BIOPROCESS TECHNOLOGY

II M.SC. MICROBIOLOGY

UNIT I

INDUSTRIALLY IMPORTANT MICROBES AND THEIR

DEVELOPMENT: SCREENING METHODS FOR INDUSTRIAL MICROBES –

DETECTION AND ASSAY OF FERMENTATION PRODUCTS – CLASSIFICATION

OF FERMENTATION TYPES – STRAIN SELECTION AND IMPROVEMENT.

MUTATION AND RECOMBINANT DNA TECHNIQUES FOR STRAIN

DEVELOPMENT

UNIT II

FERMENTER – TYPES AND FUNCTION

FERMENTERS – BASIC DESIGN, DESIGN COMPONENTS – ASEPSIS AND

CONTAINMENT REQUIREMENTS – BODY CONSTRUCTION AND

TEMPERATURE CONTROL – AERATION AND AGITATION SYSTEMS –

STERILIZATION OF FERMENTER, AIR SUPPLY AND MEDIUM; ASEPTIC

INOCULATION METHODS – SAMPLING METHODS, VALVE SYSTEMS – A

BRIEF IDEA ON MONITORING AND CONTROL DEVICES AND TYPES OF

FERMENTERS

UNIT III

FOOD MICROBIOLOGY

MICROBIOLOGY OF FERMENTED MILK – STARTER CULTURES, BUTTER

MILK, CREAM, YOGHURT, KAFIR, KUMISS, ACIDOPHILIC MILK, AND

CHEESE. MICROBES AS SOURCE OF FOOD (SPIRULINA, SACCHAROMYCES

CERVICEAE, RHIZOPUS SP.) FOOD AND WATER BORNE INFECTIONS AND

FOOD MICROBIAL POISOINING. FOOD SANITATION IN FOOD

MANUFACTURE AND IN THE RETAIL TRADE. FOOD CONTROL AGENCIES

AND ITS REGULATIONS

UNIT IV

FERMENTATION IN BATCH CULTURE:

MICROBIAL GROWTH KINETICS, MEASUREMENT IN GROWTH(CELL

NUMBER, DIRECT AND INDIRECT METHODS) GROWTH AND NUTRIENT,

GROWTH AND PRODUCT FORMATION, HEAT EVOLUTION, EFFECT OF

ENVIRONMENT(TEMPERATURE, PH, HIGH NUTRIENT CONCENTRATION)

MEDIA FORMULATION STERILIZATION, KENETICS OF THERMAL DEATH

OF MICROORGANISMS, BATCH AND CONTINUOUS STERILIZATION ( ALL IN

RELATION TO FERMENTATION)

UNIT V

LEGAL PROTECTION AND IPR:

GATT AND IPR, FORMS OF IPR, IPR IN INDIA, WTO ACT, CONVENTION ON

BIODIVERSITY(CBD), PATENT COOOPERATION TREATY(PCT), FORMS OF

PATENTS AND PATENTABILITY, PROCESS OF PATENTING, INDIAN AND

INTERNATIONAL AGENCIES INVOLVED IN IPR & PATENTING, GLOBAL

SCENARIO OF PATENTS AND INDIA’S POSITION, PATEN TING OF

BIOLOGICAL MATERIALS

REFERENCES:

1. DEMAIN, A.L. AND DAVIES, J.E (1999) MANUAL OF INDUSTRIAL

MICROBIOLOGY AND BIOTECHNOLOGY, ASM PRESS

2. GLICK, B.R. AND PASTERNAK (1994) MOLECULAR BIOTECHNOLOGY,

ASM PRESS

3. STANBURY, P.F., WHITAKER.A, AND HALL, S.J. PRINCIPLES OF

FERMENTATION TECHNOLOGY, PERGAMON PRESS

4. GLICK, B.R. AND PASTERNAK JJ (1998) MOLECULAR

BIOTECHNOLOGY, II EDITION, ASM PRESS, NEW YORK.

5. MITTAL, D.P (1999) INDIAN PATENTS LAW, TAXMANN, ALLIED

SERVICES (P) LTD.

6. TORTORA, G.J.FERNKE B.R. AND CASE C.L (2001) MICROBIOLOGY –

AN INTRODUCTION, BENJAMIN CUMMINGS

7. VENKATARAMAN, L.V.(1983) A MONO0GRAPH ON SPIRULINA

PLANTENSIS, CFTRI, MYSORE

8. KALAICHELVAN, P.T AND ARUL PANDI (2007) BIOP[ROCESS

TECHNOLOGY, MJP PUBLISHERS, CHENNAI

APRIL 2007 2 mark question

1. Mention two water borne infections

2. Total count

3. Thermal death time

4. GATT

5. Patents

5 mark question

6. Differentiate between food intoxication and food infection

7. Write on the role of food quality control agencies to provide safe food to

consumers

8. Discuss the merits and demerits in direct methods of microbial growth

measurements

9. Write a brief account on media formulation for large scale fermentation

10. Explain different forms of IPR

11. Give a brief account of convention on biodiversity

10 mark question

12. Describe the various methods for measurement of microbial growth

13. Write an essay on forms of patents and patentability and process of

patenting

APRIL 2007

2 mark question

1. Rind rots

2. Aero tolerant anaerobes

3. Antiport

4. Dunkel

5. WIPO

5 mark question

6. Briefly explain the causes for slowness of starter cultures and add a note

on the remedial measures

7. Enlist National and International agencies that regulate food quality

8. How will you measure microbial growth by online estimation method

9. Tabulate the advantages of batch and continuous sterilization

10. Discuss the patent case history of Turmeric

11. Comment on CBD

10 mark question

12. Comment on various forms of IPR

APRIL 2008 2 mark question

1. Humilin

2. Vaccum Breaker

3. Food infection

4. Continuous sterilization of media

5. GATT

6. Trade mark

5 mark question

7. Write short notes on botulism

8. Write a brief account on food control agencies and its regulations

9. Explain the media formulation for fermentation

10. Discuss the IPR in India

11. List out various Indian and International agencies involved in IPR

10 mark question

12. Discuss media formulation and sterilization

13. Write an essay on global scenario of patents

APRIL 2009 2 mark question

1. Mention two food intoxicating organisms

2. GATT

3. Biodiversity

5 mark question

4. Describe the effect of pH on fermentation microorganisms

5. Comment on thermal death of microorganisms

6. Write a brief account on five principles of trading system established by

WTO

7. Comment on patenting microorganisms

10 mark question

8. Discuss on botulism, causative organism, toxigenic types, symptoms and

its control

9. Discuss microbial growth kinetics with respect to nutrient concentration

and product formation

10. Write an essay on global scenario of patents and Indian position

1. Mention two water borne infections:

Many important human pathogens are maintained in association with living organisms

other than humans, including many wild animals and birds. Some of these bacterial and

protozoan pathogens can survive in water and infect humans. When waters are used for

recreation or are a source of sea food that is consumed uncooked, the possibility for disease

transmission certainly exists.

♣ Enteric fever ( Typhoid) - Salmonella typhi

♣ Shigellosis - Shigella dysenteriae

♣ Cholera - Vibrio cholerae

♣ Diarrhea by Campylobacter - Campylobacter jejuni

♣ Legionnaires disease - Legionella pneumophila

♣ Leptospirosis - Leptospira icterohaemorrhagiae

♣ Infectious Hepatitis - Hepatitis A virus

♣ Viral Gastroenteritis - Rota virus, calici virus

♣ Poliomyelitis - Polio virus, Echo virus

♣ Giardiasis - Giardia lamblia

♣ Amoebiasis - Entamoeba histolytica

♣ Cryptosporiodiosis - Cryptosporidium parvum

♣ Meningoencephalitis - Naegleria gruberi

♣ Schistosomiasis - Schistosoma

2. Total count:

♣ Increases in the total cell mass, as well as in cell numbers, accompany population growth.

Therefore techniques for measuring changes in cell mass can be used in following growth.

♣ The most direct approach is the determination of microbial dry weight. Cells growing in

liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an

especially useful technique for measuring the growth of fungi. It is time consuming, however,

and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several

hundred milliliters of culture to collect a sufficient quantity.

♣ More rapid, sensitive techniques depend on the fact that microbial cells scatter light

striking them. Because microbial cells in a population are of roughly constant size, the amount

of scattering is directly proportional to the biomass of cells present and indirectly related to cell

number. When the concentration of bacteria reaches about 10 million cells per ml, the medium

appears slightly cloudy or turbid. Further increases in concentration result in greater turbidity

and less light is transmitted through the medium. The extent of light scattering can be measured

by a spectrophotometer and is almost linearly related to bacterial concentration at low

absorbance levels. Thus population growth can be easily measured spectrophotometrically as

long as the population is high enough to give detectable turbidity.

3. Thermal death time:

♣ The heat resistance of microorganisms usually is expressed in terms of thermal death time,

which is defined as the time it takes at a certain temperature to kill a stated number of organisms

or spores under specified conditions.

♣ This is sometimes referred to as the absolute thermal death time to distinguish it from the

majority thermal death time for killing most of the cells or spores present and the thermal

death rate, expressed as the rate of killing.

♣ The factors affecting the heat resistance of cells or spores are (1) temperature – time

relationship, time for killing cells or spores under a given set of conditions decreases as the

temperature is increased (2) initial concentration of spores or cells, more spores or cells

present the greater the heat treatment necessary to kill all of them (3) previous history of the

vegetative cells or spores, the conditions under which the cells have been grown and spores

have been produced and their treatment thereafter will influence their resistance to heat

4. GATT:

♣ The General agreement of tariffs and trade (GATT) was framed in 1948 by developed

countries to settle the disputes among the countries regarding share of world trade. It is decided

by tariffs rates and quantitative restrictions on imports and exports.

♣ The primary objective of GATT was to expand international trade by liberalizing trade so as

to bring about all round economic prosperity. The preamble to the GATT mentioned the

following as its important objectives:

a) Raising standard of living

b) Ensuring full employment and a large and steadily growing volume of real

income and effective demand

c) Developing full use of the resources of the world

d) Expansion of production and international trade

♣ Havana round at Cuba, Annecy round at France, Torquay round at England, Geneva round at

Geneva, Dillon round at Geneva, Kennedy round at Geneva, Tokyo round at Tokyo, Uruguay

round at Uruguay, Doha round are some of the rounds of GATT which decided the tariffs rates

and restrictions on imports and exports

5. Patents:

♣ Patent is a special right to the inventor that has been granted by the government through

legislation for trading new articles, a patent is a personal property which can be licensed or sold

by the person / organization just like any other property.

♣ For example, Alexander Graham Bell obtained patent for his telephone. This gave him the

power to prevent engine from making or using or selling a telephone elsewhere.

♣ A patent consists of three parts: the grant, the specification and claims.

♣ The grant is filled at the patent office which is not published. It is a signed document which

is actually the agreement that grants patent right to the inventor. However, the specification and

claims are published as a single document which is made public at a minimum charge from the

patent office.

♣ The specification part is narrative in which the subject matter of invention is described how

the invention was carried out.

♣ The claim section specifically defines the scope of the invention to be protected by the patent

to which the others may not practice.

♣ The maximum duration of patent is for 5 years from the date of grant and 7 years from the

date of filing the patent application.

6. Dunkel:

♣ The GATT was framed in 1948 by developed countries to settle the disputed

among the countries regarding share of world trade.

♣ It is decided by tariffs rates and quantitative restrictions on imports abd

exzoirts

♣ For a long time benefits from GATT was achieved obnly by developed

countries.

♣ In 1988 the US Congress enacted a law ‗ the Omnibus Trade and

Competitiveness Act‘ (OTCA)

♣ As a result of which the USA became powerful to investigate the laws related

to trade and check them if not beneficial to its interest

♣ After warning, if the investigated country does not change its law within the

desired period the US takes action against that country

♣ In 1992, the US gave warning to India to change some of its laws of IPR,

patents and copyrights

♣ India had certain inhibitions to sign on GATT draft

♣ Therefore there was much debate through out the country on this issue and

bad intension of the US

♣ The professionals, politicians and scientific argued that the total package of

TRIPS must guarantee for economic and technological subjugation of the country

♣ The director general of GATT, A. Dunkel came to India for discussion on this

issue.

♣ Certain provisions were suggested to include in GATT draft that India will not

give any kind of subsidies for the production of oil seeds and pulses as the

international price is more than that in India

♣ India assured to change its patent laws by 2003. In the changed patent it will

introduce the product patent and enhance patent duration

♣ It will open the market to foreign patent holders also and open the agriculture

to patent technology

♣ Even there are several groups and organizations that have rejected this draft of

suggestion and opposed the decision taken by the government

7. WIPO:

♣ The WIPO is one of the specialized agencies of the united nations

♣ It has provided that the intellectual property shall include rights relating to the

following

♣ Literacy, artistic and scientific works, performance of artists, phonograms,

broadcast innovation in all fields of human endeavor, scientific discoveries, trade

marks, service marks, and commercial names, industrial designs, protection

against unfair competition and all other rights resulting from intellectual activity

in the areas of industrial, scientific literary or artistic

♣ The intellectual property is protected by and governed by appropriate national

legislation. The national legislation specifically describes the inventions which

are the subject matter of protection and those which are exclude form a protection

for example, methods of treatment of humans or therapy and invention whose use

would be contrary to law or invention which are injurious to public health are

excluded from patentability in the India legislation

8. Mention two food intoxicating organisms: ♣ Bacillus cereus: (Food Poisoning)

ONSET: 1-16 Hours

FOOD INVOLVED: Grains including rice, flour, dry-mix products, i.e. for

soups, gravies, puddings and dried potatoes.

SYMPTOMS: Diarrhea, abdominal pain, nausea, and vomiting.

PREVENTION: Do not hold prepared foods at room temperature; refrigerate

quickly after preparation; keep dry foods and mixes dry.

♣ Botulism: (Food Poisoning)

ONSET: 12-36 Hours

FOOD INVOLVED: Inadequately processed, usually home canned, low acid

foods, meat and fish.

SYMPTOMS: Difficulty in swallowing, weakness, dizziness, voice changes

PREVENTION: Toxin destroyed by boiling; cook foods thoroughly; use a

pressure cooker in processing.

♣ Clostridium perfringens: (Food Poisoning)

ONSET: 6-24 Hours

FOOD INVOLVED: Stews, meat pies or meat gravies held at warm

temperatures.

SYMPTOMS: Nausea, sometimes vomiting, colicky, pains, diarrhea.

PREVENTION: Thoroughly cook foods; refrigerate at 40 degrees Fahrenheit

or less; hold at 150 Fahrenheit or more.

♣ Staphylococcus aureus : (Food Poisoning)

ONSET: 1-6 Hours

FOOD INVOLVED: Cooked ham, salads of protein food, custard pastries,

Hollandaise sauce, warmed over food.

SYMPTOMS: Nausea, vomiting, diarrhea, acute prostration, abdominal

cramps.

PREVENTION: Cook foods thoroughly; refrigerate at 40 degrees Fahrenheit

or less; hold at 150 Fahrenheit or more; keep hands clean and skin sore-free.

9. Biodiversity:

♣ Biodiversity is a new name for species – richness (plants, animals and microorganisms)

occurring as an interacting system in a given habitat

♣ Biodiversity cannot be replaced because the species becomes adapted in a given habitat after

a long course of time

♣ That is why, due to plausticity in their nature and unsustainable resource utilization over 2.5

lakhs species are lost and thousands are threatened to extinction

♣ If a species extinct it means whole of the gene pool extinct.

♣ The real value of biodiversity lies in the information‘s that are enclosed in the genes.

Therefore there is urgent need for future to protect the genes from destruction

♣ Biodiversity may be defined as the inherent and externally imposed variability within and

among the living organisms present in the terrestrial, marine and other ecosystems at the specific

time

♣ Diversity includes the variability in genes, genotype, species, genera, family and ecosystem

at a particular time in a specific region.

♣ Biodiversity is an expression of both numbers and differences and differences can be seen as

a measure of complexity

10. Humilin:

♣ Humulin is synthesized in a special non-disease-producing laboratory strain of

Escherichia coli bacteria that has been genetically altered to produce human insulin.

♣ Humulin 70/30 is a mixture of 70% Human Insulin Isophane Suspension and 30%

Human Insulin Injection (rDNA origin).

♣ It is an intermediate-acting insulin combined with the more rapid onset of action

of Regular human insulin.

♣ The duration of activity may last up to 24 hours following injection.

♣ The time course of action of any insulin may vary considerably in different

individuals or at different times in the same individual.

♣ As with all insulin preparations, the duration of action of Humulin 70/30 is

dependent on dose, site of injection, blood supply, temperature, and physical activity.

♣ Humulin 70/30 is a sterile suspension and is for subcutaneous injection only.

♣ It should not be used intravenously or intramuscularly. The concentration of

Humulin 70/30 is 100 units/mL (U-100).

11. Food infection:

♣ The food serves as a vehicle for the transfer of the pathogen to the consumer, in whom the

pathogen grows and cause disease

♣ The causative organisms are ingested and gives rise to the disease

♣ It produces symptoms after the ingestion of the pathogen followed by growth in the host,

including tissue invasion and / or the release of toxins

♣ Toxins are released only after the growth of the pathogen in the host intestinal tract

♣ Sources of contamination of food may be animals infected during its life time, human

carriers

♣ Divided into two types:

a) food does not support the growth of the pathogens but carries them e.g.

Tuberculosis, diphtheria, dysenteries, typhoid, brucellosis, cholera, infectious

hepatitis, Q fever

b) food serve as culture medium for the growth of the pathogens eg. Salmonellosis,

Bacillus cereus gastroenteritis, Yersiniosis, Clostridium perfringens

gastroenteritis, E.coli infections, Shigellosis

12. Continuous sterilization of media:

♣ The two main disadvantages of batch sterilization just mentioned, culture

medium damage and high energy consumption, can be largely avoided by use of a

continuous sterilization procedure.

