Determination of Cell Mass for Measurement of microbial Growth

 The growth results into synthesis of new cellular material. Therefore, it results into the increase in cell mass. Hence, methods used to determine cell mass can be useful to measure the growth.

  Further, every cell possesses a specific amount of cell mass. Hence, this can be also useful to find out probable number of organisms present in the growth culture.

Criteria used to determine cell mass

Every cell possesses a specifie chemical composition, dry weight as well as the volume. Hence various methods based on determination of chemical constituents of cell, volume and dry weight are applied to measure cell mass.

1]. Determination of cellular nitrogen and protein content

  • Every cell possesses a fixed proportion of protein and nitrogen. To determine them, cells are first harvested, washed and then used for quantitative determination.
  • Commonly micro Kjeldahl method is used to measure cellular nitrogen. This method is much laborious.
  • Nitrogenous matter of the medium is also estimated together. Therefore this method is not used for routine cell mass determination.

2] Determination of cellular nucleic acid content

  • Like nitrogen and protein, each bacterial cell possesses a fixed proportion of RNA and DNA. Hence their estimfuion can also be a useful criterion to measure cell mass.

3] Determination of cellular phosphorus and sulphur contents

  • Phosphorus and sulfur are other major elemental constituents of cell, apart from carbon and nitrogen. They also occur in a specific proportion in cell.
  •  Hence, chemical estimation of cellular phosphorus and sulfur can be useful to measure cell mass.

4] Determination of dry weight of cell

  • This is one of the highly useful methods to measure growth. However, it can be used only when cell population is much dense in the culture.
  • The cells are separated from culture by centrifugation or filtration, washed thoroughly and then dried in oven at 50-55°C. Then their dry weight is determined.
  • However in some instances, dry weight of cells may not be indicative of living matter.
  • E.g. Bacteria can accumulate large amount of poly B hydroxy butyric acid as reserve material during late log phase without causing any increase in other cellular constituents. It may account for 70% of total dry weight in some instances.

5] Determination of packed cell volume (PCV)

  • Each bacterial cell belonging to a particular species has a specific volume. Increase in cell mass and cell number results in to overall increase in cell volume.
  • Hence, methods determining cell volume of bacteria can be used to measure growth.
  • This is achieved by centrifugation of a fixed volume of growth culture and then the settled cell mass is measured as packed cell volume.

6] Determination by turbidimetric method

  • Another method of measurement of cell mass is an optical method, by determining turbidity resulting due to growth in broth cultures.
  • When, a beam of light is allowed to pass through a suspension of bacteria, the bacterial particles coming in the path of light cause scattering and absorption of light depending upon the cell density as per Beer-Lambert's law.
  • Therefore amount of light transmitted through the culture suspension is reduced.
  • The reduction in the amount of transmitted light, therefore, depends on the cell density or cell mass in the culture suspension.
  • It is measured as % transmittance or optical density (O.D.) by the use of photoelectric colorimeter or spectrophotometer or densitometer.
  • Usually the O.D. is measured at 540 nm wavelength of light. This is because at this wavelength light absorption by medium will be maximum and hence it will give more correct value of O.D. of the bacterial suspension.
  • Another method of measuring cell density by applying same principle is through use of Nephelometer.
  • Nephelometer does not measure transmitted light, passing through the culture suspension, but it measures intensity of light scattered by bacterial particles.
  • The diagrammatic presentation of measurement of cell density by both photoelectric colorimeter and nephelometer is shown in  figure below.

Diagrammatic presentation of measurement of cell density by photoelectric colorimeter.
  • a] Adjustment of instrument to read zero optical density by use of uninocculated culture medium as "blank" tube. 
  • b] Measurement of optical density of the growth tube, more the turbidity, greater the optical density.

Advantages and disadvantages of methods for determination of cell mass

  1. Of all the methods, cell mass determination by turbidimetric method is most convenient and reliable.
  2. Other methods especially involving determination of cell constituents such as cellular nitrogen, protein nucleic acids, phosphorous or sulfur has only academic value. In routine, these methods are complex, and impractical. Media constituents may interfere with estimation.
  3. The methods provide information about total cell mass; both living and non-living.

Electronic count by coulter counter

Electronic count by coulter counter

This is a highly sophisticated technique for counting bacterial cells present in the culture. The method is named so after its Inventor. 

The electronic device consists of tube having small orifice of about 10aua30 mm in diameter, which connects two compartments of the counter, which contain conductive medium. 

  The interior and exterior of the tube is connected with electrodes. The conductive medium completes the circuit.

  When, bacteria pass through the small orifice, the conductivity between two compartment drops momentarily.

  This is electronically recordcd in the counter. Thus as the bacteria pass through the orifice from one compartment to the other, each time they give electronic pulse to be counted. Thus the method involves a much rapid and accurate count.

