from Rosewell park memorial institute

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Complete Culture Media
The complete culture media for animal cell contains amino acids, vitamins, inorganic salts, glucose other
organic compound, growth factors, hormones, and antibiotics and finally serum.

Amino acids:
• Essential amino acids and some non-Essential amino acids are added in various concentration (Alanine,
Arginine, Asparagine, Cystine, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine,
Phenyl alanine, etc)

Vitamins:
• Water soluble vitamins (B- complex, choline, inositol)
• Fat Soluble vitamins also (A, D, E and K)
Salts:
• Calcium ions - for cell adhesion
• Sodium, Potassium and Chloride ions – for regulate osmololity
• Phosphate, Sulphate and Bicarbonate ions – for ATP synthesis
Glucose:
• Important source of energy.
Hormones and growth factors:
• They are usually added in Serum free media
Other Organic Supplements:
• Proteins, peptides, lipids, nucleosides
Antibiotics:
• These are added to prevent the contamination of microorganism to the animal cell.
• Eg., Ampicillin, Penicillin, Gentamycin, tetracycline, Erythormycin, Kanamycin and Neomycin
Serum:
• Serum is a natural biological fluid. Eg. Calf serum, Fetal bovine Serum, Horse Serum and Human Serum.
• It should be free from diseases. Approximately 5-20% of serum is mostly used.
Proteins:
• Serum protein is involved in promoting attachment and growth eg. Fetuin, fibronectin. It will increase the
viscosity of the medium and maintain the buffering action
Nutrient and its Metabolites:
• Amino acids, Glucose, Phospholipids, fatty acids, nucleosides are collectively called as nutrients
• Nutrients metabolic intermediates like pyruvic acid, lactic acid also involved in the animal cell growth.
Growth Factors:
Growth factors present in the serum which stimulate the proliferation of cells in the culture. They are,
• Platelet derived growth factor (PDGF)
• Fibroblast growth factor (FGF)
• Epidermal growth factor (EGF)
• Insulin like growth factor (IGF)
Hormones:
• Hydrocortisone – Promotes Cell attachment
• Insulin – for glucose uptake
• Growth hormone – Promotes cell proliferation
Inhibitors:
• Bacterial toxins, antibodies, serum contains cell inhibitors namely transforming growth factor β.
• These are removed by heat (at 56 °C for 30 mins)

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Supplementation of the medium with Tissue extracts:
• Culture media can be supplemented with certain tissue extracts and microbial culture extracts
• eg., Chick embryo extract, proteolytic digest of beef heart, tryptose etc

Serum Free Media:

Addition of Serum to the culture media was an old practice. In recent years, serum free media is developed.
Disadvantages of Serum in media:
• Variable Composition – There is no uniformity in the composition of serum, variable to batch to batch.
• Contamination – By pathogens
Advantages of Serum Free Media:
• It is easily control growth of the cells as desired.
• Using different set of factors, can achieve differentiation of cells.
Development of Serum free media:
1. Growth factors: Erythropoietin, Interleukins, Hepatocyte growth factor, Lipopolysaccharide
2. Hormones: Combination of steroid hormones, Growth hormones are mostly added
3. Nutrients: Choline, ethanolamine, linoleic acid, iron, copper, selenium are added in serum free media
4. Proteins: Bovine serum albumin added. Promotes cell survival and growth
5. Polyamines: Putrescine is a polyamines which promote cellular growth and differentiation
6. Protease inhibitors: Eg. Soybean trypsin inhibitor is added for the trypsin mediated subcultures.

TYPES OF ANIMAL CELL CULTURE

Based on cell growth, the animal cell culture is divided into,
Anchorage dependent cells or Adherent layer cells:

- These cells will grow by adhere to the walls of the container (by cellular matrix principle). These are
immobile. Eg. Fibroplasts and Epithelial cells.
- Normally Divided cells uniform in size ie., all cells have one cell thickness.
- So it is called Monolayer cells.

Suspension Cells:

- These do not attach to the surface of the culture container.

- It will grow by floating on the surface of the culture medium. So it is called suspension cells.
- Eg. Stem cells (Blood, spleen and bone marrow) and Tumor cells. It will grow very fast.

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Based on Cell division, the animal cell culture is divided into,
1. Primary Cell culture:
- Primary culture is the cell culture system that is formed by culture cells directly obtained from the
parental tissue of multicellular organism.
- Mostly they are dividing into new cells only for a limited time.
Process to obtain primary cell culture:
➢ Primary cell culture is obtained from fresh tissues.
➢ Tissues are removed from animal organs aseptically using sterile razor.
➢ Tissues then dissociated by proteolytic enzymes (trypsin) that break the cells and separated.
➢ Thus obtained cell suspensions are washed with buffer solution (to remove proteolytic enzymes)
➢ Washed cell suspension are then spread on the surface of petridish containing culture medium
➢ When the petridish incubated, this cell will grow by adhere to the wall of the petridish & divide.

Confluent state and the need of subculture:

→ Confluent state means primary cell culture utilize all the culture nutrients and attain the full growth which
cover the whole container (Too Crowd of the container), this generally leads to death of the cells
(Senescence stage).

