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General Microbiology

Principles of Microbial Nutrition and Growth

Dr. Sunita Aggarwal Department of Microbiology Institute of Home Economics

New Delhi – 1100 16

17 May 2007 CONTENTS GrowthGrowth of microbial population (Growth curve)Measurement of microbial growthFactors influencing the growthCulture mediaLife cycle of different microorganisms Sulfolobus Escherichia coli Bacillus Chlamydomonas Ectocarpus Rhizopus Saccharomyces Neurospora Plasmodium Paramecium Keywords Binary fission, Budding, Fragmentation, Growth curve, Generation time, Colony Forming Units, Cardinal temperatures, Psychrophiles, Psychrotrophs, Thermophiles, Hyperthermophiles, Acidophiles, Neutrophiles, Alkalophiles, Obligate aerobes, Microaerophiles, Facultative anaerobes, Aerotolerant, Obligate Anaerobes, Barotolerant,Barophiles,Photolithoautotrophs, Photoorganoheterotrophs, Chemolithoautotrophs, Divisome, Endospore, Zoospore, Aplanospore, Hypanpospore, Palmella Stage, Isogamous, Anisogamous, Oogamous, Heterothallic, Calyptogametes, Gymnogametes, Isogamy, Homothallic, Zygospore, Pleurilocular sporangium, Unilocular sporangium, Microgamete, Macrogamete, Coenocytic, Sporangiophore, Oidia, Sporangiospore, Ascospore, Microconidia, Macroconidia, Trichogyne, Perithecium, Digenetic, Gametogony, Sporogony, Schizogony, Metacryptozoites, Schuffner’s granules, Merozoites, Exflagellation, Ookinete, Oocyst, Conjugation, Exconjugate, Autogamy, Cytogamy, Endomixis.

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Growth

Growth refers to proportional increase in the quantity of all the major constituents in an organism. Since microorganisms are so small that it is difficult and not easily possible to quantify the increase in its major constituents, growth in their context refers to increase in the number of cells or microbial population. In higher eukaryotes, growth refers to the development of an individual organism. Microbial growth is closely associated with their reproduction and they reproduce more rapidly as compared to complex organisms. Reproduction in prokaryotic microorganisms

There are a number of ways by which microorganisms increase in their population (Fig. 1). These are - (i) Binary Fission

Binary fission is the most common method of asexual reproduction in prokaryotes. It is a symmetrical process in which the cells grow in size and divide in two halves to form two daughter cells of almost equal size.

Fig. 1: Types of reproduction in microorganisms In dividing bacteria, generally two or more copies of its DNA are present per cell. This is because of continuous synthesis of DNA, that is required due to slower replication rate of DNA as compared to cell division. With the continuous DNA replication, it is ensured that each of the

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daughter cell acquires a complete copy of the genome. This is in contrast to eukaryotes where DNA replication occurs at a particular phase of the cell cycle, i.e. S phase. With few exceptions, in most of the prokaryotes, progeny cells usually separate after cell division. However, various types of cell arrangements are formed in many microbes as the daughter cells remain together (Fig. 2) e.g. In Staphylococcus cells form grape like clusters.

Fig. 2: Cell arrangement in microorganisms

(ii) Budding

It is an asymmetric process where a small protrusion called the bud arises from the parent cell. The bud receives the cytoplasm and nucleus from the parental cell and pinches off from it to form an individual organism after growth (Fig. 1). Examples include some types of Gram negative bacteria, yeasts, etc. (iii) Fragmentation

It is a process which involves breaking of a long filament or a trichome into fragments (Fig.1). Each fragment then grows to become an individual organism. It is also a type of asymmetric division. Examples include some photosynthetic bacteria. (iv) Spores or conidia

Reproductive spores or conidia are produced by the parental organism (fig.1), which on germination give rise to another individual e.g. Actinomycetes. Reproduction in eukaryotic microorganisms

Contrary to prokaryotes, the genome in eukaryotes is present in the nucleus. Multiplication of the cell involving a complex cell cycle include four phases : G1, S, G2, and M (Fig. 3). Duration of these phases vary depending on the species. Of these, while DNA replication occurs only in S or synthesis phase, synthesis of most of the cellular materials, doubling of cytoplasm and

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preparation for nuclear division occur during the gap periods (G1 and G2). Nuclear division occur during M phase which is followed by cytokinesis, resulting in the formation of two daughter cells. In many cases such as fungi cytokinesis may not occur after mitosis and results in a multinucleated cell.

Fig. 3: Cell cycle in eukaryotes Growth of microbial population (Growth curve)

Microbial growth can be studied by analysing the growth curve. When microbes are is cultivated in batch culture or closed system (where nutrients in the medium are supplied only once at the beginning of the growth and there is no replenishment thereafter) it passes through four distinct stages (Fig. 4). These are:

i. Lag phase ii. Log or exponential phase

iii. Stationary phase, and iv. Death or decline phase

All these four phases together constitute a growth curve.

i. Lag phase

Lag phase is the period during which no increase in the cell number or mass occurs. Though the cells do not divide, these are physiologically active, adapting themselves to the environmental conditions. The duration of the lag phase varies depending on the existing environmental conditions, the state of the bacterial cells and their previous growth conditions e.g. if the bacteria from old culture is used for growth, the lag phase is longer as cells take time to repair cellular components and replenish intracellular materials. When the vigorously growing culture is used for inoculation of the same culture medium, the lag phase is shorter or even absent.

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Fig. 4: Bacterial growth curve

ii Log or exponential phase

Once the lag phase is over, the cells start dividing, with an increase in their number and mass. During exponential phase, the cellular metabolic activities are at their peak. Rate of growth remains constant and the cell number becomes double with every cell division. Mathematically, it can be represented as –

Nt = No 2n

Where Nt is the number of microbes at time t, No is the number of microbes at time zero and n

is the number of generations. During log phase the cell number increases exponentially and the curve between time and log

of number of cells is a straight line. The time taken by a cell to divide is termed as generation time ‘g’. It varies with the type of organism and the environmental conditions e.g. under favourable conditions, generation time for E.coli is 20 minutes whereas for Mycobacterium tuberculosis, it is more than 12 hours.

Exponential phase is a phase of balanced growth, i.e., all the cellular constituents are formed

at constant rate and the population is uniform in terms of chemical and physiological properties.

The rate of growth is constant during exponential phase. It increases with the increase in

concentration of a limiting nutrient and then reaches a plateau.

iii Stationary Phase

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Exponential phase cannot continue indefinitely because of the following reasons which operate in concert:

a. Exhaustion of essential nutrients in the medium. b. Accumulation of toxic (exuded) cellular metabolites in the environment or medium. c. Achieving the level of critical population conditioned by the above two reasons.

These factors eventually lead to decrease in population growth and the growth curve becomes horizontal. This marks the beginning of the stationary phase, during which there is no net increase in the population size. Here, the number of cells formed equals the number of cells that die. The population size is at its peak during stationary phase. It varies with the type of microorganisms, the nutrient availability and other environmental conditions. Physiological changes also start appearing in the cell e.g. formation of inclusion bodies, compromised cellular metabolism, disintegration or lysing of the cell wall, etc. The duration of stationary phase also varies. It may be very short lived in many microorganisms while in others it may persist for an extended period of time.

iv. Death or decline phase

Just like exponential phase, stationary phase also cannot persist indefinitely and the microbial population eventually enters into a decline phase. This is due mainly to a continuous depletion of cell nutrients and accumulation of toxic products. The cells no longer being able to repair the cell constituents, maintain the integrity of cell wall and cell membrane and can not function properly. The rate of cell death exceeds the rate of cell formation and this leads to a gradual decrease in the population size. Though the rate of death varies largely with the environmental conditions and the microbial type, decline in the population occur exponentially as it happens during log phase. For some microbes, all the cells in a population die within few hours whereas others may take days to die out completely. Few microbes do form endospores or cysts to escape death. For many microbes, the death rate may also decrease after the population size has reduced drastically because of the presence of resistant cells.

Measurement of microbial growth

Microbial growth can be analyzed by measuring their cell numbers, cell mass or any other cellular component. Different methods can be employed for this purpose. Each method has its own advantages and disadvantages. The most appropriate method and approach depends on the experimental situation. The various methods are – i. Measurement of cell number by

a. Total count, and b. Viable count using plate counts or membrane filters

ii. Measurement of cell mass by a. Dry weight method, and

b. Turbidometric method

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iii. Biochemical analysis of cellular components and metabolic products.

Measurement of cell number

Total count

It is simple, inexpensive and quick method which gives the count for both viable and nonviable cells. Here, the cell number in a sample is determined by direct counting of cells under microscope using counting chamber like Petroff - Hausser counting chamber and hemocytometer (Fig. 5). The number of cells in the sample is determined by considering the volume of the chamber and the dilution of the sample used. The method gives the idea regarding size, shape and arrangement of cells in a population. However, it is not possible with these methods to distinguish between dead and viable cells, with these methods.

Fig. 5: Measurement of cell number by Petroff- Hausser Chamber Besides counting chambers, electronic counters such as Coulter Counter can also be used to get the total count. Microbial sample is passed through the orifice. Each time the cell passes through the hole, there is a drop in conductivity which is counted as the number of cells. However, presence of debris particles also interfere with the counting of bacteria in this case.