The other modification of the technique is the use of a photodiode. Near the orifice, a photodiode and sensor are placed on the opposite sides. 

  As the organism passes through the orifice, it obstructs the path of light. This is sensed by the sensor and recorded in the counter.

Figure : Electronic coulter current counter method determining bacterial cell numbers. 

  1. Flow of the growth medium through a tube is allowed through a narrow capillary tube with a small orifice. Both ends of the tube are connected to electronic circuit through electrode. 
  2. Through the orifice only one cell is able to pass at a time. Passage of cell through orifice causes momentary drop in conductivity, which is gives electronic pulse, to be counted.

Limitations of electronic count method

1. However, the method has got a major limitation that it cannot distinguish between dead or living organisms as well as dust particle. Every particulate matter can give rise to pulse to be counted as the organisms. 

  2. Many times if the organisms are larger than the diameter of the orifice, they may plug into It. Similarly clumps of bacteria cannot pass through. Hence the specimen should be initially treated to separate the clumps and then used for the count.

Microbiology of Air: Sources of Micro-organisms in Air

  Air is the mixture of gases that makes up the Earth's atmosphere. It is a mixture of about
- 78% Nitrogen,
- 21% Oxygen,
- 0.9% Argon,
- 0.04% Carbon dioxide and very small amounts of other gases.
Air also contains the variable amount of water vapours.

  Air microbiology is concerned with the study of living microbes in the air. They are usually referred to as bioaerosols. Atmosphere is actually unsuitable for growth of microorganisms due to extreme temperature variations, solar radiations, lack of nutrients, low amount of available water etc.

  Therefore, the number of microorganisms in air is less than in the other natural environments. However, there is still a large enough number that they can affect the atmosphere.

Microbes in air have an opportunity to travel long distances with the help of wind. They are ecologically significant because they can be associated with disease in humans, animals and plants.

  Most of the organisms are found in the lower region of atmosphere. Atmosphere is usually occupied by those forms of microorganisms that are resistant to adverse conditions in the air. They can be bacterial spores or cysts, capsulated bacteria, fungal spores, enveloped viruses etc.

Sources of Micro-organisms in Air

  • Entry of microorganisms in air takes place through various ways.   
  • Natural sources such as soil, lakes, oceans, animals and humans.
  • Unnatural sources such as sewage treatment plants, animal rendering, fermentation processes and agricultural activities.
  • Soil is one of the source.
  • Wind blow disturbs soil surface and liberates the soil microorganisms into the air.
  • These microorganisms remain suspended in air for long time.
  • Manmade actions like plugging and digging.
  • Plant and animal surfaces.
  • Large water bodies like oceans and bays.
  • Water droplets or aerosols produced by wind or tidal actions at surface of these water bodies.
  •  Most droplets are produced from top 0.1 mm water surfaces called as 'micro layer'.
  •  It consists of many more micro-organisms than from deep layers.
  • Bubbles from surface add thousands of microbes in air.
  • Most significant source is the man himself. Organisms from oral, nasal, rectal passages of man and animals come in air.
  • Human activities like coughing, sneezing, laughing and even talking.
  • Such biological aerosoles may spread bacteria upto distance of about 15 feet.
  • Infected persons release pathogens.
  • Respiratory pathogens are drought resistant and can survive in air for long period.
  • Air in the hospital is a site where microbial load is always very high.
  • Many human infections are caused by microorganisms from atmosphere.
  • For example - Epidemics of Legionnaires disease (a severe form of pneumonia caused by bacterium Legionella) were due to aerosols generated from contaminated water of air conditioning equipments.
  • Aerosols formed by high speed drills in dental clinics add a large population of bacteria.
  • Various industrial, agricultural, municipal are responsible for the generation of bioareosols.
  • For example - Sprinkler irrigation of crops and forest land with effluent from sewage treatment plants, trickling filter beds in sewage treatment plants, slaughter houses, spray washing processes etc.

Infectious dust, Droplets & Droplet Nuclei

(i) Infectious dust:

  • Dust in air arises from sand, soil, ash, lint from bedding, clothing, carpets.
  • It usually contains all saprophytic organisms.
  • Dust loaded with saprophytic organisms is harmless for human. But if it is loaded with air borne pathogens, it is called as 'infectious dust.
  • Air borne pathogens are added to the dust by body secretions.
  • When secretions are dried , pathogens remain in dust and form infectious dust.
  • Nasal or throat secretions on the handkerchief eventually dry and leave some residual material.
  • During sneezing or coughing, large aerosol droplets get expelled and settle on floor or bed clothes.
  • Moisture content of these droplets evaporates leaving behind residue.
  • These residues are then disturbed during the handling of handkerchiefs, sweeping of floor, bed making This makes the dust infectious.
  • If it is inhaled by healthy persons, they may get infected.

