1 Pharmaceutical Microbiology Unit 1st introduction, history of microbiology, its branches

Scope and its importance

Introduction of Prokaryotes and Eukaryotes

Study of Ultra Structure and morphological classification of bacteria

Nutritional requirements, raw materials used for culture media and Physical Parameters for Growth, Growth Curve

Isolation and preservation methods for pure cultures

Cultivation of anaerobes

Quantitative of Anaerobes

Quantitative measurements of bacterial growth

Study of different types of phase contrast microscopy dark fieldmicroscopy and electron microscopy

Introduction

The word MICROBIOLOGY was derived from Greek word MIKROS which means small SMALL

and BIOS which means Life.

for eg-VIRUSES , BACTERIA, FUNGI, ALGAE etc

MICRO-SMALL

BIO-LIVING

LOGY - SCIENCE

HISTORY OF MICROBIOLOGY

Early history of microbiology. Historians are unsure who made the first observations of microorganisms, but the microscope was available during the mid‐1600s, and an English scientist named Robert Hooke made key observations. He is reputed to have observed strands of fungi among the specimens of cells he viewed. In the 1670s and the decades thereafter, a Dutch merchant named Anton van Leeuwenhoek made careful observations of microscopic organisms, which he called animalcules. Until his death in 1723, van Leeuwenhoek revealed the microscopic world to scientists of the day and is regarded as one of the first to provide accurate descriptions of protozoa, fungi, and bacteria.

After van Leeuwenhoek died, the study of microbiology did not develop rapidly because microscopes were rare and the interest in microorganisms was not high. In those years, scientists debated the theory of spontaneous generation, which stated that microorganisms arise from lifeless matter such as beef broth. This theory was disputed by Francesco Redi, who showed that fly maggots do not arise from decaying meat (as others believed) if the meat is covered to prevent the entry of flies. An English cleric named John Needham advanced spontaneous generation, but Lazzaro Spallanzani disputed the theory by showing that boiled broth would not give rise to microscopic forms of life.

Louis Pasteur and the germ theory. Louis Pasteur worked in the middle and late 1800s. He performed numerous experiments to discover why wine and dairy products became sour, and he found that bacteria were to blame. Pasteur called attention to the importance of microorganisms in everyday life and stirred scientists to think that if bacteria could make the wine "sick," then perhaps they could cause human illness.

Pasteur had to disprove spontaneous generation to sustain his theory, and he therefore devised a series of swan‐necked flasks filled with broth. He left the flasks of broth open to the air, but the flasks had a curve in the neck so that microorganisms would fall into the neck, not the broth. The flasks did not become contaminated (as he predicted they would not), and Pasteur's experiments put to rest the notion of spontaneous generation. His work also encouraged the belief that microorganisms were in the air and could cause disease. Pasteur postulated the germ theory of disease, which states that microorganisms are the causes of infectious disease.

Pasteur's attempts to prove the germ theory were unsuccessful. However, the German scientist Robert Koch provided the proof by cultivating anthrax bacteria apart from any other type of organism. He then injected pure cultures of the bacilli into mice and showed that the bacilli invariably caused anthrax. The procedures used by Koch came to be known as Koch's postulates (Figure ). They provided a set of principles whereby other microorganisms could be related to other diseases.

The development of microbiology. In the late 1800s and for the first decade of the 1900s, scientists seized the opportunity to further develop the germ theory of disease as enunciated by Pasteur and proved by Koch. There emerged a Golden Age of Microbiology during which many agents of different infectious diseases were identified. Many of the etiologic agents of microbial disease were discovered during that period, leading to the ability to halt epidemics by interrupting the spread of microorganisms.

Despite the advances in microbiology, it was rarely possible to render life‐saving therapy to an infected patient. Then, after World War II, the antibiotics were introduced to medicine. The incidence of pneumonia, tuberculosis, meningitis, syphilis, and many other diseases declined with the use of antibiotics.

Work with viruses could not be effectively performed until instruments were developed to help scientists see these disease agents. In the 1940s, the electron microscope was developed and perfected. In that decade, cultivation methods for viruses were also introduced, and the knowledge of viruses developed rapidly. With the development of vaccines in the 1950s and 1960s, such viral diseases as polio, measles, mumps, and rubella came under control.