Viable count

Viable count give the number of cells in a population, which are capable of reproducing. Plating technique is a simple and widely used approach to obtain viable counts. It can be performed in two ways – Spread plate and Pour plate method. In the Spread plate method, 0.1 ml of the appropriate dilution of the sample is spread uniformly over the solid agar surface and the surface colonies are obtained on incubation. Pour plate method involves mixing of 0.1 or 1 ml of the appropriate dilution of the sample with warm molten agar in a sterile plate with a gentle swirling motion. Both surface and subsurface colonies are formed in the Pour plate method. Either of the two methods can be used for plating. Generally, Spread plates gives higher counts as compared to Pour plate method.

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Special colony counters can be used for accurate counting of colonies which appear on spread and pour plates (Fig. 6). The results are expressed in the form of Colony Forming Units (CFU) per ml. Mathematically, CFU/ml can be expressed as – Colonies present on the Plate X Dilution Factor CFU/ml = -------------------------------------------------------------------- Volume sampled Dilution factor is the reciprocal of the dilution used to calculate CFU/ml. Only those plates, which have colony number between 30 and 300 are considered for calculations. Membrane filters can also be used for viable count. In this case sample is passed through the filter which is then placed on nutrient agar plate or on a pad soaked with liquid media. Each cell forms a separate colony after incubation (Fig. 6). Membrane filter can also be used for obtaining total count by staining the cells on filter by fluorescence dye and observing the same under epi fluorescence microscope.

Fig. 6: Viable count by plating technique

Measurement of Cell Mass

Dry weight method One way to determine microbial growth is to measure the cell dry weight, as balanced growth results in an increase in total cell mass. The cells growing in liquid media are collected by centrifugation or filtration, washed and dried in oven. The weight of the dried mass is then taken. The method is commonly and generally used for fungus.

Turbidometric Method

The growth of microbes makes the broth turbid or cloudy. Turbidity depends on the microbial concentration. When the concentration of bacteria is around 107 cells/ml, the medium is slightly turbid. Further increase in bacterial concentration increases the turbidity, which can be measured by using spectrophotometer. The culture medium is kept in the spectrophotometer and a beam of monochromatic light is passed through it (Fig.7). The interference to the incident beam due to scattering or absorption of light by the bacterial cells is interpreted in terms of percent transmission or absorption. Mathematically it can be represented as –

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Io/It = % Transmission Where Io is the intensity of beam entering the broth and It is the intensity of beam leaving the broth.

Optical Density (O.D) = 2 – log%T As the bacterial concentration increases, turbidity also increases and %T decreases with increase in optical density. Microbial count is measured with the help of previously made calibration curves. The method is simple and does not require any incubation. Results are obtained immediately provided population is large enough to give desirable turbidity.

Fig. 7: Measurement of cell mass by turbidometric method

Biochemical Analysis

During balanced growth, there is a proportional increase in all the characteristic cellular constituents and metabolic products like DNA, RNA, Peptidoglycan, amino acids, alcohol etc. of a cell. These cellular components can be used as indicators of cell growth, as the total quantity of the cell constituent would be directly related to the total microbial cell mass e.g. cells can be analyzed for protein or nitrogen. An increase in microbial population will be reflected in higher concentration of total protein level. Similarly, growth can also be determined by measuring the depletion of various nutrients e.g. carbon during the growth.

Factors influencing the growth

The growth of the microorganisms is affected both by the physical and chemical factors in the environment and also on availability of nutrients. Microorganisms can grow over a wide range of environmental conditions and even in extreme inhospitable environments, where most of other organisms get killed. Microorganisms growing in such harsh conditions are called extremophiles. The various factors which influence the microbial growth are:

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i. Temperature

Environmental temperature dramatically influences the microbial growth by affecting the enzyme catalyzed reactions. Each microorganism has a characteristic minimum, maximum and optimum temperature - called cardinal temperatures for growth (Fig. 8).

Fig. 8: Relation of growth rate to temperature No growth occurs below the minimum or above the maximum growth temperature. Below the minimum temperature, cell membrane solidifies inhibiting enzyme activity. As the temperature increases above minimum level, rate of chemical reaction increases (almost doubles with every 10oC rise in temperature) which makes the microorganisms grow faster. Maximum growth occurs at optimum temperature above which rise in temperature slows down the growth. High temperatures denature enzymes and proteins and disrupt the cell membrane. Optimal temperature is always closer to the maximum temperature. Cardinal temperatures vary with the microorganisms. Microorganisms as a whole can grow at a temperature as low as –20oC to above 120oC e.g.Geogemma sp. (archaebacteria) can grow at 121oC. Very high temperature and high pressure is required to completely kill all bacterial cells. The principle is used in autoclaves for sterilization. Based on the temperature requirement, microorganisms can be categorized into five groups (Fig. 9) – a. Psychrophiles

These are the organisms whose minimum growth temperature is less than 0oC, optimum around 15oC and maximum 20oC. Example – Polaromonas vacuolata. Psychrophiles can be isolated from Arctic and Antarctic habitats. b. Psychrotrophs or facultative psychrophiles

These are low temperature growing microorganisms with optima at 20-30oC and maximum at 35oC .

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c. Mesophiles

These are the microorganisms that require a minimum temperature of 15-20oC, optimum temperature around 20-45oC and maximum about 45oC for their growth. Example - E. coli and most of the human pathogens. d. Thermophiles Thermophiles are the organisms having minima around 45oC and optima between 55-65oC. Maximum temperature is 80oC. It includes habitats like hot springs, compost, etc. Most of the thermophiles are prokaryotes and few are algae and fungi. Example - Bacillus stearothermophilus. e. Hyperthermophiles

Hyperthermophiles grow at very high temperature with maxima above 100oC. These do not grow below 55oC and have growth optima between 80oC and 113oC. Example - Pyrococcus abyssi, Pyrodictium occultum

Fig. 9: Temperature ranges for microbial growth ii. pH

pH indicates the concentration of hydrogen ions H+ in the solution and is represented as negative logarithm of H+ concentration. Its’ value extends from zero to seven in the acidic region and from seven to fourteen in the alkaline range.Neutral solutions have pH seven. Like temperature, microorganisms can grow over a wide range of pH, i.e., from pH of value zero to around pH of 10, though each microorganism has a definite pH range for its growth. Different microbial groups also have specific pH preferences. With few exceptions, most of the bacteria and protozoa generally grow near neutral pH, i.e., 6.5 to 7.5. Whereas fungi and yeasts prefer acidic environments (pH 2 to 4), algae grow better in slightly acidic surroundings. These microbial growth conditions and specificities form the foundation of the science of food preservation and industrial food processing. Depending upon the pH requirement, microorganisms can be grouped as: -

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a. Acidophiles

These thrive well at very low pH values with optima between pH 0 and 5.5. e.g. Cyanidium caldarium and Sulfolobus acidocaldarius – grow at acidic pH of 1 to 3 of hot springs. Ferroplasma acidarmanus and Picrophilus oshimae are growing around pH of zero. b. Neutrophiles

These organisms prefer pH in near neutral range.These can grow within a pH range of 5.5 to 8 e.g. Staphylococcus aureus. c. Alkalophiles

These organisms grow well within pH range of 8.5 to 11.5. Extreme alkalophiles have optima at pH 10 or higher e.g. Bacillus alcalophilus. Microorganisms often change pH of their surrounding by producing metabolites, e.g. acid is produced during fermentation. Thiobacillus reduces sulfur to produce sulfuric acid. Some microbes produce ammonia and make the surrounding alkaline. Inhibition to growth due to changes in pHcan be prevented by adding buffers in the medium. Commonly used buffer is a phosphate buffer.

iii. Oxygen concentration

Majority of microbial groups vary in their requirement for atmospheric oxygen. Algae are always aerobic. Most of fungi are normally aerobic except some like the yeast. Bacteria and Protozoa may or may not use oxygen for growth depending upon the species. Microorganisms can be categorized into five categories on the basis of the use of oxygen (Fig.10). These are as follows: a. Obligate aerobes

Oxygen is a must for their growth. It is used as terminal electron acceptor for electron transport chain during aerobic respiration and also in synthesis of sterols and unsaturated fatty acids by eukaryotic microbes Example – Micrococcus luteus. b. Microaerophiles These organisms need oxygen for growth at a level of 2 to 10%. Exposure to 21% atmospheric level of oxygen damage these organisms. Example – Campylobacter. c. Facultative anaerobes These are the organisms which can grow both in presence or absence of oxygen. However, growth is better when the oxygen is present. Depending on the presence of oxygen, these use either the aerobic respiration or fermentation for generating energy. Example – E.coli. d. Aerotolerant anaerobes These organisms do not use oxygen even if it is present. They grow equally well in the presence or absence of oxygen. Example – Enterococcus faecalis, Lactobacillus plantarum.

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e. Strict or obligate anaerobes These do not need oxygen at all and in fact die if it is present. Example – Bacteroides, Fusobacterium, Clostridium, Methanococcus, etc. Both aerotolerants and strict anaerobes generate energy by fermentation and anaerobic respiration.