(ii) Droplets:

  • Sneezing or coughing activity of respiratory tract infection persons expels (exhales) millions of micro droplets of saliva and mucus along with varying number of micro-organisms.
  •  Such particles are called as droplets. Thus, droplets contain thousands of living micro- organisms along with saliva and mucus.
  • Most of the droplets are larger in size (0.1 mm in diameter).
  •  Such droplets settle rapidly, within shorter distance from their source of origin before drying.
  • Even if such droplets are inhaled by a healthy person before their settling, they are immediately trapped in nasal baffles and in the nasopharynx material and are prevented from reaching upto lungs.
  • Thus, droplets are not that much dangerous as far as the spread of infection is concerned.

(iii) Droplet Nuclei :

  •  In warm and dry atmosphere, small droplets are dried due to evaporation of moisture content leaving behind dried mucous with live bacteria.
  • They are called as 'droplet nuclei'. They are very much small in size with diameter less than 0.1 mm.
  • Such droplet nuclei can remain in air for longer period of time (upto hours to days).
  •  The dried mucus protects the organisms present at the center.
  • They are also carried away to a longer distance.
  • If these are inhaled by health persons, they directly enter into the lungs, without capturing in mechanical traps of nasal baffles and mucus of nasopharynx.
  •  The organisms from droplet nuclei directly settle in the alveoli of lungs.
  • Chances of spread of infection via droplet nuclei increases when people are crowded together.
  • Frequency of air borne infections is more in winter when people prefer to live in crowd places.

   In modem days, the chances of spreading infection are increased because of modern urban areas like buses, aero planes, restaurants and bars which are always crowded.
 The diseases caused by Klebsiella pneumoniae, Streptococcus pneumoniae and Mycobacterium tuberculosis may spread through droplet nuclei.

Sampling Methods for Microbiological Examination of Air

Sampling Methods for Microbiological Examination of Air

Microorganisms in air cause a problem of contamination in food industries, milk processing plants, industrial processes, pathological and research laboratories and mainly in operation theatres.

   Therefore, measurement of no. of organisms in air at such places is important. Microorganisms in air may be present in free form or in the form of clumps or chains.

  They may adhere to the dust particles or to saliva or to mucus of man. Several methods have been developed for the microbiological examination of air.

  Simplest method is exposure of sterile nutrient medium plates to air for different time periods The plates are then incubated during which the settled organisms grow and form colonies.

  The number of organisms can be calculated from the number of colonies. But this gives a rough idea about the numbers and kinds of microorganisms in air. Because the volume of air coming in contact with plate is not known.

  Therefore, the advanced techniques like solid impaction, liquid impingement, filtration, sedimentation, centrifugation, electrostatic precipitation, thermal precipitation etc. are used. Among these, solid impaction and liquid impingement techniques are most commonly used.

1. Solid Impaction (Sieve Device) :

  • Number of sampling devices which work on the principle of impaction of airborne particles.
  • In these, an aspirator sucks a known volume of air which collides with nutrient agar in plate.
  • This causes the microbes in the air to stick to the surface of an agar medium.
  • The plates are then incubated at specific temperature for a specific time to form the colonies.
  • These are the most often used methods of detection and enumeration of microbes in air.
  • The most widely known device that is based on the solid impaction is the Sieve Device.
  • The sieve device is of two  types : Single Stage, Multistage Device

a). Single Stage Device :

  • It is mechanically very simple.
  • It consists of a metal box with a metal cover.
  • The cover has a large number of evenly spaced small holes.
  • Under this a petriplate containing agar medium can be kept.
  • A measured volume of air is drawn through these hole. The particles containing microorganisms are allowed to settle on the agar surface.
  • Plates are then incubated for specific time. Then microbial colonies on agar surface are counted.

Andersen's Air sampler & Single Stage Sieve Device

b). Multistage Device :

  • It consists of perforated plates with progressively smaller holes at each stage.
  • Allows particles to be separated according to size.
  • Best known multi-stage samplers is Andersen's air sampler
  • In this, air is drawn through a series of six circular plates each perforated with 400 holes.
  • The plates in series have progressively smaller holes.
  • Therefore, the largest particles being deposited in the first while the smallest in last petri dish.
  • Viable particles can be collected on a variety of bacteriological agar media.
  • Incubated for counting and identification.
  • All the particles collected are sized aerodynamically and can be directly related to human lung deposition.

Advantages of Sieve Device

  1. The volume of air to be analyzed can be measured.
  2. An air is drawn with high speed. Therefore, the particles of smaller size that might be escaped can also be settled on agar surface.
  3. There will be even distribution of air sample on agar surfaces that results in formation of isolated colonies.
  4. This is helpful to take a colony count.
  5. The methods are very simple and efficient.
  6. Therefore, they are most widely used.