Modern microbiology. Modern microbiology reaches into many fields of human endeavor, including the development of pharmaceutical products, the use of quality‐control methods in food and dairy product production, the control of disease‐causing microorganisms in consumable waters, and the industrial applications of microorganisms. Microorganisms are used to produce vitamins, amino acids, enzymes, and growth supplements. They manufacture many foods, including fermented dairy products (sour cream, yogurt, and buttermilk), as well as other fermented foods such as pickles, sauerkraut, breads, and alcoholic beverages.

One of the major areas of applied microbiology is biotechnology. In this discipline, microorganisms are used as living factories to produce pharmaceuticals that otherwise could not be manufactured. These substances include the human hormone insulin, the antiviral substance interferon, numerous blood‐clotting factors and clotdissolving enzymes, and a number of vaccines. Bacteria can be reengineered to increase plant resistance to insects and frost, and biotechnology will represent a major application of microorganisms in the next century.

Antoni van Leeuwenhoek (1632-1723), a cloth trader from Delft, is the founding father of microbiology.

Microbiology has had a long, rich history, initially centered in the causes of infectious diseases but now including practical applications of the science. Many individuals have made significant contributions to the development of microbiology.

BRANCHES OF MICROBIOLOGY

 branches of microbiology can be classified into pure and applied sciences. Microbiology can be also classified based on taxonomy, in the cases of bacteriology, mycology, protozoology, and phycology. There is considerable overlap between the specific branches of microbiology with each other and with other disciplines, and certain aspects of these branches can extend beyond the traditional scope of microbiology .In general the field of microbiology can be divided in the more fundamental branch (pure microbiology) and the applied microbiology (biotechnology). In the more fundamental field the organisms are studied as the subject itself on a deeper (theoretical) level. Applied microbiology refers to the fields where the micro-organisms are applied in certain processes such as brewing or fermentation. The organisms itself are often not studied as such, but applied to sustain certain processes.Pure microbiology

Bacteriology: the study of bacteria

Mycology: the study of fungi

Protozoology: the study of protozoa

Phycology/algology: the study of algae

Parasitology: the study of parasites

Immunology: the study of the immune system

Virology: the study of viruses

Nematology: the study of nematodes

Microbial cytology: the study of microscopic and submicroscopic details of microorganisms

Microbial physiology: the study of how the microbial cell functions biochemically. Includes the study of microbial growth, microbial metabolism and microbial cell structure

Microbial pathogenesis: the study of pathogens which happen to be microbes

Microbial ecology: the relationship between microorganisms and their environment

Microbial genetics: the study of how genes are organized and regulated in microbes in relation to their cellular functions Closely related to the field of molecular biology

Cellular microbiology: a discipline bridging microbiology and cell biology

Evolutionary microbiology: the study of the evolution of microbes. This field can be subdivided into:

Microbial taxonomy: the naming and classification of microorganisms

Microbial systematics: the study of the diversity and genetic relationship of microorganisms

Generation microbiology: the study of those microorganisms that have the same characters as their parents

Systems microbiology: a discipline bridging systems biology and microbiology.

Molecular microbiology: the study of the molecular principles of the physiological processes in microorganisms

Phylogeny: the study of the genetic relationships between different organisms[4]

Applied microbiologyEdit

Medical microbiology: the study of the pathogenic microbes and the role of microbes in human illness. Includes the study of microbial pathogenesis and epidemiology and is related to the study of disease pathology and immunology. This area of microbiology also covers the study of human microbiota, cancer, and the tumor microenvironment.

Pharmaceutical microbiology: the study of microorganisms that are related to the production of antibiotics, enzymes, vitamins, vaccines, and other pharmaceutical products and that cause pharmaceutical contamination and spoil.

Industrial microbiology: the exploitation of microbes for use in industrial processes. Examples include industrial fermentation and wastewater treatment. Closely linked to the biotechnology industry. This field also includes brewing, an important application of microbiology.

Microbial biotechnology: the manipulation of microorganisms at the genetic and molecular level to generate useful products.

Food microbiology: the study of microorganisms causing food spoilage and foodborne illness. Using microorganisms to produce foods, for example by fermentation.

Agricultural microbiology: the study of agriculturally relevant microorganisms. This field can be further classified into the following:

Plant microbiology and Plant pathology: The study of the interactions between microorganisms and plants and plant pathogens.

Soil microbiology: the study of those microorganisms that are found in soil.

Veterinary microbiology: the study of the role of microbes in veterinary medicine or animal taxonomy.