Fig. 10: Bacterial growth in relation to oxygen requirement The presence of oxygen is detrimental to some microorganisms because oxidation inactivates many enzymes and produce toxic products like superoxide, hydrogen peroxide and hydroxyl radicals, etc. which readily destroy cellular constituents. Some microorganisms possess enzymes to nullify these toxic products like superoxide distmutase, catalase, peroxidase and provide protection to the organism. iv. Water activity and osmotic pressure

Water is essentially required by microorganisms, as most of the nutrients are taken by them in a soluble form. Availability of the water in a substance is reduced by binding of water to the solute molecules and also by its adsorption to the solid surface . The degree of water availability can be expressed as water activity (aw) which is defined as the vapor pressure of the substance to the vapor pressure of the pure distilled water, i.e., aw = Vapor pressure of a substance (Psoln) Vapor pressure of water (Pwater ) Water activity of the pure distilled water is 1.0, which reduces with addition of salts and sugar. Most of the microorganisms generally require aw values between 0.9 and 1.0, though fungi and yeasts have a lower aw as compared to bacteria. Water activity is inversely related to osmotic pressure. With few exceptions, most of the microorganisms do not tolerate high osmotic pressure. These are osmophiles and osmotolerants which can grow over a wide range of water activity. Whereas osmophiles require high osmotic

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pressure for growth osmotolerants do not require high osmotic pressure but can withstand it, e.g. Staphylococcus aureus can grow in environment with upto 3M NaCl. Some microorganisms are halophiles (salt loving), i.e., require high concentration of sodium salts (20-30%) in environment, e.g., Halobacterium is present in dead sea and salt piles along seacoast. It makes the salt piles bright pink. Halophiles have adapted to their environment because of modified cell membrane, protein structure and high amount of potassium ions required for the stability of enzymes, ribosomes, transport and other proteins. v. Pressure

Many microorganisms living in deep sea are called barophiles and barotolerants which can withstand a pressure of more than 400-500 atmospheres. Barophiles need high pressure for growth. Increased pressure does adversely affect barotolerants but not as much as it does the nonbarotolerant bacteria. Barophiles have been found among several bacterial genera, e.g., Photobacterium, Shewanella, Colwellia. Many archaea are thermobarophiles e.g. Pyrococcus sp,. Methanococcus jannaschii.

vi. Light

Presence of sunlight is another requirement for many microorganisms to grow. The microorganisms that require light for growth are called phototrophs (photo-light, troph- attracted to). Intensity as well as the wavelength of light exert important effects on the microbial growth. Different microorganisms harvest light of different wavelengths depending on their photo pigments. vii. Nutrients

Besides physical factors, presence of nutrients also affect the growth of microorganisms. These elements or nutrients are grouped into two types- macro- and micro- elements depending on the amount in which these are required by a cell. a. Macroelements

Macroelements usually make up about 95% of the dry weight of the cell and are required in large amounts. These include carbon, oxygen, hydrogen, nitrogen, sulphur, phosphorus, potassium, calcium, magnesium and iron. The most abundant element in the cell is carbon, which makes the backbone of all the macromolecules, i.e., carbohydrates, lipids, proteins and nucleic acids. It constitutes about 50% of the cell and is obtained either as CO2 as the sole carbon source (autotrophs) or in the form of reduced, complex organic molecules (heterotrophs). Other five elements, i.e., N, O, H, S and P are also important elements in constituting the lipids, nucleic acids, carbohydrates and proteins. Macroelements can be assimilated either in inorganic or organic form as their salts or in solution depending upon the microbial type. Potassium and magnesium are required for the activity of enzymes. Calcium gives heat resistance to endospores and Iron forms the part of cytochromes.

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b. Micronutrients or trace elements

Trace elements are required in such a little amount that these are generally supplied as contaminants from water, glassware and other media components. Important micronutrients are metals like manganese, zinc, cobalt, molybdenum, nickel and copper, most of which are the structural components of various enzymes and other proteins. These are also involved in catalysis of reactions and maintenance of protein structure e.g. molybdenum (Mo++) is required for nitrogen fixation. c. Growth factors

Most of the microorganisms are able to synthesize all the compounds needed for their survival and sustained growth. Since due to absence of essential enzymes, many microorganisms cannot manufacture them, these compounds or their precursors have to be externally supplied from the environment. Such organic compounds are called growth factors and include amino acids, purines, pyrimidines and vitamins. These growth factors are needed in very small amount. Some microorganisms have specific nutritional requirements and are called fastidious organisms. Besides, the nutrient elements and growth factors, microorganisms also require the source of electrons and energy. There are two sources of electrons – inorganic substances (lithotrophs i.e. rock eaters) and organic compounds (organotrophs). Similarly, energy can also be obtained from two sources i.e. light energy from sun and chemical energy from inorganic and organic compounds. Organisms using light energy are called phototrophs and those deriving it from oxidation of chemical compounds are called chemotrophs. Nutritional types of Microorganisms

Based on the source of carbon, electrons and energy, microbes can be placed under four nutritional types – a. Photolithoautotrophs

They use light as source of energy, inorganic compounds as electron source and CO2 as carbon source. Examples – green and purple sulphur bacteria, algae and cyanobacteria. b. Photoorganoheterotrophs

They use light as energy source, and organic compounds as electron as well as carbon source. Examples – purple and green nonsulphur bacteria. c. Chemolithoautotrophs

They use inorganic compounds as source of energy as well as electrons. Carbon-dioxide is used as a source of carbon. Examples- sulphur oxidizing bacteria, hydrogen bacteria, and nitrifying bacteria.

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d. Chemoorganoheterotrophs

They use organic compounds as the source for all i.e. carbon, electrons and energy. Examples – fungi and many non-photosynthetic bacteria. Among all these microorganisms, the most prevalent ones are the photolithoautotrophs and chemoorganoheterotrophs. The other two nutritional types, i.e., photoorganoheterotrophs and chemolithoautotrophs are relatively very few. Many microorganisms show metabolic flexibility depending on the environment and change from one nutritional type to another. These are called mixotrophs because they combine chemolithoautotrophs and heterotrophs, e.g. purple non sulphur bacteria. Culture Media

Microorganisms can be grown and maintained in the laboratory by providing all the nutrients and environmental conditions needed for their optimal growth. These nutrients can be supplied in the form of solid or liquid preparations. Such a nutrient preparation which is used to grow, transport and store microorganisms is called a culture medium (Pl. media). The precise composition of a medium depends on the species to be cultivated and is usually based on nutritional conditions naturally encountered by the microorganisms. There are two categories of media depending on the source of nutrients incorporated in the media. i. Defined or synthetic media

The media where exact composition and amount of each ingredient is known precisely. It is commonly used for growth of microorganisms having simple nutritional requirements e.g. BG-II medium used for cyanobacteria. ii. Undefined or complex media

In complex media, the exact chemical composition and the quantity of each component of the ingredient is not known. It contains nutrients of undefined composition like peptone, beef extract, yeast extract, etc. providing a wide range of nutrients (i.e. amino acids, peptides, nucleotides, vitamins, minerals, carbon source etc.) for better growth of different kinds of microorganisms. It is routinely used in the laboratory e.g. Nutrient agar, Potato dextrose agar. Culture media can be grouped as a general purpose media and specialized media. General purpose media is called so because these support the growth of many microorganisms. Specialized media are used for isolation and identification of microorganisms. Depending on the purpose for which these specialized media are used these can further be categorized as enriched media, selective media, differential media and so on. i. Enriched media These are specially fortified media and are used to encourage the growth of fastidious heterotrophs e.g. blood agar which is prepared by adding 5% defibrinated blood to the general purpose media.

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ii. Enrichment media

These media encourage preferential growth of a desired microorganism without inhibiting the growth of others. iii. Selective media

It selects a particular microorganism by inhibiting the growth of all the microorganisms except the interested one e.g., incorporation of dyes like eosin, methylene blue etc. in the media, selects the Gram negative microorganisms. iv. Differential media

Differential media is used to distinguish between different microorganisms without selecting for the growth of a particular type. It favors the appearance of traits that are characteristics of particular microorganism e.g. blood agar which is both a differential and enriched medium distinguishes between haemolytic and nonhaemolytic bacteria. Haemolytic bacteria produces greenish (partial haemolysis) or clear (complete haemolysis) zone around the colonies because of destruction of red blood cells. v. Selective and differential media

Such media have a dual role. These medias’ first select a particular group of microorganisms by inhibiting the growth of others and then differentiate among the members of the selected group e.g. Eosin Methylene Blue (EMB) agar, MacConkey agar etc. While presence of lactose and pH indicators in EMB and MacConkey agar differentiates lactose fermenters from nonlactose fermenters, ingredients like eosin and crystal violet selects for Gram negative bacteria. Life cycle of different microorganisms

Sulfolobus

Genus Sulfolobus belongs to a group of crenarchaebacteria. These are included in volume 1 of Bergey’s Manual of Systematic Bacteriology edition 2. Type species is Sulfolobus acidocaldarius. Other species are S. solfataricus, S. shibatae. Sulfolobus species are distributed worldwide. These are found in continental solfatara fields like geothermal mud hole, Japan; geothermal springs, Iceland; volcanic hot springs, Italy; acid hot spring, USA etc. Sulfolobus species are located almost invariably where there is a volcanic activity. These strive well in acidic environment having high temperature and presence of sulphur. Structure

Morphologically, these are irregular lobed cocci, which usually occur singly (Fig. 11). Sometimes pilus like and pseudopodium like structures may form. Cell size is about 0.8 – 2 µm in diameter.