♣ Although continuous sterilization is the logical preliminary step for continuous

fermentations in industrial scale, it is also of value in batch fermentations, making

greater yields possible for the time and space allotted.

♣ The reason for this is because of the exponential relationship‘ between death rate

and temperature, making the time required for the complete elimination of life shorter

if higher temperatures are used. While batch sterilization is carried out in 30-60

minutes at 121°C, continuous sterilization is normally accomplished in 30- 120

seconds at 140°C.

♣ The heating of culture media for continuous sterilization can be done either by

injection of steam or by means of heat exchangers.

♣ Sterilization with steam injection is done by injecting steam into the nutrient

solution. The temperature is raised quickly to 140°C and is maintained for 30-120

seconds.

♣ Due to the formation of condensate, the nutrient solution becomes diluted; to

correct this, the hot solution is pumped through an expansion valve into a vaporizer

and the condensate is removed via vacuum pumps so that the sterilized nutrient

solution has the same concentration after the cooling process as before.

♣ The disadvantage of this process is the sensitivity it exhibits to changes in the

viscosity of the medium and to pressure variations.

♣ In the continuous process using heat exchangers the nutrient solution in the first

heat exchanger is preheated to 90-120°C within 20-30 seconds by the exiting

previously sterilized nutrient solution. Then in the second heat exchanger, it is heated

indirectly with steam to 140°C.

♣ This temperature is maintained for 30-120 seconds in a holding pipe before it is

placed in the first exchanger for preliminary cooling and then in a third exchanger for

cooling to the temperature of the fermenters. The cooling phase is only 20-30

seconds.

♣ In the process using heat exchangers, 90% of the energy input is recovered.

♣ The disadvantage of this method is that with some nutrient solutions, insoluble

salts (e.g., Calcium oxalate) are formed and crusts the first heat exchanger, due to the

temperature differences between the nutrient solution and the cold incoming.

♣ Starch-containing solutions which viscous when heated are difficult to use in

continuous sterilization processes.

♣ The advantages of continuous sterilization of media are as follows:

1. Increase of productivity since the short period of exposure to heat

minimizes damage to media constituents

2. Better control of quality

3. Leveling of the demand for process steam

4. Suitability for automatic control

13. Trade mark:

♣ A trade mark is an identification symbol which is used in the course of trade to enable the

public to distinguish on traders goods from the simi9lar goods of the other traders.

♣ The public makes use of these trade works in order to choose whose goods they will have to

buy

♣ If they are satisfied with the purchase they can simply repeat their order by using the trade

mark, for example KODAX for photography goods, IBM for computers, zodiac for readymade

cloths

♣ Trade marks rights are very important. Trade mark laws vary in every country. Through

agreement it is ensured that the trademark of one country must be protected in another country.

♣ India trade and merchandise rights are very important. Therefore multinational companies

spend large amount of money to maintain their trademarks throughout the world

14. Differentiate between food intoxication and food infection:

S.No. Food intoxication Food infection

1.

2.

3.

4.

5.

6.

The microorganisms grow in the food

and produce toxins that can then affect

the consumer

The toxin is ingested and gives rise

to the disease

It produces symptoms shortly after

the food is consumed because

growth of the disease causing

microorganism is not required

Toxins produced in the food can be

associated with microbial cell or can

be released from the cells

Sources of contamination of food may

be human carriers, soil, improper

refrigeration, eating raw foods and

animal carriers

Divided into two types:

a) Botulism - neurotoxin

b) Staphylococcal intoxication –

enterotoxin

The food serves as a vehicle for the transfer of

the pathogen to the consumer, in whom the

pathogen grows and cause disease

The causative organisms are ingested and gives

rise to the disease

It produces symptoms after the ingestion of the

pathogen followed by growth in the host,

including tissue invasion and / or the release of

toxins

Toxins are released only after the growth of the

pathogen in the host intestinal tract

Sources of contamination of food may be

animals infected during its life time,

human carriers

Divided into two types:

a) food does not support the growth of the

pathogens but carries them e.g. Tuberculosis,

diphtheria, dysenteries, typhoid, brucellosis,

cholera, infectious hepatitis, Q fever

b) food serve as culture medium for the growth

of the pathogens eg. Salmonellosis,

Bacillus cereus gastroenteritis,Yersiniosis,

Clostridium perfringens gastroenteritis,

E.coli infections, Shigellosis

15. Write on the role of food quality control agencies to provide

safe food to consumers:

♣ The objectives of the control, regulation and inspection of food are primarily to give

assurance that foods received by the consumer will be pure, healthful and of the quality claimed.

♣ International Agencies: The food and agricultural organization (FAO), the world health

organization (WHO) and the international children’s emergency fund (UNICEF): They are

not major enforcement or control agencies; their common interest in adequate, healthful and safe

foods is noteworthy.

♣ FAO is primarily concerned with food production through improved methods of production,

processing, preservation and distribution of foods.

♣ WHO activities are more related to the health of the consumer and the maintenance of food

wholesomeness.

♣ The joint FAO / WHO food standards commission is forum for the cooperation among

nations to develop or agree upon various international standards for the food industry. These

finalized international standards were to be published in a food Codex Alimentarius as either

regional or worldwide standards. As of 1986 there were about 150 international standards

adopted by the commission.

♣ Subsidiary groups of this organization have published a code of practice on general principles

of food hygiene and several codes of hygienic practice for specific commodities.

♣ The international commission on microbiological specifications for foods (ICMSF) is a

voluntary body which has focused primarily on establishing international sampling plans and

methods of analysis. This group has been responsible for organizing collaborative studies on

sampling plans and laboratory methodology with other groups, including the international

dairy federation (IDF), and the association of official analytical chemists (AOAC).

♣ Federal agencies: the authority of U.S. federal enforcement agencies is confined to foods

shipped interstate or foods produced in or shipped into territories. The federal register, published

almost daily contains news announcements of agencies of the federal government pertaining to

food standards and inspection.

♣ Food and Drug Administration (FDA): The food and drug administration of the

department of health, education and welfare (HEW) enforces the federal, drug and cosmetic act

as amended in 1980.

♣ Basically, the functions of the FDA relate to the agency‘s responsibility for ensuring that all

foods are safe and wholesome and that all foods are honestly and informatively labeled.

♣ Two of the main operational programs include the FDA compliance policy guides (FDA,

1982 a) and FDA food defect action levels (FDA, 1982b).

♣ The compliance policy guides contain specific microbiological criteria for various foods and

feeds. Specifications are given for total or aerobic plate counts, coliforms, coagulase positive

staphylococci, food borne pathogens, mycotoxins, bacterial indicators and E.coli.

♣ The food defect actions levels deal mostly with the level of natural defects that may be

tolerated in particular food or commodity. The natural defects are not necessarily related to any

particular health hazards. Rather, they are specified levels of natural defects above which the

FDA may remove products form the market place.

♣ United States Department of Agriculture (USDA): Through the Agricultural marketing

act, the egg products inspection act, the wholesome meat act (Federal Meat Inspection Act) and

the wholesome poultry products act (Poultry inspection act), the USDA has legislative authority

to promote the marketing of safe, high quality agricultural products, egg and egg products, meat

and poultry products and dairy products are covered by the USDA agricultural marketing

service (AMS) and the food safety and inspection service (FSIS).

♣ Specific microbiological criteria have been established by the AMS under the authority of

the Agricultural Marketing Act – for processed milk products and raw milk; salmonella free egg

and egg products. Federal Meat Inspection Act and the Poultry inspection act give the USDA to

carry out programs in the meat and poultry processing industries. The food safety inspection

service (FSIS) has responsibility for all these products

♣ National Marine fishery service (NFMS): the United States department of commerce/

National Marine fishery service (USDC / NFMS) is fee for service fisheries products inspection

program. Most of the criteria applied have to do with organoleptic evaluation of the product and

examinations of the processing facility.

♣ United States army Natick research and development center: Food procurement and

food protection for military purposes involve problems that are not always encountered in

civilian food service, therefore, the department of defense, through the defense standardization

and specification program, has established numerous microbiological criteria for the

procurement of foods. The Natick microbiology branch is concerned with microbiological

problems involved in military ration development and the establishment of microbiological

criteria for initial procurement

♣ State Agencies: State food laws usually are enforced through state department of public

health, agriculture or sanitary engineering. Some states have microbiological standards or

guidelines for food

♣ Commercial Agencies: Food association or institutes make recommendations or even

attempt regulation within their own industries. Thus the national food processors association has

set microbiological standards for sugar and starch for canning, American dry milk institute has

established bacteriological standards for dry milk, the American bottlers of carbonated beverages

has bacteriological standards for sweetening agents used in soft drinks

♣ Professional societies: The American public health association has published many

recommended methods for the bacteriological and microbiological examination of foods and

international association of milk, food and environmental sanitarians has also published

recommended procedures for the investigation of food borne illnesses. (international association

of milk, food , and environmental sanitarians, 1976)

♣ Private agencies: several private agencies approve and list tested foods, e.g. the good

House keeping Institute

♣ The processing Industry: Many specific food processing industries and / or companies

have established their own in house microbiological criteria for finished products and raw

materials and ingredient. Usually the larger the company, the more sophisticated its

microbiological testing program.

♣ Agency cooperative programs:

a) The food and drug administration, state agencies, and the private shellfish

industry voluntarily cooperate in the national shellfish sanitation program

b) The FDA and USDA cooperative is a salmonella surveillance program for dry

milk products

c) The retail food protection program (FDA) is another example of a federal – state

cooperative program

d) Several publications including the vending of food and beverages (FDA, 1978),

the retail food store sanitation code (FDA, 1982c) and the food service sanitation

manual (FDA, 1976) serve as a source of technical assistance to various state

agencies

e) The united states public health service and the FDA have established the grade A

pasteurized milk ordinance(PMO)

f) The national marine fishery service (NFMS) through the united states department

of commerce interacts and cooperates with other federal agencies, including the

FDA and department of defense

16. Discuss the merits and demerits in direct methods of microbial

growth measurements:

The various methods used in the measurement of microbial growth and also the

synchronous growth and continuous culture techniques. Microbial growth which

results in increase in cell number and also the cell mass. The word cell number

implies to the viable cell which has ability to metabolize and reproduce while the cell

mass indicates the total mass of viable and non-viable cells.

Measurement / Enumeration of Micro-organisms is required for

1. assessing the rate of microbial reproduction,

2. determining the potability of water,

3. determining the suitability of food for human

4. consumption,

5. evaluating the necessity for antimicrobial therapy, and

6. for industrial, research and medical reasons.

♣ Petroff—Hausser counting in which a special slide and cover glass are used to

contain a sample of known volume.

♣ Advantages :

1. they are more rapid,

2. equipments and requirements needed are minimum,

3. Morphology of microbes can also be studied.

♣ Disadvantages : These methods count both the viable as well as non- viable cells,

the accuracy also declines with very dense sample and diluted sample leading to

statistical errors

♣ Electronic cell counters / coulter counters:

This method is valuable for heat labile samples and it is rapid also. But the

main drawbacks are both the dead and living cells are counted. Even the inanimate

particles may be counted giving errors. Counting the microbial cells is more difficult

because the cells are very small in size and if the tube is narrowed to allow only one

cell to pass through it, the culture does not flow well and eventually clogs.

♣ Viable Cell Count:

1. Standard Plate Count: Drawbacks

a) The major limitation of this method is selectivity. There is no common set of

incubation conditions and medium composition that permits the growth of all

types of organisms present in sample.

b) The pipetting and transferring of samples at various points may give errors.

c) If the bacterial suspension is not homogeneous and contains aggregates of

cells, may give lower CFU than the actual one.

2. Membrane Filter Technique: The disadvantage of this method is that if the

sample is very dense / turbid, it may clog the filter.

♣ Dip-slide Culture Technique

It is a semi quantitative method mainly used by doctors to quickly identify and count

the micro-organisms from body fluids such as urine, CSF etc. This technique helps

the doctor to gather information about the nature of infection.

♣ Most Probable Number ( MPN ) Technique

It is one of the indirect methods used to count the viable cells. Conventionally

important in water microbiology to determine the potability of water sample. This

method provides statistical number of bacteria present in the sample.

♣ Biomass Determination :

Most of the viable cell counting techniques are very accurate but time

consuming. In order to perform a more rapid cell population count, indirect methods

have been developed. One common method is to determine the biomass and correlate

that value with the number of cells per ml. previously measured by a standard plate

count. Actually the word biomass refers to the total weight of a particular type of

organism and related to increase in protoplasmic constituents.

♣ Turbidimetric method

Turbidimetric measurements are based on the principle that the number of

microbial cells in a suspension is inversely proportional to the amount of light

transmitted. Such methods are rapid and reproducible but the O.D. values do not

directly inform us about the number of cells in the suspension.

♣ Measurement of Dry or wet Weight of Biomass:

Such methods are based on increase in protoplasmic constituents during the

microbial growth. The practical difficulties are no methods are available by which we

can measure the dry or wet weight of single microbial cell, hence, the procedures are

used for microbial population.

♣ Advantages and Applications of Continuous Culture Technique:

1. They provide a constant source of active cells for the study of physiology and

genetics of the organism.

2. Cultures in the exponential phase are highly useful in fermentation industry

because of their high physiological activities.

3. As the cells are grown continuously, it is possible to study synthesis or catabolism

of limiting substrates and also to select various classes of mutants.

4. The system can be coupled with selective enrichment to isolate.

5. They are used in industry for the production of single cell protein (SCP)

♣ Application Of Synchrony

It is not possible to analyze a single bacterium because of its small size. It is

very important to know about cell organization, differentiation and macromolecule

synthesis. The synchronous cultures provide cell population in the same stage of

growth or division cycles. Results made for synchronized cultures are equivalent to

the results made for individual cells. But, the synchrony lasts only for a few

generations, since even the daughter cells of a single mother cell soon get out of phase

with one another.

♣ Techniques for Synchronous Growth :

In any large microbial population, the cells are growing randomly. By certain

techniques it is possible to impose synchronous growth, lasting for three to four

generations and again enter into the asynchronous growth.

♣ The important techniques for inducing synchrony are :

1. Manipulation of environmental conditions,

2. Separation by physical means of cells in population that are at the same stage

of the cell cycle,

3. Helmstetter—Cummings technique.

17. Write a brief account on media formulation for large scale

fermentation :

♣ Medium formulation is an essential stage in the design of successful laboratory

experiments, pilot scale development and manufacturing process

♣ The constitutents of a medium must satisfy the elemental requirements for cell

biomass and metabolite production and there must be an adequate supply of energy

for biosynthesis and cell maintenance

♣ The first step to consider is an equation based on the stoichiometry for growth

and product formation

♣ Thus for an aerobic fermentation:

carbon and energy source + biomass + products +

nitrogen source + CO2 + H2O

oxygen + other requirements

♣ This equation should be expressed in quantitative terms which is important in the

economical design of media if component wastage is to be minimal

♣ Thus it should be possible to calculate the minimal quantities of nutrients which

will be needed to produce a specific amount of biomass.

♣ Knowing that a certain amount of biomass is necessary to produce a defined

amount of product, it should be possible to calculate substrate concentrations

necessary to produce required product yields.

♣ There may be medium components which are needed for product formations

which are not required for biomass production

♣ Unfortunately it is not always easy to quantify all the factors very precisely

♣ A knowledge of the elemental composition of a process microorganism is

required for the solution of the elemental equation

♣ This information may not be available so that data will serve as a guide to the

absolute minimum quantities of N, S, P, Mg and K to include in an initial medium

recipe

♣ Trace elements (Fe, Zn, Cu, Mn, Co, Mo, B) may also be needed in smaller

quantities.

♣ An analysis of relative concentrations of individual elements in bacterial cells and

commonly used cultivation media quoted by Cooney (1981) showed that some

nutrients are frequently added in substantial excess of that required, e.g., P, K,

however others are often near limiting values (Zn, Cu)

♣ The concentration of P is deliberately raised in many media to increase the

buffering capacity

♣ The points emphasize the need for considerable attention to be given to medium

design

♣ Some microorganisms cannot synthesize specific nutrients (amino acids, vitamins

or nucleotides)

♣ Once a specific growth factor has been identified it can be incorporated into a

medium in adequate amounts as a pure compound or as a component of a complex

mixture

♣ The carbon substrate has a dual role in biosynthesis and energy generation.

♣ The carbon requirement for biomass production under aerobic conditions may be

estimated from the cellular yield coefficient (Y) which is defined as

Quantity of cell dry matter produced / Quantity of carbon substrate utilized

♣ Thus for bacteria with a Y for glucose of 0.5 which is 0.5 g cells / g glucose, the

concentration of glucose needed to obtain 30g/dm3

cells will be 30 / 0.5 – 60 g / dm3

♣ One litre of this medium would also need to contain approximately 3 g N, 1 g K,

0.3 g S and 0.1 g Mg.

