Isolation of Rhizobium

 Isolation of Rhizobium 

Aim

To isolate Rhizobium species from root nodules of legumes

Principle

Nitrogen fixation is the phase of the nitrogen cycle during which enzymatically competent microorganisms (with nitrogenase) convert atmospheric nitrogen into nitrogenous compounds. This process replenishes the soil with usable nitrogen that is rapidly removed by plant use, denitrification and leaching. Nitrogen fixation is mediated by two microbial systems. One consists of non-symbiotic free living microorganisms such as members of Azotobacter, Beijerinckia, Clostridium and Cyanobacteria, which are capable of using Nitrogen gas as their nitrogen source. The second system involve symbiotic microbial forms such as members of Rhizobium, which following infection grow in a nodule in the root of leguminous plants. A mutually benefited association is established in which nutrients in the plant sap is used by Rhizobium as it fixes atmospheric nitrogen to ammonia which can be assimilated by the plants into various proteins. Rhizobium finds application as biofertilizer in recent times.

Rhizobium species are generally cultured aerobically at 25-30⁰C in Yeast extract mannitol agar (YEMA) medium. Fast growing Rhizobia develop colonies in 3-6 days and slow growing Rhizobia in 7-10 days. Congo Red is used as a dye to enable differentiation of Rhizobium species from other bacteria. Rhizobium appear as pale coloured colonies while soil bacteria such as Agrobacterium species and others absorb the red dye. YEMA is widely used for the cultivation of Agrobacterium, Rhizobium and other soil microorganisms. It contains mannitol as a carbon source and yeast extract as a source of readily available amino acids, vitamin B complex and growth factors for Rhizobia. Congo red. Colonies of Rhizobia stand out as colourless, translucent, glistening and elevated, with entire margins

Rhizobium can fix atmospheric nitrogen only in root nodules of legumes where it is in the bacteroid stage of its life cycle. It possesses the entire complement of genes for nitrogen fixation, which are normally latent and become active only under special conditions. Rhizobium makes nitrogen available to the plant and in turn, the bacteria derive nutrients from the tissues of the plants. A crushed leguminous root nodule may be used as the source of a stained slide preparation. Rhizobium appear as bacteroids within the nodule, and are distinguished by their pleomorphism, wherein they appear in a variety of shapes, such as x, y, t, v etc.

Materials Required

Freshly picked leguminous plants with root nodules as source of Rhizobium, Yeast extract mannitol agar (YEMA) plates (pH 6.8-7), 0.1% acidified mercuric chloride, 95% ethanol, methylene blue, crystal violet, safranin, Gram’s Iodine

Equipments – Bunsen burner, inoculating loop, glass slide, staining tray, glassware marking pencil, blade, glass rod.

Procedure

1.      Healthy root nodules of a young leguminous plants were obtained by cutting with a blade

2.      The nodules were washed thoroughly first with tap water, then with sterile distilled water, under aseptic conditions so as to remove contaminants and adhering soil particles

3.      Thereafter the nodules were immersed into 0.1% acidified mercuric chloride for 5 minutes

4.      The nodules were then transferred into a sterile beaker containing 10 ml of 95% ethanol and kept for 2-3 minutes and blot dried

5.      The nodules were finally washed for 5 minutes with sterile tap water using sterile blotting paper

6.      Nodules were aseptically crushed with glass rod

7.      The nodules were streaked on the YEMA medium incorporated with Congo Red

8.      Plates were incubated in an inverted position at 28⁰C for 48-72 hours

Microscopic Observation

1.      A root nodule was thoroughly rinsed and crushed between two slides.

2.      A loopful of the material from the nodule was used for preparing smears on 2 slides

3.      Gram staining was done

4.      The slides were observed under the microscope

Observation and Result

            On YEMA plates, growth was obtained on 2-3 days of the incubation.  Rhizobium appeared as gummy cream coloured colonies as it does not absorb Congo red dye. Agrobacteria and other soil bacteria appeared red since they take up the dye strongly.

On microscopic observation, Rhizobia appeared as Gram negative, non-motile, rod shaped bacteria

 

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Isolation of Azotobacter

 Isolation of Azotobacter 

Aim

To isolate Azotobacter species from soil

Principle

The free living bacteria having the ability to fix molecular nitrogen can be distinguished into obligate aerobic and facultative anaerobic organisms. Members of the genus Azotobacter of the family Azotobacteriaceae, are heterotrophic obligate aerobic nitogen fixers. Azotobacter species are Gram- negative, aerobic soil-dwelling bacteria. They are grouped into three genera namely Azotobacter, Beijerinckia and Derxia. Several species of Azotobacter are recognized in neutral and alkaline soils, in water, and in the rhizosphere of some plants. Azotobacter chroococcum, Azotobacter agilis, Azotobacter vinelandii are some common examples.