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Fig. 11: Electron micrograph of Sulpholobus sp.

Growth conditions

Organism is thermoacidophile (high temperature and acidic environment) and strictly aerobic. Temperature range is 55-87oC, the optimum being 80oC. pH range is 1 to 6, though optimally it grows at pH 2 to 3. Presence of tetraethers in cell membrane helps Sulfolobus to survive extreme temperatures and in acidic pH. Physiology

Sulfolobus grows both lithotrophically and organotrophically. Lithotrophic growth occurs by utilizing sulfide or tetrathionate. Complex organic molecules like yeast extract, sugars or amino acids are used during organotrophic growth. Studies have indicated that Sulfolobus have a TCA cycle, which is similar to that of mitochondria of eukaryotes. Principle metabolic pathways include a glycolytic pathway, a pentose phosphate pathway and the TCA cycle. Life cycle

Sulfolobus specially grows fast with the generation time of about 4 to 6 hours. Its DNA content is found to be identical to other crenarcheal orders and is contained in a single circular chromosome. However, most of the cells in a population are found to possess two complete copies of the chromosome showing dominance of the post replicative state. In Sulfolobus during exponential phase, DNA replication starts soon after the completion of cell division and there is a long interval between termination of chromosome replication and cell division (i.e. long post replicative stage). Even during stationary phase, the cells contain two genome equivalents, indicating that the cells enter in stationary phase during post replication period. During stationary phase the changes occur in cell size, morphology and composition. Escherichia coli E.coli has been included in Volume 2 of Bergey’s Manual of Systematic Bacteriology edition 2. It belongs to the class Gammaproteobacteria and Order Enterobacteriales. Structure E. coli is a Gram negative, facultative anaerobic, rod shaped (Fig. 12) bacteria which is commonly present in large intestine of human and warm blooded animals and may play a nutritional role in intestinal tract by synthesizing vitamins. It is a opportunistic pathogen which

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can cause number of diseases like urinary tract infections, gastroenteritis, septicemia, kidney and gall bladder infections. There are four major types of E.coli, which cause gastroenteritis. These are:

a. Enteropathogenic E.coli (EPEC) b. Enterotoxigenic E.coli (ETEC) c. Enteroinvasive E.coli (EIEC) d. Enterohemorrhagic E.coli (EHEC)

Fig. 12: Photomicrograph of Escherchia coli E.coli is routinely identified by serological tests based on detection of somatic cell wall (o), flagellar (H) and surface (k) antigens. E.coli possess a single circular chromosome. About 88% of the genome represent functional ORF while 10% of it contains regulatory sequences. Life cycle

E. coli life cycle is relatively fast. It multiplies by the process of binary fission with a generation time of about 20 minutes under best nutritional conditions (Fig. 13). During multiplication, the cell elongates to approximately twice its length without change in diameter by addition of new cell wall materials to the pre-existing cell wall and then divides into two daughter cells with the formation of a septum in the centre of the cell. Multiplication begins with the replication of DNA. The newly formed DNA attaches to the plasma membrane at adjacent sites close to the center of the cell. All the cellular constituents also increase in amount during the process. Once DNA synthesis is over, it gives the signal for the formation of a structure called divisome by the proteins called Fts (Filamentous temperature sensitive proteins). The key protein involved is Fts Z which attaches to the center of the cell cylinder in the space between the duplicated nucleotides. These polymerise to form a Z ring to which other Fts proteins bind and form a divisome. Proteins like those involved in peptidoglycen synthesis may also be involved in formation of divisome. This spot marks the cell division plane and it triggers the synthesis of new cell wall and cell membrane material in both the directions until the cell doubles in size. The cell elongation pulls two chromosome copies apart. Par A, Par B, Muk B and other proteins are involved in separation of DNA. After the chromosomes have been separated, the constriction starts at the site of divisome. As constriction occurs, the Fts Z ring depolymerise and wall material grows inward so as to separate the two daughter cells. The process is then repeated.

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Fig. 13: Binary fission in E. coli Bacillus

Bacillus is a large and diverse genus of bacteria, which belongs to a family Bacillaceae. The family is characterized by production of refractile resting structure called endospores. Earlier species were included in genus Bacillus on the basis of two characteristics, i.e., aerobic growth and endospore formation which resulted in the heterogeneity in the genus with respect to physiology, ecology and genetics. In first edition of Bergey’s manual of systematic bacteriology, the G+C content of the species in Genus Bacillus varies from 32 to 69%. In its second edition, phylogenetic classification scheme landed Bacillus into the class ‘bacilli’, order Bacillales and the family Bacillaceae. There are many new genera along with Bacillus in this family. Bacillus is found to show a kinship with certain non-spore forming species like Planococcus, Lactobacillus and Staphylococcus on the basis of 16s rRNA analysis. Bacillus is ubiquitous in nature. It can be found in a wide range of habitats and predominates in soil environment. Few are pathogenic to both vertebrates and invertebrates. Structure

The cells are Gram positive, straight rods of size about 0.5 – 2.5 X 1.2 – 10 µm (Fig.14) . These are present in pairs or in chains and have round (B.sphaericus, B. globisporus) to oval (B. subtilis, B.cereus) endospores (one endospore per cell). Endospore formation is a mechanism of survival under adverse conditions rather than a mechanism of reproduction and it shows unusual resistance to chemicals and physical agents. Most of the Bacillus species are motile by peritrichous flagella. Bacillus is aerobic or facultative anaerobes, having a fermentative or respiratory metabolism. It is chemoorganotroph which can degrade most of the residue substances from plant and animal sources.

21

Fig. 14: Photomicrograph of Bacillus sp Life cycle

Under optimal conditions of growth, Bacillus species exhibit a generation time of about 25 minutes. It multiplies by the process of binary fission during favourable conditions. However, when a population passes out of the exponential phase of growth, endospore formation begins (fig.15). This process occur in several stages and takes about 8 to10 hours depending on the species, and involving about 200 genes in the sporulation. The process beings with the formation of an axial filament of nuclear material, followed by inward folding of the cell membrane which encloses a part of the DNA in double layer to form an immature spore. Then cortex and protein coat is laid down between the two membranes resulting in the formation of a mature spore, which is released with destruction of sporangium by the lytic enzymes. With onset of the ideal conditions, the endospore transforms into an active vegetative cell. The process involves activation of the endospore, breaking spore dormancy stage and emergence of spore protoplast from the spore coat. The spore protoplast then develops again into an active growing bacteria.

Fig. 15: Endospore formation in Bacillus

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Chlamydomonas

Chlamydomonas belongs to the division Chlorophyta, the members of which are grass green in color due to the abundance of chlorophyll a and b over carotene and xanthophylls. Chlamydomonas is widely found in fresh water bodies like ponds, ditches, swimming pools, lakes, etc. and also on moist soil. Few of its species are even marine. One species, C.nivalis is found in snow in arctic and alpine regions, imparting red colour to snow environment making ‘red snow’. All species except one (C.dysosmos) have obligate autotrophic nutrition. . Structure

Chlamydomonas is a biflagellate, unicellular oval to oblong green algae with 20-30 µm in diameter (Fig.16). The cell is bounded by a cellulosic cell wall and a capsule enclosing a protoplast which contains a cup shaped choloroplast, two contractite vacuoles, a red eye spot and other cell organelles. The Nucleus is present in the center or near to the anterior end of the cell. Chloroplast is cup shaped, which almost fills the cell at is blunt hinder end and has one medium sized starch forming structure – the pyrenoid at its posterior end and a photoreceptive organ – eyespot at its anterior end.

Fig. 16: Structure of Chlamydomonas sp Life cycle

Chlamydomonas reproduce in two ways: i. Asexual reproduction

ii. Sexual reproduction i. Asexual reproduction

A sexual reproduction occurs through different methods. a. Zoospore formation

Under favorable environmental conditions, Chlamydomonas reproduce asexually by formation of zoospores (Fig.17). During the process, the parental cell discards the flagella and comes to rest. In some species, however, the cell remains motile. The vacuole disappear and the protoplast is withdrawn from the cell. It divides mitotically by several successive bipartitions (all longitudinal and simultaneous) to produce 4,8 or 16 uninucleated daughter protoplasts. The

23

chloroplast along with pyrenoid is also halved with each successive division. Parental eyespot is received by only one of the daughters, whereas in others it is formed a new. All daughter protoplasts are aflagellate and lie parallel to each other within the parental cell wall. These daughter protoplasts become mitozoospores or zoospores by secreting a cell wall, acquiring a pair of flagella and a contractile vacuole, and gets liberated with rupturing or gelatinizing of cell wall. The mitozoospores are exactly like the parental cell except that these are smaller in size. These grow in size and repeat the process almost daily under ideal conditions. The process takes only few hours. b. Aplanospore formation

Chlamydomonas can also reproduce by formation of aplanospores. The parental cell resorbs the flagella and comes to rest. Its protoplast gets round up and develops a thin wall around itself, to become an aplanospore. At times, it may develop a thick cell wall with red coloration. Such resting spores are called hypanospore. c. Palmella stage

Under unfavourable conditions, Chlamydomonas develops into a palmella stage. Just like zoospore formation, here also the protoplast divides successively by bipartition method to produce 2, 4 or 8 cells without development of flagella and gets released. These remain together within the gelatinized parental cell wall. Daughter cells may also divide further and the process of division may continue to produce a colony of considerable size in which numerous aflagellate daughter protoplasts remain embedded within a common mucilaginous matrix formed from the cell wall of the parent and daughter cells of successive generations. This assemblage of cells which is short lived is known as Palmella stage (Fig.17). With the recurrence of ideal growth conditions,these cells transform in zoospores by acquiring a flagella. Sometimes cells in a palmella stage convert into a hypanospore by secreting a thick cell wall.

