Friday, January 29, 2021

Methylene Blue Reduction Test

Aim: To determine the quality of milk by Methylene blue reductase test

Principle: Methylene Blue Dye Reduction Test, commonly known as MBRT test is a quick method to assess the microbiological quality of milk. This test is based on the fact that the methylene blue dye solution added to the milk get decolorized when the oxygen present in the milk get exhausted due to microbial activity. The sooner the decolorization, it is assumed that the more inferior is the bacteriological quality of milk.

Methylene blue is a redox indicator, that lose its color under the absence of oxygen and is thought to be reduced to colorless leucomethylene blue. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the color to disappear. The greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear. Thus, the time of reduction is taken as a measure of the number of organisms in milk.

Milk is a good medium for the growth of microorganism.  A variety of microorganism can be found in both raw milk and pasteurized milk. Normally the milk is contaminated with microorganisms such as Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterobacter spp., Bacillus spp., etc.  Contaminated milk is one of the important sources for transmission of diseases from animals to humans. The main reason for this contamination is the un-proper handling of milk. Normally milk is contaminated during the milking process by the microorganisms present in the exterior surface of the animals, pipelines such as udder and adjacent areas. Unsterilized dairy utensils such as milking machines, milk cans are also a good source of contamination by the microorganism.

Methylene Blue Dye Reduction Test is widely used at the dairy reception area, processing units and milk chilling centres where it is followed as acceptance/rejection criteria for the raw and processed milk

Material Required

1.      Milk samples (raw, boiled, pasteurized)

2.      Reagent: Methylene Blue dye (1:25000)

3.      Sterile screw capped tubes

4.      Water bath

5.      Bunsen burner

Procedure

1.     1 mg of Methylene Blue dye was dissolved in 250 ml of sterile distilled water

2.     Each of the milk samples were thoroughly mixed

3.    3 ml of Methylene Blue dye was added to appropriately labelled MBRT tubes.

        Using different  pipettes, transfer about 30 ml of each type of milk into the above tubes and they were closed with stopper. 

6.     The contents of the tubes were mixed gently by inverting each test tube.

7.     The tubes were incubated in water bath at 37oC for 5-6 hours

Observation

Milk samples observed for Methylene Blue reduction every five minutes for the first 30 minutes and then every 30 minutes for the rest of the time. Reduction is demonstrated by decolorization of the dye.

Result

The tubes which contained the samples of packet milk showed colour change from blue to white within 30 minutes, showing Methylene Blue dye reduction test positive. Raw milk showed Methylene Blue dye reduction within 5 minutes indicating very poor quality of the milk. Disappearance of colour within the limited time indicates the absence of oxygen in milk. Hence it shows packet milk and raw milk are of poor quality. Pasteurized milk showed Methylene blue reduction within 130 minutes, indicating fair quality of the sample. Boiled milk showed Methylene blue reduction in 450 minutes indicating good quality. 



Observations (left hand side)

Quality of milk sample by Methylene Blue Dye Reduction Test

Milk sample

Methylene Blue Dye Reduction Time

Quality

Packet milk

Within 30 minutes

Very poor

Raw milk

Within 30 minutes

Very poor

Pasteurized milk

130 minutes

Fair

Boiled milk

450 minutes

Good

 

Classification of Milk Quality

Methylene Blue Dye Reduction Time

Quality of milk

Approximate number of Bacteria

Within 30 minutes

Very poor quality

>2,00,00,000

31-120 minutes

Poor quality

>40,00,000 to 2,00,00,000

121-360 minutes

Fair quality

>5,00,000 to 40,00,000

361-450 minutes

Good quality

Below 500000

Friday, January 22, 2021

Isolation and screening of amylase producing microorganisms from soil

 Aim

To isolate bacteria producing amylase, capable of degrading the polysaccharide starch from the soil

Principle

Polysaccharides are large molecular weight polymers of monosaccharide units joined by glycosidic bonds. Polysaccharides can be structural and nutritional in nature. Nutritional polysaccharides are formed by both plant and animal cells and they are used as reserve food supplies. Glycogen is the reserve polysaccharide of animal cells and starch serves a similar function in plant cells.

Starch is a high molecular weight polysaccharide containing single monomeric units of glucose. Starch contains two forms of glucose polymers- amylose and amylopectin. Amylose consists of long unbranched chains of D-glucose units connected by α-1,4 glycosidic linkages. Amylopectin is branched with one branch for every thirty glucose units. The monomeric glucose units are linked by α-1,4 glycosidic linkages in the unbranched region and α-1,6 glycosidic linkages in the branched regions in amylopectin.

