eChapters

Introduction

The motile bacteria show movements with the help of flagella. The motility of bacteria can be observed by hanging drop method under microscope.

Requirements

1. Bacterial culture
2. Cavity slide
3. Vaseline
4. Coverslip

Procedure

1. Place a very small drop of 24 hours old bacterial culture in the center of a coverslip.
2. Put a small amount of Vaseline at each cover of the depression side of the cavity.
3. Invert the cavity slide over the coverslip. The coverslip adheres to glass slide. When the coverslip is inverted, the hanging drop is suspended in the well.

Introduction

A haemocytometer is a single piece of glass with an H-shaped trough forming two counting areas. Hemocytometers are used to determine the total cell count and viability of many different cell types The full grid on a hemocytometer contains nine squares, each of which is 1 mm¹. Each counting area is defined by a grid system. It has supports that hold the cover glass at the proper distance (0.1 mm) above these areas for the calculation of the concentration per volume of liquid.

Requirements

1. Inoculum
2. Inoculation needle
3. Test tubes

Introduction

Photosynthetic bacteria are autotrophs and anaerobes. These bacteria produce Hydrogen sulphide. These are predominant in the bottom of stagnant water enriched with mud.

Requirements

1. Mud sample
2. Enrichment medium of Photosynthetic bacteria or salt solution (1.0g/lt. of (NH⁴)₂SO⁴ 1 g; K²HPO⁴, 1 g; MgSO⁴,0.2g; NaCl, 5g; pH 7.2)
3. Sterilized bottle with plug
4. Fluorescent tube

Introduction

Rhizobia are Gram-negative soil bacteria , aerobic nitrogen fixers and adhere to the roots of leguminous plants. Fixed nitrogen is assimilated to proteins and is transported along the plants. A part of proteins is also released in rhizosphere region.

Requirements

1. Yeast Extract Mannitol Agar (YEMA)
2. Sterile saline
3. HgCl₂ solution (1%)
4. Ethanol (70%)
5. Root nodules of Mung bean
6. Water blanks
7. Pipettes
8. Petri plates
9. BOD Incubator

Introduction

Antibiotics are the substances produced by one microorganism and are required to inhibit the growth of other microorganisms. Antibiotics are used to combat infectious diseases. The antibiotic sensitivity is very significant due to development of resistance among various microorganisms.

Requirements

1. Swab
2. Nutrient agar
3. Petri plates
4. Bacterial cultures
5. Forceps
6. Antibiotic discs
7. BOD Incubator

Procedure

1. Prepare the bacterial lawn on Nutrient agar plate.

Introduction

Most of the bacteria, excepting the photosynthetic bacteria, are damaged by ultraviolet (UV) radiation. UV rays have lower energy content and are capable of producing lethal effect in cells exposed to wavelengths in the range of 200 nm to 300 nm. Nucleic acids absorb these radiations leading to formation of thymine dimers which distort the structure of DNA. Distortion in DNA brings the interferences in DNA replication and transcription. The germicidal effect of UV can be used to determine the minimum amount of exposure required to produce 100% mortality of micro-organisms.

Requirements

1. Nutrient agar medium
2. Card board
3. Incubator
4. Petri plates
5. Inoculating loop
6. Bacterial cultures

Introduction

The most important factor that influences the activity of bacterial enzymes is temperature. Most ofthe organisms grow under mesophilic conditions (at25⁰C). If the optimum temperature is increased, the rate of growth of organism is affected due to changes in protein structure which results denaturation of critical cell protein and ultimately inhibitsthe growth. If temperature is below critical temperature, then there is complete stoppage of growth as less activation energy is available for critical process to occur. However, there are certain organisms which can tolerate extreme temperatures viz.

1. Psychrophiles: Tolerate and grow at > 20⁰C temperature
2. Mesophiles: Tolerate and grow at between 20⁰C and 45⁰C temperature
3. Thermophiles: Tolerate and grow at <45⁰C temperature.

Introduction

The water potential plays an important role on growth of bacteria. If the medium has higher concentration of solute, it is said to be hypertonic. Due to this, the cytoplasm dehydrates and shrinks, resulting in inhibition of growth of bacteria. If solute concentration is less, the medium is hypotonic and hence maximum osmotic pressure on the cell.

Requirements

1. Cultures of E.coli and Bacillus
2. Nutrient agar
3. Sodium Chloride (stock solution at 5 M concentration)
4. Inoculating loop
5. BOD incubator

Procedure

Introduction

The pH (hydrogen ion concentration) of the environment provides the maximum effect on the growth of bacteria. Each organism grows at its optimal pH. Due to variation in other factors such as temperature, osmotic pressure etc marked changes in pH have also been observed.