Fig. 17: Life cycle of Chlamydomonas

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ii. Sexual reproduction

Sexual reproduction can be isogamous, anisogamous or oogamous depending upon the species and environmental conditions like deficiency of nutrition, nitrogen supply, presence of calcium etc. All the anisogamous and oogamous species are heterothallic. Isogamous species may be homo or heterothallic in nature. The gametes produced are biflagellate and are with (calyptogametes) or without a cell wall (gymnogametes). In isogamous species, gametes may be gymno - or calypto - gamete whereas in others it is only calyptogametes. a. Isogamy

Here, sexual reproduction involves the fusion between morphologically identical gametes (Fig. 17). In homothallic species these gametes come from the same parental cell, which comes to rest, retracts its flagella and undergoes successive bipartitions to produce 16, 32 or even 64 daughter protoplasts. Each of the protoplast develops a pair of flagella and becomes a pear shaped gamete. These gametes are released into the surrounding water with rupture of the parent cell wall and fuses in pairs either sideways or end to end. A quadriflagellate zygote is produced which swims for a while before coming to rest. It withdraws its flagella, becomes round and develops a thick cell wall to become a zygospore. Gametes in heterothallic species are morphologically alike but physiologically and functionally show disparity. These come from parents of two different mating types, i.e., + (Plus) and – (Minus) by repeated division of the protoplast. In case of C. moewusii, vegetative cells themselves function as calyptogametes. The calyptogametes of opposite mating types get attracted because of a glycoprotein substance – isoaglutinins – and forms clump through entanglement of flagellar tips (Fig.18). The calyptogametes then emerge out from the clump in pairs (+ & -). These swim for some time with the help of the flagella of + strain and then the protoplasts of the paired individuals slip out from their cell wall. The two protoplasts fuse to form a zygote which develops a thick cell wall and becomes a zygospore. b. Anisogamy

Starting of anisogamy can be seen in C. monoica where gametes are morphologically alike (physiological anisogamy). After pairing of gametes, the protoplast of one flows entirely into the cell wall of another and fuses with it (Fig. 18). Well-defined anisogamy can be seen in C. braunii. The gametes produced are of different size. The male gamete (micro-gamete) is about half the size of the female (macorgamete) gamete and is produced in larger number (8 or 16 per cell) as compared to female gametes, which count 2 to 4 per cell. Also, microgametes are more motile than macrogamete. The microgamete swims towards the macrogamete, gets attached to it and pass on its protoplasm into the cell wall of the macrogamete. Fusion of cytoplasm followed by nuclear and chloroplast fusion results in the formation of a zygote. Zygote loses its flagella and becomes zygospore. In both isogamy and anisogamy, gametes fuse in surrounding water.

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c. Oogamy

Primitive type of oogamy can be seen in few species of Chlamydomonas, e.g. C. coccifera. Here, female mother cell discards its flagella and directly transforms into a round non-motile single macrogamete or egg, which is never shed. On the other hand, microgametes are produced from the male parent cell by repeated divisions. About 16 biflagellated microgametes are formed per cell, which are shed in water. Male gametes swim and one of them attaches itself to the macrogamete (Fig.-18). Once the intervening wall dissolves, its protoplast pass over to the macrogamete and fusion of the two results in the formation of a non-motile zygote.

Fig.18: Reproduction types in Chlamydomonas Zygospores formed during the sexual reproduction represent the resting period during unfavourble conditions. It is orange red, spherical in structure with a thick, smooth or stellate wall. It also contains fat, reserve food material and germinates in water. At the time of germination, its color changes from red to green and diploid nucleus undergoes meiosis to produce 4 or rarely 8 haploid nuclei which develop into meiospores with a thick cell wall and a pair of flagella and are liberated with rupturing of the zygospore wall. Released meiospores grow in to an adult. Sexual reproduction increases vitality and vigour in the species and also helps in survival under adverse conditions. Thus, life cycle of Chlamydomonas comprises of two phases –

i. Haploid Phase – represented by vegetative cell and the gametes. ii. Diploid Phase – represented by the zygote.

However, there are no regular alternations of haploid and diploid generations.

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Ectocarpus

Ectocarpus belongs to the division phaeophyta, a group of brown marine algae, found in both littoral and sub littoral zones attached to rocks and stones. Some species are present in fresh water while few others are epiphytes, endophytes or endozoic. Structure

Ectocarpus has the simple type of thallus (Fig. 19), which has two parts:

i. Prostrate or creeping part ii. Erect or projecting part

Prostrate portion is sparsely or profusely branched and is attached to the substratum usually by branched rhizoids or sticks as epiphytic species.

Erect portion is profusely branched and consists of numerous upright uniseriate (cells are arranged one above the other in a single strand) filaments arising from the prostrate part and moving freely in water. At the end of the filaments, series of elongated, tapering, hyaline vacuolated cells are present as colorless hair.

Fig.19: Structure of Ectocarpus filament Thallus of Ectocarpus may be haploid or diploid which are morphologically similar. The haploid thalli involve in sexual reproduction whereas diploid thalli reproduce asexually. Nutritionally, Ectocarpus is obligatory photoautotrophic. Life Cycle

Ectocarpus reproduces both by the sexual and asexual means. i. Asexual reproduction Asexual reproduction involves the production of biflagellate zoospores within sporangia, which are of two types:

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a) Pleurilocular (Multicellular) sporangium b) Unilocular (Unicellular) sporangium

Both the kinds of sporangia are born terminally and singly on the lateral branchlets of diploid thallus (sporophyte). a. Pleurilocular sporangium

It is formed from the terminal cell of the branchlet (sporangial mother cell) which enlarges and undergoes in several transverse and vertical mitotic divisions to produce elongated cone like structure (Fig.20). It is called pleurilocular sporangium, which may be sessile or stalked containing several hundred cubical cells. Subsequently, the protoplast of each cell is converted into a single biflagellate, pear shaped, diploid zoospore (mitospore) with single nucleus. The two flagella are laterally located and are of unequal lengths. The longer flagella (tinsel) in motion is directed forward and the shorter one (whiplash) is directed backward. Eyespot is usually situated near the point of origin of flagella and brown chromatophore is present at the hinder end. Liberation of zoospores occur through the apical aperture. Septa between the cells disappear and zoospores are released one by one in a slow stream through the apical aperture.

Fig. 20: Development of pleurilocular sporangium in Ectocarpus sp Zoospores swim for a while and then settle down on a substratum with their anterior end downside. Zoospores become round and their flagella are resorbed. A tubular prolongation comes out from the zoospore and forms the prostrate system. This then grows to form a diploid sporophyte. Here, there is no alternation of generations. b. Unilocular sporangium

It is also formed by a terminal cell of the branchlet, which grows in size to become ellipsoidal or globose in shape with numerous chromatophores. The diploid nucleus is divided by meiosis to produce 4 haploid nuclei which further undergoes mitosis to form 32 to 64 daughter nuclei. This is followed by division of the cytoplasm into number of daughter protoplasts, each with a single nucleus and chromatophore. Each daughter protoplast then converts into a biflagellate haploid zoospore called meiozoospore (gonozoospores). It is exactly like mitospore in structure except

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that it is haploid. Meiospores are also provided with large tinsel and short whiplash flagella. All the meiospores are liberated simultaneously in a gelatinous mass through a small apical aperture. Meiospores also swim for a short period of time and then settles on a substratum. It withdraws its flagella and on germination produces a gametophyte. Thus, it results in the alternation of generations. These morphologically similar gametophytes are physiologically of two kinds in many species. In many species (E.reptans) only one kind of sporangia - either unilocular or pleuriloculor - is present. ii. Sexual reproduction

Ectocarpus species are either homothallic or heterothallic. Sexual reproduction is isogamous in most of the species (e.g. E.globifer) though in some it is morphologically (E. secundus) or physiologically (E.siliculosus) anisogamous (Fig.21). Sexual reproduction of oogamous type is not present in Ectocarpales.