Starch is not capable of permeating the cell membrane because of its high molecular weight. To assimilate starch for energy and catabolic reactions, it must be degraded into basic glucose units by starch hydrolyzing enzyme, amylase. Starch is converted to dextrins and then maltose, which is broken down to soluble glucose molecules that can be transported into the cell for energy production, through glycolysis. Since enzymes are catalysed and are not consumed in chemical reactions, a few molecules of enzymes are used over and over to break down a great deal of starch.



Amylases are commercially used in bread making, brewing, for production of sugar syrups, for mash preparations, in spot removers, for the removal of silver from used photographic films, in instant hot breakfast cereals and also in dry cleaning process.

Amylases give a deep blue colour and amylopectin gives a red to brown colour when they react with iodine depending on the source of starch. Once starch is completely hydrolyzed the oligo and disaccharides do not form a colour complex with iodine. The zone of hydrolysis is evident by the lack of colour.

Materials Required

Starch agar medium, Gram’s Iodine solution, sterile petridish, inoculation loop, Bunsen burner

Procedure

1. 1 gm soil was suspended in 100 ml sterile distilled water in a conical flask

2. Serial dilution was performed in tubes containing 9 ml sterile distilled water.

2. Aliquots of  0.1 ml was transferred from 10-5 and 10-6 into starch agar plates and spread plate performed

3. The inoculated plates were incubated at 37oC for 24-48 hours

4. After incubation, the plates were analyzed with Gram’s Iodine solution and then observed for starch hydrolysis

Observation

Presence of colorless hydrolysis zone around the colony after treatment with Gram’s Iodine solution is positive evidence for degradation of starch. Absence of clear zone indicate a negative reaction.

Result

A clear zone around amylase producing colonies indicated the presence of amylase producers which were isolated from the soil sample.

 



 

 

Monday, January 18, 2021

INDIAN INITIATIVES FOR ENVIRONMENT CONSERVATION

 Today, more than ever before, there is a great need to create public awareness about conservation of the environment and to ensure the full participation of society in the mitigation of environmental problems. Environment and sustainable development are interlinked and should be regarded as national priorities. The Government, environmental monitoring agencies, NGOs, academic and research institutions and the media must work synergistically to combat pollution and thus create a healthy environment. 

GOVERNMENT INITIATIVES 

Degradation of the environment and its disastrous consequences have been a matter of great concern for governments all over the world. Several legislations have been passed to provide a legal framework for the management and preservation of the environment. 

Environment was first discussed on June 5, 1972 at the United Nations Conference on “Human Environment” held at Stockholm leading to the declaration of June 5 as World Environment Day. Within four years of the historic Stockholm Conference, India enacted legislation aimed at protection of the environment which later formed a part of the Indian Constitution. 

The 42nd Amendment Article 48Aof our Constitution provides “The State shall endeavour to protect and improve the environment and to safeguard forests and wildlife in the country” and Article 51A (g) provides “It shall be the duty of every citizen of India to protect and improve the natural environment including forests, lakes, rivers and wildlife and to have compassion for all living creatures”. Thus environment was given a pride of place in the Directive Principles of State Policy and Fundamental Rights. 

The need for integrating environmental factors into the process of planned sustainable development was first highlighted in the Fourth Five Year Plan (1969-1974). Thus, in our country the debate between environments versus development was triggered as early as in the 1960s. 

The other important milestones in the path of environment conservation are: 

1. In 1972 the National Committee on Environmental Planning and Coordination (NCEPC) was set up to serve as a high level advisory body to the government. 

2. In 1980 the N.D. Tiwari Committee was appointed which recommended the setting up of an independent Department of Environment under the Government of India, which subsequently became a full-fledged Ministry of Environment and Forests in 1985. 

3. The Wildlife Protection Act was passed in 1972(and subsequently amended in 2002) for setting up National parks and sanctuaries. 

4. The Wildlife Protection Rules were passed in 1973 for the protection of all bird and animal species irrespective of their habitat. As a result, conservation projects were launched for endangered species which have contributed in a large measure to the increase in wildlife in India especially Project Tiger. 

5. Forest Conservation Act of 1980 for protection of all types of forests and prevention of non-forest activities was amended in 1998. 

6. Environment Rules for Siting of Industrial projects were passed in 1999. 

7. The Biological Diversity Act of 2002 laid down clear guidelines for conservation of the biological diversity of the country. 