♣ An adequate supply of the carbon source is also essential for a product forming

fermentation process

♣ In a critical study, analyses are made to determine how the observed conversion of

the carbon source to the product compares with the theoretical maximum yield

♣ This may be difficult because of limited knowledge of the biosynthetic pathways

♣ Cooney (1979) has calculated theoretical yields for penicillin G biosynthesis on

the basis of material and energy balances using a biosynthetic pathway based on

reaction stoichiometry

♣ Using a simple model for a batch culture penicillin fermentation it was estimated

that 28, 61, 11% of the glucose consumed was used for cell mass, maintenance and

penicillin respectively

♣ When experimental results of a fed batch penicillin fermentation were analysed

26% of the glucose has been used for growth and 70% for maintenance and 6% for

penicillin

♣ The maximum experimental conversion yield for penicillin was calculated to be

0.0053 g/g glucose (88.5 units /mg)

♣ Thus the theoretical conversion value is many times higher than the experimental

value

♣ Herbach etal (1984) concluded that there were six possible biosynthetic pathways

for penicillin production and two possible mechanisms for ATP production NADH

and FADH2

♣ They calculated that conversion yields by different pathways varied from 638 to

1544 units of penicillin per mg glucose

♣ At that time the best quoted yields were 200 units penicillin per mg glucose

♣ This gives a production of 13 to 29 % of the maximum theoretical yield

♣ The other major nutrient which will be required is oxygen which is provided by

aerating the culture

♣ The design of a medium will influence the oxygen demand of a culture in that the

more reduced carbon sources will result in a higher oxygen demand

18. Explain different forms of IPR:

♣ Intellectual property rights is protected by different ways: patents, copyrights, trade secrets

and trade marks, designs, geographical indications

PATENTS:

♣ Patent is a special right to the inventor that has been granted by the government through

legislation for trading new articles, a patent is a personal property which can be licensed or sold

by the person / organization just like any other property.

♣ For example, Alexander Graham Bell obtained patent for his telephone. This gave him the

power to prevent engine from making or using or selling a telephone elsewhere.

♣ In some European countries the monopoly rights were granted only to the inventors so that

they may develop new articles beneficial for the society.

♣ In the USA the maximum limit of this monopoly is for 17 years

♣ In India, the Indian Patent Act (1970) allows the process patents but not the product patent

and the maximum duration of patent is for 5 years from the date of grant and 7 years from the

date of filing the patent application.

♣ The patents in terms give the inventor the rights to exclude the others from making, using or

selling his invention.

♣ A patent consists of three parts: the grant, the specification and claims.

♣ The grant is filled at the patent office which is not published. It is a signed document which

is actually the agreement that grants patent right to the inventor. However, the specification and

claims are published as a single document which is made public at a minimum charge from the

patent office.

♣ The specification part is narrative in which the subject matter of invention is described how

the invention was carried out.

♣ The claim section specifically defines the scope of the invention to be protected by the patent

to which the others may not practice.

COPY RIGHTS:

♣ The copy protection is only a form of expression of ideas. One of the best examples is books.

The authors, editors, publishers or both publisher and author / editor have copy rights. The

materials of the book cannot be reprinted or reproduced without written permission from copy

right holders

♣ Patent and trade secrets provide protection of only basic know-how, whereas copy rights

protect the expressed materials viz., materials in printed, video-recorded or taped forms.

Biotechnological materials subject to copyright include database of DNA sequence or any

published forms, photomicrographs, etc.

♣ In India, copyright Act 1957 was amended in 1994 brought enforce in 1999. It includes

computer programme, tables and databases; the computer programme is defined as set of works

expressed in words, codes, schemes, or any other form including a machine readable medium

capable of causing computer to perform a particular task or achieve a particular result. In India,

the ministry of human resource and development look after the copyright act.

TRADE SECRETS (KNOW HOW):

♣ The private propriety information that benefits the owners is called trade secret.

♣ It may be of any type, from process to product yield.

♣ The most popular example is Coca Cola that has covered its best kept secrets of its formula

under this law

♣ India does not have any trade secrets. Therefore, it allows any company to register and

protect the details of formulae

♣ Usually a patent runs out for 10 – 20 years but under the law of trade secrets a company will

have no obligation to reveal the trade secrets

♣ In India the limit of trade secrets is at least five years and two years in the USA

♣ If the trade secrets become public before the granted period the intellectual is paid

compensation and unauthorized users are punished by the court

♣ The trade secrets in the area of biotechnology may comprise of hybridization conditions, cell

lines, processing, designing, consumers list

TRADE MARKS:

♣ A trade mark is an identification symbol which is used in the course of trade to enable the

public to distinguish on traders goods from the simi9lar goods of the other traders.

♣ The public makes use of these trade works in order to choose whose goods they will have to

buy

♣ If they are satisfied with the purchase they can simply repeat their order by using the trade

mark, for example KODAX for photography goods, IBM for computers, zodiac for readymade

cloths

♣ Trade marks rights are very important. Trade mark laws vary in every country. Through

agreement it is ensured that the trademark of one country must be protected in another country.

♣ India trade and merchandise rights are very important. Therefore multinational companies

spend large amount of money to maintain their trademarks throughout the world

ADVANTAGES OF PATENTS AND OTHER FORMS OF IPR:

a) Encouraging and safeguarding intellectual and artistic creations

b) Disseminating new ideas and technologies quickly and widely

c) Promoting the investment

d) Providing consumers with the result of creation and invention

e) Providing increased opportunities for the distribution of the above effects across

the countries in a manner proportionate to national levels of economic and

industrial development

19. Give a brief account of convention on biodiversity:

♣ Biodiversity is a new name for species – richness (plants, animals and microorganisms)

occurring as an interacting system in a given habitat

♣ Biodiversity cannot be replaced because the species becomes adapted in a given habitat after

a long course of time

♣ That is why, due to plausticity in their nature and unsustainable resource utilization over 2.5

lakhs species are lost and thousands are threatened to extinction

♣ If a species extinct it means whole of the gene pool extinct.

♣ The real value of biodiversity lies in the information‘s that are enclosed in the genes.

Therefore there is urgent need for future to protect the genes from destruction

♣ Biodiversity may be defined as the inherent and externally imposed variability within and

among the living organisms present in the terrestrial, marine and other ecosystems at the specific

time

♣ Diversity includes the variability in genes, genotype, species, genera, family and ecosystem

at a particular time in a specific region.

♣ Biodiversity is an expression of both numbers and differences and differences can be seen as

a measure of complexity

LEVELS OF BIODIVERSITY:

♣ Genetic diversity: Genes are the functional entities of all organisms because they determine

the physical and biological features of organism. Genes of one organism differ from that of the

other organisms. Besides variations arise due to mutation brought about in genome which is the

cause of genetic diversity

♣Species diversity: A species is a group of organisms which are genetically identical and

interbreed to produce progeny. In contrast horses and zebra are two different species but

genetically similar. They can interbreed but produce all infertile offspring. Usually species

differ in appearance and thus one can differentiate one species from the other. Thus species

diversity is estimated on the basis of total number of species within the discrete geographical

boundaries

♣ Ecosystem diversity: There is diverse ecosystems and organisms living in such ecosystems

are adapted to its respective ecosystems. Therefore, there arises diversity among them. Under

ecosystem diversity two phenomenons are frequently referred: a) the variety of species within

different ecosystems i.e. more diverse ecosystems contain more species and (b) the variety of

ecosystems found within a certain bio- geographical boundaries

BIODIVERSITY IN INDIA:

♣ India is rich in biodiversity to agriculture, animal husbandry, fisheries and forestry. Much

has been described in Ayurved and other ancient literature about the indigenous system of

medicine, knowledge and wisdom of people. These are supported by a very strong scientific and

technological base.

♣ In India over 1,15,000 species of plants and animals have already been identified and

described. The country is an important Centre of Diversity and origin of over 167 important

cultivated plant spices and domestic animals.

♣ A few crops which arose in India and spread throughout the world are: rice, sugarcane,

vignas, jute, mango, citrus, banana, millets, spices, medicinal aromatics and ornamentals.

♣ No country in the world is as rich in biodiversity as ours. Himalayas itself has the natural

wealth of plants many of which are still unknown and many endangered

CONSERVATION OF BIODIVERSITY:

♣ There is an urgent need for biodiversity rich countries to save it against destruction.

♣ However in most developing countries, biodiversity is attached to environment and forest

agencies which have no idea about it. If such countries are not aware of conserving it for

sustainable utilization they would be compelled to export biodiversity and import products for

well being of their people

♣ The agreement between Institute of Biodiversity (INBio) in Costa Rica and Merck (USA) is

hailed through out the industrial world. Under this agreement, extracts from the wild plants,

insects and microorganisms from Costa Rica are supplied Merck. In return, INBio receives from

Merck over 1.35 million US dollars, and expects royalty on the commercial products. INBio has

to contribute 50% of royalty to the government of Costa Rica for National park service. The

Government has given INBio rights to bioprospects and share conservation work. Thus the

INBio represents an alliance between biologists / biochemists and businessmen

♣ In India, a large number of institutions are involved in conservation and utilization of

biodiversity which come under Ministry of Environment and forest, Agriculture and Science and

Technology.

♣ They deal conservation of biosphere reserve, national parks, wildlife, sanctuaries field gene

banks, etc

♣ The country needs more expertise and methodologies besides tiger – bird – wild life

syndrome

♣ India is predominantly an agricultural country. Therefore, the policy makers have to realize

that conservation and sustainable utilization of biodiversity must be placed on the top of all

developmental planning.

20. Discuss the patent case history of Turmeric:

♣ Turmeric root is actually a tropical rhizome related to ginger and galangal. It has a

thin papery skin that can be scraped off to reveal the bright orange, somewhat dry

flesh inside. Turmeric is an important mainstay of cooking in Asia and gives the

yellow colour to all curry blends.

♣ A few years ago the US government granted an American company a patent on

Turmeric when it was discovered that the spice had antitumour properties against

numerous different cancers.

♣ The Indian government had to sue the US Patent office to have the patent reversed

as Ayurvedic medicine has recognized the extraordinary curative powers of turmeric

for more than 5000 years!

♣ Turmeric is now one of the most researched plants for anticancer properties.

♣ Turmeric oil is primarily CO2 or solvent extracted to retain the maximum amount

of the bright yellow curcumin, which is thought by science, to be the most active

compound in the plant.

♣ This hydrosol was distilled from a biodynamically grown crop, yielding just 1

pound of hydrosol from 3 pounds of rhizome. Its rapid action on ‗suspect‘ skin spots

and everything else, makes me quite certain that the anticancer properties of this plant

reside in many of its chemical constituents not just the one or two currently of interest

to patent hungry drug companies.

♣ Smell and Taste: the hydrosol has a fresh, quite bright top note followed by an

earthy, green, slightly masculine, bass note. The taste is unique, spicy without being

sharp or warm, mildly sweet but with the same earthy bottom note as the odour. I

always taste the soil when I visit distilleries and plant growers, turmeric brings the

soil to you

♣ Therapeutics: Turmeric has shown efficacy against cancers of the skin, lungs,

colon and breast as well as leukemia, neurodegenerative, cardiovascular, pulmonary,

metabolic, autoimmune and neoplastic diseases. (No wonder a corporation wanted a

patent!)

♣ On a less dramatic level turmeric is one of the best skin healers being powerfully

antioxidant.

♣ Use it for acne, eczema, psoriasis, hives, sunburn, rosacea or any inflammative

condition.

♣ Internally it purifies the liver making it a wonderful digestive aid and detoxifier.

Take ½ tbsp to relieve bloat, gas and other side effects caused by excess.

♣ It is neutral in effect, neither heating nor cooling so it can be used by all

constitutional types.

♣ Medical research almost daily finds new modes and areas of action for this lowly

little root and an ounce of turmeric is worth a pound of cure in my book.

♣ Energetics: Turmeric sends shivers up my spine with the level of information it

contains and confers in a very direct fashion.

♣ Turmeric has a habit of drawing our attention to certain areas of the body, or to

certain emotions or thought processes, those that require our undivided attention, now.

♣ If turmeric prompts you to look at something, don‘t ignore the nudge, it may be an

early warning sign or even an alert.

♣ Turmeric holds the power of the sun as it translates to electromagnetic energy. Its

bright orange colour provides ‗warmth‘ without heat, it‘s pungency keeps us aware

and present, it‘s thin skin and shallow growth make it vulnerable but its dense foliage

provides protection. This is the voice of the earth and it is talking about healing.

♣ The phenomenon of biopiracy does not seem to let up despite public outrage and

the striking down of illegitimate patents as happened in the case of turmeric and to

some extent, basmati rice.

♣ This is not just grossly unethical; it is in violation of international agreements like

the Convention on Biological Diversity (CBD) and the International Treaty on Plant

Genetic Resources (ITPGR) which recognize the rights of the farming community

over the genetic wealth used in agriculture.

♣ Both CBD and ITPGR also require that this genetic wealth can only be used after

seeking permission and by sharing part of the profits with rural communities in a

‗benefit sharing‘ agreement.

♣ Indian laws have in fact provided for such benefit sharing and have designated a

National Gene Fund into which revenues from such use should flow. Despite action at

the national level, the phenomenon of biopiracy shows no signs of abating.

♣ Genetic resources, the valuable raw materials of the booming herbal industry and

the threshold agri-biotechnology industry have become coveted and sought after.

♣ The dilemma is that these resources are the property of the worlds‘ farming and

tribal communities, located largely in the south and the industries are largely

multinational or located in the industrial north.

♣ Biopiracy is the expedient with which the industry has decided to help itself to

valuable resources without paying anything for them.

♣ This clearly unethical situation continues even as national governments chase

patent grants in the US and more recently, Europe, and spend thousands of dollars to

present a challenge.

♣ In the case of turmeric, the patent was struck down and in the case of Basmati rice;

there was a sort of compromise. Chasing patents in foreign patent offices is a clearly

untenable situation.

♣ Developing country governments need to join hands to force an international

agreement against patents on biological materials that clearly belong to communities

in the south.

♣ A start has been made by a joint submission in the TRIPS Council by India and

other developing countries that under the WTO patent regime, it should be mandatory

to declare the source of the biological material, proof of prior informed consent and

agreements for benefit sharing, before the grant of a patent.

♣ Not surprisingly, the US, backed by the MNCs is opposing this move but this is

where future negotiations and actions must focus.

21. Discuss the IPR in India:

Copyright & related rights:

♣ The rights of authors of literary and artistic works (such as books and other

writings, musical compositions, paintings, sculpture, computer programs and films)

are protected by copyright, for a period of 60 years after the death of the author.

♣ Also protected through copyright and related (sometimes referred to as

―neighboring‖) rights are the rights of performers (e.g. actors, singers and musicians),

producers of phonograms (sound recordings) and broadcasting organizations.

♣ The main social purpose of protection of copyright and related rights is to

encourage and reward creative work.

International Agreements

Trade Related Aspects of Intellectual Property Rights: (TRIPs)

♣ India is a signatory of TRIPs in the Uruguay Round agreement of 1995.

♣ It is now bound to amend her existing laws in order to make it to make it TRIPs-

compliant.

♣ The government has initiated action to bring in the requisite changes.

♣ In the last few years, India has enacted fully TRIPs-compliant Trademarks Act,

Copyright Act, Designs Registration Act, Geographical Indications Act and

Protection of Layouts for Integrated Circuits Act. A novel Plant Varieties Protection

and Farmers Rights Act 2001 and the Bio-diversity Act 2002 are also in Place.

Patent Co-operation Treaty (PCT)

♣ On 7th September 1998, India deposited its instrument of accession to the PCT

and is bound by Patent Co-operation treaty as 98th contracting State of PCT from 7th

December 1998.

♣ Furthermore, nationals and residents of India are entitled to file International

applications for patents under PCT at Patent office, Kolkatta as receiving office.

♣ With effect from 19th November 1999 Patent office branches at Mumbai, Chennai

& New Delhi are also receiving the PCT applications allowing the applicants to file

application at their regional Patent office.

♣ As on date of India‘s accession to PCT, there were nine International Search

Authorities (ISA‘s) & Eight International Patent Examination Authorities (IPEA‘s).

Govt. of India opted for all of them as competent ISA‘s & IPE‘s, providing maximum

options to its applicants. As on today India is the only State having maximum options.

Modernization of Patent Offices:

Government has taken up comprehensive modernization of intellectual Property

administration to complement legislative matters.

Modernization Initiatives:

♣ A completely modernized and computerized Designs Office in Kolkata has been

made operational.

♣ Modernization of infrastructure in all the Patent offices

♣ Integrated IP offices have been set up in each of the four metro cities so as to house

all activities under a single roof

♣ A logo for IP administration has been designed and put into use

♣ Re-designing of website (http://www.ipindia.nic.in) to make it more user-friendly

♣ Computerization of Patent database

♣ Human Resource Development for activities to meet International Standards

♣ An Intellectual Property Training Institute (IPTI) has been established at Nagpur to

provide training

♣ Online search facilities & connectivity to international databases CII – APTDC

Ready Rec5koner for IPR in India

♣ Work manuals prepared for patent offices to ensure uniformity in the operation.

♣ Introduction of request system for examination of all applications address to the

problem of backlog of pending patent applications

♣ Digital database of over 1, 00,000 patent records and 48,000 design records

prepared so far. A searchable database will be put on the website.

Patents:

♣ Patent Act of 1970

♣ The Patent Act, 1970 came into force on 20.4.1972 replacing Indian Patents and

Designs Act, 1911. The Patents Act, 1970 in comparison with Indian Patents and

Designs Act, 1911 has far more reaching effect in some areas such as food, Drug and

Medicines where all the patents granted under this category are deemed to be

endorsed with the words “Licence of Right”.

♣ Further the term of Patents related to Food, Drug & Medicines is seven years

from the date of Patent or 5 years from its grant whichever is less.

♣ More elaborate definition of invention has been provided. Product Patents for

Drugs & Medicines including certain class of chemicals, which are produced by

chemical reactions, have been abolished.

♣ However, inventions relating to methods or processes of their manufacture are

patentable. The area of search for novelty has been extended to any documents

published anywhere in the world.

Safeguards Provided In the Legislation

♣ Exclusions from patentability

♣ Protection of traditional knowledge

♣ Redrafting of Compulsory Licenses provisions

♣ Provisions to check prices and ensure prompt availability of patented products after

the expiry of the term of patent

♣ Protection of Biodiversity

Some of the changes made in the Patents Amendment Act 2002 are:

♣ Inventions relating to method of treatment of plants are to be made patentable.