Environmental microbiology: the study of the function and diversity of microbes in their natural environments. This involves the characterization of key bacterial habitats such as the rhizosphere and phyllosphere, soil and groundwater ecosystems, open oceans or extreme environments (extremophiles). This field includes other branches of microbiology such as:

Microbial ecology

Microbially mediated nutrient cycling

Geomicrobiology

Microbial diversity

Bioremediation: use of micro-organisms to clean air, water and soils.

Water microbiology (or aquatic microbiology): The study of those microorganisms that are found in water.

Aeromicrobiology (or air microbiology): The study of airborne microorganisms.

biotechnology: related to recombinant DNA technology or genetic engineering.

SCOPE AND ITS IMPORTANCE

Scope of Microbiology

Microorganisms are present everywhere on earth which includes humans, animals, plants and other living creatures, soil, water and atmosphere.

Microbes can multiply in all three habitats except in the atmosphere. Together their numbers far exceed all other living cells on this planet.

Microorganisms are relevant to all of us in a multitude of ways. The influence of microorganism in human life is both beneficial as well as detrimental also.

For example microorganisms are required for the production of bread, cheese, yogurt, alcohol, wine, beer, antibiotics (e.g. penicillin, streptomycin, chloromycetin), vaccines, vitamins, enzymes and many more important products.

Microorganisms are indispensable components of our ecosystem. Microorganisms play an important role in the recycling of organic and inorganic material through their roles in the C, N and S cycles, thus playing an important part in the maintenance of the stability of the biosphere.

They are also the source of nutrients at the base of all ectotropical food chains and webs. In many ways all other forms of life depend on the microorganisms.

Microorganisms also have harmed humans and disrupted societies over the millennia. Microbial diseases undoubtedly played a major role in historical events such as decline of the Roman empire and conquest of the new world.

In addition to health threat from some microorganisms many microbes spoil food and deteriorate materials like iron pipes, glass lenses, computer chips, jet fuel, paints, concrete, metal, plastic, paper and wood pilings.

There is vast scope in the field of microbiology due to the advancement in the field of science and technology.

The scope in this field is immense due to the involvement of microbiology in many fields like medicine, pharmacy, diary, industry, clinical research, water industry, agriculture, chemical technology and nanotechnology.

The study of microbiology contributes greatly to the understanding of life through enhancements and intervention of microorganisms. There is an increase in demand for microbiologists globally.

Genetics: Mainly involves engineered microbes to make hormones, vaccine, antibiotics and many other useful products for human being.

Agriculture: The influence of microbes on agriculture; the prevention of the diseases that mainly damage the useful crops.

Food science: It involves the prevention of spoilage of food and food borne diseases and the uses of microbes to produce cheese, yoghurt, pickles and beer.

Immunology: The study of immune system which protect the body from pathogens.

Medicine: deals with the identification of plans and measures to cure diseases of human and animals which are infectious to them.

Industry: it involves use of microbes to produce antibiotics, steroids, alcohol, vitamins and amino acids etc.

Agricultural microbiology – try to combat plant diseases that attack important food crops, work on methods to increase soil fertility and crop yields etc. Currently there is a great interest in using bacterial or viral insect pathogens as substitute for chemical pesticides.

Microbial ecology – biogeochemical cycles – bioremediation to reduce pollution effects

Food and dairy microbiology – try to prevent microbial spoilage of food and transmission of food borne diseases such as botulism and salmonellolis. Use microorganisms to make foods such as cheese, yogurt, pickles and beers.

Industrial microbiology – used to make products such as antibiotics, vaccines, steroids, alcohols and other solvents, vitamins, amino acids and enzymes.

Microbial physiology and Biochemistry – study the synthesis of antibiotics and toxins, microbial energy production, microbial nitrogen fixation, effects of chemical and physical agents on microbial growth and survival etc.

Microbial genetics and Molecular biology – nature of genetic information and how it regulated the development and function of cells and organisms. Development of new microbial strains that are more efficient in synthesizing useful products.

Genetic engineering – arisen from work of microbial genetics and molecular biology. Engineered microorganisms are used to make hormones, antibiotics, vaccines and other products. New genes can be inserted into plants and animals.

Applications of Microbiology

Microbiology is one of the largest and most complex of the biological sciences as it deals with many diverse biological disciplines.

In addition to studying the natural history of microbes, it deals with every aspects of microbe-human and environmental interaction. These interactions include: ecology, genetics, metabolism, infection, disease, chemotherapy, immunology, genetic engineering, industry and agriculture.