2. Liquid Impingement Device :

  • In this, a known volume of air is passed in a sterile broth medium, where micro-organisms from air are trapped.
  • The aliquots of broth are then used for plating.
  • The plates are incubated at room temperature for specific time and the colony count is taken to determine the number of micro-organism.
  • There are several devices, among which the most important one is Bead Bubbler Device.

Bead Bubbler Device

  • It consists of 250 ml capacity suction flask with side arm linked to suction pump and flow meter.
  • The flask is filled with sterile nutrient broth.
  • The sterile glass beads of size 5 mm in diameter are also placed in the flask.
  • A glass bubbler is fitted in the flask with rubber stopper.
  • Diameter of glass bubbler is approximately 14 mm.
  • It has many small openings at the base with3 mm diameter.
  • A suction flask is added with sterile nutrient broth medium along with sterile glass beads.

  • The bubbler is fitted with rubber stopper.
  • With help of suction pump and flow meter, known volume of air is allowed to pass through medium.
  • Air enters into broth medium in the form of bubbles.
  • Because of glass beads bubbles are formed and the clumps or chains of organisms are broken down.
  • Aliquots of medium are used to culture on solid agar medium & plates are incubated at room temperature.
  • Colony count is taken & number of organisms is determined.


  1. Volume of air to be analyzed can be measured
  2. Because of glass beads and bubble formed, the micro-organisms from the clumps and chains are separated from each other.
  3. Thus exact count can be drawn.

Viruses Cause Cancer

  According to Viral Gene Hypothesis, certain viruses are responsible for the oncogenesis. Virus which are causes cancer called oncogenic virus. First evidence of a relationship between viruses and cancer was obtained by demonstration of transmission of leukemia by cell free filtrate of blood of diseased chickens to healthy chickens. 

  In 1911, Rous also transmitted chicken sarcoma from diseased chicken to healthy one. The viral etiology of various cancers in animals like fishes, dogs, mice, rats, rabbits, frog, horses etc. is clearly evidenced. 

  Many viruses are found to be associated with many cancers in humans also. Viruses that are capable of producing cancer are called 'oncogenic viruses' or 'tumor viruses'.

There are two classes of tumor viruses as DNA and RNA tumor viruses. A common characteristic of both these viruses is that the viral genome is some way becomes integrated into the host cell DNA and replicates along with host cell chromosome.

I] DNA Tumor Viruses :

There are five groups of DNA viruses causing cancer in humans. They include -
  a. Papova
  b. Adeno
  c. Pox
  d. Herpes and
  e. Hepatitis viruses.

Among these, Papova viruses have been received more attention because they are known to cause tumors in a variety of animals.

a) Papova Viruses :

  • Name papova is derived from first two letters of three viruses as Papilloma, Polyoma and Simian vacuolating 40 virus ( SV 40 virus).
  • They belong to the papovaviridiae family.
  • Papovaviridiae family includes two genera - Papiloma viruses and Polyoma viruses
  • SV40 virus is now placed in the genus Polyoma viruses.
  • All papova viruses are small, naked, icosahedral, circular double stranded DNA viruses.
  • They are commonly found in humans and other mammals.
  • Their size ranges from 45nm to 55nm in diameter. 
  • They contain 5 to 7 genes. The icosahedral capsid is constructed from 2 to 3 polypeptides

i) Papilloma virus :

  • Small, non enveloped, icosahedral, circular, ds DNA viruses.
  • They are about 55 nm in diameter.
  • Their capsid contains 72 capsomers.
  • Capsomers made from two types of proteins as L,(Major)and L2(Minor).
  • These are wart causing viruses.
  • Natural infection results in formation of benign warts in humans and other animals.
  • These viruses induce tumor only in natural host.
  • They transform normal cells into cancer cells in vitro very rarely.
  • Warts produced by these viruses are usually benign But they may convert into malignant carcinomas.
  • Also found to be associated with human penile, uterine and cervical carcinomas.
  • They replicate exclusively in the keratinocytes.
  • Keratinocytes form the outermost layer of the skin as well as some mucosal surfaces of the genitals, anus and mouth.

ii) Polyoma Viruses :

  • Name polyoma refers to multiple (poly) tumors (oma) producing viruses.
  • They are also small, naked, circular, ds DNA viruses, about 45nm in diameter.
  • Capsid is made from three proteins as - VP1, VP2 and VP3.
  • These three proteins form pentameric capsomers.
  • There are about 72 pentameric capsomers.
  • These viruses also encode a protein called 'T antigen'. The polyoma viruses do not induce tumors in the natural hosts like monkey and mouse.
  • For example - Simian vacuolating virus (SV 40) replicates in the kidneys of monkeys ( natural host ) without causing any disease.
  • However, the SV 40 virus cause the sarcomas in the hamster. They also transform the cells in vitro.
  • The SV 40 virus is most thoroughly studied oncogenic DNA virus.
  • It is a monkey polyoma virus.
  • It can't induce tumor in monkey (natural host) but induce tumors in experimental hamsters as well as rodents.
  • The two other examples of human polyoma virus are JC and BK virus.