♣ The term of Patent is made 20 years

♣ Conditions for granting Compulsory Licenses

♣ Patenting micro-organisms

♣ An Appellate hear appeals from decisions of the controller.

The Amendment has major implications on the following:

♣ Introduction of product patent protection for food, pharmaceutical and chemical

inventions.

♣ Examination The ―mail box‖ applications, from January 01, 2005 Ensuring that

this next and the final amendment makes the domestic law completely TRIPs-

compliant and also safeguards national interests, pose a considerable dilemma for the

government.

♣ While there is a need to incentives domestic industry to continue to develop and

prepare themselves to face competition, protection of biodiversity and traditional

knowledge, with fair benefit-sharing arrangements for communities, also needs to be

ensured.

♣ The issue is one of finding a delicate balance a patent regime will offer to

safeguard inventor‘s interest while also check for overuse or misuse of IPR. One view

is that application of a strong competition law will ensure that the latter eventuality

does not happen, also addressing the affordability issue as under Section 83.

Trademarks

Legislation

♣ India affords full protection to trade marks under the Trade Marks and

Merchandise Act.

♣ The Indian law of trademarks is protected by the Trade & Merchandise Marks

Act, 1958.

♣ A new statute i.e. the Trade Mark Act, 1999 has been enacted in India to bring it

in conformity with the TRIPs Agreement, to which India is a signatory.

♣ Indian Trademarks Act, 1999, came into force on September 15, 2003. India has

made a step towards fulfilling its international obligations.

♣ Consequently, the Indian trademark law has now become fully compatible with

the International standards laid down in the TRIPs Agreement.

♣ The New Act primarily consolidates and amends the old Trade & Merchandise

Marks Act, 1958 and provides for better protection of goods and services.

♣ On the whole the Trademarks Act, 1999 has removed the inconvenient provisions

of the old Act and has fostered the rights of the traders and other service providers

significantly.

♣ It also sends a warning to infringers. This Act not only makes Trade Marks Law

TRIPs compatible but also harmonizes it with International systems and practices.

Convention Application and International Treaties

♣ India has declared certain countries as convention countries, which afford to

citizens of India similar privileges as granted to its own citizens.

♣ A person from a convention country, may within six months of making an

application in his home country, apply for registration of the trademark in India.

♣ If such a trademark is accepted for registration, such foreign national will be

deemed to have registered his trademark in India, from the same date on which he or

she made application in his home country

Industrial Design:

♣ In 1911 the Designs Act was passed by the then British Government in India. The

Designs Act of 1911 governs industrial Designs.

♣ The registration of a design confers on the registered proprietor the right to take

action against third parties who apply the registered design without license or consent.

♣ Under the TRIPS Agreement, minimum standards of protection of industrial

designs have been provided for

♣ As a developing country, India has already amended its national legislation to

provide for these minimal standards.

♣ The existing legislation on industrial designs in India is contained in the New

Designs Act, 2000 India had achieved a mature status in the field of industrial designs

and in view of globalization of the economy.

♣ The present legislation is aligned in view of the changed technical and

commercial scenario and made to conform to international trends in design

administration.

♣ This replacement Act is also aimed to in act a more detailed classification of

design to conform to the international system and to take care of the proliferation of

design related activities in various fields.

Geographical Indications:

♣ Geographical Indications of Goods are defined as that aspect of industrial

property which refers to the geographical indication referring to a country or to a

place situated therein as being the country or place of origin of that product.

♣ Typically, such a name conveys an assurance of quality and distinctiveness, which

is essentially attributable to the fact of its origin in that, defined geographical locality,

region or country.

♣ Under Articles 1 (2) and 10 of the Paris Convention for the Protection of

Industrial Property, geographical indications are covered as an element of IPRs.

♣ They are also covered under Articles 22 to 24 of the Trade Related Aspects of

Intellectual Property Rights (TRIPS) Agreement, which was part of the Agreements

concluding the Uruguay Round of GATT negotiations.

♣ India, as a member of the World Trade Organization, enacted the Geographical

Indications of Goods (Registration & Protection) Act, 1999 has come into force with

effect from 15th September 2003. The source of Geographical origin of the biological

material used in invention is required to be disclosed in the specification.

Copyrights:

♣ India has a very strong and comprehensive copyright law based on Indian

Copyright Act. 1957 which was amended in 1981, 1984, 1992, 1994 and 1999 (w.e.f.

January 15, 2000). The amendment in 1994 were a response to technological changes

in the means of Communications like broadcasting and telecasting and the emergence

of new technology like computer software.

♣ The 1999 amendments have made the Copyright Act fully compatible with Trade-

Related Aspects of Intellectual Property Rights (TRIPS) Agreement. & fully reflects

Berne Convention.

♣ The amended law has made provisions for the first time, to protect performers‘

rights as envisaged in the Rome Convention.

♣ With these amendments the Indian Copyright law has become one of the most

modern copyright laws in the world.

♣ Moreover, India is signatory to both the International copy- right conventions i.e.

the Berne Convention of 1886 and Universal Copyright Convention of 1952. India is

also an active member of WIPO and UNESCO

♣ Indian representatives have taken part in the international copyright conferences

putting forward the Indian point of view and thus helping in getting proper

amendments made in the interests of developing countries.

♣ Much credit goes to India for getting the 1971 Paris Act, which recognizes the

needs of developing countries and given a special treatment for that.

♣ During the last two decades the Government of India has taken a number of

measures towards the implementation and enforcement of copyright law.

♣ Under the Ministry of Human Resources Development a special Copyright

Enforcement Advisory Council has been set up in which the heads of police from all

the States of India participate and join forces to see to it that copyright is properly

enforced. These developments have made a check on copyright infringement, by and

large quite effective.

♣ The Federation of Indian Publishers has Copyright Council, which organizes

training courses in copyright for the benefit of publishers and authors all over the

country from time to time with assistance from the Ministry of Education and

sometimes World Intellectual Property Organization (WIPO).Recently, the Federation

of Indian Publishers (FIP) in collaboration with the ‗Authors Guild of India‘ have

established ‗Indian Reprographic Rights Organization‘ for the protection of the

interests of copyright holders in India and abroad and also to act as a Collecting

Society.

♣ Being a member of the International Publishers Association (IPA) Indian

publishers take an active part in the International Publishers Copyright Council

(IPCC) from time to time. In the Indian law, copyright falls into ‗public domain‘ 60

years after the death of the author.

Facing Digital and Electronic Publishing

♣ During the last couple of years the IPA, specially helped by the Association of

American Publishers (AAP), have developed a system called Digital Object Identifier

(DOI).

♣ The technocrats in India are also developing methodology by which firstly,

copyright infringement can be checked and secondly, it can detect the source form

where the infringement has originated.

♣ Such developments have deterrent effect on the pirates. The police force is also

being trained to trace the pirates in all these technical and intricate electronic devices.

♣ But over all the electronic publishing, digital and Internet being in the initial

stages in book publishing in India, not many problems have surfaced here in a big

way as in advanced countries.

♣ But times are coming when Indian publishers will have to face these challenges

and they are gearing up for that.

♣ Special cells for copyright enforcement have been set up in 23 States and Union

Territories. In addition, for collective administration of copyright, copyright societies

have been set up for different classes of works.

Biological Diversity:

♣ The Biological Diversity act was passed in 2002 and had received the assent of

the President on the 5th February 2003.

♣ The Act aims to provide for conservation of biological diversity, sustainable use

of its components and fair and equitable sharing of the benefits arising out of the use

of biological resources, knowledge and for matters connected therewith or incidental

thereto.

♣ Indian citizens, companies are allowed free access to biological resources within

the country for research purposes but are barred from transferring findings to foreign

entities without the National Biodiversity Act (NBA) approval.

Traditional knowledge:

♣ Traditional knowledge is in the public domain and should, therefore, not qualify

for patents. This is indeed what happened with turmeric and might well have occurred

with pigeon pea extracts and ngali nut oil.

♣ But for resolution of the issue, there has to be a database and computerisation of

traditional knowledge.

♣ While creating a database of knowledge handed down orally is difficult, this

constraint is responsible for many of India's patent-related problems.

♣ While existing traditional knowledge is not patentable, increments to knowledge

can indeed be patented, as they are in the US.

♣ The database of the National Innovation Foundation (NIF) demonstrates that India

is not lacking in such incremental addition to traditional knowledge.

♣ It is paradoxical that despite two amendments to the Indian Patent Act 1970-

amendment 1999 and 2002, such inventive components of traditional knowledge

cannot be patented in India.

♣ Small innovators have to file for patents in the USPTO, with higher application

fees and transaction costs.

♣ Traditional knowledge cannot be patented if documented under the TRIPS

agreement of the WTO.

♣ That is why neem and haldi products when patented by the USPTO were revoked

once India proved that these were the products of Indian traditional knowledge.

♣ One of the major issues needing resolution is the protection of traditional

knowledge under a new "sui generis" system.

♣ The CSIR has already undertaken the documentation of traditional knowledge so

that it is available for prior art search.

22. Write a brief account on five principles of trading system

established by WTO:

♣ The WTO establishes a framework for trade policies; it does not define or specify

outcomes.

♣ That is,it is concerned with setting the rules of the trade policy game, not with the

results of the game.

♣ Five principles are of particular importance in understanding both the pre-1994

GATT and the WTO: nondiscrimination, reciprocity, enforceable commitments,

transparency, and safety valves.

Nondiscrimination

♣ Nondiscrimination has two major components: the most-favored-nation (MFN)

rule, and the national treatment principle.

♣ Both are embedded in the main WTO rules on goods, services, and intellectual

property, but their precise scope and nature differ across these three areas.

♣ This is especially true of the national treatment principle, which is a specific, not a

general commitment when it comes to services.

♣ The MFN rule requires that a product made in one member country be treated no

less favorably than a ―like‖ (very similar) good that originates in any other country.

♣ Thus, if the best treatment granted a trading partner supplying a specific product is

a 5 percent tariff, this rate must be applied immediately and unconditionally to

imports of this good originating in all WTO members.

♣ In view of the small number of contracting parties to the GATT (only 23

countries), the benchmark for MFN is the best treatment offered to any country,

including countries that are not members of the GATT.

♣ National treatment requires that foreign goods, once they have satisfied whatever

border measures are applied, be treated no less favorably, in terms of internal

(indirect) taxation than like or directly competitive domestically produced goods (Art.

III, GATT).

♣ That is, goods of foreign origin circulating in the country must be subject to taxes,

charges, and regulations that are ―no less favorable‖ than those that apply to similar

goods of domestic origin.

♣ The MFN rule applies unconditionally. Although exceptions are made for the

formation of free trade areas or customs unions and for preferential treatment of

developing countries, MFN is a basic pillar of the WTO.

♣ One reason for this is economic: if policy does not discriminate between foreign

suppliers, importers and consumers will have an incentive to use the lowest-cost

foreign supplier.

♣ MFN also provides smaller countries with a guarantee that larger countries will

not exploit their market power by raising tariffs against them in periods when times

are bad and domestic industries are clamoring for protection or, alternatively, give

specific countries preferential treatment for foreign policy reasons.

♣ MFN helps enforce multilateral rules by raising the costs to a country of defecting

from the trade regime to which it committed itself in an earlier multilateral trade

negotiation. If the country desires to raise trade barriers, it must apply the changed

regime to all WTO members.

♣ This increases the political cost of backsliding on trade policy because importers

will object. Finally, MFN reduces negotiating costs: once a negotiation has been

concluded with a country, the results extend to all.

♣ Other countries do not need to negotiate to obtain similar treatment; instead,

negotiations can be limited to principal suppliers.

♣ National treatment ensures that liberalization commitments are not offset through

the imposition of domestic taxes and similar measures.

♣ The requirement that foreign products be treated no less favorably than competing

domestically produced products gives foreign suppliers greater certainty regarding the

regulatory environment in which they must operate.

♣ The national treatment principle has often been invoked in dispute settlement

cases brought to the GATT. It is a very wide-ranging rule: the obligation applies

whether or not a specific tariff commitment was made, and it covers taxes and other

policies, which must be applied in a nondiscriminatory fashion to like domestic and

foreign products. It is also irrelevant whether a policy hurts an exporter. What matters

is the existence of discrimination, not its effects.

Reciprocity

♣ Reciprocity is a fundamental element of the negotiating process. It reflects both a

desire to limit the scope for free-riding that may arise because of the MFN rule and a

desire to obtain ―payment‖ for trade liberalization in the form of better access to

foreign markets.

♣ The costs of liberalization generally are concentrated in specific industries, which

often will be well organized and opposed to reductions in protection.

♣ Benefits, although in the aggregate usually greater than costs, accrue to a much

larger set of agents, who thus do not have a great individual incentive to organize

themselves politically.

♣ In such a setting, being able to point to reciprocal, sector-specific export gains

may help to sell the liberalization politically.

♣ Obtaining a reduction in foreign import barriers as a quid pro quo for a reduction

in domestic trade restrictions gives specific export-oriented domestic interests that

will gain from liberalization an incentive to support it in domestic political markets.

♣ A related point is that for a nation to negotiate, it is necessary that the gain from

doing so be greater than the gain available from unilateral liberalization. Reciprocal

concessions ensure that such gains will materialize.

Binding and Enforceable Commitments

♣ Liberalization commitments and agreements to abide by certain rules of the game

have little value if they cannot be enforced. The nondiscrimination principle,

embodied in Articles I (on MFN) and III (on national treatment) of the GATT, is

important in ensuring that market access commitments are implemented and

maintained. Other GATT articles play a supporting role, including Article II (on

schedules of concessions).

♣ The tariff commitments made by WTO members in a multilateral trade

negotiation and on accession are enumerated in schedules (lists) of concessions. These

schedules establish ―ceiling bindings‖: the member concerned cannot raise tariffs

above bound levels without negotiating compensation with the principal suppliers of

the products concerned.

♣ The MFN rule then ensures that such compensation—usually, reductions in other

tariffs—extends to all WTO members, raising the cost of reneging.

♣ Once tariff commitments are bound, it is important that there be no resort to other,

nontariff, measures that have the effect of nullifying or impairing the value of the

tariff concession.

♣ A number of GATT articles attempt to ensure that this does not occur. They

include Article VII (customs valuation), Article XI, which prohibits quantitative

restrictions on imports and exports, and the Agreement on Subsidies and

Countervailing Measures, which outlaws export subsidies for manufactures and

allows for the countervailing of production subsidies on imports that materially injure

domestic competitors

♣ If a country perceives that actions taken by another government have the effect of

nullifying or impairing negotiated market access commitments or the disciplines of

the WTO, it may bring this situation to the attention of the government involved and

ask that the policy be changed.

♣ If satisfaction is not obtained, the complaining country may invoke WTO dispute

settlement procedures, which involve the establishment of panels of impartial experts

charged with determining whether a contested measure violates the WTO.

♣ Because the WTO is an intergovernmental agreement, private parties do not have

legal standing before the WTO‘s dispute settlement body; only governments have the

right to bring cases. The existence of dispute settlement procedures precludes the use

of unilateral retaliation.

♣ For small countries, in particular, recourse to a multilateral body is vital, as

unilateral actions would be ineffective and thus would not be credible.

♣ More generally, small countries have a great stake in a rule-based international

system, which reduces the likelihood of being confronted with bilateral pressure from

large trading powers to change policies that are not to their liking.

Transparency

♣ Enforcement of commitments requires access to information on the trade regimes

that are maintained by members. The agreements administered by the WTO therefore

incorporate mechanisms designed to facilitate communication between WTO

members on issues.

♣ Numerous specialized committees, working parties, working groups, and councils

meet regularly in Geneva. These interactions allow for the exchange of information

and views and permit potential conflicts to be defused efficiently.

♣ Transparency is a basic pillar of the WTO, and it is a legal obligation, embedded

in Article X of the GATT and Article III of the GATS.WTO members are required to

publish their trade regulations, to establish and maintain institutions allowing for the

review of administrative decisions affecting trade, to respond to requests for

information by other members, and to notify changes in trade policies to the WTO.

♣ These internal transparency requirements are supplemented by multilateral

surveillance of trade policies by WTO members, facilitated by periodic country-

specific reports (trade policy reviews) that are prepared by the secretariat and

discussed by the WTO General Council.

♣ The external surveillance also fosters transparency, both for citizens of the

countries concerned and for trading partners. It reduces the scope for countries to

circumvent their obligations, thereby reducing uncertainty regarding the prevailing

policy stance.

♣ Transparency has a number of important benefits. It reduces the pressure on the

dispute settlement system, as measures can be discussed in the appropriate WTO

body.

♣ Frequently, such discussions can address perceptions by a member that a specific

policy violates the WTO; many potential disputes are defused in informal meetings in

Geneva.

♣ Transparency is also vital for ensuring ―ownership‖ of the WTO as an

institution—if citizens do not know what the organization does, its legitimacy will be

eroded.

♣ The trade policy reviews are a unique source of information that can be used by

civil society to assess the implications of the overall trade policies that are pursued by

their governments.

♣ From an economic perspective, transparency can also help reduce uncertainty

related to trade policy.

♣ Such uncertainty is associated with lower investment and growth rates and with a

shift in resources toward nontradables (Francois 1997).

♣ Mechanisms to improve transparency can help lower perceptions of risk by

reducing uncertainty. WTO membership itself, with the associated commitments on

trade policies that are subject to binding dispute settlement, can also have this effect.

Safety Valves

♣ A final principle embodied in the WTO is that, in specific circumstances,

governments should be able to restrict trade.

♣ There are three types of provisions in this connection: (a) articles allowing for the

use of trade measures to attain noneconomic objectives; (b) articles aimed at ensuring

―fair competition‖; and (c) provisions permitting intervention in trade for economic

reasons.