The environment:

Microbes are responsible for the cycling of carbon, nitrogen phosphorus (geochemical cycles)

Maintain ecological balance on earth

They are found in association with plants in symbiotic relationships, maintain soil fertility and may also be used to clean up the environment of toxic compounds (bio-remediation).

Some are devasting plant pathogens, but others act as biological control agents against these diseases.

Medicine:

Disease causing ability of some microbes such as

Small Pox (Variola virus)

Cholera (Vibrio cholera)

Malaria (Plasmodium, protozoa) etc.

They have also provided us with the means of their control in the form of antibiotics and other medically important drugs.

Food:

Microorganisms have been used to produce food, from brewing and wine making, through cheese production and bread making, to manufacture of soy sauce.

Microbes are also responsible for food spoilage.

Biotechnology:

Commercial applications include the synthesis of acetone, organic acids, enzymes, alcohols and many drugs.

Genetic engineering – bacteria can produce important therapeutic substances such as insulin, human growth hormone, and interferon.

Research:

Because of their simple structure they are easier to study most life processes in simple unicellular organisms than in complex multicellular ones.

Millions of copies of the same single cell can be produced in large numbers very quickly and at low cost to give plenty of homogenous experimental material.

Because they reproduce very quickly, they are useful for studies involving the transfer of genetic information.



Future of Microbiology

Future challenges such as finding new ways to combat disease, reduce pollution and feed the world's population.

AIDS, hemorrhagic fevers and other infectious diseases

Create new drugs, vaccines. Use the techniques in molecular biology and rDNA to solve the problems

Host-pathogen relationships

Study the role of microorganisms as

Sources of high-quality food and other practical products such as enzymes for industrial application

Degrade pollutants and toxic wastes

INTRODUCTION OF PROKARYOTES AND EUKARYOTES----

Prokaryotic cells comprise bacteria and archaea. They typically have a diameter of 0.1–5 μm, and their DNA is not contained within a nucleus. Instead, their DNA is circular and can be found in a region called the nucleoid, which floats in the cytoplasm. Prokaryotes are organisms that consist of a single prokaryotic cell.

Eukaryotic cells are found in plants, animals, fungi, and protists. They range from 10–100 μm in diameter, and their DNA is contained within a membrane-bound nucleus. Eukaryotes are organisms containing eukaryotic cells.

Prokaryotic Cells

Eukaryotic Cells

Nucleus?

No

Yes

DNA arrangement

Circular

Linear

Size (diameter)

0.1–5 μm

10–100 μm

Unicellular?

Always

Sometimes

Multicellular?

Never

Usually

A prokaryote is a typically unicellular organism that lacks a nuclear membrane-enclosed nucleus. The word prokaryote comes from the Greek πρό (pro, 'before') and κάρυον (karyon, 'nut' or 'kernel').In the two-empire system arising from the work of Édouard Chatton, prokaryotes were classified within the empire Prokaryota.But in the three-domain system, based upon molecular analysis, prokaryotes are divided into two domains: Bacteria (formerly Eubacteria) and Archaea (formerly Archaebacteria). Organisms with nuclei are placed in a third domain, Eukaryota  In the study of the origins of life, prokaryotes are thought to have arisen beforeProkaryotes lack mitochondria, or any other eukaryotic membrane-bound organelles; and it was once thought that prokaryotes lacked cellular compartments, and therefore all cellular components within the cytoplasm were unenclosed, except for an outer cell membrane. But bacterial microcompartments, which are thought to be simple organelles enclosed in protein shells, have been discovered,along with other prokaryotic organelles.While typically being unicellular, some prokaryotes, such as cyanobacteria, may form large colonies. Others, such as myxobacteria, have multicellular stages in their life cycles.Prokaryotes are asexual, reproducing without fusion of gametes, although horizontal gene transfer also takes place.

Molecular studies have provided insight into the evolution and interrelationships of the three domains of life. The division between prokaryotes and eukaryotes reflects the existence of two very different levels of cellular organization; only eukaryotic cells have an enveloped nucleus that contains its chromosomal DNA, and other characteristic membrane-bound organelles including mitochondria. Distinctive types of prokaryotes include extremophiles and methanogens; these are common in some extreme environments.

Both eukaryotes and prokaryotes contain large RNA/protein structures called ribosomes, which produce protein, but the ribosomes of prokaryotes are smaller than those of eukaryotes. Mitochondria and chloroplasts, two organelles found in many eukaryotic cells, contain ribosomes similar in size and makeup to those found in prokaryotes. This is one of many pieces of evidence that mitochondria and chloroplasts are descended from free-living bacteria. The endosymbiotic theory holds that early eukaryotic cells took in primitive prokaryotic cells by phagocytosis and adapted themselves to incorporate their structures, leading to the mitochondria and chloroplasts.