Requirements

1. Erlenmeyer flasks (100 ml)
2. Culture of E. coli
3. Pipettes
4. Spectrophotometer
5. pH meter

Procedure

Introduction

Demonstration of biological nitrogen fixation is usually accomplished by estimating the total nitrogen through use of some modification of the Kjeldahl procedure. This technique is accurate and reliable whenever a medium initially low in nitrogen is employed.

Principle

The dried organism is digested with sulphuric acid. Organic and nitrate nitrogen is converted to ammonium sulphate and the ammonia gas is distilled into boric acid forming ammonium borate making weak boric acid alkaline. The ammonium borate is back titrated with standard sulphuric acid until the reaction mixture changes its colour from green to pink due to formation of ammonium sulphate. The standard acid used in back titration of ammonium borate is a measure of nitrogen content of the organism.

Introduction

Resazurin has high tinctorial value (related to colour), imparting characteristic blue colour. Resazurin test gives us indirect information concerning bacterial content and /or activity. Resazurin is also sensitive to the reducing action of leucocytes. The results of this test are comparatively less affected by antibiotic residues in milk than methylene blue reduction test.

Principle

Reduction of dye is indicated by a gradual colour change through various shades of purple and mauve to full pink, at which point the resazurin completely changes to resorufin.

Q    :    Ubiquinone (coq).

Q    :    Every.

Q fever      :    An acute zoonotic disease caused by the rickettsia Coxiella burnetii.

Procedure

Place a portion of your RNA sample (approximately 40 mg, hydrated) into a heavy walled Pyrex test tube. Add 1.0 ml of 1 N HCl and seal the tube.

Heat the tube in a boiling water bath for 1 hour.

Cool the tube, open it and place the contents into a centrifuge tube. Centrifuge the contents at 2,000 RPM in a clinical centrifuge to remove any insoluble residue. The supernatant contains your hydrolyzed RNA.

Prepare Whatman filter paper No. 1 for standard one-dimensional chromatography.

Using a micropipette, spot 20 µl of your hydrolyzate onto the paper, being careful to keep the spots as small as possible (repeated small drops are better than one large drop). Allow the spots to completely dry before proceeding.

Place the paper chromatogram into your chromatography tank and add the solvent (acetic acid:butanol:water). Allow the system to function for an appropriate time (approximately 36 hours for a 20 cm descending strip of Whatman #1). Remove the paper and dry it in a circulating air oven at 40° C for about 2 hours.

Locate the spots of nucleotides by their fluorescence under an ultra-violet light source. Expose the paper chromatogram to a UV light source and outline the spots using a light pencil. The order of migration from the point of origin is guanine (light blue fluorescence), adenine, cytilic acid and finally, uridylic acid.

After cafefully marking the spots, cut them out with cutters and place the paper cut outs into separately labeled 15 ml conical centrifuge tubes. Add 5.0 ml of 0.1 N HCl to each tube and allow the tubes to sit for several hours to elute the nucleotides from the paper.

Introduction

This method can be scaled to yield amounts of DNA ranging from less than ten to more than hundreds of micrograms of DNA. However, shearing forces are generated at every step, with the result that the DNA molecules in the final preparation rarely exceed 100-150 kb in length. DNA of this size is adequate for Southern analysis on standard agarose gels, as a template in Polymerase chain reactions (PCRs), and the construction of genomic DNA libraries in bacteriophage Lambda vectors.

Principle

The method described in protocol involves digesting eukaryotic cells or tissues with proteinase K in the presence of EDTA (to sequester divalent cations and thereby inhibit DNases) and solubilizing membranes and denaturing proteins with a detergent such as SDS. The nucleic acids are then purified by phase extractions (isopropanol). Contaminating RNA is eliminated by digestion with an RNase, and low molecular-weight substances are removed by dialysis (or centrifugation).

To isolate a streptomycin resistant mutant of E.coli by Indirect selection using Replica Plate Method.

Introduction

J. Lederberg & his wife E. M. Lederberg devised this technique in 1952.

U.V. light coming from the sun emits strong radiation to extent the antibacterial effect in the microbial population. However it has a very short penetration power, UV light is used as a mutagenic agent & was used by Beedle & Tatum to obtain biochemical mutation. The mutation rate is only once in 10^-9 cells per population. Then hence using mutagens like UV light by 10 to 1000 folds will increase this mutation rate.

In isolation of the mutant using UV light the dose of UV light is measured by its units/nm² organisms. The time selected for the mutation is calculated on the basis of 0.1 to 1% survival & a suitable biochemical character is used to evaluate the mutation.