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11 Jun 2018

Biological Oxygen Demand (BOD)

It is also known as Biochemical Oxygen Demand (BOD). It is defined as the amount of oxygen consumed during the process of degradation and eventual stabilization of unstable organic substances by the biochemical activities of aerobic and other microbes. This degradation of the chemical complex is a desirable process and the final product is called stabilized wastewater. The aerobic bacteria consume oxygen during the process of oxidization of the organic and other oxidizable inorganic substances. The immensity of biochemical degradation depends on the population of bacteria. An actively growing population of bacteria will consume more oxygen to quickly decompose unstable complexes. Biological/Biochemical Oxygen Demand (BOD) is reduced with the decrease in the quantity of these complexes in the wastewater. Therefore, it can be surmised that BOD is directly proportional to the level of degradable chemical complexes; high concentration of chemical substances will result in the high BOD.

The BOD is a very useful measure of the efficiency of methods of wastewater treatment. A method in which amount of BOD reduced quickly is considered as most effective and efficient method. Therefore, exactly stabilized effluent, when discharge in the body of water, does not cause reduction of oxygen in the water.

Wastewater Disposal Methods

It is a well-known fact that the wastewater should be treated properly and effectively before its disposal into receiving water bodies. Disposal of wastewater may be accomplished with or without treatment.

Disposal of Wastewater by Treatment Methods

There are different methods available for the removal of microorganisms and stabilized the putrescible organic and inorganic chemicals in the wastewater. These methods are known as wastewater treatment methods. It is a very interesting fact that usually microorganisms are used to reduce the large burden of wastewater, which is organic matter. With few exceptions, wastewater treatment plants are integrated with physical, chemical and microbiological methods to concern with the different problems related to wastewater.

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According to distinct types of treatment, they are divided into four types. Each type of treatment process has a special purpose, targeting the removal of all sources of materials and reducing the burden of microorganisms from the wastewater.

Primary Treatment of Wastewater

This process is mainly designed to remove the total solids from the wastewater by sedimentation and render it adequately free from pathogenic bacteria by chlorination. Initially, large objects are removed by bar screens from the wastewater flow. It removes a significant amount of particulate matter. The collected objects are then put into the grinder and released back into the wastewater flow.

The wastewater is then allowed to flow to a series of large primary settling compartments in which most of the organic matters and dense inorganic particles such as grit and sands are removed. Usually, there are two types of settling compartment, (a) grit compartment and (b) sedimentation tank or quiescent settling compartment. In grit compartment, wastewater flows very slow which permits large and heavy particulate matter to settle out. In the next step, the municipal and industrial wastes (particulate organic matters) in wastewater are removed in the sedimentation tank. In sedimentation tank, wastewater is allowed to stay for 1 to 3 hours during which most of the suspended organic matter settles out. The sedimented material is in the form of a semi-solid mass called sludge. The efficiency of sludge formation can be increased by the addition of various chemicals to coagulate the suspended particles which enhanced the sedimentation rate. The sludge is not allowed to remain in the bottom of sedimentation tank for a long period because of anaerobic bacteria produce gases during metabolism that tend to resuspend the settled material and increased the odor. Therefore, the sedimentation tank is equipped with scrapper mechanisms that occasionally removes the bottom sludge to a collection hopper. The underflow sludge becomes a waste product of the process. The remaining liquid portion of the wastewater which leaves the tank is called effluent.

 

To be continue...

07 Jun 2018

There are certain bacteria which cannot be stained by Gram's method. In 1882, Paul Ehrlich developed a method of staining such type of bacteria. This method was named, and still known as acid-fast staining and the bacteria were named as acid-fast bacteria. In the same year, Ehrlich's method was improved by Zehil and Neelsen. Nowadays, Ziehl-Neelsen method is believed as most important differential staining procedure used for the identification of acid-fast species of Mycobacterium, Actinomyces, and Nocardia. There are many acid-fast bacteria which are pathogenic, such as M. tuberculosis (tuberculosis), A. israelii (actinomycosis), M. leprae (leprosis), and N. asteroides (nocardiosis).

Acid-fast bacteria may be defined as those cells which keep the color of the primary dye (carbol fuchsin) even after the process of decolorization by the acid-alcohol solution. Those bacteria which fail to do so are known as non-acid-fast bacteria.

Acid-fast bacteria are coated with a thick waxy material, mycolic acid, which makes the bacterial cells highly resistant to the penetration of dyes. The penetration of dye is promoted by using heat as mordant. The heat invades the dye through the waxy coat and into the cytoplasm.