8. The Prevention of cruelty to animals Act of 1960 was later amended to Animal welfare Act in 2006. 

9. The State /Union Territory Minor Forest Produce (Ownership of Forest Dependent Community) Act of 2005 was a landmark legislature which recognised the legitimate rights of the forest dwellers to the use of forest produce. 

10. The National Green Tribunal Act of 2010 which enables creation of special tribunals for expeditious disposal of cases involving environment. 

WATER AND AIR POLLUTION –PREVENTION AND CONTROL 

'Over the years, the Government of India has passed several path breaking acts to curb and prevent water and air pollution: 

1. Water (Prevention and Control of Pollution) Act of 1974 provides institutional safeguards for the prevention and abatement of water pollution. This act resulted in the setting up of the Central Pollution Control Board. 

2. Water (Prevention and Control of Pollution) Cess Act of 1977 provides for levy and collection of cess from water consuming industries and establishments. 

3. Air (Prevention and Control of Pollution) Act of 1981 defined air pollution in the atmosphere and measures to curb air pollution including noise pollution. 

4. In 1970 the Merchant Shipping Act dealing with oil spills from ships near the coastal areas was passed. This act was primarily aimed at preventing marine pollution and to protect the vulnerable marine life. 

5. The Environment (Protection) Act of 1986 was a comprehensive legislation covering all aspects of protection of the environment. This act was passed by Parliament after the Bhopal Isocyanate gas tragedy of December3, 1984. 

6. The Coastal Regulation Zone Notification of 1991regulates all activities in the coastal areas including construction and seeks to safeguard backwaters and river estuaries. 

MINISTRY OF ENVIRONMENT AND FORESTS (MOEF) 

MoEF set up in 1985, implements the various environment protection laws all over the country through the Central and State Pollution Control Boards. MoEF has the mandate to 

1. Monitor and control pollution especially in industries, 

2. Increase the forest cover in the country, 

3. Conserve and increase wildlife and the rich biodiversity of the country, 

4. Support research in Environmental Science and Technology. 

MoEF works closely with the Ministry of Non-Conventional Energy Sources and the Department of Ocean Development (DOD). It is also the nodal agency for monitoring climate change in the country. The Forest Survey of India monitors changes in the land and forest resources and implements social forestry programmes. In 1982, MoEF instituted the Environment Information System to provide information to policy planners, decision makers, scientists, industry and the general public through workshops and seminars in regional languages and extensive use of the print and electronic media. 

The Government has made Environment Impact Assessment (EIA) compulsory for all proposed developmental projects. EIA is an environment management tool to evaluate the possible impact of projects on the environment and involves sampling of air, water and soil followed by analysis. Technical aspects like effluent emission, air pollution and noise pollution are also studied. 

The Central and State Pollution Control Boards are required to ensure the strict implementation of the policies laid down by the government to conserve the environment . 

Sadly these well intentioned rules are, more often than not, flouted and unscrupulous elements continue to ravage the fragile ecosystem of the country. 

INSTITUTIONAL INITIATIVES 

Several national level scientific institutions are engaged in research for finding feasible solutions to environmental problems facing the country. 

1. National Environmental Engineering Research Institute (NEERI), one of the 39 laboratories under CSIR with headquarters at Nagpur and five zonal laboratories, is engaged in providing innovative and cost effective solutions in the following thrust areas: 

a. Recovery and recycling of waste from industry, 

b. Waste water recycling for zero discharge, 

c. Air and water pollution, 

d. Environmental biotechnology, 

e. Toxic waste management, 

f. Impact of salinity on soil. NEERI also disseminates information regarding environmental issues through its journal, website, annual reports, publications and newsletter

 2. The Energy Research Institute (TERI) (earlier Tata Energy Research Institute), located at Delhi is a premier institute devoted to energy related studies and has handled numerous projects dealing with climate change, biodegradable materials, biodiesel and insecticides from plants and recovery of oil from oil wells using bacteria. TERI University offers PhD programmes in climate change, forest ecology, biodiversity assessment and conservation and wetland management . TERI is deeply involved in the development of clean technologies which will be energy efficient and will have minimum adverse environmental impact. 

3. National Institute of Oceanography (NIO), based at Goa is involved in the chemical, physical and biological studies of the oceans. One of the major areas of research is marine pollution and impact of land reclamation on marine life. 

 

NNRMS carries out the following tasks:

·         Mapping of forests and grasslands.