The genome in a prokaryote is held within a DNA/protein complex in the cytosol called the nucleoid, which lacks a nuclear envelope.The complex contains a single, cyclic, double-stranded molecule of stable chromosomal DNA, in contrast to the multiple linear, compact, highly organized chromosomes found in eukaryotic cells. In addition, many important genes of prokaryotes are stored in separate circular DNA structures called plasmids. Like Eukaryotes, prokaryotes may partially duplicate genetic material, and can have a haploid chromosomal composition that is partially replicated, a condition known as merodiploidy.

STUDY OF ULTRA STRUCTURE AND MORPHOLOGICAL CLASSIFICATION OF BACTERIA----

"

When arranged in cuboidal or in different geometrical or packet arrangement.

(vi) Staphylococci:

Arranged in irregular clusters like bunch of grapes.

2. Rod Shaped:

They are also called bacilli and are commonest in microbial world.

ADVERTISEMENTS:

They are of two kinds:

(i) Short rods:

Very short rods, occurring mostly singly.

(ii) Long rods:

ADVERTISEMENTS:

Cylindrical shape, are known as Bacilli or rods, occurring singly, in pairs or in chains.

3. Vibrios:

They are curved rods or comma shaped, their curvature is always less than a half turn.

4. Spirilla:

They are curved or spiral shaped cells, their curvature exceeds that of a half turn. They may be classified as either spirilla or spirochetes.

ADVERTISEMENTS:

Few bacteria actually are flate. For instance, Anthony E. Walsy has discovered square bacteria living in salt ponds. These bacteria are shaped like flate, square to rectangular box about 2 µm to 4 µm and only 0.27 µm thick.

However, some bacteria are variable in shape and have a single characteristic form. These are called Pleomorphic (corynbacterium, Arthrobacter).

Ultra-Structure of Bacterial Cell:

Examination of bacterial cells with electron microscope reveals various component structures. Some of these are outside the cell membrane; others are internal to cell membrane .

Structures Outside the Cell Membrane:

Capsule:

Some prokaryotic organisms secrete slimy or gumy materials (exopolymers) on their surface. A variety of these structures consist of polysaccharides and a few consist proteins. The more general term glycocalyx is also used.

The glycocalyx is defined as the polysaccharide containing material lined outside the cell. Composition of these layers varies in different organisms but can contain glycoprotein and different polysaccharides including polyalcohol and amino sugars

Cocci:

Simplest form of bacteria in which bacterium appears like a spherical cell.

ADVERTISEMENTS:

(i) Micrococci:

When bacterium appears singly.

(ii) Diplococci:

When they appear in pairs of cells.

(iii) Streptococci:

When they appear in chain form.

(iv) Tetrad:

Arranged in square of four.

(v) Sarcinae:

Size of a Bacterial Cell:

There are great variations in size of bacteria. They measure from 0.75 µ to 1.5 µ but on an average each cell of bacterium measures about 1.25 µ to 2 µ (a in diameter. Some times their size varies due to slide preparation.

The smallest rod shaped eubacteria is Dialister Pneum osintes and measures between 0.15 µm to 3.0 µm size. Sulphur bacteria, Thiophysa volutans has diameter of about 18 µm and is considered as largest amongst all bacteria.

NUTRITIONAL REQUIREMENTS, RAW MATERIALS USED FOR CULTURE MEDIA AND PHYSICAL PARAMETERS FOR GROWTH,GROWTH CURVE

--------

For growth and nutrition of bacteria, the minimum nutritional requirements are water, a source of carbon, a source of nitrogen and some inorganic salts. ... Bacteria which derive energy from sunlight are called phototrophs. Those that obtain energy from chemical reactions are called chemotrophs.There are four things that can impact the growth of bacteria. These are: temperatures, moisture, oxygen, and a particular pH.Nutritional requirements for the bacterial growth

Water covers 70% of the total bacterial mass. Two important gaseous constituents in microorganism are oxygen and hydrogen. Other than these, carbon, nitrogen , sulfur and phosphorus are other major elements in the bacterial cell. Potassium, magnesium and calcium are also important elements required for the growth of bacteria. There are several metal ions which are needed in the trace amount which usually acts as a cofactor in many essential enzymatic reactions. These trace elements are not needed to be added with culture media, as they can already been found as a result of contamination of water and other nutrient sources. These trace elements includes metal ions of manganese, cobalt, zinc copper and molybdenum.