♣ Category (a) includes provisions allowing for policies to protect public health or

national security and to protect industries that are seriously injured by competition

from imports.

♣ The underlying idea in the latter case is that governments should have the right to

step in when competition becomes so vigorous as to injure domestic competitors.

♣ Although it is not explicitly mentioned in the relevant WTO agreement, the

underlying rationale for intervention is that such competition causes political and

social problems associated with the need for the industry to adjust to changed

circumstances.

♣ Measures in category (b) include the right to impose countervailing duties on

imports that have been subsidized and antidumping duties on imports that have been

dumped (sold at a price below that charged in the home market).

♣ Finally, under category (c) there are provisions allowing actions to be taken in

case of serious balance of payments difficulties or if a government desires to support

an infant industry.

23. Describe the various methods for measurement of microbial

growth:

The various methods used in the measurement of microbial growth and also the

synchronous growth and continuous culture techniques. Microbial growth which

results in increase in cell number and also the cell mass. The word cell number

implies to the viable cell which has ability to metabolize and reproduce while the cell

mass indicates the total mass of viable and non-viable cells.

Measurement / Enumeration of Micro-organisms is required for

a) assessing the rate of microbial reproduction,

b) determining the potability of water,

c) determining the suitability of food for human

d) consumption,

e) evaluating the necessity for antimicrobial therapy, and

f) for industrial, research and medical reasons.

The Enumeration Methods are as follow

1. Total cell count

2. Viable cell count

3. Biomass determination

♣ Total cell count method counts both viable and non- viable cells. It is used to

determine the number of cells from milk, soil sample and vaccines. Direct

microscopic count or Breed count is a simple method. It is performed by making a

smear of the sample to be studied and staining the cells with a suitable dye. The smear

is prepared from known amount of sample. If required the original sample is diluted

appropriately. The cells are counted from different microscopic fields. The cells are

enumerated by taking the average number of cells and multiplying with the dilution

factor. It is the quick technique used to determine the total number of cells per ml. of

given sample.

♣ Petroff—Hausser counting in which a special slide and coverglass are used to

contain a sample of known volume. The slide has grid etched on its surface to make

counting easier and more effective and accurate. The total number of cells is

multiplied by the dilution factor of the sample to determine number of cells per ml. of

original sample. In short it is like use of haemocytometer slide in counting the blood

cells.

♣ Advantages :

a) they are more rapid,

b) equipments and requirements needed are minimum,

c) morphology of microbes can also be studied.

♣ Disadvantages : These methods count both the viable as well as non- viable cells,

the accuracy also declines with very dense sample and diluted sample leading to

statistical errors.

♣ Electronic cell counters / coulter counters:

An electronic device called coulter counter is used for direct enumeration of

cells in suspension. A sample of culture is injected into a reservoir and conducted

through a narrow capillary tube. The tube is only large enough to allow one cell at a

time to pass an electric- eye light beam. Every time an individual cell passes through

the light beam, an electronic circuit is triggered to register a count. This method is

valuable for heat labile samples and it is rapid also. But the main drawbacks are both

the dead and living cells are counted. Even the inanimate particles may be counted

giving errors. Counting the microbial cells is more difficult because the cells are very

small in size and if the tube is narrowed to allow only one cell to pass through it, the

culture does not flow well and eventually clogs.

♣ Viable Cell Count:

The problems which we are facing in total cell count or direct microscopic count can

eliminated by using a viable cell count.

1. Standard Plate Count

This is the most basic, effective and indirect method for cell counting. This is

the most sensitive technique as every single viable cell is counted. Serial ten fold

dilutions are prepared for a known volume of culture / sample. The diluted suspension

is spread ( surface spreading technique ) on the surface of agar plate containing

nutrients or mixed with sterile molten nutrient agar medium and the plates are poured

( Bulk seeding ). The plates are incubated at optimum temperature for sufficient time

to allow bacteria to grow and form colonies. It is assumed that each colony arises

from individual bacterial cell. Hence, counting the number of colonies ( colony

forming units- CFU ) and multiplying with dilution factor, the number of bacteria in

the original culture / sample can be determined. The plates showing colonies in the

range of 30 and 300. Less than 30 colonies are not acceptable for statistical reasons

while more than 300 colonies may be very close to each other and difficult to count.

♣ Drawbacks

d) The major limitation of this method is selectivity. There is no common set of

incubation conditions and medium composition that permits the growth of all

types of organisms present in sample.

e) The pipetting and transferring of samples at various points may give errors.

f) If the bacterial suspension is not homogeneous and contains aggregates of

cells, may give lower CFU than the actual one.

2. Membrane Filter Technique :

♣ SPC is very useful technique in isolating and counting the viable cells from the

given sample but basically that contain only a few cells. The method is time

consuming and needs a lot of preparations. An alternative method is membrane filter

technique. A sample is allowed to pass through a membrane filter, made up of

cellulose acetate, with known pore size. The microbial cells are retained by the filter,

the filter is removed aseptically from the assembly and placed on the surface of soft

nutrient agar. Once the nutrients penetrates the filter and makes contact with the cells,

each separated cell develops into a recognizable colony. The filter pads with colonies

can be preserved. The disadvantage of this method is that if the sample is very

dense / turbid, it may clog the filter.

♣ Dip-slide Culture Technique

It is a semi quantitative method mainly used by doctors to quickly identify and

count the micro-organisms from body fluids such as urine, CSF etc. A dried and

compressed sponge like membrane filter is fixed into the slot of a specially designed

slide called culture stick is used for the detection of causative agents. This technique

helps the doctor to gather information about the nature of infection.

♣ Most Probable Number ( MPN ) Technique

It is one of the indirect methods used to count the viable cells. Conventionally

important in water microbiology to determine the potability of water sample. Three or

five tubes of DSLB and SSLB medium are inoculated with 10, 1 and 0.1 ml. of water

sample to be tested. The tubes are incubated at 37 c for 24 to 48 hours. This method

provides statistical number of bacteria present in the sample.

♣ Biomass Determination :

Most of the viable cell counting techniques are very accurate but time

consuming. In order to perform a more rapid cell population count, indirect methods

have been developed. One common method is to determine the biomass and correlate

that value with the number of cells per ml. previously measured by a standard plate

count. Actually the word biomass refers to the total weight of a particular type of

organism and related to increase in protoplasmic constituents.

♣ Turbidimetric method

Turbidimetric measurements are based on the principle that the number of

microbial cells in a suspension is inversely proportional to the amount of light

transmitted. Turbidity is measured in optical density ( O.D.) units that are inversely

proportional to the percentage of trasmittance ( % T ). Colorimeter or

spectrophotometer is a device that measure the amount of light absorbed or scattered

by a suspension. It consists of a light source, a chamber in which the specimen is

placed, and a photoreceptor to measure the amount of transmitted light. Such methods

are rapid and reproducible but the O.D. values do not directly inform us about the

number of cells in the suspension. The O.D. values are converted to cell numbers or

cell mass by using a calibration curve, which is constructed by plotting the O.D.

against some other parameter that reflects microbial population like total viable count.

♣ Measurement of Dry or wet Weight of Biomass:

Such methods are based on increase in protoplasmic constituents during the

microbial growth. The practical difficulties are no methods are available by which we

can measure the dry or wet weight of single microbial cell, hence, the procedures are

used for microbial population. To determine the dry weight, the microbial suspension

is passed through a membrane filter and the biomass is dried in an oven at 100 c or in

vaccum at 80 c.

♣ Estimation of Chemical Constituents of Cells

A more precise biomass may be determined by estimating one of the chemical

constituents from the dried cell material. While talking about microbial growth, we

have already stated that during growth, organisms synthesize the macromolecules and

accumulate them within the cells. As nitrogen is typically found in amino acids and

nitrogenous bases of all the cells, the nitrogen content is measured by Kjedahal‘s

method for biomass determination. The increase in nitrogen content reflects the

microbial growth. The metabolic changes in the medium can be used to determine

microbial growth and commonly known as Biological Assay or Bioassay. The

increase in acid production or carbon di-oxide during growth are easily detectable.

♣ Continuous Culture Technique

In Batch Cultures, the nutrients are not renewed and so growth remains

exponential for only few generations. Microbial population can be maintained in a

state of exponential phase for a long time if the causes of auto inhibition are

eliminated. This is called continuous culture technique. Such growth is Steady State

or Balanced Growth The growth chamber is connected to a reservoir of sterile

medium. Once growth has been initiated, fresh medium is added continuously from

the reservoir. The volume of liquid in growth chamber is maintained constant by

allowing excess volume to be removed continuously through siphen overflow. If the

fresh medium enters at constant rate, the concentration of bacteria in growth chamber

remains constant. Continuous culture environments are common in nature. e. g. the

human G.I.T. supports a microbial population as residential flora. These organisms

are in a constant state of reproduction, as fresh nutrients are supplied and G.I.T.

contents are flushed out of the body with the faeces.

♣ Chemostat

A simple device, chemostat consists of a vessel in which the culture is grown.

Devices are connected to the growth vessel that control the amount of air and fresh

medium to be added to the culture as well as the overflow of medium containing cells

and waste products. The culture reaches to a constant volume and density when

equilibrium between inflow of fresh medium and outflow of medium reaches. All the

constituents of medium, except one, are in excess in chemostat. The deficient nutrient

will support the growth of only limited number of micro-organisms. Thus the density

of culture in chemostat is controlled by nutrient limitation. Glucose as a carbon

source or nitrogen source can be used in setting up chemostat. By controlling the rate

at which nutrients are added to the chemostat, the rate of bacterial growth is also

controlled. The rate of addition of nutrients is known as dilution rate, if dilution rate

increases, more bacteria are washed out of culture vessel than are produced by cell

division.

♣ Turbidostat

Continuous culture systems can be operated as Turbidostats in which cell

density is controlled by increasing or decreasing the flow of culture medium into the

growth vessel. There is no limiting nutrient like used in chemostat. A photoelectric

device is attached to the turbidostat which measures the cell density. Any change in

cell density is passed on as signal to a pump that increases or decreases the flow of

growth medium into growth vessel. Maximum sensitivity and stability are achieved at

high dilution rates, within a range over the culture biomass changes rapidly with

dilution rates.

♣ Advantages and Applications of Continuous Culture Technique:

1. They provide a constant source of active cells for the study of physiology and

genetics of the organism.

2. Cultures in the exponential phase are highly useful in fermentation industry

because of their high physiological activities.

3. As the cells are grown continuously, it is possible to study synthesis or catabolism

of limiting substrates and also to select various classes of mutants.

4. The system can be coupled with selective enrichment to isolate.

5. They are used in industry for the production of single cell protein ( SCP )

♣ Synchronous Growth

In the batch culture or normal growth curve all the organisms are present at the

different stages of growth or the cells are growing randomly. Such bacterial

populations do not give conclusive studies about the growth behaviour of individual

cells, as the distribution of cell size and cell age is random. In order to study growth

behaviour of individual cell, it is necessary to grow them synchronously. Synchronous

cultures are composed of populations of cells that are at the same stage of their cell

cycle. Bacteria normally reproduce by transverse binary fission, a process in which a

cell divides to produce two equal sized progeny cells. The major events included in

bacterial cell cycle are 1) growth or coordinated synthesis of macromolecules and

cellular components in the cells, 2) septum formation and 3) division or the process in

which each daughter cell receives a copy of DNA. All the cells in the synchronous

culture will divide at the same time, all will grow for the same generation time, and all

will divide again at the same time. Thus the entire cell population is uniform with

respect to growth and division.

♣ Application Of Synchrony

It is not possible to analyze a single bacterium because of its small size. It is

very important to know about cell organization, differentiation and macromolecule

synthesis. The synchronous cultures provide cell population in the same stage of

growth or division cycles. Results made for synchronized cultures are equivalent to

the results made for individual cells. But, the synchrony lasts only for a few

generations, since even the daughter cells of a single mother cell soon get out of phase

with one another.

♣ Techniques for Synchronous Growth :

In any large microbial population, the cells are growing randomly. By certain

techniques it is possible to impose synchronous growth, lasting for three to four

generations and again enter into the asynchronous growth.

♣ The important techniques for inducing synchrony are :

4. Manipulation of environmental conditions,

5. Separation by physical means of cells in population that are at the same stage

of the cell cycle,

6. Helmstetter—Cummings technique.

♣ Induction Technique

Induction technique in which several chemical or physical parameters are used

to induce synchrony. The technique to be employed is often related to the type of

organism used in the experiment. The cyclic shifts of temperature can be used for

Salmonella typhi. The culture is subjected to fixed intervals of alternate cycles of

growth at 25 c ( sub-optimum ) and 37 c ( optimum ) for a time span equivalent to the

generation time of that test organism. As shown by viable counts, division occurs

during each exposure to 37c whereas, the population level remains same during each

exposure to 25 c. Scientists have proved that one of the steps in cell division is

particularly temperature dependent. At lowe r or higher temperatures the cells show

only the vegetative growth. Photosynthetic micro-organisms can be synchronized by

subjecting a culture to alternate cyclic exposures to light and dark. A frequently used

technique is to starve cell by placing them in a medium in which one of the nutrients

is in short supply. It is known that during the starvation period the cell population

approaches a uniform state of growth in cell cycle. Later the cells are placed in a

nutritionally complete medium to get synchrony.

♣ Selection Technique:

Synchronous population can be selected from random population by physical

separation of cells that are at the same stage of cell cycle either by filtration or by

density centrifugation. For physiological studies, such selection techniques are oftenly

used. Membrane filtration technique involves passing the asynchronous culture

through the membrane filter having pore size equivalent to the size of daughter cells.

Only the daughter cells with same size can pass through it while other cells of

variable size will be retained by the filter. Such separated daughter cells after

inoculation into the fresh medium show synchronous growth. An asynchronous

growth culture is subjected for sucrose density gradient. During centrifugation, the

large and heavy cells will settle at the bottom of the centrifuge tube while the smaller

and recently formed cells remain at the top.

♣ Helmstetter Cumming Technique

An excellent technique to obtain synchrony and it is based on selective separation

methodology. The Cellulose Nitrate Membrane Filter is used to filter the

asynchronized culture and then inverting the filter, allowing fresh medium to flow

through it. The loosely associated bacteria are washed out from filter, the only

bacterial cells is effluent stream of medium are those that arise through division. All

cells in the effluent medium are newly formed and at the same stage of growth. The

number of cells remains constant, in this period the cells metabolize, grow and

increase in size. Then, abruptly, the number of cells doubles. After few cell cycles,

synchrony is lost rapidly as various cells from a population do not divide and grow at

a time.

24. Write an essay on forms of patents and patentability and

process of patenting:

♣ A patent is an exclusive right granted by a country to the owner of an invention to

make, use, manufacture and market the invention, provided the invention satisfies

certain conditions stipulated in the law.

♣ Exclusive right implies that no one else can make, use, manufacture or market the

invention without the consent of the patent holder.

♣ This right is available for a limited period of time.

♣ In spite of the ownership of the rights, the use or exploitation of the rights by the

owner of the patent may not be possible due to other laws of the country which has

awarded the patent.

♣ These laws may relate to health, safety, food, security etc.

♣ A patent in the law is a property right and hence, can be gifted, inherited,

assigned, sold or licensed.

♣ As the right is conferred by the State, it can be revoked by the State under very

special circumstances even if the patent has been sold or licensed or manufactured or

marketed in the meantime.

♣ The patent right is territorial in nature and inventors/their assignees will have to

file separate patent applications in countries of their interest, along with necessary

fees, for obtaining patents in those countries.

♣ A new chemical process or a drug molecule or an electronic circuit or a new

surgical instrument or a vaccine is a patentable subject matter provided all the

stipulations of the law are satisfied.

♣ Patent is a special right to the inventor that has been granted by the government through

legislation for trading new articles

♣ A patent is a personal property which can be licensed or sold by the person / organization

just like any other property.

♣ For example, Alexander Graham Bell obtained patent for his telephone. This gave him the

power to prevent engine from making or using or selling a telephone elsewhere.

♣ A patent consists of three parts: the grant, the specification and claims.

♣ The grant is filled at the patent office which is not published. It is a signed document which

is actually the agreement that grants patent right to the inventor. However, the specification and

claims are published as a single document which is made public at a minimum charge from the

patent office.

♣ The specification part is narrative in which the subject matter of invention is described how

the invention was carried out.

♣ The claim section specifically defines the scope of the invention to be protected by the patent

to which the others may not practice.

♣ The maximum duration of patent is for 5 years from the date of grant and 7 years

from the date of filing the patent application

The Indian Patent Act:

♣ The first Indian patent laws were first promulgated in 1856. These were modified

from time to time.

♣ New patent laws were made after the independence in the form of the Indian

Patent Act 1970.

♣ The Act has now been radically amended to become fully compliant with the

provisions of TRIPS.

♣ The most recent amendment was made in 2005 which were preceded by the

amendments in 2000 and 2003.

♣ While the process of bringing out amendments was going on, India became a

member of the Paris Convention, Patent Cooperation Treaty and Budapest Treaty.

Definition of invention

♣ A clear definition has now been provided for an invention, which makes it at par

with definitions followed by most countries.

♣ Invention means a new product or process involving an inventive step and capable

of industrial application.

♣ New invention means any invention or technology which has not been anticipated

by publication in any document or used in the country or elsewhere in the world

before the date of filing of patent application with complete specification i.e., the

subject matter has not fallen in public domain or it does not form part of the state of

the art.

♣ Inventive step means a feature of an invention that involves technical advance as

compared to existing knowledge or having economic significance or both and that

makes the invention not obvious to a person skilled in the art. ―capable of industrial

application means that the invention is capable of being made or used in an industry"

Novelty:

♣ An invention will be considered novel if it does not form a part of the global state

of the art.

♣ Information appearing in magazines, technical journals, books, newspapers etc.

constitutes the state of the art.