·         Monitoring of forest encroachment and shifting cultivation Forest fire surveillance.

·         Mapping and monitoring of biosphere reserves.

·         Eco-system management related studies for environmentally fragile/sensitive regions like the Himalayas, Western Ghats etc.

·         Development of Environment/Forest Resources Information System (ERIS/FRIS).

·         Establishment of State Forest Data Management Centres (S17DMCs), with linkages to National System.

·         Resources assessment of fuel wood.

·         Environmental Impact Assessment (EIA) involving land, water and air pollution

·         Environmental hazard related studies (landslide, volcano, earthquake).

·         Mapping/monitoring of Coastal Regulation Zone/coastal areas, etc.

 

 NNRMS helps in:

·         Identifying zones which could yield ground water and the suitable locations for recharging water

·         Monitoring command areas.

·         Estimating crop areas and yields

·         Assessing deforestation

·         Mapping urban areas for planning purposes

·         Delineating ocean areas with higher fish catch potential and

·         Monitoring of environment and scene specific spot imagery

·         Training educationists about GIS technologies.

Different day observations to promote awareness and participation in environment conservation

June 5 World Environment Day On this day, in 1972, the Stockholm Conference on human environment was held in Sweden. There was a large gathering from all over the world and people expressed their concerns about the increasing environmental problems. The ‘World Environment Day (WED) is the United Nations’ most important day for encouraging worldwide awareness and action for the protection of our environment. Since it began in 1974, it has grown to become a global platform for public outreach that is widely celebrated in over 100 countries.

June 17 World Day to Combat Desertification and Drought The World Day to combat desertification, since 1995 promotes public awareness relating to international cooperation to combat desertification and the effects of drought. The United Nations General Assembly declared in 1994, June 17 as the ‘World Day to Combat Desertification and Drought’ to promote public awareness of the issue. Implementation of the United Nations Convention to Combat Desertification in countries experiencing serious drought and/or desertification, particularly in Africa, began.

September 16 World Ozone Day The United Nations declared in 1994 this day as the ‘International Day for the Preservation of the Ozone Layer’. It is the day the Montreal Protocol was signed. The Montreal Protocol on Substances that Deplete the Ozone Layer (a protocol to the Vienna Convention for the Protection of the Ozone Layer) is an international treaty designed to protect the ozone layer by phasing out the production of numerous substances that are responsible for ozone depletion. On August 26, 1987, it was proposed and entered into force on 26 August 1989. Montreal is the most populous city in Quebec and the second most populous municipality in Canada. It is named after Mount Royal, the triple-peaked hill in the heart of the city.

October 2-8 National Wildlife Week In order to orient the common man towards the protection of wildlife, the Indian Board of Wild Life (IBWL) decided to observe Wildlife Week and since then from October 2 – 8, every year organize different activities related to wildlife conservation to commemorate this week. Wildlife Week celebrated all over the country, offers to preserve fauna means the animal life of India.

Reference 

INDIAN INITIATIVES FOR ENVIRONMENT CONSERVATION D. V. Prabhu Department of Chemistry, Wilson College (University of Mumbai) Mumbai, Chief Editor Green Chemistry & Technology Letters, India

Green Chemistry & Technology Letters eISSN: 2455-3611, Vol 4, No 1, 2018, pp 01-05 

https://geographyandyou.com/17-important-days-indias-environment-buffs/

Sunday, January 17, 2021

CANDIDIASIS

 

  • Candidiasis
  • Candidiosis
  • Candidosis
  • Moniliasis

-          Infection of skin, mucosa and rarely of internal organs

-          Caused by a yeast like fungus, Candida albicans and occasionally by other Candida species

-          Candida albicans- Ovoid, spherical, budding yeast cell, which produces pseudomycelia both in culture and tissues

       Dimorphic fungi, existing in both mold and yeast forms.

 




-       

-          Candida species are normal inhabitants of the skin and mucosa - gastrointestinal tract, respiratory tract, vagina, and mouth.

-          Candidosis is an opportunistic endogenous infection

      -          A common predisposing factor is diabetes

-          Candida infections are rarely serious in  healthy people

-          Their growth is naturally suppressed by other normal microbiota but when these microbiotas are disrupted, Candida multiplies rapidly, producing candidiasis.

-          Also cause nosocomial blood infections in hospitalized patients.

-          In rare cases, it may spread through other parts of the body if the patient's immune system is not functioning properly-in severe cases it can affect the blood, the membrane lining the heart muscle (endocardium), or membranes around the brain (meninges).