JC Virus - It causes progressive multifocal leukoencephalopathy – damage or inflammation of white matter of brain at multiple locations. Symptoms include- weakness, paralysis, loss of vision, impaired speech, coma and death.

BK virus - Associated with renal diseases in kidney transplanted patients.

II] RNA Tumor Viruses :

  • Oncogenic RNA containing viruses are called "OncoRNA viruses'.
  • They are classified as 'retroviruses' because the viral RNA produces intermediate DNA by using reverse transcriptase enzyme.
  • From this DNA copy, viral RNA are produced which are accommodated in viral capsid while DNA copies are integrated into host genome.
  • Retroviruses have been isolated from many vertebrates like fishes, reptiles, birds and mammals.
  • They cause leukemia and sarcoma in chicken while lymphoma and mammary carcinoma in mice.

Based on the host range and disease type, there are five categories of Oncorna viruses They include -
a) Avian leukosis complex -

  • e.g. Rous sarcoma virus of chickens.

b) Murine leukosis virus -

  • e.g. Murine leukemia & sarcoma virus.

c) Mammary tumor virus of Mice -  

  • e.g. Bittner virus that causes breast cancer and multiplies in mammary glands. It is transmitted to the offsprings through the breast milk. 

d) Leukosis sarcoma virus -

  • It produces sarcoma in the animals such as cat, hamster, guinea pigs and monkey. 

e) Human T cell lymphotrophic virus ( HTLV) -

  • It causes T cell lymphoma.
  • There are two types of HTLV as HTLV type 1 and HTLV type 2.
  • Type Icauses adult T cell lymphoma and Type 2 causes hairy cell leukemia.
  • In hairy cell leukemia - the bone marrow has difficulty in producing enough normal cells like WBCs, RBCs and platelets.

Retrovirus :

  • Retroviruses are icosahedral with envelope. They have diploid genome i.e. they carry two identical segments of RNA genome.

  • RNA molecules are plus sense single stranded. Consists of 3 important genes- gag, env & pol.  
  • Name retroviruses refers to participation of reverse transcriptase in their multiplication. 
  • Multiplication of these viruses requires the transcription of RNA into DNA and its subsequent integration into a host genome. 
  • Only an integrated viral DNA can be transcribed into viral RNA.

Retrovirus replication

  • Replication of positive sense ssRNA occurs as shown in figure.
  • Virus multiplication is not lytic and does not kill the cell. 
  • Integrated genome of the retroviruses replicate along with host genome and therefore, it is present in all cancer cells. 
  • Oncogenic development is due to the expression of a viral gene known as 'src' gene. 
  • Base sequence of viral 'src' gene is similar to that of the host cell gene whose product plays a role in the regulation of growth of the normal cells. 
  • This suggests that the oncogenes of retroviruses are of cellular origin. Thus, the retroviruses cause the cancer as they introduce the new genetic material into host DNA and cause transformation.
  • Three theories have been proposed which are concerned with the oncogenesis by RNA viruses in human beings. 
• These are
  a). Oncogene theory
  b). Protovirus theory and
  c). Provirus theory

a) Oncogene Theory :

Oncogene Theory is Proposed by Huebner & Todaro.

  • According to this, the genetic information for cancer is present in each of the body cell and it is transmitted from parent to offspring through the germ cells.
  • Infection of body cells by RNA viruses has occurred millions of years ago during evolution.
  • Therefore, every cell is assumed to contain oncogene i.e. viral gene responsible for oncogenesis. 
  • This oncogene is normally kept in repressed condition (prevented from functioning by the regulatory genes of the host cell). 
  • When the oncogene becomes derepressed either by a physical, chemical or biological agent, it gets expressed itself by the formation of a transforming protein. 
  • This protein could change the normal cell into a malignant one although no virus could be recovered from it.

b) Protovirus Theory :

Protovirus Theory was Proposed by Temin

  • According to this, certain DNA regions of normal cells are transcribed to RNA & reverse transcribed to DNA by reverse transcriptase enzyme. 
  • Such newly formed DNA segments get back integrated into original DNA either at the same site or at different sites.
  • This leads to the amplification of genetic regions.
  • In this flow of genetic information from DNA to RNA & back to DNA, the genetic information becomes independent of cell genome. 
  • At this stage with or without effect of mutations, the protoviruses may arise which would direct synthesis of RNA tumor viruses or alternatively the integration of newly formed DNA could produce new gene alignment leading to oncogenesis.
  • Thus, this theory postulates that the normal back & forth flow of genetic information during amplification process, with or without the effect of mutation or due to alignment of new gene sequences may generate RNA tumor viruses or genes necessary to induce oncogenesis.