ELEMENT% OF DRY WEIGHTCarbon50%Oxygen20%Nitrogen14%Hydrogen8%Phosphorus3%Sulfur1%Potassium1%Magnesium0.5%Calcium0.5%Iron0.2%ManganeseTrace amountCobaltTrace amountZincTrace amountCopperTrace amountMolybdenumTrace amount

Factors affecting growth

pH: Bacteria grow best at their optimum pH. Most of the bacteria can grow in the neutral environment at pH=7 (known as neutrophiles) such as Vibrio cholreae. But some may have optimum pH in acidic region (known as acidophiles) such as Lactobacillus, while others may grow in highly basic regions (Alkaliphiles) such as Agroacterium.

Temperature: Bacteria grow best at optimum temperature. Most of the bacteria shows optimum growth above 35oC, but many have optimum temperature below freezing temperature and also above boiling temperature of water. Some bacteria can only grow at particular temperature and are called as obligate for that temperature, while others may tolerate changes in temperature and thus called, facultative for that temperature or condition.

Bacteria which have optimum temperature below 20oC are known as Psychrophiles. Bacillus globisporus is an obligate psychrophile, while Xanthomonas pharmicola is a facultative psychrophile. Listeria monocytogenes is known to cause spoilage in refrigerated food. T

hose bacteria which shows best growth at temperatures between 25-40oC are known as Mesophiles. Most of the human pathogens are under this category.

Some bacteria shows best growth above 50oC and are known as Thermophiles. Bacillus stearothermophilus   is an example of obligate thermophile. Some archaeobacteria are known to show growth at 115oC.

Oxygen: Bacteria which require oxygen for its growth are known as aerobes. An example of obligate aerobe is Pseudomonas. Those bacteria which cannot grow in the presence of oxygen are known as anaerobes. Clostridium botulinum, C. tetani and Bacteroids are the examples of obligate anaerobes. Microaerophiles such as Campylobacter shows best growth in the presence of very little oxygen.

Bacteria which usually carry aerobic metabolism in the presence of oxygen, but may also grow in anaerobic conditions are known as facultative anaerobes. For example:Staphylococcus and E.

Moisture: Water is required for all the bacteria to grow. They usually gets this water from the moisture present in the environment. in conditions of dryness, bacteria forms spores which can survive by remaining in the dormant stage for long time.

Hydrostatic pressure: It is the pressure exerted by the water, which increases as the depth increases. Some of the bacteria can only survive in deep into the sea under high pressures. Such bacteria are called barophiles. On decreasing pressure, the structure of their enzymes changes and bacteria cannot survive.

Osmotic pressure: Bacterial cell undergo plasmolysis when kept in hyperosmotic environment. When bacterial cells are kept in an environment having lesser osmotic pressure, water moves inside the cell which makes the cell tugid. Due to presence of cell wall, cell does not burst. Those bacteria which so not require high osmotic conditions are nonhalophiles.

Marine bacteria are generally moderate halophiles, which some bacteria like Halobacterium are extreme halophiles.

Radiations: Most of the bacteria cannot survive the radiations such as gamma rays and UV rays. But there are few bacteria which can withstand very high dose of radiations, such as, Deinococcus radiodurans can survive upto 10,000 Grays of radiations.

Nutritional factors: Bacteria requires proper nutrition for the growth. Nutrients which discussed in the starting are required for the proper growth of bacteria.

Peptones and Extracts for Culture Media: The Properties that Matter

A variety of peptones and extracts are available from Merck for producing culture media to be used in a wide range of industrial microbiology applications.

Peptones are a mixture of water soluble polypeptides, peptides, amino acids and other substances remaining after the digestion of protein material. The quality of the peptones is determined by the quality of the selected raw materials, their storage conditions and digestion parameters. Raw materials must stored in a way that prevents the growth of spoilage organisms.

Peptones are used in a wide range of microbiological applications. Applications may require different peptones: A peptone that is suitable for optimal growth of one organism may not be satisfactory for other organisms or for the production of a compound or cell culture. A range of visual, physical, biochemical (USP) and composition properties characterize peptones. However, if the composition, appearance, biochemical, chemical and physical test parameters are satisfactory, the peptone may still be unsuitable for use in culture media if biological tests yield abnormal results. For selecting a cell culture or optimizing its performance, or for fermentation applications it is often necessary to test various peptones at different concentrations.