Fig. 21: Sexual reproduction in Ectocarpus

Process of formation of gametes resembles to that of zoospores formation in pleurilocular sporangium. The gametes are formed in large, elongated, conical, multicellular sex organs called the gametangium. The gametangia are produced from the terminal cells of lateral branchlets of the gametophytes by a process just similar to the developmental process of pleurilocular sporangia. The terminal cell of the lateral branchlet becomes enlarged and undergoes repeated transverse and longitudinal divisions to produce a structure of several hundred cubical cells arranged in 24-40 transverse tiers. These cells get converted into biflagellate pyriform gametes, the structure of which is just like zoospores. Sexual fusion between the gametes may be iso – or aniso-gamous depending upon the species. In isogamous, fusion of the gametes occur from the same plant or even from the same gametangium. Anisogamous type of sexual reproduction can be seen in E. siliculosus and E. secundus. In case of E. siliculosus the gametes are morphologically similar but physiologically

29

different. Male gametes are relatively more active and motile as compared to female gamete, which is passive and sluggish. Female gamete turn motionless after sometime and get surrounded by several male gametes with their longer flagella attached to its surface. A volatile substance called ectocarpin secreted by a female gamete is responsible for the attraction of male gametes to the surface of the female gamete. Such clustering of male gametes around the passive female gamete is called clump formation. Ultimately, flagella of one of the surrounding male gamete are resorbed and it fuses with the female gamete to produce a zygote. The remaining active gametes however swim away. In E. secundus both the gametes are produced in separate gametangia on the same thallus. Both the gametes are motile but of unequal length. Male gametes (microgamete) are smaller in size and are produced in microgametangium whereas female gametes (macrogametes) are large in size and are produced in megagametangia. Both types of gametangia are present on the same plant. Macrogamete soon comes to rest and several microgametes get attached to it. One of them fuses with it to form zygote and others swim away. Zygote germinates without going into the resting stage. As there is no meiosis during germination, zygote always produces diploid sporophyte. These sporophytes bear either both or one of the two sporangia, i.e., uni- and pleuri-locular sporangia. In pleurilocular sporangium, diploid zoospores are formed which play no role in alternation of generations. From unilocular sporangium, meiospores are produced. Gametes, which fail to fuse germinate directly into a new plant. Alternation of generation is seen in the life cycle of Ectocarpus. Rhizopus

Rhizopus belongs to the class zygomycetes, the members of which are characterized by the production of thick walled sexual spores called zygospores (Gr. Zygos-yoke spora-seed) Rhizopus is cosmopolitan in distribution and can be isolated from variety of substrates like vegetables, fruits, pickles, jams etc. Commonly, it is known as bread or black mold, it lives primarily as a saprobe, though few species may be weak parasites. Structure

The thallus is a well developed, branched, white fluffy mass called mycelium which later becomes greyish due to appearance of sporangia (fig.22), hence popularly called black mold. The mycelium consists of numerous slender , aseptate , coenocytic hyphae which are of three kinds: i. Stolons - These grow horizontally over the substratum in all directions. Each stolon arise from the point of contact of the mycelium with the substratum. ii. Holdfast or Rhizoids - These brown, branched rootlike hyphae help in anchoring the fungus to the substratum and also in absorbing water and nourishment from the substratum. iii. Sporangiophores – These vertically grown hyphae, arise just opposite to the rhizoids and are

involved in asexual reproduction.

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Fig. 22: Rhizopus sp. mycelium showing sporangia

Life cycle

Rhizopus reproduce both by asexual and sexual means. i. Asexual reproduction

It can occur through fragmentation, oidia formation and through sporulation. a. Fragmentation

It is the common method under favourable conditions. Each of the hyphae fragment separated from the mycelium grows to form a new mycelium. b. Oidia formation

This occur when mycelium grows under submerged conditions in a nutritive medium. The young coenocytic hyphae develops septa and produces multi-nucleated segments which separate and become rounded oidia or oidiospores. Each oidia grows into a new mycelium in a nutritive medium. c. Sporulation

Rhizopus also reproduce asexually by producing large number of sporangiospores within round black sac like structure called sparangia that are formed at the tip of vertical aerial hyphae i.e. sporangiophores (Fig. 23) . During sporulation, the tip of the sporangiophore swells in a vesicle in which cytoplasm and nuclei flows from the sporangiophore. As the development proceeds, sporangium gets divided into two zones separated by a wall

• Columella - highly vacuolated dome like central zone and • Sporiferous – peripheral, dense, spore bearing zone.

The protoplast of sporiferous zone divides into a large number of round to oval shaped multinucleated, thick walled, bluish or brown sporangiospores. These spores are liberated in the air with the bursting of sporangial wall due to excess internal pressure exerted by the increased quantity of fluid within the columella and sporangiophore.

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Under suitable conditions sporangiospores germinate by forming a germ tube which then develops into a Rhizopus mycelium by apical growth.

Fig. 23: Life cycle of Rhizopus sp.

ii. Sexual reproduction

Towards the end of the growing cycle Rhizopus undergoes in sexual reproduction (fig.23) . Being heterothallic sexual reproduction involves gametangial copulation between two morphologically similar but genetically distinct and compatible mycelia, designated as ‘+’ and ‘-’. The process starts with the formation of special hyphae called zygophores near the tip of the growing aerial hyphae. This, as suggested by Burgeff (1924) is induced by a diffusible harmone secreted by compatible strains. Zygophores from compatible strains get attracted towards each other and adhere in pairs at their tips to form a fusion septum. Thereafter, the tips of these zygophores swell to form progametangium which separates into two cells by formation of gametangial septum near the tip- terminal gametangium and a subterminal suspensor cell. The fusion septum then dissolves resulting in plasmogamy followed by karyogamy where nuclei of opposite strains fuse. The nuclei that do not fuse, subsequently degenerate. In the meantime the fused cell, called prozygosporangium enlarges and develops a thick wall to become zygosporangium which bears a single zygospore containing a number of diploid nuclei. Zygosporanium is set free with the withering of the suspensor. It needs a resting period of several months before germination. Under suitable conditions, meiosis takes place in zygospore just prior to germination to produce haploid nuclei. Zygospore then absorb water and cracks open the zygosporangium wall.Thereafter, zygospore content comes out of it to form a small hyphae called germ tube which later develops a sac like structure called germ sporangium at its tip. Latter contains either one type (+ or -) or a mixture of two types of spores (+ and -) which are then liberated. Sometimes without gametangial fusion, zygospore like structure called azygospore is formed parthenogenitically.

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Saccharomyces

Saccharomyces cerevisiae although widely distributed is particularly abundant in substrata containing sugar. It lives as a saprophyte and is commonly known as brewer’s or baker’s yeast as it is extensively used in bread or wine making. Structure

Saccharomyces is a unicellular organism which are globose or oval in shape (2-8 µ D x 3-15 µ L), however, some species are even elongated, cylindrical or rectangular. Cells are colorless and produce white to cream coloured colonies on artificial media. These consist of a tiny mass of protoplasts surrounded by a thin chitinous cell wall and having a large vacuole occupying a significant portion of the cytoplasm. Life Cycle

Yeast reproduce both sexually and asexually. i. Asexual reproduction

Saccharomyces normally reproduce asexually by the process of budding and therefore is called a budding yeast. It is the most common mechanism of reproduction under favourable conditions. During budding, a bud is formed at predetermined spot (Fig. 24). The cell wall in this region becomes soft and the protoplast bulges out as a protuberance which gradually increases in size. Nucleus and vacuole then divide in a parent cell and one copy of each migrates into the enlarging bud. Various organelles also accumulate in the newly formed bud. When the bud is fully formed, it gets separated from the parental cell by centripetal formation of septa (primary septum of chitin and a secondary septum of glucan) at the joint of the bud with the parental cell. This leaves scars on both the cells - convex bud scar on the parental cell and a concave birth scar on the daughter cell. The detached bud grows and starts budding again. By counting the bud scars on a cell, one can know the number of times the cell has undergone in a budding process. ii. Sexual reproduction

Sexual reproduction process is extremely simple in yeast. No sex organs like artheridia and oogonia are involved, rather 2 different mating types of haploid somatic cells (heterothallism) - α and a - fuse to form a zygote (Fig. 24). Sometime, two ascospores of opposite strains also act as copulating gametangia and fuse to give a zygote. Each mating type produces a sex harmone which changes the shape of opposite strain in to an elongated or pear shaped structure besides altering the cell wall to facilitate fusion between cells of two strains. This leads to the fusion of protoplast (plasmogamy) of the two cells to produce a dikaryon or a large fusion cell with 2 nuclei. Later on karyogamy occurs resulting in the formation of a large diploid, ellipsoidal zygote. Under favourable conditions, the zygote can multiply by the budding process, but during unfavourable conditions it becomes spherical in shape and behave as an asci. A large central vacuole disappears in the latter and meiosis occurs in diploid nucleus so as to produce four haploid nuclei, two of each strain α and a. Each of the

33

nuclei with some cytoplasm gets enclosed within the envelope and becomes thick walled, globose or ovoid, haploid ascospores. Under favourable conditions, ascospore swell and gets released by rupturing the ascus wall. These ascospores germinate by producing a small protuberance or bud which later on give rise to a typical somatic cell. Sometimes two ascospores of opposite mating types fuse to form a zygote.

Fig. 24: Life cycle of Saccharomyces cerevisiae In presence of abundant food supply, budding process occur very rapidly, producing pseudomycelium. The buds often produce new buds before separation from the mother cell resulting in formation of branched or unbranched pseudomycelium. The cells later separates from pseudomycelium. The life cycle of yeast is therefore diplobiontic and has two distinct phases:

i. Diplo -or saprophyte phase that include zygote and ii. Haplo – or gametophyte phase that include ascospores and haploid somatic cells.