PURPOSE

  • Differentiation between acid-fast and non-acid-fast bacteria.
  • Diagnosis of pulmonary tuberculosis from sputum smear. See also: Examination of Sputum

NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

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Reagents

  1. Carbol fuchsin
  2. Acid-alcohol solution
  3. Methylene blue

Equipment

  1. Bunsen burner
  2. Wire loop
  3. Glass Slide
  4. Spirit lamp
  5. Microscope

PROCEDURE

Acid-Fast staining by Ziehl-Neelsen method

  1. Take a clean glass slide and prepare a thin smear from the specimen using sterile technique. The smear should be extremely thin covering a large area of the slide.
  2. Dry the smear is air and then fix the slide by passing the slide through the flame.
  3. Cover the slide with carbol fuchsin. Keep it for 5 minutes over a spirit lamp with constant heating but not boiling. Do not allow the stain to dry over the slide.
  4. When the slide is cooled, wash it with tap water.
  5. Flood the slide with acid-alcohol and leave it for 3 minutes. Wash the slide with tap water and drain.
  6. Counterstain with methylene blue for 2 minutes.
  7. Wash the slide with tap water and keep it for dry.
  8. Observe the slide under oil immersion objective.

OBSERVATION

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved bright red rods. Non-acid-fast cells appear blue.

Acid-Fast Staining by Mobin Method

In 1985, a Pakistani microbiologist, Abdul Mobin Khan developed a method for the staining of acid-fast bacteria. In this method, heating of flooded primary dye on smear is not required. However, initial fixing of the smear over the flame is necessary in order to increase the permeability of the cell wall and promote the newly formulated primary dye to penetrate the cell.

Needs

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

Reagents

  1. Mobin stain
  2. 1% H2SO4
  3. Crystal violet

Equipment

  1. Wire loop
  2. Bunsen burner
  3. Glass slides
  4. Microscope

Procedure

  1. Using sterile technique, prepare a thin smear from the specimen covering a large area of the glass slide.
  2. Dry the smear in the air and then fix it by passing the slide 20 times through the flame.
  3. Place the smear on the staining rack and cover it with Mobin stain. Keep it for 10 minutes.
  4. Pour off the stain and wash the slide with tap water.
  5. Decolorize the smear by 1% H2SO4 solution till it is light pink.
  6. Wash the slide with tap water and keep it for dry.
  7. Observer the slide under oil immersion objective.

Observation

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved red rods while non-acid-fast bacteria as blue.

07 Jun 2018

In 1883 (originally published in 1884), Dr. Hans Christian Gram (1853-1938) developed a technique for the classification of bacteria into two broad groups, Gram-positive and Gram-negative. It is the most important staining technique for the classification and differentiation of bacteria.

The Gram stain consist of four reagents; crystal violet (use as a primary dye), Gram's iodine (use as a mordant), ethyl alcohol (use as a decolorizer), and safranin (use as a counterstain). The Gram-negative, on the other hand, lose the primary dye (crystal violet) when decolorized and, thus, take the color of counterstain (safranin).

The Gram-reaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 11-22% in Gram-negative cell wall and 1-4% in the Gram-positive cell wall. In the Gram-negative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell wall. The dehydrating result of alcohol cannot fill these pores which cause the liberate of primary stain making the cell colorless. Such cells take the color or counterstain (safranin). On the contrary, the amount of lipid is very low in the Gram-positive cell wall and easily dissolved by in ethyl alcohol, causing the formation of very small pores. These pores are further closed by the dehydrating effect of alcohol which does not permit the primary dye (crystal violet) to leave the cell.

PURPOSE

Identification, differentiation, and classification of the bacteria.

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NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria or fungi grown and isolated in culture.

Reagents

  1. Crystal violet
  2. Gram iodine
  3. Ethyl alcohol (95%)
  4. Safranin
  5. Ceder wood oil

Equipment

  1. Bunsen burner
  2. Wire loop
  3. Glass slides
  4. Microscope

PROCEDURE

  1. Prepare a smear of the specimen, dry in air and then fix it in low flame.
  2. Flood the smear with crystal violet, and keep it for 1 minute. Wash the smear with running tap water.
  3. Pour Gram's iodine on smear and after 1 minute, wash it with tap water.
  4. Pour alcohol on the smear until the purple color no longer comes from the smear.
  5. Pour safranin on the smear and after 45 seconds, wash it with tap water and keep the smear dry in air.
  6. Observe the stained smear under oil immersion lens and note down the arrangement, shape, and Gram-reaction of the cells.

OBSERVATION

The Gram-positive bacteria appear in purple color and Gram-negative bacteria appear in pink color.

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