♣ Oral description of the invention in a seminar/conference can also spoil novelty.

Novelty is assessed in a global context.

♣ An invention will cease to be novel if it has been disclosed in the public through

any type of publications anywhere in the world before filing a patent application in

respect of the invention.

♣ Therefore it is advisable to file a patent application before publishing a paper if

there is a slight chance that the invention may be patentable.

♣ Prior use of the invention in the country of interest before the filing date can also

destroy the novelty.

♣ Novelty is determined through extensive literature and patent searches. It should

be realized that patent search is essential and critical for ascertaining novelty as most

of the information reported in patent documents does not get published anywhere else.

♣ For an invention to be novel, it need not be a major breakthrough. No invention is

small or big.

♣ Modifications to the existing state of the art, process or product or both, can also

be candidates for patents provided these were not earlier known. In a chemical

process, for example, use of new reactants, use of a catalyst, new process conditions

can lead to a patentable invention.

Inventiveness (Non-obviousness):

♣ A patent application involves an inventive step if the proposed invention is not

obvious to a person skilled in the art i.e., skilled in the subject matter of the patent

application.

♣ The prior art should not point towards the invention implying that the practitioner

of the subject matter could not have thought about the invention prior to filing of the

patent application.

♣ Inventiveness cannot be decided on the material contained in unpublished patents.

♣ The complexity or the simplicity of an inventive step does not have any bearing

on the grant of a patent.

♣ In other words a very simple invention can qualify for a patent. If there is an

inventive step between the proposed patent and the prior art at that point of time, then

an invention has taken place.

♣ A mere 'scintilla' of invention is sufficient to found a valid patent. It may be often

difficult to establish the inventiveness, especially in the area of up coming knowledge

areas.

♣ The reason is that it would depend a great deal on the interpretative skills of the

inventor and these skills will really be a function of knowledge in the subject area.

Usefulness:

♣ An invention must possess utility for the grant of patent. No valid patent can be

granted for an invention devoid of utility.

♣ The patent specification should spell out various uses and manner of practicing

them, even if considered obvious.

♣ If you are claiming a process, you need not describe the use of the compound

produced thereby. Nevertheless it would be safer to do so. But if you claim a

compound without spelling out its utility, you may be denied a patent.

Non patentable inventions:

♣ An invention may satisfy the conditions of novelty, inventiveness and usefulness

but it may not qualify for a patent under the following situations:

1. An invention which is frivolous or which claims anything obviously contrary to

well established natural laws e.g. different types of perpetual motion machines.

2. An invention whose intended use or exploitation would be contrary to public order

or morality or which causes serious prejudice to human, animal or plant life or

health or to the environment e.g., a process for making brown sugar will not be

patented.

3. The mere discovery of a scientific principal or formulation of an abstract theory

e.g., Raman Effect and Theory of Relativity cannot be patented.

4. The mere discovery of a new form of a known substance which does not result in

enhancement of the known efficacy of that substance or the mere discovery of any

new property or new use of a known substance or the mere use of a known

process, machine or apparatus unless such a known process results in a new

product or employs at least one new reactant. For the purposes of this clause, salts,

esters, polymorphs, metabolites, pure form, particle size, isomers, mixtures of

isomers, complexes, combinations and other derivatives of known substance shall

be considered to be the same substance unless they differ significantly in

properties with regard to efficacy.

5. A substance obtained by a mere admixture resulting only aggregation of the

properties of the components thereof or a process for producing such substance.

6. The mere arrangement or rearrangement or duplication of features of known

devices each functioning independently of one another in a known way. If you put

torch bulbs around an umbrella and operate them by a battery so that people could

see you walking in rain when it is dark, then this arrangement is patentable as

bulbs and the umbrella perform their functions independently.

7. A method of agriculture or horticulture. For example, the method of terrace

farming cannot be patented.

8. Any process for medical, surgical, curative, prophylactic, diagnostic, therapeutic

or other treatment of human beings, or any process for a similar treatment of

animals to render them free of disease or to increase economic value or that of

their products. For example, a new surgical technique for hand surgery for

removing contractions is not patentable.

9. Inventions relating to atomic energy;

10. Discovery of any living thing or non-living substance occurring in nature;

11. Mathematical or business methods or a computer program per se or algorithms;

12. Plants and animals in whole or any part thereof other than microorganisms but

including seeds, varieties and species and essentially biological processes for

production and propagation of plants and animals;

13. A presentation of information;

14. Topography of integrated circuits;

15. A mere scheme or rule or method of performing mental act or method of playing

games;

16. An invention which, in effect, is traditional knowledge or which is aggregation or

duplication of known component or components.

♣ Computer program per se as such has not been defined in the Act but would

generally tend to mean that a computer program with out any utility would not be

patentable. Protection of seeds and new plant varieties is covered under a different

Act, which provides a protection for a period of 10 years. Similarly, topography of

integrated circuits is protected through yet a different Act.

Term of the patent:

Term of the patent will be 20 years from the date of filing for all types of inventions.

Application:

♣ In respect of patent applications filed, following aspects will have to be kept in

mind:-

1. Claim or claims can now relate to single invention or group of inventions

linked so as to form a single inventive concept

2. Patent application will be published 18 months after the date of filing

3. Applicant has to request for examination 12 months within publication or 48

months from date of application, whichever is later

♣ No person resident in India shall, except under the authority of a written permit

sought in the manner prescribed and granted by or on behalf of the Controller, make

or cause to be made any application outside India for the grant of a patent for an

invention unless (a) an application for a patent for the same invention has been made

in India, not less than six weeks before the 6 application outside India; and (b) either

no direction has been given under the secrecy clause of the Act or all such directions

have been revoked.

Provisional Specification:

♣ A provisional specification is usually filed to establish priority of the invention in

case the disclosed invention is only at a conceptual stage and a delay is expected in

submitting full and specific description of the invention.

♣ Although, a patent application accompanied with provisional specification does

not confer any legal patent rights to the applicants, it is, however, a very important

document to establish the earliest ownership of an invention.

♣ The provisional specification is a permanent and independent scientific cum legal

document and no amendment is allowed in this.

♣ No patent is granted on the basis of a provisional specification. It has to be a

followed by a complete specification for obtaining a patent for the said invention.

♣ Complete specification must be submitted within 12 months of filing the

provisional specification. This period can be extended by 3 months.

♣ It is not necessary to file an application with provisional specification before the

complete specification.

♣ An application with complete specification can be filed right at the first instance.

Complete Specification:

♣ It may be noted that a patent document is a techno-legal document and it has to be

finalized in consultation with an attorney. Submission of complete specification is

necessary to obtain a patent. Contents of a complete specification would include the

following.

a) Title of the invention.

b) Field to which the invention belongs.

c) Background of the invention including prior art giving drawbacks of the

known inventions & practices.

d) Complete description of the invention along with experimental results.

e) Drawings etc. essential for understanding the invention.

f) Claims, which are statements, related to the invention on which legal

proprietorship is being sought. Therefore the claims have to be drafted very

carefully.

Compulsory license:

♣ Any time after three years from date of sealing of a patent, application for

compulsory license can be made provided

1. Reasonable requirements of public have not been met

2. Patented invention is not available to public at a reasonably affordable price

3. Patented invention is not worked in India 7 among other things

♣ Reasonable requirements of public are not satisfied if working of patented

invention in India on a commercial scale is being prevented or hindered by

importation of patented invention.

♣ Applicant's capability including risk taking, ability of the applicant to work the

invention in public interest, nature of invention, time elapsed since sealing, measures

taken by patentee to work the patent in India will be taken into account.

♣ In case of national emergency or other circumstances of extreme urgency or

public non commercial use or an establishment of a ground of anti competitive

practices adopted by the patentee, the above conditions will not apply.

♣ A patentee must disclose the invention in a patent document for anyone to

practice it after the expiry of the patent or practice it with the consent of the patent

holder during the life of the patent.

Patenting of microbiological inventions:

♣ The Indian Patent Act has now a specific provision in regard to patenting of

microorganisms and microbiological processes. It is now possible to get a patent for a

microbiological process and also products emanating from such processes.

♣ As it is difficult to describe a microorganism on paper, a system of depositing

strain of microorganisms in some recognized depositories was evolved way back in

1949 in USA.

♣ An international treaty called "Budapest Treaty" was signed in Budapest in 1973

and later on amended in 1980.

♣ India became a member of this Treaty, with effect from December 17, 2001. This

is an international convention governing the recognition of deposits in officially

approved culture collections for the purpose of patent applications in any country that

is a party to this treaty.

♣ Because of the difficulties and virtual impossibility of reproducing a

microorganism from a description of it in a patent specification, it is essential to

deposit a strain in a culture collection centre for testing and examination by others.

♣ An inventor is required to deposit the strain of a microorganism in a recognized

depository, which assigns a registration number to the deposited microorganism.

♣ This registration number needs to be quoted in the patent application dealing with

the microorganism.

♣ Obviously a strain of microorganism is required to be deposited before filing a

patent application.

♣ It may be observed that this mechanism obviates the need of describing a

microorganism in the patent application.

♣ Further, samples of strains can be obtained from the depository for further

working on the patent.

♣ There are many international depositories in different countries such as ATCC,

DSM etc. which are recognized under the Budapest Treaty.

♣ The Institute of Microbial Technology(IMTEC), Chandigarh is the first Indian

depository set up under the Budapest Treaty.

Mail box provision:

♣ TRIPS requires that countries, not providing product patents in respect of

pharmaceuticals and chemical inventions have to put in a mechanism for accepting

product patent applications with effect from 1 January 1995.

♣ Such applications will only be examined for grant of patents, after suitable

amendments in the national patent law have been made.

♣ This mechanism of accepting product patent applications is called the "mail box"

mechanism. This system has been in force in India and now such applications are

being taken up for examination.

Exclusive Marketing Right:

TRIPS requires that member countries of the WTO not having provision in their laws

for granting product patents in respect of drugs and agrochemical, must introduce

Exclusive Marketing Rights (EMR) for such products, if the following criteria are

satisfied:

1. A patent application covering the new drug or agrochemical should have been filed

in any of the WTO member countries after 1 January 1995

2. A patent on the product should have been obtained in any of the member countries

(which provides for product patents in drugs and agrochemical) after 1 January 1995

3. Marketing approvals for the product should have been obtained in any of the

member countries

4. A patent application covering the product should have been filed after 1 January

1995 in the country where the EMR is sought

5. The applicant should apply seeking an EMR by making use of the prescribed form

and paying requisite fee.

♣ EMR is only a right for exclusive marketing of the product and is quite different

from a patent right.

♣ It is valid up to a maximum period 5 years or until the time the product patent

laws come into effect. The necessary amendment to: the Patents Act, 1970 came into

force on 26 March 1999.

♣ The provision is applicable with retrospective effect from 1 January 1995.

♣ As per the 2005 amendments in the Patents Act, the provision of EMR is no

longer required.

♣ These rights were awarded in India from time to time and there have been some

litigations as well where the courts came up with quick decisions.

Timing for filing a patent application:

♣ Filing of an application for a patent should be completed at the earliest possible

date and should not be delayed.

♣ An application filed with provisional specification, disclosing the essence of the

nature of the invention helps to register the priority by the applicant.

♣ Delay in filing an application may entail some risks like

(i) Other inventors might forestall the first inventor by applying for a patent for the

said invention

(ii) There may be either an inadvertent publication of the invention by the inventor

himself/herself or by others independently of him/her.

♣ Publication of an invention in any form by the inventor before filing of a patent

application would disqualify the invention to be patentable.

♣Hence, inventors should not disclose their inventions before filing the patent

application.

♣The invention should be considered for publication after a patent application has

been filed.

♣Thus, it can be seen that there is no contradiction between publishing an inventive

work and filing of patent application in respect of the invention.

Protecting new plant variety:

♣ New plant varieties can now be protected in India under the New Plant Variety

and Farmers Rights Protection Act in 2001.

♣ New plant varieties cannot be protected through patents. However, the Act has not

become operational as subsidiary legislation is yet to be put in place.

♣ India has enacted the which, in addition to meeting the technical features of

UPOV, provides rights to farmers to use the seeds from their own crops for planting

the next crop.

♣ Further, there are provisions for benefit sharing with farmers, penalty for

marketing spurious propagation material and protecting extant varieties. There is a

provision for protecting extant variety and farmers‘ varieties as well.

♣ The total period for protection is 10 years from the date of registration.

♣ There are 5 main criteria to arrive at a decision whether a plant variety is really

new or not. These are distinctiveness, uniformity, stability, novelty and denomination.

♣ The variety shall be deemed to be distinct if it is clearly distinct from any other

variety whose existence is a matter of common knowledge at the time of filing of the

application.

♣ The variety shall be deemed to be uniform if, subject to the variation that may be

accepted from the particular features of its propagation, it is sufficiently uniform in its

relevant characteristics.

♣ The variety shall be deemed to be stable if its relevant characteristics remain

unchanged after repeated propagation or, in the case of a particular cycle of

propagation at the end of each such cycle.

♣ The variety shall be deemed to be new if, at the date of filing of the application for

breeders right, propagating or harvesting material of the variety has not been sold or

otherwise disposed of to others, by or with the consent of the breeder for the purpose

of exploitation of the variety.

♣ The variety shall be designated by a denomination, which will be its generic

designation.

♣ The premise that the variety denomination must be its generic designation class

for a requirement that 'denomination must enable the variety to be identified.

25. Discuss on botulism, causative organism, toxigenic types,

symptoms and its control:

♣ Botulism is a severe illness affecting primarily the nervous system (neuroparalytic

disorder) caused by the botulism toxin produced by Clostridium botulinum.

♣ Botulism can be classified into the following categories: foodborne, infant, wound

and undetermined.

♣ The botulinum toxins are a group of seven related neurotoxins produced by the

bacillus Clostridium botulinum.

♣ Botulism and tetanus toxins are very similar in structure and function, but differ

dramatically in their clinical effects because they target different cells in the nervous

system.

♣ Botulinum toxins are the most lethal toxins known. For type A toxin, the toxic

dose is estimated at 0.001 mcg/kg; the lethal dose for a 70-kg person by the oral route

is estimated at 70 mcg, by the inhalation route 0.80 to 0.90 mcg and by the

intravenous route 0.09 to 0.15 mcg.

♣ The toxins are identified by neutralization with type-specific antitoxin; minor

cross-neutralization between types C and D and between types E and F have been

observed. The toxins are produced by vegetative cells (i.e., germination of spores),

and released by cell lysis. Some toxins are fully activated by the bacteria that produce

them (proteolytic strains of type A, B and F), and some require exogenous proteolytic

activation (types E and non-proteolytic types B and F).

Botulinum toxins are colorless, odorless and presumably tasteless. The toxins are

inactivated by heating (>85°C for 5 minutes).

♣ Toxins are differentiated according to their antigenic differences: types A to G.

Human botulism is almost always caused by neurotoxins A, B, E and F. Type A

botulism is found most commonly in the west and type B is more common in the east.

Type E is associated with fish. Types C and D are associated primarily with botulism

in birds and mammals. Almost all cases of infant botulism are caused by types A and

B.

♣ Botulinum toxin A produced longer paralysis than botulinum toxin B, consistent

with human observations.

1. For type A, duration of paralysis was exponentially related to toxin dose; the

paralysis time doubled with every 25% increase of the toxin concentration.

2. For type B, the duration of paralysis was linear relative to the toxin dose.

3. Types C and D cause natural disease in birds, horses and cattle; strains that

produce these types reside in the intestinal tract of certain animals.

Contaminated silage has been reported to cause botulism outbreaks among

cattle.

4. Type E toxin had the shortest duration of action, but unlike the other two

toxins, the dose of toxin did not influence recovery time.

5. Toxin type G has never clearly been shown to cause human disease.

♣ Aerosolized particles of toxin are approximately 0.1 to 0.3 mcm in size. In the

event of an intentional release of botulinum toxin, the causative organisms may or

may not be present These toxins could be delivered by aerosol. When inhaled, these

toxins produce a clinical picture very similar to foodborne intoxication. However, the

botulinum toxin is so inherently toxic that this characteristic does not limit its

potential as a biological weapon. Exposure to botulinum toxin occurs through the

following mechanisms (toxin is not absorbed through intact skin):

Epidemiology:

♣ Food botulism is caused by the ingestion of a preformed toxin in contaminated

food. The toxin is produced when the bacteria grow in food that has been improperly

preserved or stored under anaerobic conditions.

♣ Most poisonings in the U.S. are due to home-canned vegetables and fruits.

Botulinum spores are often present in the environment; therefore identification of the

organism in food is not necessarily diagnostic.

♣ Not all food borne botulism results from ingestion of improperly prepared home-

canned food as demonstrated by restaurant-associated outbreaks from foods such as

patty-melts, potato salad and aluminum foil–wrapped baked potatoes. The word

―botulism‖ comes from the Latin word botulus, or sausage.

♣ The following steps are necessary for a food item to cause botulism:

1. The food item must be contaminated with C. botulinum spores, which are

normally found in soil.

2. The spores must survive food preservation methods.

3. Adequate conditions for spore germination and neurotoxin production must be

present

4. The food must not be reheated adequately (>85°C for 5 minutes) to inactivate

the heat-labile toxin before the food is consumed.

5. Generally, adequate conditions for germination and neurotoxin production

include the following:

6. An anaerobic environment

7. Non-acidic pH (generally 4.6 to 4.8; pockets of different pH may be present

within a single food source and allow toxin to be produced in a food that

overall has an acidic pH)

8. Minimum temperature of 10°C (the optimum temperature for growth of

proteolytic strains is close to 35°C; some non-proteolytic strains of types B, E

and F can produce toxin at refrigeration temperatures [3°C to 4°C]

9. Availability of water with limited solute concentration

Infant botulism:

♣ In contrast to classical foodborne botulism, which is intoxication due to ingestion

of preformed botulinum toxin, infant botulism occurs after infants eat spore

contaminated food. The spores grow in the intestines and then release the toxin in the

body.