These two phases alternate in the life cycle of Saccharomyces and are equally important. Both these life cells are perpetuated by budding process. Neurospora

Neurospora which belongs to the class Ascomycetes are characterized by the presence of septate hyphae and production of ascospores Neurospora is commonly known as bakery or red bread mold and is the common environmental contaminant because of its rapidly growing hyphae and easily dispersed conidia. Commonly isolated species are N. sitophila, N. crassa and N. tetrasperma.

34

Structure

has a haploid mycelium, which is composed of a mass of multinucleated, branched,

ife cycle

reproduce by both sexual and asexual process.

Asexual Reproduction

rs through the production of a large number of asexual reproductive

uring germination the conidia swell markedly by absorption of water and the germ tube

tudies suggest that a conidium may differentiate several other conidia from itself, which

Neurosporaseptate hyphae having a chitinous cell wall. At the tip of the hyphae, a pyramidal cap like apical body is present which probably corresponds to the growing point. Large number of oval shaped, pink conidia develop from the aerial hyphae. L

Neurospora i.

Asexual reproduction occuspores, i.e., conidia on short aerial hyphae – the conidiophores (Fig.25a). These conidia are of two types- multinucleated macroconidia and uninucleated microconidia. Microconidia usually arose singly from a specialized short hyphae – the microconidiophore. Macroconidia arise directly from a vegetative hyphal conidiophores and seems to be acropetally formed by repeated constriction of hyphae. Demerges from it to produce a mycelium. Sdisperse and repeat the asexual cycle on germination under favourable conditions.

Fig. 25a: Life cycle of Neurospora crassa

ii. Sexual reproduction

three types of life cycles –

Neurospora species show

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• Heterothallic, example, N.crassa

oensis, and

n heterothallic species, two mating types MAT A and MAT a are involved which

omothallics do not need both MAT idiomorphs. Here any haploid individual strain goes

• Homothallic, example, N. galapag• Pseudohomothallic, example N. tetrasperma

Imorphologically look alike but have totally different DNA sequences at one chromosomal locus. These are not alleles and are called idiomorphs. Hthrough the sexual cycle itself without pairing with another species (Fig.25b). Diploid nucleus is formed by fusion of two haploid nuclei.

Fig. 25b: Homothallic and pseudohomothallic cycle in Neurospora sp.

In Pseudohomothallic species both MAT idiomorphs are required to complete the sexual cycle

xual reproduction in Neurospora occurs through gametangial contact. Female part is

fter fertilization, protoperithecium rapidly enlarges and gets melonized to become perithecium.

and after meiosis each ascospore receive both MAT A and MAT a nucei .These ascospores produce a mycelium having a mixture of nuclei of dual mating type. Here also, no pairing with another individual occurs (Fig. 25 b). Seprotoperithecia, which contains multinucleated ascogonium (Gr askos - sac; gennao – give birth). Long hyphal branches called trichogynes are produced in ascogonium. The male part is a microconidia, which supply nuclei to the rarely branched receptive trichogynes. Nuclei fuse to form transient diploid nucleus, which undergoes meiosis. The four haploid products of meiosis stay together in a sac called ascus. These nuclei undergo mitotic division to produce octad of ascospores within each ascus. N.tetrasperma contains only four ascospores in the ascus. AAscospores escape through ostium, a definite opening at the apex of perithecium. Ascospores which have characteristic nerve like ribs on outer wall are initially uninucleate but later contain two haploid sister nuclei. When ejected from perithecia these are covered with a mucilagenous

36

layer which helps in their attachment to the substratum. Ascospores also have a well-defined germination pore at each end through which these germinate by producing germ tubes from one or both the ends. The germ tube is constricted at the pore and then swells like a balloon which again narrows to an average hyphal diameter. These hyphae form then forms mycelial colonies. Plasmodium

elongs to Sub-phylum sporozoa, which include parasite protozoans on both

• P. vivax – Cause tertian, benign tertian or vivax malaria,

Plasmodium binvertebrates and vertebrates and cause various dreadful diseases. One of the most important member is Plasmodium – the causative agent of malaria (Italian-malo-bad, aria-air). About 60 species of Plasmodium are known of which the following four are responsible for causing malaria in human.

• P. ovale - Cause ovale or mild tertian malaria, • P. malariae – Cause quartan malaria, and • P. falciparum – Cause malignant tertian malaria or pernicious malaria. It is the most fatal

lasmodium has digenetic life cycle (Fig. 26), i.e., it has two hosts:

i. The primary or definite host - These are the mosquitoes where sexual reproduction

ii. iate host – These are vertebrate animals which are involved in asexual

among all the above four types. Recurrence of fever and chill occur after every 48 hours in all the types except in quartan malaria wherein it occurs at each 72 hours interval.

P

occurs. Intermedreproduction.

Fig. 26: Plasmodium life cycle

Mosquitoes also act as vector of Plasmodium o exchange malarial parasite from an infected

asmodium is widely distributed all over the world. Among the four Plasmodium species

t

human to a normal human being. Plinfecting human beings, P.vivax is the most widely distributed while P.ovale is the rarest one.

37

Life Cycle

e most common cause of malaria in human beings, which completes its life cycle in

sexual reproduction

or schizogony occurs in the intermediate host (Fig. 27) which includes

i. Infection and exo-erythrocytic schizogony

i ony, and

Infection and exo-erythrocytic schizogony

of sporozoites – the infective form of Plasmodium –

he duration between the initial sporozoite infection and the first appearance of the parasite in

etacryptozoites are of two types –

a) Micrometacryptozoites - These are smaller, more numerous and enter into the RBCs. al

ii. Erythrocytic schizogony

ter into the blood and start erythrocytic phase. These invade

P. vivax is thtwo hosts i.e. female Anopheles mosquito (Primary host) and man (intermediate host). Gamete production and fusion (gametogony) followed by post-zygotic multiplication (sporogony) occur in the primary host. This results in the production of sporozoites and completion of sexual life cycle. A

Asexual reproduction following stages:

ii. Erythrocytic schizogony ii. Post-erythrocytic schizogiv. Formation of gametocytes.

i.

Asexual cycle starts with the introductioninto human through the saliva of mosquito, when it bites. Sporozoites are small spindle shaped (11-12 µm length and 0.5 – 1 µm width), unicellular organisms which are present in the salivary glands of female anopheles. After entering into the human body, sporozoites remain in circulation for about half an hour. These then enter into the liver and invade hepatocytes. Inside the liver cell, each sporozoite grows to form a large spherical schizont, which divides by multiple fission (schizogony) to form thousands of uninucleated cryptomerozoites or cryptozoites. Liver cells lyse and liberate cryptomerozoites. This phase of reproduction is called pre-erythrocytic schizogony. These cryptozoites infect other normal hepatic cells and repeat the process several times to produce enormous number of metacryptozoites or phanerozoites. Their formation is called the exo-erythrocytic phase. Tthe blood is called pre-patent period and it varies from one species to other. The period between infection and the appearance of first malarial symptoms is called incubation period. In P.vivax, prepatent period is of 8 days and incubation period is about 14 days. M

b) Macrometacryptozoites - These are large but less in number. These invade the normliver cells and continue exo-erythrocytic schizogony.

Micrometacryptozoites enerythrocytes and convert into a round form called trophozoites. As trophozoite grows, a central vacuole develops in its cytoplasm pushing its nucleus to one side. This stage is called signet ring stage. The trophozoites feed on haemogloblin of erythrocytes. These take a large volume of

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erythrocytic cytoplasm and form a food vacuole. Digestive enzymes are secreted in food vacuole, which break down haemoglobin in to two parts – protein component and hematin. The protein component is absorbed by the trophozoite whereas hematin is changed to a toxic form – haemozoin. These trophozoites now convert from signet ring stage to amoeboid trophozoite. RBCs get enlarged and acquire red eosinophilic granules called schuffner’s granules. The amoeboid trophozoites grow in size and become rounded schizonts. The nucleus of each

i. Post erythrocytic schizogony

may invade liver cells and undergo schizogony.

. Formation of gametocytes

ythrocytic cycles, the merozoites increase in size inside RBCs and

a) Microgametocytes (male) are smaller in size, have clear cytoplasm and a large diffused

b) etocytes (female) are large in size, round with food laden cytoplasm and a

oth the gametocytes contain large amount of haemozoin. The gametocytes do not divide or

exual Cycle in mosquito

cks the blood of infected person, gametocytes also enter into it along

Gametogony

etocytes produce male and female gametes. Due to change in temperature (from

divides and forms 12-24 nuclei which get arranged at periphery and are surrounded by cytoplasm. Each cytoplasmic mass with one nucleus then becomes oval shaped merozoites which are liberated into the blood plasma with the rupture of RBCs. The toxins are also released into the blood along with merozoites. The bursting of schizonts is synchronous. The toxins are carried to all body parts, deposited in spleen, liver and under the skin and produce symptoms of malaria, i.e., high fever, chill sweating, profuse shivering, etc. The liberated merozoites enter into normal erythrocytes and repeat the erythrocytic schizogony, the duration of which is about 48 hours in P. vivax. ii

In many cases some merozoites iv

After entering into many erbecome rounded gametocytes. These gametocytes are of two types –

nucleus. Megagamcompact nucleus.