An extract is an infusion, i.e. the water soluble fraction obtained by soaking a substrate in water for a period of time, followed by filtration to clear the solution. The substrates are often digested by a weak proteolysis with pancreatin (of porcine origin) before being filtered and concentrated. For high-yield fermentation, many media need to be supplemented with extracts.

Industrial Microbiology

Raw Materials & Supplements

Dehydrated Raw Materials

Supplements for Culture Media

All Dehydrated Raw Materials Products

All Industrial Microbiology Products

All Products

Peptones, Extracts and Agar-Agar

Biological Raw Materials for Culture Media

Biological raw materials (peptones, extracts, agar-agar), core ingredients of many culture media, are available from Merck in a considerable variety of products to meet a wide range of industrial microbiology applications.

All our peptones, agar-agar and extracts enable optimal performance due to our strict quality control and assurance standards. The format in which Merck manufactures most of these raw materials is as dehydrated granules. Working with granulated raw materials produces less dust contamination of the laboratory environment. The granules dissolve quickly to ensure optimal flow properties

growth curve is a graphical representation of how a particular quantity increases over time. Growth curves are used in statistics to determine the type of growth pattern of the quantity—be it linear, exponential, or cubic. Once the type of growth is determined, a business can create a mathematical model to predict future sales. An example of a growth curve is a country's population over time.

A growth curve is a way to visually represent the growth of some phenomena over time, either in the past or into the future or both.

Growth curves are typically displayed on a set of axes where the x-axis is time and the y-axis quantifies the phenomenon in question.

ISOLATION AND PRESERVATION METHODS FOR PURE CULTURE-----

Pure culture, in microbiology, a laboratory culture containing a single species of organism. A pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample manyfold before inoculating the new medium. Both methods separate the individual cells so that, when they multiply, each will form a discrete colony, which may then be used to inoculate more medium, with the assurance that only one type of organism will be present. Isolation of a pure culture may be enhanced by providing a mixed inoculum with a medium favouring the growth of one organism to the exclusion of othersThese methods include refrigeration, paraffin method, cryopreservation, and lyophilization (freeze drying).03-Nov-2016The following points highlight the top four methods used for maintenance and preservation of pure cultures. The methods are: 1. Refrigeration 2. Paraffin Method 3. Cryopreservation 4. Lyophilisation.

Method # 1. Refrigeration:

Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic activities of the microorganisms are greatly slowed down but not stopped.

ADVERTISEMENTS:

Thus their growth continues slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the microbes after sometime.

Method # 2. Paraffin Method:

This is a simple and most economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture and stored upright at room temperature.

The layer of paraffin ensures anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure culture to remain in a dormant state and, therefore, the culture is preserved for several years.

Method # 3. Cryopreservation:

Cryopreservation (i.e., freezing in liquid nitrogen at-196°C) helps survival of pure cultures for long storage times. In this method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol, that prevent the formation of ice crystals and promote cell survival.

Method # 4. Lyophilisation (Freeze-Drying):

ADVERTISEMENTS:

In this method, the culture is rapidly frozen at a very low temperature (-70°C) and then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and their metabolic activities are stopped; as a result, the microbes go into dormant state and retain viability for years.

Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in refrigerators. Freeze- drying method is the most frequently used technique by culture collection centres.

CULTIVATION OF ANAEROBES----

An anaerobic bacteria culture is a method used to grow anaerobes from a clinical specimen. Obligate anaerobes are bacteria that can live only in the absence of oxygen. Obligate anaerobes are destroyed when exposed to the atmosphere for as briefly as 10 minutes. Some anaerobes are tolerant to small amounts of oxygen. Facultative anaerobes are those organisms that will grow with or without oxygen. The methods of obtaining specimens for anaerobic culture and the culturing procedure are performed to ensure that the organisms are protected from oxygen.

Anaerobic bacterial cultures are performed to identify bacteria that grow only in the absence of oxygen and which may cause human infection. If overlooked or killed by exposure to oxygen, anaerobic infections result in serious consequences such as amputation, organ failure, sepsis, meningitis, and death. Culture is required to correctly identify anaerobic pathogens and institute effective antibiotic treatment.