♣ The infant‘s large intestine vulnerability to spore germination and toxin production

is not yet understood. Honey is the one identified and avoidable source of botulinum

spores.

♣ By a process of exclusion (testing over the years of hundreds of foods, beverages

and other items placed in infants' mouths with negative results), it was concluded that

most infant botulism patients acquired their spores by swallowing microscopic dust

particles that carry the spores.

♣ In most cases the precise source is not identified. Honey has been identified as one

vehicle and should not be given to children under one year of age.

♣ Botulism is not transmitted from person to person. C. botulinum spores are found

throughout the world in soil samples and marine sediment.

Wound botulism:

♣ C. botulinum is a natural contaminant of soil throughout the United States.

♣ Wound botulism has been recognized with increasing frequency among injecting

drug users. Wound botulism in drug abusers can be misdiagnosed as drug

intoxication.

♣ It should be considered in injecting drug users who present with dysarthria and

dysphagia.

♣ Wound botulism may occur following traumatic injury to an extremity, such as a

compound fracture, laceration, puncture wound, gunshot wound, severe abrasion

("road rash"), or crush injury.

♣ Sinusitis associated with intranasal cocaine use has been the source of wound

botulism in a few cases. A few cases have occurred postoperatively (usually following

intra-abdominal procedures) and an abscessed tooth was the source of C. botulinum

infection in one case.

The usual incubation period is:

♣ for foodborne botulism 12 to 36 hours (range, 6 hours to 10 days)

♣ for wound botulism, 4 to 14 days between the time of injury and the onset of

symptoms

♣ for infant botulism, the incubation period is estimated at 3 to 30 days from the

time of exposure to spore-containing honey or other food.

Clinical Description:

♣ Botulinum neurotoxins predominantly affect the peripheral neuromuscular

junction and autonomic synapses, and its effects are primarily manifested as

weakness. It is NOT associated with mental status or sensory changes.

♣ Except for infant botulism, onset of symptoms occurs abruptly within a few hours

or evolves gradually over several days. Botulism is an acute, afebrile symmetric,

descending, flaccid paralysis. Fatigue, dizziness, dysphagia, dysarthria, diplopia, dry

mouth, dyspnea, ptosis, ophthalmoparesis, tongue weakness and facial muscle paresis

are early findings seen in more than 75% of cases. Progressive muscular involvement

leading to respiratory failure ensures

♣ Infant botulism occurs in infants younger than six months of age. The disease

infant botulism is first suspected based on clinical features of the infant patient (12

months of age or younger). Symptoms such as:

1. poor feeding

2. droopy eyelids

3. constipation

4. lethargy

5. bulbar palsies, hypotonia, weakness and loss of head control

6. The spectrum of disease ranges from mild (eg, constipation, slow feeding) to

rapidly progressive (eg, apnea, sudden infant death).

Laboratory Tests:

♣ A toxin neutralization bioassay in mice is used to identify botulinum toxin in

serum, stool, or suspect foods. To increase the likelihood of diagnosis, both serum and

stool should be obtained from all persons with suspected botulism.

In infant and wound botulism, the diagnosis is a two step process:

1-Demonstrate C.botulinum organisms

2-Demonstrate the presence of toxin in feces, wound exudate or tissue

samples.

♣ The toxin has been demonstrated in serum in approximately one percent of infants

with botulism.

♣ In foodborne cases, serum specimens collected more than three days after

ingestion of toxin usually are negative, at which time stool and gastric aspirates are

the best diagnostic specimens for culture. Since obtaining a stool specimen may be

difficult because of constipation, an enema using sterile nonbacteriostatic water can

be given. Enriched and selective media are used to culture C.botulinum from stool and

foods. C. botulinum is a large, usually gram-positive, strictly anaerobic bacillus that

forms a subterminal spore.

Treatment:

♣ Adults with botulism are treated with an antitoxin derived from horse serum

distributed by CDC.

♣ Treatment with Botulism Immune Globulin (BIG) should be started as early in the

illness as possible and should not be delayed while awaiting laboratory confirmation.

♣ Antitoxin therapy is usually carried out with a trivalent (types A, B and E) equine

serum. The antitoxin is only effective against toxin unattached to nerve endings. Early

injection of antitoxin (within 24 hours of onset of symptoms) can preserve nerve

endings, prevent progression of disease and reduce mortality.

♣ Equine botulinum antitoxin also is obtainable and can be administered to adults

after testing for hypersensitivity to equine sera if BIG is not available. Approximately

nine percent of treated persons experience some degree of hypersensitivity reaction to

equine sera.

Prevention:

♣ When a food item has been identified by epidemiologic evidence or laboratory

tests, immediate recall of the product is necessary.

♣ Education to improve home-canning methods should be promoted, but cases also

may be restaurant-acquired.

♣ Use of a pressure cooker (at 116°C = 240.8°F) is necessary to kill spores of C.

botulinum. Boiling for ten minutes will destroy the toxin. Time-temperature-pressure

requirements vary with the product being heated.

♣ In addition, food containers that appear to bulge may contain gas produced by C.

botulinum and should be discarded. Other foods that appear to be spoiled should not

be tasted.

♣ The only known prevention measure for infant botulism is to avoid feeding honey

to infants twelve months of age or younger.

♣ Breastfeeding may slow the onset of illness if it develops.

26. Discuss media formulation: ♣ Medium formulation is an essential stage in the design of successful laboratory

experiments, pilot scale development and manufacturing process

♣ The constituents of a medium must satisfy the elemental requirements for cell

biomass and metabolite production and there must be an adequate supply of energy

for biosynthesis and cell maintenance

♣ The first step to consider is an equation based on the stoichiometry for growth and

product formation

♣ Thus for an aerobic fermentation:

carbon and energy source + biomass + products +

nitrogen source + CO2 + H2O

oxygen + other requirements

♣ This equation should be expressed in quantitative terms which is important in the

economical design of media if component wastage is to be minimal

♣ Thus it should be possible to calculate the minimal quantities of nutrients which

will be needed to produce a specific amount of biomass.

♣ Knowing that a certain amount of biomass is necessary to produce a defined

amount of product, it should be possible to calculate substrate concentrations

necessary to produce required product yields.

♣ There may be medium components which are needed for product formations

which are not required for biomass production

♣ Unfortunately it is not always easy to quantify all the factors very precisely

♣ A knowledge of the elemental composition of a process microorganism is required

for the solution of the elemental equation

♣ This information may not be available so that data will serve as a guide to the

absolute minimum quantities of N, S, P, Mg and K to include in an initial medium

recipe

♣ Trace elements (Fe, Zn, Cu, Mn, Co, Mo, B) may also be needed in smaller

quantities.

♣ An analysis of relative concentrations of individual elements in bacterial cells and

commonly used cultivation media quoted by Cooney (1981) showed that some

nutrients are frequently added in substantial excess of that required, e.g., P, K,

however others are often near limiting values (Zn, Cu)

♣ The concentration of P is deliberately raised in many media to increase the

buffering capacity

♣ The points emphasize the need for considerable attention to be given to medium

design

♣ Some microorganisms cannot synthesize specific nutrients (amino acids, vitamins

or nucleotides)

♣ Once a specific growth factor has been identified it can be incorporated into a

medium in adequate amounts as a pure compound or as a component of a complex

mixture

♣ The carbon substrate has a dual role in biosynthesis and energy generation.

♣ The carbon requirement for biomass production under aerobic conditions may be

estimated from the cellular yield coefficient (Y) which is defined as

Quantity of cell dry matter produced / Quantity of carbon substrate utilized

♣ Thus for bacteria with a Y for glucose of 0.5 which is 0.5 g cells / g glucose, the

concentration of glucose needed to obtain 30g/dm3

cells will be 30 / 0.5 – 60 g / dm3

♣ One litre of this medium would also need to contain approximately 3 g N, 1 g K,

0.3 g S and 0.1 g Mg.

♣ An adequate supply of the carbon source is also essential for a product forming

fermentation process

♣ In a critical study, analyses are made to determine how the observed conversion of

the carbon source to the product compares with the theoretical maximum yield

♣ This may be difficult because of limited knowledge of the biosynthetic pathways

♣ Cooney (1979) has calculated theoretical yields for penicillin G biosynthesis on

the basis of material and energy balances using a biosynthetic pathway based on

reaction stoichiometry

♣ Using a simple model for a batch culture penicillin fermentation it was estimated

that 28, 61, 11% of the glucose consumed was used for cell mass, maintenance and

penicillin respectively

♣ When experimental results of a fed batch penicillin fermentation were analysed

26% of the glucose has been used for growth and 70% for maintenance and 6% for

penicillin

♣ The maximum experimental conversion yield for penicillin was calculated to be

0.0053 g/g glucose (88.5 units /mg)

♣ Thus the theoretical conversion value is many times higher than the experimental

value

♣ Herbach etal (1984) concluded that there were six possible biosynthetic pathways

for penicillin production and two possible mechanisms for ATP production NADH

and FADH2

♣ They calculated that conversion yields by different pathways varied from 638 to

1544 units of penicillin per mg glucose

♣ At that time the best quoted yields were 200 units penicillin per mg glucose

♣ This gives a production of 13 to 29 % of the maximum theoretical yield

♣ The other major nutrient which will be required is oxygen which is provided by

aerating the culture

♣ The design of a medium will influence the oxygen demand of a culture in that the

more reduced carbon sources will result in a higher oxygen demand

27. Discuss media Sterilization:

♣ In virtually all fermentation processes, it is mandatory for a cost-effective

operation to have contamination-free seed cultures at all stages, from the preliminary

culture to the production fermenter.

♣ A bioreactor can be sterilized either by destroying the organisms with some lethal

agent such as heat, radiation, or a chemical, or by removing the viable organisms by a

physical procedure such as filtration.

♣ Microorganisms can be removed from fluids by mechanical methods, for example,

by filtration, centrifugation, flotation, or electrostatically. They may also be destroyed

by heat, chemical agents, or electromagnetic waves. Although cells may be disrupted

and killed by mechanical abrasion on a small scale, this method is not satisfactory

industrially. Similarly x-rays, beta rays, ultra-violet light, and sonic radiations, while

useful for laboratory purposes are not applicable to the sterilization of large volume of

fluids. Gamma rays, on the other hand, may

prove useful, particularly in food industry.

Sterilization of Culture Media:

♣ Nutrient media as initially prepared contain a variety of different vegetative cells

and spores, derived from the constituents of the culture medium, the water, and the

vessel.

♣ These must be eliminated by a suitable means before inoculation.

♣ A number of means are available for sterilization, but in practice for large scale

installations, heat is the main mechanism used.

Heat Sterilization:

♣ This is the most useful method for the sterilization of nutrient media. A number of

factors influence the success of heat sterilization:

1. the number and type of microorganisms present

2. the composition of the culture medium

3. the pH value

4. the size of the suspended particles

♣ Vegetative cells are rapidly eliminated at relatively low temperatures, but for

destruction of spores, temperatures of 121°C are needed.

♣ Spores of Bacillus stearothermophilus are the most heat resistant. Therefore they

are used as assay organisms for testing the various procedures used to sterilize

equipment.

♣ Radiation (UV, X Rays, or y rays) Although occasionally used in the food

industry, these agents are not used in industrial fermentation.

♣ Chemical methods Although a number of chemical disinfectants are known, they

cannot be used to sterilize nutrient media because there is a risk that inhibition of the

fermentation organism could occur from the residual chemical.

Mechanical Removal of Organisms

1. Centrifugation, adsorption to ion exchange, adsorption to activated carbon, or

filtration are possible.

2. Filtration is the only method in practical use. Filter sterilization is often used

for components of nutrient solutions which are heat -sensitive and would thus

be denatured through the steam sterilization process normally used in

industrial fermentation.

3. Deep filters (plate filters) are some time used to filter complex nutrient

solutions. The disadvantages of filtration are: 1) certain components of the

nutrient solution may be adsorbed on the filter material, and 2) high pressures

must be used (up to 5 bar), which are undesirable in industrial practice.

Batch Sterilization:

♣ Most nutrient media are presently sterilized in batch volumes in the bioreactor at

121°C.

♣ Approximate sterilization times can be calculate from the nature of the medium

and the size

♣ Not only the nutrient media, but also the fittings, valves and electrodes of the fer-

menter itself must be sterilized.

♣ Therefore actual sterilization times are significantly longer than calculated ones

and must be empirically determined for the specific nutrient solutions in the

fermenter.

♣ One method of sterilization is to injecting n into the fermenter mantle or interior

coils red sterilization.

♣ Another method is to inject steam into the nutrient solution itself. With direct

steam injection, condensate accumulates in the fermenter and the volume of liquid

increases during the sterilization process.

♣ It takes 2-3 s to reach the sterilization temperature of 121C depending on the

steam conduction and enter size. Once the proper temperature has , reached, another

20-60 minutes are required for the actual killing process, followed by cooling for

about one hour.

♣ Another disadvantage of heat sterilization (and from the standpoint of

microbiology the most significant shortcoming) is that heating, sterilization and

cooling phases not only kill microorganisms but also severely alter nutrient solutions.

Discoloration and changes in the pH

value result from carameli-zation and Maillard reactions. Vitamins are destroyed and

the quality of the culture medium deteriorates. The extent to which the subsequent

fermentation is affected depends on the organism and the process.

Continuous Sterilization:

♣ The two -main disadvantages of batch sterilization just mentioned, culture medium

damage and high energy consumption, can be largely avoided by use of a continuous

sterilization procedure.

♣ Although continuous sterilization is the logical preliminary step for continuous

fermentations in industrial scale, it is also of value in batch fermentations, making

greater yield possible for the time and space allotted.

♣ The reason for this is because of the exponential relationship between death rate

and temperature, making the time required for the complete elimination of life shorter

if higher temperatures are used. While batch sterilization is carried out in 30-60

minutes at 121°C, continuous sterilization is normally accomplished in 30- 120

seconds at 140°C.

♣ The heating of culture media for continuous sterilization can be done either by

injection of steam or by means of heat exchangers.

♣ Sterilization with steam injection is done by injecting steam into the nutrient

solution. The temperature is raised quickly to 140°C and is maintained for 30-120

seconds.

♣ Due to the formation of condensate, the nutrient solution becomes diluted; to

correct this, the hot solution is pumped through an expansion valve into a vaporizer

and the condensate is removed via vacuum pumps so that the sterilized nutrient

solution has the same concentration after the cooling process as before.

♣ The disadvantage of this process is the sensitivity it exhibits to changes in the

viscosity of the medium and to pressure variations.

♣ In the continuous process using heat exchangers , the nutrient solution in the first

heat exchanger is preheated to 90-120°C within 20-30 seconds by the exiting

previously sterilized nutrient solution. Then in the second heat exchanger, it is heated

indirectly with steam to 140°C.

This temperature is maintained for 30-120 seconds in a holding pipe before it is

placed in the first exchanger for preliminary cooling and then in a third exchanger for

cooling to the temperature of the fermenter. The cooling phase is only 20-30 seconds.

♣ In the process using heat exchangers, 90% of the energy input is recovered.

♣ The disadvantage of this method is that with some nutrient solutions, insoluble

salts (e.g., calcium calcium oxalate) are formed and crusts a the first heat exchanger,

due to the perature differences between the nutrient solution and the cold incoming.

♣ Starch-containing solutions which viscous when heated are difficult to use in

continuous sterilization processes.

The advantages of continuous sterilization of media are as follows:

1. Increase of productivity since the short period of exposure to heat minimizes

damage to media constituents,

2. Better control of quality,

3. Leveling of the demand for process steam,

4. Suitability for automatic control

Sterilization of Fermentation Air

♣ Most industrial fermentations are operated under vigorous aeration and the air

supplied to the fermenter must be sterilized.

♣ To prevent contamination of either the fermentation by airborne.

♣ Microorganisms or the environment by aerosols generated within the fermenter,

both air input and air exhaust ports have air filters attached.

♣ These filters are designed to trap and contain microorganisms. Filters are made of

glass fibre, mineral fibers, poly tetra-fluoroethylene (PTFE) or polyvinyl chloride

(PYC), and must be steam sterilizable and easily changed.

♣ In some circumstances, particularly where pathogenic organisms are being grown,

fermenter exhaust may also undergo dry heat sterilization (incineration) as an

additional safety measure.

♣ Methods available for sterilizing gases include filtration, gas injection (ozone), gas

scrubbing, radiation (UV), and heat.

♣ Of these_filtration and heat are practical at an industrial scale

♣ Previously air was sterilized by passing it over electrically heated elements. Due to

high cost of electricity this method has become obsolete.

♣ Filter sterilization by glass wool filters are used. Particles are trapped by a

combination of physical effect which includes inertial effects, blocking effect,

diffusion, gravity separation and electrostatic attraction.

♣ The disadvantages are shrinkage and solidification during steam sterilization.

♣ Glass Fiber filter cartridges has replaced glass wool filter as these do not have the

drawback mentioned.

Media and Vessel Sterilization:

♣ For pilot-scale and industrial aseptic fermentations the fermenter‘ can be sterilized

empty.

♣ The vessel is then filled with sterile medium, prepared in a batch or continuous

medium ‗cooker‘ that may supply several fermentations.

♣ Alternatively, the fermenter is filled with formulated medium and the two are

sterilized together in one operation.

♣ However, some industrial fermentation are not aseptic, but microbial

contamination is still maintained at a minimum level by boiling or pasteurization of

the media.

♣ Otherwise, a fast-growing contaminant could outgrow the industrial

microorganism, or at least utilize some valuable nutrients.