Azotobacter species are large, usually oval, but may appear in different shapes from rods to spheres, Gram negative. The size of the cells ranges from 2-10 µm long and 1-2 µm wide. They are typically polymorphic, i.e. of different sizes and shapes. In microscopic preparations, the cells can be dispersed or in irregular clusters or chains of varying lengths. In fresh cultures, cells are motile due to peritrichous flagella. Later, the cells lose their mobility, become almost spherical, and produce a thick layer of mucus, forming the cell capsule. Azotobacter are free-living in soil and water. The nitrogen fixing enzyme, nitrogenase is oxygen-sensitive. The high respiration rate of Azotobacter uses up free oxygen within the cells and protects the nitrogenase. Azotobacter species are relatively easy to isolate from soil by growing on nitrogen free media, where the bacteria are forced to use atmospheric nitrogen gas for cellular protein synthesis. Cell proteins are mineralized in soil after the death of the Azotobacter cells, enhancing the nitrogen availability of the crop plants.

Azotobacter species can be used as an important biofertilizer. Inoculation of soil or seeding with Azotobacter is effective in increasing crop yield. It is also known to synthesize biologically active substances such as vitamins, IAA, and Gibberellins in pure culture. Fungistatic properties against certain pathogenic fungi such as Alternaria, Fusarium species etc. is also reported. These attributes explain the observed effects of Azotobacter in improving seed germination and plant growth.

Materials Required

Soil sample, Jensen’s medium, 10 ml and 9 ml sterile water blanks, Sterile 1 ml pippete, sterile petridishes, Bunsen burner, glass marking pencil

Procedure

1. Jensen’s medium was prepared and sterilized by autoclaving at 121⁰C 15 lbs per inch² pressure for 15 min.

2. 1 gm of sieved soil was added to 10 ml sterile water blanks and shaken for 15-20 min.

3. Serial dilution was performed to obtain 10^-2, 10^-3. 10^-4, 10^-5 dilutions.

4. 1 ml aliquots of various dilutions were pipetted onto sterile petriplates and pour plated by adding molten medium at bearable warmth.

5. The plates were rotated for uniform distribution and incubated at 28⁰C for 3 days.

Observation

The plates were observed after 3 days incubation for the appearance of colonies on the agar surface.

Result

On Jensen’s medium, Azotobacter colonies appeared as milky and mucoid. On microscopic observation, short rods of Azotobacter were confirmed.

Bacterial Growth Curve and its significance

 

Bacterial Growth Curve and its significance

Bacterial growth curve:

When a fresh medium is inoculated with a given number of cells, and the population growth is monitored over a period of time, plotting the data by putting number of cells along Y-axis and time along with X-axis will yield a bacterial growth curve.

The growth of a bacterial population can be expressed in various phases of a growth curve.

In the first phase, called the lag phase, the population remains at the same number as the bacteria become accustomed to their new environment. Metabolic activity is taking place, and new cells are being produced to offset those that are dying.

In the logarithmic phase, or log phase, bacterial growth occurs at its optimal level and the population doubles rapidly. This phase is represented by a straight line, and the population is at its metabolic peak.

During the next phase, the stationary phase, the reproduction of bacterial cells is offset by their death, and the population reaches a plateau. The reasons for bacterial death include the accumulation of waste, the lack of nutrients, and the unfavorable environmental conditions that may have developed. If the conditions are not altered, the population will enter its decline, or death phase. The bacteria die off rapidly, the curve turns downward.

Phases of bacterial growth curve:

Four phases:

1.     Lag phase or preparatory phase or phase of adjustment.

2.     Log phase or exponential growth phase.

3.     Stationary phase.

4.     Decline or death phase or lysis.

Lag phase:

When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase.  This is the time required for the inoculated bacteria to get adjusted in the new environment (temp, pH, nutrients etc.). Although cell division does not take place immediately and there is no net increase in mass, the cell is synthesizing new components.

The lag phase varies considerably in length with the condition of the microorganisms and the nature of the medium. A lag phase is necessary for a variety of reasons.

·           The cells may be old and depleted of ATP, essential cofactors, and ribosome.  All these will be synthesized during the lag phase.

·           The medium may be different from the one the microorganism was growing in previously. So new enzymes would have to be synthesized to use the different nutrients.

·           There are chances that the microorganisms in the inoculum have been injured, cell wall or membrane damages which need to be repaired.

·           The size of the inoculum – if the inoculum contains high number of cells, lag phase would be smaller

Lag phase will be quite long if the inoculum is from an old culture or a refrigerated culture, or if it is inoculated into a chemically different medium.

Lag phase will be shorter if a young, vigorously growing culture is transferred to fresh medium of the same composition.

During the lag phase, the cells retool, begin to increase in mass, replicate their DNA and finally divide.  

Characteristics of lag phase:

-         Metabolic activity of the cells increases, cells starts synthesizing various proteins, RNA, etc and they will be growing in volume or mass

-         Repair damaged parts of bacterial cell.

-         No appreciable multiplication of bacteria occurs.

-         Required time: 1-4 hrs.

Importance of lag phase - During lag phase, the organisms will be susceptible to membrane acting antibiotic (such as Polymyxin, Amphotericin-B etc.) or Detergents, soaps and other surface acting agents.

Log phase:

Once the metabolic machinery is running properly, bacteria starts dividing by binary fission and double their number. This is the exponential or log phase and during this phase microorganism are growing and dividing at the maximal rate possible.  Growth rate depends on their genetic potential, nature of medium, and conditions under which they are growing.