Bdevelop further because of high body temperature in man. These remain in blood for several weeks until they die or get ingested by the vector in which they continue their development. S

When female mosquito suwith the blood. Gametocytes reach to the stomach where their development begins. The sexual cycle involves following stages: i.

Developing gamwarm blooded man to cold blooded mosquito), the nucleus of the male gametocyte divides mitotically to produce several haploid nuclei, each of which enters into long, thin, flagella like projections of cytoplasm. These projections break away as mature male or microgametes. The process is called exflagellation and occurs in the midgut of mosquito.

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Female gametocyte reorganizes to become female (megagamete) gamete. It produces a small cytoplasmic projection called cone of reception or fertilization cone. The nucleus of the megagamate comes near to this receptive cone.

Fig. 27: Detailed life cycle of Plasmodium vivax

ii. Fertilization

Fertilization is anisogamous and it occurs when microgamete attaches itself to the receptive cone of a megagamete. The two fuse to form a round zygote with a diploid nucleus. iii. Ookinete and encystment

Zygote remain motionless for a short period before it gets converted to a form called ookinete or vermicule. It is elongated, vermi form and motile structure (15-22 µm length and 3 µm width) which penetrate through the wall of the midgut and settles under the thin membrane that separates midgut from the haemocoel. Here, it becomes spherical and get enclosed in a thin membranous cyst formed partly from the ookinete and partly derived from the midgut tissues of the mosquito. The encysted zygote is called oocyst. iv. Sporogony

The oocyte grows in size and enters into asexual multiplication cycle. Its nucleus first divides by meiosis and then by mitosis to form a large number of haploid nuclei. Cytoplasm separates into irregular cytoplasmic masses with the appearance of large number of vacuoles into it. From these masses thousands of finger like projections arise and daughter nuclei migrate into each of them. These become sporozoites, which are slender, spindle shaped having tapering ends and a broad middle part containing a single nucleus. Each oocyte may produce 10,000 sporozoites which are liberated into the haemocoel on rupturing of the oocyte and move to different body

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parts including salivary glands. More than 2,00,000 sporzoites may be present in the salivary gland. The whole cycle is completed in about 10-20 days. Thousands of sporozoites enter into the body of a healthy individual as mosquito bite and the asexual cycle in man starts again. Paramecium

Paramecium belongs to the Phylum Protozoa which include unicellular microscopic organisms. Paramecium has a cosmopolitan distribution and is abundantly found in water bodies rich in decaying organic matter like, ponds, streams, lakes, pools etc. Few species of Paramaecium are also marine. Structure

Paramecium has a slipper or cigar shaped elongated body with a size varying from 80 µ to 350 µ in length and 45 µm to 75 µm in width. It is pointed and broadest in the posterior half and rounded and blunt at its anterior end (Fig. 28). The body has a ventral (oral) and dorsal (aboral) surface.

Fig. 28: Paramaecium cell structure

Paramecium is surrounded by a thin, firm membrane called pellicle which helps in maintaining the shape. The entire body is covered by numerous small hair like projections called cilia, which helps it in locomotion and food collection. Cytoplasm is bounded by a pellicle and is differentiated into two regions:

- Outer ectoplasm (Cortex), which is clear, narrow and dense. - Inner endoplasm (medulla), which is large, semi fluid and granular.

Various organelles like mitochondria, golgi bodies, ribosomes, reserve food granules, etc. are present in endoplasm. Two contractile vacuoles are present at each end of the body, close to

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dorsal surface which are involved in osmoregulation. Certain strains of Paramecium may have particles like Kappa, Pi and Mu. Paramecium is a heterokaryon, i.e., have two types of nucleus.

a) Macronucleus (somatic) - It is large kidney shaped and a triploid. It controls the feeding and vegetative activities of a cell.

b) Micronucleus - It is small, diploid, and usually spherical. It controls the reproductive

activities of the organism. On the ventral surface of the body oral apparatus is present which is a shallow depression comprises of oral groove, vestibule, buccal cavity, cytopharynx (gullet) and cytostome (mouth). Mode of nutrition in Paramecium is holozoic and feeds on bacteria, small protozoa, unicellular algae, yeast, diatoms and also on small bits of animals and plants. Life cycle

Paramecium reproduces both sexually and asexually. Asexual reproduction involves binary fission while sexual one includes conjugation, autogamy, cytogamy and endomixis. i. Asexual reproduction

Transverse (Horizontal) Binary Fission – Here the parent cell divides at right angle to the longitudinal axis of the body (Fig.-29). During division, Paramecium stops feeding, oral groove disappears and micronucleus divides mitotically (nuclear membrane remains intact). Macronucleus also separates amitotically into two by simply becoming elongated and constructed in the middle. After division of both the nucleus, two oral grooves are formed, one each in anterior and posterior halves. Whereas the parental cytopharynx is retained by the anterior half, a new one is formed in the posterior half. Two new contractile vacuoles also appear one in each half. Parental contractile vacuoles remain in the body. Two new buccal structures are formed. This is followed by constriction in the middle of the body separating two halves as two daughter Paramecia. Anterior one is called proter and the posterior one is known as opisthe. These then grow in size and again divide by the same process. Paramecium divides twice or thrice in every 24 hours. Time taken for division is about 30 minutes but separation of daughter paramecia takes more than an hour. After certain period of time the binary fission slows down and the cells have to be rejuvenated by conjugation. ii. Sexual reproduction

a. Conjugation

It is a type of sexual phenomenon which involves temporary union of two individuals of different mating types (Fig. 29). Each of the Paramecium species exist in a number of varieties or syngens and each syngens in turn, has many mating types. These mating types are morphologically similar but have physiological differences. Conjugation never occurs between same mating types.

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During conjugation two individuals called preconjugants of different mating types unite vertically by their oral grooves. A substance produced by cilia helps in this union. These stop feeding and their buccal structures disappear. The ectoplasm and pellicle degenerate at the point of contact and an endoplasmic bridge is formed between the two to facilitate material exchange. These two, now called conjugants, remain united for about 12 to 48 hours, swim actively and undergo nuclear changes.

Fig. 29: Life cycle of Paramaecium The Macronucleus simply breaks down into fragments and is absorbed by the cytoplasm. Micronucleus undergoes meiosis and produces four haploid micronuclei of which three degenerate. The remaining one undergoes in unequal mitosis to produce a small migratory and a large stationary gamete nuclei. The former migrates through protoplasmic bridge into other individual and fuses with its stationary nucleus to form a diploid zygotic nucleus. The conjugants now separates as exconjugants. In each exconjugant, zygote nucleus divide three times mitotically to produce eight nuclei, of which four becomes macro and four micronuclei. Three micronuclei disintegrate. The remaining one divides into two and at the same time exconjugant also undergoes in binary fission to produce two daughter paramecia each receiving two macro and one micronuclei. Further binary fission of these daughter exconjugants finally result in four daughter paramecia from each exconjugant, each possessing one macro and one micronuclei. Conjugation occurs under adverse living conditions and individuals must have passed through about 200 generations asexually by binary fission before undergoing conjugation. Conjugation seems to be necessary for the continued vitality and vigor of the species and for ensuring inherited variations. b. Autogamy

It resembles conjugation but it take place within a single individual only. During the process, macronucleus disintegrates and two micronuclei undergo meiosis to produce eight micronuclei of which seven disintegrate. Remaining haploid micronucleus undergoes a mitotic division to form two gamete nuclei, which fuses to form homozygous diploid zygote. The latter divide

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twice to produce four nuclei, two of which become micro and other two macronuclei. The cell and the micronuclei then divide to form two daughter individuals. Autogamy rejuvenates Paramecium and always results in homozygosity. c. Cytogamy

Two paramecia fuse here temporarily by their oral surfaces. Nuclear division occurs similar to conjugation but no nuclear exchange occurs (Fig. 30). Two haploid gamete nuclei fuses like autogamy.

Fig. 30: Autogamy in Paramaecium

d. Endomixis

Here macronucleus degenerate and micronucleus divide to produce eight daughter nuclei, of which six degenerates. Paramecium then divides to produce two daughter paramecia, each receiving one micronucleus, which undergoes in two successive divisions to produce two macro and two micronuclei. The micronuclei divide again along with binary fission of Paramecium to produce daughters each having one macro - and two micro-nuclei (Fig. 31).

Fig. 31: Endomixis in Paramaecium

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Suggested readings 1. Pelczar MJ, Chan ECS, Krieg NR . Microbiology . McGraw Hill Book Co. 2. Prescott LM , Harley JP, Klein DA . Microbiology . Wm. C Brown Publishers. 3. Atlas RM. Principles of Microbiology. Macmillan Publishing Co. New York. 4. Stanier RY, Ingraham JL, Wheelis ML, Painter PR. General Microbiology .Macmillan Publishing Co. New

York . 5. Alexopoulos CJ, Mims CW, Blackwell M . Introductory Mycology. John Wiley and Sons , Inc. 6. Kumar HD. Introductory Phycology. Affiliated East Western Press N.P. 7. Kotpal RL, Agarwal SK, Khetarpal RP. Invertebrates. Rastogi Publications.