c) Provirus theory :

This theory was Proposed by Spiegelman

  • It states that some cancers are caused by direct infection with oncogenic viruses. 
  • It further states that tumor viruses are transmitted horizontally from individual to individual entering in provirus states. 
  • They may cause the cancer as a result of chance activation of their transforming genes.
  • Occasionally, they may be transmitted vertically i.e. from one generation to other. 
  • Thus, this theory is different from the oncogene and protovirus theory which state that the spontaneous mutation in animals and humans cells may cause oncogenic events. 
  • Among these three hypothesizes, the oncogene hypothesis is consistent with number of observations. They include -
  • Spontaneous intracellular appearance of oncogenic RNA viruses after repeated sub-culturing of cells.
  • This gave no indication of previous virus infection.
  • The appearance of oncogenic RNA viruses upon exposure to oncogenic agents in the cells.
  • The demonstration of the occurrence of oncogenic viral genetic material in the cells by hybridization studies.

Pasteurization of Milk : Temperature, Types, Advantages and Disadvantages

 Pasteurization process developed in 19th century in France by Louis Pasteur, for the preservation of wine. Its application resulted in coining a new term 'Pasteurization'.
  Louis Pasteur was pioneer in its use for the preservation of wine Dr. Soxhlet of Germany introduced this process for preservation of milk in 1886.

Definition of Pasteurization

Pasteurization refers to - The process of heating of each and every particle of milk to at least 62.8° C (145°F) for 30 min. or 71.7° C (161ºF) for 15 seconds or to any temperature-time combination, which is equally efficient, in an approved and properly operated equipment.
  After pasteurization the milk is immediately cooled to 5°C or below. The term pasteurization is mainly applied to the market milk today.

Objectives of Pasteurization :

• To render the milk safe for human consumption by destruction of all pathogenic microorganism.
• To improve the keeping quality of milk by destruction of almost all spoilage organisms.
• Thus, the pasteurization does not kill all micro-organisms in milk and therefore, it is not a method of milk sterilization but it is a process of disinfection.
• Pasteurization is also intended to inactivate some enzymes in the raw milk.

Relationship Between Time & Temperature :

• The efficiency of pasteurization depends upon time-temperature relationship.
• Initially the pasteurization temperature was set to kill Mycobacterium tuberculosis, which was supposed to be the most heat resistant organism in milk.
• This organism gets destroyed at a temperature of 140°F in 10 minutes.
• Thus, the initial temperature of pasteurization was set to 143°F (61.6°C) for 30 min.
• Later on, it was discovered that another pathogen namely Coxiella burnetii (causative agent of Q fever), also occurs in the milk and it is able to survive at the temperature of 143ºF.
• Thus, to ensure the destruction of Coxiella, temperature of pasteurization was raised to 145°F (62.8°C).

Methods of Pasteurization

There are three methods of Pasteurization in common practice.
1. Low temperature holding (LTH) 2. The high temperature short time (HTST)
3. Ultra high temperature (UTH).

1. Low Temperature Holding  (LTH) :

• This is also called as low temperature long time (LTLT) method or batch or vat pasteurization method.
• In this process, the milk is heated to 62.8° C(145°F) for 30 minutes and promptly cooled to 5° C or below.
• It is a batch method and thus it takes a little more time. The heating of milk in this method is carried out by three different types of pasteurizers -

i]. Water-Jacketed Vat -

• This is double walled tank in which hot water or steam under partial vacuum circulates for heating and cold water for cooling.
• The outer wall is usually insulated to reduce heat loss.
• The heat exchange takes place through the wall of the inner lining.
• The milk is agitated by slowly moving paddles or propellers.
• When heating, the vat cover is left open for escape of off - flavours, and when holding, the cover is closed.

ii]. Coil Vat Type -

• The heating or cooling medium is pumped through a coil placed in either a horizontal or vertical position.
• Such coil is turned through the milk.
• The turning of coil agitates the milk.

iii]. Water Spray Type -

• A film of hot water is sprayed over the surface of the tank holding the milk.

2. High Temperature Short Time Method (HTST) :

• It is the modern method of pasteurization of milk
• It is used for large volume of milk.
• In this method, the milk is heated to 71.7°C(161ºF)for 15 second and cooled promptly to 5°C or below. This method gives the continuous flow of milk.
• Milk is heated either in tubes or in thin metal plates or in heat exchangers by electricity or hot water.
• Raw cold milk is allowed to enter in heat exchanger and hot, pasteurized milk is taken out.
• It is comparatively faster method. There are different designs of HTST pasteurization system.