The rate of growth of the bacterial cells is constant during the exponential phase, they are dividing at regular intervals. This phase is termed as exponential phase since, rapid multiplication and increase of cell numbers occur geometrically or exponentially.

The microbial population is most uniform in terms of chemical and physiological properties during this phase and thus exponential phase cultures are usually used in biochemical and physiological studies. 

Characteristics of log phase:

-         Exponential or geometrical increase in population size.

Exponential : 2⁰    2¹           2²           2³           2⁴           2⁵           2⁶           2⁷       etc.

No. of cells  : 1      2        4        8        16      32      64      128    etc.

-         Active synthesis of cell wall.

-         Metabolic activity very high.

-         Time required: 1-4 hrs.          

Importance of log phase – cells are more susceptible to Antibiotics that inhibits cell wall synthesis, protein synthesis, DNA replication, etc. The virulence or disease causing capability of pathogenic bacteria is highest.

During exponential phase each microorganism is dividing at constant intervals. This interval is known as the generation time.  Generation time or doubling time is the time required for a bacterial cell to divide into two or it is the time required for a bacterial population to double in its size.   

If a culture tube is inoculated with one cell that divides every 20 minutes, the population will be 2 cells after 20 minutes, 4 cells after 40 minutes, 8 cells after 80 minutes and so forth. Because the population is doubling every generation, the increase in population is always 2ⁿ where n is the number of generations. The resulting population increase is exponential or logarithmic

Let N₀ is the initial population number

N_t is the population at time t

n is the number of generations in time t

N_t = N₀ X 2ⁿ

log N_t = log N₀ + n log 2

The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate constant (k). This is the number of generations per unit time, expressed as the generations per hour.

The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g), can be calculated.

If the population doubles (t = g), then

                N_t    = 2 N₀

                   

The mean generation time is the reciprocal of the mean growth rate constant.

           

The mean generation time (g) can also be determined directly from a semilogarithmic plot of the growth curve and the growth rate constant can be then calculated from the g value. This could be done by extrapolating the population size (y axis) in the curve to the time (x axis).  

Generation times vary markedly with species of microorganism and environmental conditions. They range from less than 10 minutes (0.17 hours) for a few bacteria to several days with some eukaryotic microorganisms. Generation times in nature are usually much longer than in culture.

Microorganism	Temperature (0C)	Generation time (minutes)
Bacillus subtilis	37	25
Bacillus stearothermophilus	60	8
Escherichia coli	37	20
Staphylococcus aureus	37	30
Streptococcus lactis	37	26
Mvcobacterium tuberculosis	37	720
Treponema pallidum	37	1980
Saccharomyces cerevisiae	30	120

 

Stationary phase:

In this phase, some bacteria begin to die, some still continue to multiply. The growth rate decreases and the number of bacteria stabilizes. During stationary phase the total number of viable microorganisms remains constant due to the balance between cell division and cell death.

Bacteria produce secondary metabolites, such as antibiotics during stationary phase.

Even though the population size depends on nutrient availability, type of microorganism and other factors, stationary phase usually is attained by bacteria at a population level of around 10⁹ cells per ml.

Many bacteria respond with morphological changes such as endospore formation, production of starvation proteins, increased peptidoglycan cross-linking with more cell wall strength, formation of DNA-binding proteins to protect DNA and production of Chaperones to prevent protein denaturation.  As a result of these, the cells become more resistant to starvation, temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine.  Some bacteria can survive starvation for years.

Microbial populations reach the stationary phase for several reasons.

·        Nutrient limitation - if an essential nutrient is depleted, population growth will slow down.

·        O₂ availability – Growth of aerobic microorganisms are limited by the level of dissolved Oxygen. Since solubility of Oxygen is very low, only the surface of a culture will have an adequate O₂ concentration for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated.

·        Space limitation – increase in population size will result in space constraints, decrease of biological space required for bacteria

·        Accumulation of metabolic end products - Population growth also may cease due to the accumulation of toxic waste products. For example, streptococci produce lactic acid and other organic acids from sugar fermentation that their medium becomes acidic and growth is inhibited.

Characteristics of stationary phase:

-         The net increase in number of cells is zero.

-         Rate of cell division is balanced with rate of cell death are:

-         Exotoxin production starts.

-         Time required: few hours to few days.

Importance of stationary phase -

-         Release of exotoxin starts.

-         Endospore forming bacteria start formation of spore.

-         Erosion of peptidoglycan layer in Gram positive bacteria occurs, and such cells will be shown as gram negative upon staining.  

Decline or death phase or lysis:

In this phase, the total number of viable cells decrease rapidly.  Detrimental environmental changes like nutrient deprivation and the buildup of toxic wastes will lead to the decline in the number of viable cells.  The death of microbial population is usually logarithmic, that is, a constant proportion of cells die every hour.

Characteristics:

-         The death rate is greater than the multiplication rate.

-         Accumulation of significant amount of toxic metabolites occurs.

Importance:

-         Sporulation occurs in some bacteria.

-         Some bacterial release endotoxins