♣ Microbial contaminants may also metabolize the target product, produce toxic

compounds or secrete products that may block filters and interfere with downstream

processing.

♣ If the contaminant is a bacteriophage it may lyses the culture, as can occur in

fermentations involving lactic acid bacteria.

Process of Sterilization:

♣ Small laboratory-scale fermenters of 1-5L capacity are usually filled with medium

and then sterilized in a steam al1toclave.

♣ Here sterilization is normally per-formed using pressurized steam to attain a

temperature of 121°C for 15 min.

♣ Care must be taken to avoid any pressure build-up inside the fermenter by venting

without contaminating the contents.

♣ For pilot-scale -and industrial fermenters more rigorous sterilization is necessary,

involving increased sterilization time and/or higher temperature.

♣ Steam sterilization involves passing the steam under pressure into the vessel jacket

and/or internal coils.

♣ Stream may also be injected directly into the headspace above the fermentation

medium.

♣ This aids sterilization, but can result in media volume changes. Steam sterilization

is effective and cheaper than dry heat sterilization.

♣ However, certain media constituents may be heat labile and destroyed by

excessive heat, e.g. glucose, some vitamins and components of animal cell culture

media. Such heat sensitive ingredients are often filter sterilized before use;

alternatively; some can be heat sterilized with minimal degradation using a high

temperature for a very short time, e.g. 140°C for 50s.

♣ This is usually a continuous operation where the holding time is controlled by

the flow rate through the sterilizer and the material is then rapidly cooled in a heat

exchanger.

28. Discuss microbial growth kinetics:

♣ Microbial growth can be defined as an orderly increase in cellular components,

resulting in cell enlargement and eventually leading to cell division. This definition is

not strictly accurate as

it implies that a consequence of growth is always an increase in cell numbers.

However, under certain conditions growth can occur without cell division, for

example, when cells are synthesizing storage compounds, e.g. glycogen or poly-b-

hydroxybutyrate. In this situation the cell numbers remain constant, but the

concentration of biomass continues to increase. This is also true for coenocytic

organisms, such as some fungi, that are not divided into separate cells. Their

growth results only in increased Size.

Bacterial Growth Kinetics

♣ Growth kinetics of homogeneous unicellular suspension cultures can be modeled

using differential equations in a continuum model. However, filamentous growth and

growth in heterogeneous cell aggregates and assemblage, particularly biofilms,

colonies, flocs, mats and pellicles, is much more complex. In fact, heterogeneous

systems require a very different approach using cellular automaton and Swarm system

models, e.g. BacSim. The growth kinetics of filamentous organisms and

heterogeneous systems will not be discussed here.

♣ Batch Growth

During batch fermentations the population of microorganisms goes through several

distinct growth phases: lag, acceleration, exponential growth, deceleration, stationary

and death.

♣ In the lag phase virtually no growth occurs and the microbial population remains

relatively constant.

♣ Nevertheless, it is a period of intense metabolic activity as the microbial inoculum

adapts to the new environment. When cells are inoculated into fresh medium they

may be deficient in essential enzymes, vitamins or cofactors, etc., that must be

synthesized in order to utilize available nutrients, prior to cell division taking place.

♣ The chemical composition of the fermentation media influences the length of the

lag phase. It is usually longer if the inoculum was grown up using a carbon source

different from that in the fresh medium, because the cells must synthesize enzymes

required to catabolize the new substrate. Physiological stress may also have an effect,

especially as cells are often transferred from an inoculum medium of low osmotic

pressure (low solute concentration) to fresh medium of higher osmotic pressure (high

solute concentration). Other factors influencing the length of the lag phase are the age,

concentration, viability and morphology of the inoculum. inocula prepared from cells

harvested in the exponential growth phase (period of most rapid growth) exhibit

shorter lag phases than those harvested from subsequent stages.

♣ Once the cells have adapted to their new environment they enter the acceleration

phase. Cell division occurs with increasing frequency until the maximum growth rate

(mmax) for the specific conditions of the batch fermentation is reached. At this point

exponential growth begins and cell numbers/biomass increase at a constant rate.

Mathematically, this exponential growth can be described by two methods; one is

related to biomass (x) and the other to cell numbers (N). For cell biomass, growth can

be considered as an autocatalytic reaction. Therefore, the rate of growth is dependent

on the biomass concentration, i.e. catalyst, that is present at any given time. This

can be described as follows:

Rate of change of biomass is dx/dt= mx (5.1)

where x = concentration of biomass (g/L), m= specific growth rate (per hour) and t=

time (h).

♣ When a graph is plotted of cell biomass against time, the product is a curve with a

constantly increasing slope

Equation 5.1 can also be rearranged to estimate the specific growth rate (m):

m= 1/x * dxldt (5.2)

♣ During any period of true exponential growth, equation 5.1 can be integrated to

provide the following equation: xt=xoemt (5.3)

where xt = biomass concentration after time‘ t, xo = biomass concentration at the start

exponential growth, and e = base of the natural logarithm.

♣ Taking natural logarithms, loge (In), gives In Xt= In Xo + mt (5.4)

♣ This equation is of the form y = c (intercept on y axis) + mx where m = gradient (m

in equation 5.4), which is the general equation for a straight-line graph. For cells in

exponential phase, a plot of natural log of biomass concentration against time, a

semilog plot, should yield a straight line with the slope (gradient) equal to m (Fig.

5.2b), or m= (In xt -In xo)/t (5.5) (note: when plotting log10 values instead of the

natural log, the gradient of the semilog plot is equal to m/2.303; see Fig. 5.2c).

♣ A second approach is to examine growth in relation to cell number, where the

number of cells at the start of exponential growth is No

Consequently, after a period of exponential growth, time (t), the number of cells (Nt)

is given by

Nt = No 2n (5.6)

where n=the number of divisions, No = initial cell number.

♣ Taking natural logarithms gives

In Nt = lnNo+ nln2 (5.7)

♣ Therefore, the number of divisions (n) that have taken place is

given by In Nt -In No n = In2 (5.8)

♣ The number of divisions per unit time during this period of exponential growth is

determined by dividing by the time period (t):

n = lnNt-lnNo t tln2 (5.9)

where n/t = division rate constant (average number of generations per hour).

♣ Often, we are not really interested in the number of divisions per hour, unit time,

but rather in the mean generation time or doubling time (td), that is, the time required

to undergo a

single generation that doubles the population. Thus, td = t = t ln2

n In Nt -In No (5.10)

♣ During exponential growth, when all nutrients are supplied in excess and are

therefore non-limiting, there is a direct relationship between cell numbers and

biomass concentration,

assuming that mean cell size is constant.

♣ This is balanced growth, and a direct relationship between specific growth rate and

doubling time can also be established. However, under conditions where an essential

nutrient becomes limiting, unbalanced growth arises, and variations in cell numbers

(N) and biomass (x) concentration occur, as during the synthesis of cell storage

compounds.

♣ If we consider a situation where at time zero, the cell biomass is xo, then after a

fixed period of time (t) of exponential growth, equivalent to one doubling time (td),

the microbial biomass will double to 2xo, i.e. xt=2xo, when t=td,

♣ Substituting these parameters into equation 5.3 gives

2xo = xoe mt d (5.11)

♣ Taking natural logarithms produces In2xo = In xo + mtd (5.12) or mtd = In 2.

(5.13)

Therefore, in this case td = 0.693 (5.14) m

♣ Equation 5.1, rate of change of biomass (dx/dt=mx), predicts that growth will

occur indefinitely. However, during batch growth the microorganisms are

continuously metabolizing the finite supply of nutrients available in the fermentation

broth. After a certain time the growth rate decreases and eventually stops. This

cessation of growth can be due to depletion of essential nutrients (carbon source,

essential amino acids, etc.) or the build-up of toxic metabolites, such as ethanol and

lactic acid, or a combination of nutrient depletion and toxin accumulation. Monod

showed that growth rate is an approximate hyperbolic function of the concentration of

the growth-limiting nutrient(s) (Fig. 5.3). This impact of essential nutrient depletion

on- growth can be described mathematically by the Monod equation, in a form similar

to that used in biochemistry, where Michaelis-Menten kinetics define the rate of an

enzymecatalysed reaction in relation to its substrate concentration.

m = mmaxS

Ks +S (5.15)

where mmax = maximum specific growth (per hour) of the cells,

i.e. when substrate concentration is not limiting; S = concentration of limiting

substrate (g/L); Ks = saturation constant, concentration (g/L) of limiting nutrient

enabling growth at half the maximum specific growth rate, i.e. m.=1/2 mmax and is a

measure of the affinity of the cells for this nutrient.

♣ When a microorganism is provided with the limiting substrate at a concentration

much greater than the Ks, and with all other nutrients in excess, the microorganism

will grow exponentially at its maximum rate, i.e. when S» Ks, then m.=m.max.

However, as the level of this substrate decreases, it eventually becomes limiting and

can no longer sustain mmax. This is the beginning of the deceleration phase. As the

residual concentration of the limiting substrate approaches Ks and then falls below

this concentration, there is an accompanying gradual decrease in growth rate (m.).

The growth rate of a microorganism with a very high affinity for a rate-limiting

substrate (i.e. a low Ks) will not be affected until the substrate concentration becomes

very low. However, where there is a low affinity for the limiting substrate (i.e. a high

Ks), the growth rate will begin to fall even at relatively high substrate concentrations

and the organism exhibits a longer deceleration phase.

♣ The specific growth rate of the microorganism continues decelerating until all of

the available limiting substrate is metabolized. Growth is no longer sustainable and

the cells enter the stationary phase. At this point, the overall growth rate has declined

to zero and there is no net change in cell numbers/biomass (rate of cell division equals

rate of cell death). However, the microorganisms are still metabolically active,

involved in metabolizing intracellular storage compounds, utilizing nutrients released

from lysed cells, and in some cases producing secondary metabolites. The duration of

the stationary phase varies depending on the microorganisms involved and the

environmental conditions. For cells unable to survive by forming spores, this is

followed by an exponential death phase when the cells die at a constant rate and often

undergo lysis.

29. Primary sources of microorganisms in food:

♣ Internal tissues of healthy plant (fruits and vegetables) and animals (meat) are

essentially sterile.

♣ Yet raw and processed (except sterile) foods contain different types of moulds,

yeasts, bacterial and viruses

♣ Microorganisms get into foods both natural (including internal) and external

sources to which food comes into contact from the time of production until the

time of production

♣ Natural sources of plant origin– surface of the fruits, vegetables and grains

and the pores in some tubers (radishes and onions)

♣ Natural sources of animal origin – skin, hair, feathers, gastrointestinal tract,

respiratory tract and milk ducts in udders of milk

♣ Natural micro flora exist in ecological balance with their hosts and their types

and levels vary greatly with the type of plants and animals

♣ Food can be contaminated with different types of microorganisms coming

from outside sources such as air, soil, sewage, water ,humans, food

ingredients, equipments, package and insects

♣ Microbial types and their levels from these sources getting into foods vary

widely and depend upon the degree of sanitation used during the handling of

foods.

♣ Sources of microorganisms in food is important in order

a) to develop methods to control access of some microorganisms in the

food

b) to develop processing methods to kill them in foods

c) to determine the microbiological quality of foods as well as to set up

microbiological standards and specifications of foods and food

ingredients.

Microorganism in Different Sources

Plants (Fruits and Vegetables)

♣ inside tissues from plants are essentially sterile except porous vegetables

(radishes and onions) and leafy vegetables (cabbage)

♣ plants produce natural metabolites – can limit the presence of microorganism

♣ harbor microorganisms on the surface, type and level vary with the soil

condition, type of fertilizers and water used and air quality

♣ moulds, yeasts, lactic acid bacteria, bacteria such as Pseudomonas,

Micrococcus, Erwinia, Clostridium and Enterobacter

♣ pathogens especially enteric types can be present if the soil is contaminated

with untreated sewage.

♣ Diseases of the plants, damage of the surface before, during and after harvest,

long delays between harvesting and washing and unfavorable storage and

transport conditions after harvesting and before processing can greatly

increase the microbial numbers.

Animals, Birds, Fish and Shellfish

♣ Carry many types of microorganisms in the digestive, respiratory as well as in

the skin, hooves, hair and feathers

♣ Carriers can carry pathogens such as Salmonella spp., pathogenic Escherichia

coli, Campylobacter jejuni, Yersinia enterocolitica and Listeria monocytogenes

♣ Laying birds – suspect of carrying Salmonella enteritidis in ovaries and

contaminating yolk during ovulation

♣ Poor husbandry resulting in fecal contamination on the body surface (skin,

hair, feathers and udder) and supplying contaminated water and feed

(contaminated with Salmonellae) can also change their normal microbial

flora.

♣ Milk – contaminated with fecal materials on the udder surface

♣ Egg shells – contaminated with fecal material

♣ Meat- contaminated with the intestinal contents during slaughtering

♣ Enteric pathogens from fecal materials common are Staphylococcus aureus,

Micrococcus spp, moulds and yeasts.

♣ Prevention

• Effective husbandry

• Testing animals and birds for pathogens and culling the carriers will

be important in reducing incidence in foods

• Using good quality water during slaughter, defeathering, removing

digestive and respiratory organs

• Proper sanitation during processing to keep the microbial quantity

and desirable levels

• Proper cleaning of udder prior to milking

• Immediate cooling of milk after milking

• Fish and marine products should be harvested from polluted and

recommended water.

• Carcasses should be stored in proper temperatures to prevent

further contamination and microbial growth.

Air

♣ Microorganisms are present in dust in air

♣ Do not grow in dust but are transient and variable depending upon the

environment

♣ Their level is controlled by the degree of humid, size and level of dust

particles, temperature and air velocity and resistance of microorganisms to

drying

♣ Dry air with low dust content and higher temperature has a low microbial

level.

♣ Spores of Bacillus spp. Clostridium spp., mold and Gram-positive bacteria

(Micrococcus spp and Sarcina)

♣ Microbial contamination in the air can be reduced by removing the potential

sources, controlling dust particles in the air (using filtered air) using positive

air pressure, reducing the humidity level and installing UV light.

Soil

♣ Soil contains several varieties of microorganisms

♣ Can multiply in soil, their numbers can be very high

♣ Moulds, yeasts and bacteria genera (Enterobacter, Pseudomonas, Proteus,

Micrococcus, Enterococcus, Bacillus and Clostridium) can get into foods from

the soil

♣ Soil contaminated with fecal materials can be source of enteric pathogenic

bacteria

♣ Sediments where fish and marine foods are harvested can also be a source of

microorganisms in those foods

♣ Prevention - removal of soil (and sediments) and avoiding soil contamination

are used to reduce microorganisms in foods

Sewage

♣ sewage when used as fertilizer in crops can contaminate food with

microorganisms

♣ types are enteropathogenic bacteria and virsus

♣ major concern with organically grown foods and many imported fruits and

vegetables where untreated sewage may be used as fertilizers

♣ Prevention – not to use sewage as fertilizers, or should be efficiently treated to

kill the pathogens. Wash foods following harvesting is important

Water

♣ used to produce, process and in some cases store foods

♣ used for irrigation of crops, drinking by food animals

♣ raising fishery and marine products, washing foods, processing

(pasteurization, canning and cooling of heated foods), washing and sanitation

of equipment , processing and transportation facilities

♣ water is used as an ingredient in many processed foods thus can greatly

influence the microbial quality of foods

♣ wastewater can be recycled for irrigation

♣ chlorine-treated potable water should be used in processing, washing,

sanitation and as an ingredient

♣ although potable water does not contain coliforms and pathogens, it can

contain other bacteria capable of causing food spoilage (including

Pseudomonas, Alcaligenes and Flavobacterium)

♣ improperly treated water can contain pathogen and spoilage microorganisms

Humans

♣ between production and consumption foods come in contact with different

people handling the foods

♣ not only people the people working in a food processing plant, but those

handling foods at restaurants, catering services, retail stores and at home.

♣ Source of pathogenic microorganisms in food that later cause foodborne

disease

♣ Improperly cleaned hand, lack of aesthetic sense and personal hygiene, dirty

clothes and hair can be a major sources of microbial contamination in foods

♣ Pathogens such as Staphylococcus aureus, Salmonella spp. Shigella spp.

Pathogenic E. Coli and hepatitis A can be human sources.

Food Ingredients

♣ prepared or fabricated foods many ingredients or additives are included in

different quantities

♣ can be a source of both spoilage and pathogenic microorganisms

♣ various spices can possess very high populations of mold and bacterial spores

♣ starch, sugar and flour can have spores of thermophilic bacteria

♣ prevention: ingredients should be produced under sanitary conditions and

given antimicrobial treatments. Setting up acceptable microbial specifications

for the ingredients will be important in reducing microorganisms in foods

from this source

Equipments

♣ wide variety of equipment is used in harvesting, transportation, processing

and storage of foods

♣ microorganisms from air, raw foods, water and personnel can get into the

equipment and contaminate foods

♣ depend on environment and time, microbes can multiply from low initial

population to reach high level and contaminate large volumes of foods

♣ processing used continuously for a long period of time, microorganisms resent

initially can multiply and act as a continuous source of contamination in the

product

♣ small parts, inaccessible sections and certain materials may not be efficiently

cleaned and sanitized therefore can serve as sources of both pathogenic and

spoilage microorganisms in food

♣ small equipment such as cutting boards, knives, spoons due to improper

cleaning can be source of cross-contamination

♣ Salmonella, Listeria, Escherichia, Enterococcus, Micrococcus, Pseudomonas,

Lactobacillus, Listeria and yeasts and moulds can get into food from

equipment

♣ Proper cleaning and sanitation of equipment at prescribed intervals are

important

♣ Many types of packaging materials are used in food. Since they are used in the

products ready for consumption and in some cases without further heating,

proper microbiological standards (or specifications) for packaging materials

are necessary

30. :