Essential Components of a Standard HTST System

1. Raw milk tank : It is a tank where chilled raw milk is stored
2. Balance tank : It maintains a constant head for the incoming milk
3. Milk feed pump : It creates suitable pressure that is necessary for efficient flow
4. Flow control system : It controls the flow rate of milk
5. Filters and clarifiers : They remove the dirt (if any) from the milk
6. Homogenizer : It divides fat globules into micro globules to avoid fat separation in standing milk.
7. Plate Heat Exchanger (PHE) with regeneration section, heating section, holding section and cooling sections : It facilitates an efficient pasteurization
8. Flow diversion valves :
•They ensures that all the conditions for pasteurization have been met.
• It diverts the milk to head tank if it is not properly heated to pasteurization temperature for reprocessing.
• If milk is properly pasteurized milk, it passes forward through the flow diversion valves into the regeneration where it is cooled.

Plate Heat Exchanger

• The plate heat exchanger is a compact and simple unit.
• Its plate may be used for regeneration, heating, holding and cooling.
• A space of approximately 3 mm is maintained between plates by a non-absorbent rubber gasket.
• These plates are designed to provide a uniform turbulent flow of milk with rapid heat transfer.

Regeneration (heating) -

• The raw incoming milk is partially and indirectly heated by the hot outgoing milk.
• This reduces the cost of HTST process.
For example -
• Milk entering at 4°C
• Heated in regenerator to 34°C
• Heated in heating section to 74°C
• Cooled in regenerator to 44°C
• Cooled in cooling section to 4°C

• Here, the increase from 4 °C to 34°C is a change of 30°C and the decrease from 74°C to 44°C is also a change of 30°C.
• Without regeneration, the milk would need to be heated by hot water/steam from 4°C to 74°C, a difference of 70°C
• With regenerative heating, however, hot water or steam need not be used for the temperature change between 4°C and 34°C.
• This temperature change is brought about by use of the outgoing hot milk.
• Thus, here regeneration saves the heat.

• On the other hand, without regeneration the milk would need to be cooled by chilled water from 74°C to 4°C, a difference of 70°C.
• With regenerative cooling, however, chilled water need not be used for the temperature change from 74°C to 44°C, a difference of 30°C.
• This temperature change is brought about by use of cold incoming milk.
• Here also the savings due to regeneration takes place.
• The milk to be heated flows across one side of the plate and heating or cooling medium flows across the other side in the opposite direction.

Advantages of Regenerative Heating :

• Utilization of the incoming chilled milk to cool the outgoing hot pasteurized milk increases the efficiency of the PHE.
• Smaller amount of energy is required to heat the milk to pasteurization temperatures since the heating does not start from 4°C of the chilled milk.
• Reduces the amount of time required to pasteurize milk.

Advantages of HTST :

• The milk can be pasteurized quickly and effectively
• Initial cost of the method is less.
• Milk packaging can be started as soon as pasteurization begins.
• This permits more efficient utilization of labour for packaging and distribution.
• The system can be easily cleaned and sanitized Lower operating cost.
• Reduced milk losses. Development of thermophiles is not a problem.

Disadvantages of HTST :

• This system is not well adopted for small quantities milk or milk products.
• Gaskets require constant attention for possible damage and lack of sanitation.
• Complete drainage is not possible.
• Raw milk with high thermoduric bacterial count is not efficiently pasteurized as compared to LTH method.

3. Ultra High Temperature Method (UHT) :

• Milk is heated at 135ºC to 150°C (average 141°C) for just two seconds or no hold time.
• It can be carried out by the direct or indirect means of heat treatment
• Direct treatment is either injection of steam into milk or infusion of milk into a steam chamber
• Indirect treatment includes the use of heat exchangers.
• The success of method depends on immediate aseptic packaging. • The temperature and time combination is much more lethal to bacteria and kills all bacteria

UHT Pasteurization by Steam Injection Technique :

• In this technique, the steam is injected into the milk.
• The steam at a pressure higher than that of the pressure of milk flow is injected into the milk stream through a suitable nozzle. • There are many different injector designs.
• In all of these, the steam and milk are kept in thermally separate zone until they reach the mixing zone.

UHT Pasteurization by Steam Infusion Technique :

• It is very similar to injection system, except for the method of mixing of milk with steam.
• The infusion is done by dropping heated milk into a steam pressure vessel with a conical base.
• The heating is instant
• The milk is then cooled rapidly by evaporative cooling with exposure to a slight vacuum.
• This removes the water that is added to the milk due to the condensation of the steam.
• Steam infusion can be used to pasteurize a variety of dairy products like cream and special dairy products but now it is popular for milk.