→ So, to maintain the cell growth, there is need to go for subculture (Secondary cell culture) to provide
more space and nutrients for continuous growth of the cells.

Passage number: It refers that how many times a cell has subculture.

Growth Requirements for primary cell culture:

- Culture medium and its condition will vary for different cell types.
- Media commonly contains Amino acids, Vitamins, Salts, Glucose, a bicarbonate buffer system, growth
factors, hormones, O2 and CO2. To obtain best growth, small amount of blood serum is added usually and
several antibiotics to prevent bacterial contamination.
- Temperature maintained at 37 °C for normal animal cells.
- Temperature maintained at 15 – 26 °C for cold – blooded animal cells.

Aseptic techniques to be followed during animal cell culture:

➢ Bacterial infections like Mycoplasma and fungal infections contaminated in the animal cell cultures will
decrease the growth of animal cells.
➢ So, the animal cell culturing work is done in sterile environment with proper aseptic techniques. Eg.,
HEPA filter laminar airflow cabinet.

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Cryopreservation of cell culture:
➢ Due to subculturing, surplus of cells is produced. So to maintain the cells, these are treated with the
appropriate protective agent (DMSO, glycerol) and stored at temperatures below -130 °C
➢ This prevents loss of original cell and its characteristics. And thus it prevents the finite cells reaching
senescence stage and minimize the risk of mutation
➢ After Cryopreservation, to obtain the normal cells is achieved by thawing the cells (Warming) in warm
water and then rinsed with medium and serum and transferred to culture medium.

2. Secondary Cell culture or Cell lines or Subclone:

- Sub culturing of Primary cell culture is known as Secondary cell culture or Cell line or subclone.
Procedure for obtaining Secondary cell culture:
- Adhered cells of primary cell culture are dissociated or separated by enzymatically.
- Then, subculture the dissociated cells in fresh nutrient medium leads to generation of new cell lines.
- At initial sub culturing, the genes of the cells are uniform and same in character.
- But at continuous sub culturing leads to mutation of the cells and the cells are not uniform and have
different phenotype and genotype character.
- These mutated cells or cancerous cells will proliferate infinitely and the growth rate also very fast.
Based on life span the animal cell culture is divided into,
1. Finite cell lines or Normal cell lines:
- Limited life span.
- When these cells are cultured, they proliferate (divide) only for certain times. After which they are
gradually decreased in their growth and leads to death. Eg: Liver cells, kidney cells, Chick embryo.
- These are Monolayer and Anchorage dependent cells.
- Growth rate is very slow and so the yield also very low.
- Requires high serum for growth.
- Cell doubling timing is 24 -96 hrs.
2. Continuous cell lines or transformed cell lines or Established cell lines:
- During continuous sub culturing, few cells in the culture medium may get mutated to form different
morphology and phenotype characteristics. They are known as continuous cell lines. Cancerous cells
which are obtained from cancerous tissue (mutated tissues) also called as continuous cells. Eg: Hela
cells - cervical cancer cells, 293 T cells – Embryonic kidney cells, A-549 cells – Lung cancer cells.
- Their proliferation is immortal i.e. Cell is not died and grows indefinitely.
- These cells are anchorage independent or suspension cells.
- They grow very faster.
- Require less serum for growth.
- Doubling time is 12-24 hrs

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 APPLICATION OF CELL CULTURES
1. Virology:
- It is used for the identification, multiplication and isolation of virus and also to know how the virus affect
the other organisms.
2. Vaccine production:
- Animal cell culture has the ability to grow large amounts of virus in culture medium, this led to the
production of many vaccines (like).
- In Industries many vaccines are produced like Polio vaccine, Rabies, chicken pox, hepatitis B etc.
- Continuous cell lines are not used in vaccine production since these cell lines are obtained from cancer
tissues.
3. Cancer research:
- To study the growth and proliferation (increase in number) between cancer & normal cell.
- To identify the causes of cancer development, by treating the normal cells to radiation, chemicals, viruses
etc.
- To determine the drugs used to treat or destroy the cancer cells
4. Cell based manufacturing:
- Many industries produce vaccines and proteins like hormones, insulin, monoclonal antibodies in large
scale production by cell culturing.
- Artificial skin production for treating burns and diabetic skin ulcers.
- Also Artificial organs like pancreas, liver and kidney are under testing.
- Replacement of cells and tissues by study of embryonic and adult stem cells.
5. Genetic counselling:
- Amniocentesis is a procedure in which amniotic fluid is removed from the uterus of pregnant women for
testing or treatment of disorders of fetus. Fetal cells abnormalities found out by examining their
chromosomes and genes using chromosome studies like Karyotyping, chromosome painting and other
molecular techniques. There by we can reduce the chances of defective fetal birth.
6. Genetic Engineering:
- Ability to reprogram the cultured cells with new genetic material (DNA and genes) which provide the
new cellular effects.
7. Gene therapy:
- Ability to alter the defective genes of the cell and to treat the patient lacking a functional gene or
damaged gene by replacing new genes.
8. Drug screening and development:
- Cell based assay, Cytotoxicity testing, Screening of potential compounds of the drug are developing
nowadays are great important to pharmaceutical industry to skip or reducing the clinical trials.

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