Precautions

It is crucial that the health care provider obtain the sample for culture via aseptic technique. Anaerobes are commonly found on mucous membranes and other sites such as the vagina and oral cavity. Therefore, specimens likely to be contaminated with these organisms should not be submitted for culture (e.g., a throat or vaginal swab). Some types of specimens should always be cultured for anaerobes if an infection is suspected. These include abscesses, bites, blood, cerebrospinal fluid and exudative body fluids, deep wounds, and necrotic tissues. The specimen must be protected from oxygen during collection and transport and must be transported to the laboratory immediately. The health care team member who performs the collection should follow universal precautions for the prevention of transmission of bloodborne pathogens.

QUANTITATIVE MEASUREMENTS OF BACTERIAL GROWTH---

two most common classroom methods to determine bacterial growth are the Standard Plate Count (SPC) technique and turbidimetric measurement. Examples of other methods include: microscopic count, membrane filter count, nitrogen determination, cellular weight determination, and biochemical activity measurement

Spectrophotometry is an indirect method for calculating cell concentrations by measuring the changes in turbidity. Bacteria can also be counted by using the plating method, which is based on the number of colonies formed in Petri dishes containing specific growth media

As bacteria are unicellular and divide asexually the growth of the population can be followed either by the changes in number of cells or weight of cell mass. Examples of methods are turbidimetric measurements, direct microscopic count or viable count.

As bacteria are unicellular and divide asexually the growth of the population can be followed either by the changes in number of cells or weight of cell mass. Examples of methods are turbidimetric measurements, direct microscopic count or viable count.

A suitable standard for measuring microbes is the micrometer which is six times smaller than a meter (one-millionth of a meter). There are 106 µmeters in one meter, and it is these units that are used to measure the size of bacteria. Typically, bacteria range from about 1 µm to about 5 µms.

STUDY OF DIFFERENT TYPES OF PHASE CONSTRAST MICROSCOPY, DARK FIELDMICROSCOPY AND ELECTRON MICROSCOPY------

Phase-contrast microscopy is particularly important in biology. It reveals many cellular structures that are invisible with a bright-field microscope, as exemplified in the figure. These structures were made visible to earlier microscopists by staining, but this required additional preparation and death of the cells. The phase-contrast microscope made it possible for biologists to study living cells and how they proliferate through cell division. It is one of the few methods available to quantify cellular structure and components that does not use fluorescence.After its invention in the early 1930s, phase-contrast microscopy proved to be such an advancement in microscopy that its inventor Frits Zernike was awarded the Nobel Prize in 

When light waves travel through a medium other than a vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength-dependent and may give rise to colors. Photographic equipment and the human eye are only sensitive to amplitude variations. Without special arrangements, phase changes are therefore invisible. Yet, phase changes often convey important information.

UsesMicroscopic observation of unstained biological materialInventorFrits ZernikeManufacturerLeica, Zeiss, Nikon, Olympus and othersModelkgtRelated itemsDifferential interference contrast microscopy, Hoffman modulation-contrast microscopy, Quantitative phase-contrast microscopy

Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.As bacteria are unicellular and divide asexually the growth of the population can be followed either by the changes in number of cells or weight of cell mass. Examples of methods are turbidimetric measurements, direct microscopic count or viable count.

DARK FIELDMICROSCOPY--

Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

In optical microscopes a darkfield condenser lens must be used, which directs a cone of light away from the objective lens. To maximize the scattered light-gathering power of the objective lens, oil immersion is used and the numerical aperture (NA) of the objective lens must be less than 1.0. Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the NA. Often these objective lenses have a NA that is variable from 0.7 to 1.25.

ELECTRON MICROSCOPY--

Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological specimens. It is used in biomedical research to investigate the detailed structure of tissues, cells, organelles and macromolecular complexes. The high resolution of EM images results from the use of electrons (which have very short wavelengths) as the source of illuminating radiation. Electron microscopy is used in conjunction with a variety of ancillary techniques (e.g. thin sectioning, immuno-labeling, negative staining) to answer specific questions. EM images provide key information on the structural basis of cell function and of cell disease.  

There are two main types of electron microscope – the transmission EM (TEM) and the scanning EM (SEM). The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc) through which electrons can pass generating a projection image.   The TEM is analogous in many ways to the conventional (compound) light microscope.  TEM is used, among other things, to image the interior of cells (in thin sections), the structure of protein molecules (contrasted by metal shadowing), the organization of molecules in viruses and cytoskeletal filaments (prepared by the negative staining technique), and the arrangement of protein molecules in cell membranes (by freeze-fracture).

THANK YOU

FOR MORE UPDATES

MAIL ME - rv110824@gmail.com