Pasteurized Milk

• Packaging of ultra-pasteurized milk is also carried out under sterile conditions to prevent the recontamination with spoilage bacteria.
• Ultra-pasteurization makes milk free of spoilage and harmful bacteria, but it is not considered sterile and, thus, it requires refrigeration.
• Ultra-pasteurized milks have more "cooked" flavor as compared to conventionally pasteurized milks.
• Average shelf-life of ultra-pasteurized milk products is 30-90 days if held under refrigeration and in packed condition
• Once an the product is opened, it may become contaminated with spoilage bacteria. Thus, after opening, ultra-pasteurized milk should be kept well refrigerated and consumed within 7-10 days for best quality and taste.

Advantages of Pasteurization :

1. Pasteurized milk is safe for consumption.
2. Pasteurization is designed to decrease microbial count.
3. Pasteurization has little effect on the nutritive value of milk.
4. There is some loss of vitamin C and B group vitamins, but this is insignificant.
5. With Pasteurization, keeping quality of milk remains unaltered. 6. Pasteurization does not reduce the fat content of milk.

Disadvantages of Pasteurization :

1. Cooked taste may be developed, for which consumer may complain.
2. Pasteurization reduces cream layer of the milk.
3. Vitamin content may get changed. 

Properties of Immunogenicity

  Immunogenicity is an substance that induces an immune response and antigen is any substance that is capable of binding specifically to the components of the immune system such as to the antibodies.
Nowadays the terms antigen and immunogens are used interchangeably by immunologists.

Properties of a substance which determine its immunogenicity

  Immunogenicity of a substance is determined by following four main properties.

  • Foreignness
  • Molecular size
  • Chemical complexity and
  • Stability or degradeability

1). Foreignness

By the term foreign we mean non-self. Our immune system is able to distinguish between self and non-self. To be immuno genic the substance should be genetically foreign to the host. It should be different from the hosts own substances.

  Immunogen's are mostly peptides or proteins in chemical composition. If the peptide of immunogens have similar nature of the peptides of host organism, there will be no immune response against the antigen.

  For example bovine serum albumin (BSA). It is a protein which is found in all cows. So if BSA is injected from one cow into another there will be no immune response, because the protein is identical and for the immune system of every cow.

This BSA is a self molecule but if the BSA is injected into a rabbit an immune response is induced. This is because BSA is foreign to rabbits immune system. So for a molecule to be immunogenic it must be recognised as foreign by the immune system. More foreign and molecule is more is its immunogenicity.

2) Molecular size

  Second property which determines the immunogenicity of a substance is its molecular size. It has been found that relatively small substances have decreased immunogenicity whereas large substances have increased immunogenicity.

  • The most potent immunogens are molecules with molecular weight above 10,000 Dalton's.
  • Highly immunogenic molecules have a molecular weight of 1 lakh Dalton's or more.
  • Molecules with weight than 10,000 Dalton's are weakly immunogenic or not immunogenic at all.
So greater the molecular weight more immunogenic it is.

3). Chemical Composition and Molecular Complexity

  Antigens can be carbohydrates, lipids, nucleic acids and proteins chemically but their immunogenicity varies.

Carbohydrates are immunogenic only if they have relatively complex polysaccharides structure or when associated with protein carriers. For example blood group antigens.

Lipids and Nucleic acids are not immunogenic by themselves but they become immunogenic when they are conjugated to protein carriers. Proteins are generally the powerful immunogens and this is because of their molecular complexity in size.

The molecular complexity of proteins is a consequence of the variety of units known as amino acids of which proteins are made of. Besides having number of different amino acids proteins have four levels of protein structure.
- Primary structure
- Secondary structure
- Tertiary structure and
- Quaternary structure.
This also contributes to the immunogenicity of a molecule.

Let's take an example, A homo polymer of an amino acid which has a molecular weight of 30,000 Dalton's. Now if we look at its molecular weight it should be immunogenic but this is not the case because of the absence of chemical complexity. Now a copolymer which is composed of different amino acids in its molecular weight is more than 10,000 Dalton's. So it is immunogenic.

Thus molecules with complex nature are more immunogenic when compared to simple molecules.

4). Stability or Degradeability

  To stimulate immune response a substance should bind with B-cell receptors or T-cell receptors. B cells interact with the substance on its own that is via its B-cell receptors.

But T-cell receptors do not interact with the substance as a whole they interact with a distinct portion of the substance which are short peptides. These small molecules are result of antigen processing and presentation.

  Large insoluble macromolecules generally are more immunogenic than small soluble ones. Because larger molecules are more readily degraded and processed and presented to the immune system.

This also explains why proteins are excellent immunogens on the other hand highly stable and non degradable substances are not immunogenic. For example silicon. Thus to be immunogenic a substance must be degradable, highly stable and non degradable substances are not immunogenic.