Subdivisions of Candidiasis

  • Candida Granuloma
  • Candida Infection around the Nail-Paronichia
  • Candidiasis of the Skin-Cutaneous Candidiasis
  • Mucocutaneous Candidiasis, Chronic
  • Oral Candidiasis
  • Penis infections
  • Systemic Candidiasis
  • Thrush
  • Vulvovaginitis, Caused by Candida

  

Signs & Symptoms

A very wide range of symptoms can be caused by the Candida infection, from the mildest and more common forms that usually affect the mouth and vagina, to the most rare and severe forms which may affect the heart or brain:

1) Candidiasis of the Skin (Cutaneous Candidiasis or Cutaneous Moniliasis)

-          Superficial skin infection is a common location for this fungal infection.

-          The lesions appear as red, sometimes itchy patches of varying sizes and shapes. The lesions are usually rimmed with small pustules and they commonly appear in folds of the skin; i.e., the underarms, under the breasts, the navel, groin or the folds of the buttocks (e.g. diaper rash)- Crusts may form on the scalp, possibly causing hair loss.

-          The infection may spread to the face, fingertips or the trunk. When the area around the anus is involved, the infection is called Perianal Candidiasis.

-           2) Vulvovaginitis or Vaginitis caused by Candida. Pregnant women or women with diabetes are more prone -usually first appears as a thick white or yellow acidic, vaginal discharge (leukorrhea) with itching and redness of the vagina and vulva.

3) Penis, infected by Candida. Infection of the tip of the penis (Glans Penis) with Candida -less common than Candidal Vaginitis. This infection may be seen in men whose sexual partners have Candidal vulvovaginitis and in men with Diabetes.

4) Oral Candidiasis (Thrush). Common in bottle fed infants and the aged and weak - affects the mouth. Creamy white patches or sores appear on the tongue or mucous membranes of the mouth. The corners of the mouth can become red (inflamed) and cracked.

5) Candida infection around the nails (Candidal Paronichia). Begin as a painful swelling that later develops pus. The infections may occur under the nails (subungual) possibly causing loss of fingernails or toenails. Common in occupations that require frequent immersion of hands in water (dishwasher, bartender etc)

6) Chronic Mucocutaneous Candidiasis (Candida Granuloma). This is a rare and severe form of Candidiasis, characterized by chronic infection of the skin, nails, scalp, and mucous membranes. This type of Candidiasis usually develops during infancy. It is characterized by red, pustular, crusted and thickened lesions, especially on the nose and forehead.

7) Systemic Candidiasis. Most serious Candida infection-affects many parts of the body and is usually caused by an immune deficiency. Inflammation of the membrane lining the heart (endocarditis), the membrane lining the skull (meningitis), or rarely inflammation of the bone (osteomyelitis) may also occur.

Bronchopulmonary candidosis is a rare complication of preexisting pulmonary or systemic disease

 8) Intestinal Candidiasis occurs commonly following an antibiotic treatment, as diarrhea not responding to treatment

Diagnosis

Microscopy and Culture

  • Wet films or Gram stained smears from lesions/exudates show budding gram positive cells

-Candida is seen normally on skin/mucosa- its abundant presence is only of significance

-Demonstration of mycelial forms- indicate colonisation and tissue invasion –greater significance




  • Cultures on SDA- creamy white colonies, smooth with a yeasty odour

-Candida albicans can be differentiated from other Candida species by growth characteristics, sugar assimilation and fermentation tests. 

-C. albicans alone forms chlamydospores on corn meal agar cultures at 20 degree Celsius

  • Germ tube formation – ability to readily form germ tubes- within 2-3 hours, when incubated in human serum at 37 degree Celsius (Reynold’s Braude phenomenon)

  • Serology- Antibodies/agglutinins appear in sera of patients but not helpful in diagnostic, since they occur in normal persons

Delayed hypersensitivity to Candida is universal- skin testing with Candida extracts indicates active functional cell mediated immunity-CMI

Treatment

  • Management of Candidosis is by removal of predisposing causes.
  • Amphotericin B, 5-fluorocytosine and clotrimazole used for disseminated candidosis
  • Nystatin is effective, but poorly absorbed from gut- so not useful in systemic diseases

Penicillin Fermentation

  Antibiotic  - defined by  Selman Waksman  as “an organic compound produced by one microorganism that,  at great dilutions , inhibits the g...