Urease test

 Aim

To determine the abilty of microorganisms to degrade urea by the production of the enzyme urease.

Principle

Urea is a major organic waste product of protein digestion in most vertebrates and is excreted in the urine.  Some microorganisms have the ability to produce the enzyme urease.  The urease is ahydrolytic enzyme which attacks the carbon and nitrogen bond amide compounds (eg: urea) with the liberation of ammonia as shown below:

Urea + 2H₂O      urease        Carbondioxide + water + ammonia

Urease test is performed by growing the test organisms on urea agar medium containing the pH indicator phenol red (pH 6.8).  During incubation, microorganisms possessing urease will produce ammonia that raises the pH of the medium.  As the pH becomes higher, the phenol red changes from a yellow color to a red or deep pink color.  Failure of the development of  a deep pink color due to lack of ammonia production is evidence of the absence of urease production by the microorganisms.

Requirements

24 hr nutrient agar cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Urea broth, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  Using sterile techniques inoculate each organism into appropriately labeled tubes by means of streak       inoculation.  One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

 

Observations

Tubes inoculated with Staphylococcus showed color change of the medium from yellow to red indicating a positive result.  Tubes inoculated with  Streptococcus, Pseudomonas, Bacillus and E.coli  maintained the yellow color of the medium, indicating a negative result.

 Result 

Staphylococcus is urease positive where as Streptococcus,Pseudomonas, Bacillus and E.coli, are urease negative.

 

Catalase test

 

Aim

To determine the ability of microorganisms to degrade hydrogen peroxide by producing the enzyme catalase.

Principle

During aerobic respiration, microorganisms produce hydrogen peroxide.  Accumulation of these substances will result in death of the organism unless they can be enzymatically degraded.  These substances are produced when aerobes, facultative anaerobes, and microaerophiles use the aerobic respiratory pathway, in which oxygen is the final electron acceptor, during degradation of carbohydrates for energy production.  Organisms capable of producing catalase rapidly degrade hydrogen peroxide as follows:

2H₂O₂                                                  2H₂O    +    O_{2 ↑}

(Hydrogen peroxide)                        (Water)        (Free Oxygen)

 

Catalase production can be determined by adding the substrate H₂O₂ to the culture of the organism to be tested.  If catalase is present, the chemical reaction is indicated by bubbles of free oxygen gas.  This is a positive catalase test.  The absence of bubble formation is the negative catalase test.

Requirements

24 hr nutrient agar cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

3% Hydrogen peroxide, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  A loopful of cuture is placed on a clean glass slide and 1-2 drops of 3% hydrogenperoxide is added.

2.  Mixed well and immediately observed for the bubble formation.

 

Observations

On addition of hydrogen peroxide Bacillus, Staphylococcus, Pseudomonas and E.coli cultures produced bubbles within minutes, indicating the formation of free oxygen gas.  Streptococcus sp. produced no characteristic reaction.

 Result

Bacillus, Staphylococcus, Pseudomonas sp. and E.coli are catalase positive whereas Streptococcus sp. is catalase negative.

Carbohydrate fermentation test

 

Most microorganisms obtain their energy through a series of orderly and integrated enzymatic reactions leading to the biooxidation of a substrate, frequently a carbohydrate.  The major pathways by which this is accomplished are aerobic/anaerobic cellular respiration and fermentation.  Fermentation is an anaerobic metabolic process in which the final electron acceptor is an organic substrate instead of oxygen.  The actual end products depend on the factors like organism involved, nature of substrate, temperature of incubation and acidity produced. The most commonly produced end product is lactic acid even though many other substances are also produced as a result of different fermentation pathways.  In fermentation, substrates such as carbohydrates and alcohols undergo anaerobic dissimilation and produce an organic acid (lactic, formic or acetic acid) that may be accompanied by gases such as hydrogen or carbon dioxide.  Many bacteria are facultative anaerobes and are able to carry out fermentation.

Aim

To determine the ability of microorganisms to degrade and ferment carbohydrates with the production of acid and gas.

Principle

Fermentative degradation under anaerobic conditions is carried out in a fermentation broth tube containing a Durham tube, an inverted inner vial for the detection of gas production.  A typical carbohydrate fermentation medium contains (1) nutrient broth ingredients for the support of growth of all organisms (2) a specific carbohydrate that serves as the substrate for determining the organism’s fermentative capabilities and (3) the pH indicator phenol red, which is red at the neutral pH (7) and changes to yellow at a slightly acidic pH of 6.8, indicating that slight amounts of acid will cause a color change.  Following incubation with organisms, carbohydrates that have been fermented with the production of acidic wastes will cause phenol red to turn yellow, thereby indicating a positive reaction.  In some cases, acid production is accompanied by the evolution of gas that will be visible as a bubble in the inverted tube.  Cultures that are not capable of fermenting a carbohydrate substrate will not change the indicator, and the tubes will appear red; there will not be a concomitant evolution of  gas.  This is a negative reaction.     

Requirements

24 to 48 hr broth culture of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Phenol red dextrose, maltose, mannitol, sucrose and lactose broths.

Test tubes, Durham tubes, bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1. Using sterile techniques, inoculate each experimental organism into the appropriately labeled fermentation tubes using inoculation loop. Do not shake the tubes as it may cause the air bubbles to get trapped in Durham tube. 2.      Incubate all tubes for 24 hrs at 37^O C.

 Result

Microorganisms able to ferment the given sugars changed the color of the medium into yellow indicating the acid production.  In some cases, it was accompanied by gas bubble formation in the Durham tubes.Microorganisms unable to ferment the given sugars did not change the color of the medium indicating a negative reaction. The detailed result is given in table- 1.

 

 Table 1. Production of acid or acid/gas in fermentation tubes by different bacterial species

 

Bacterial species	Carbohydrate
	Glucose	Maltose	Mannitol	Sucrose	Lactose
Bacillus	AG	A	__	A	__
Streptococcus	AG	AG	__	AG	AG
Staphylococcus	A	A	__	A	A
Pseudomonas	__	__	__	A	__
E. coli	AG	AG	AG	V	AG

Note: A = acid only (broth turned yellow)

       AG = acid and gas (broth turned yellow + bubble trapped in Durham tube)

       V = variable

        _ = no change

§    Phenol red dextrose/maltose/mannitol/sucrose/lactose broth

            Trypticase                                                                    10.0 g

            dextrose/maltose/mannitol/sucrose/lactose                 5.0 g

            Sodium chloride                                                          5.0 g

            Phenol red                                                                   0.018 g

            Distilled water                                                            1 litre

            pH                                                                               7.3

 (Note: Autoclave at 12 lb pressure for 15 minutes)

Air borne fungal diseases-Dr C R Meera

 Diseases transmitted through air and caused by fungus are called air borne  fungal diseases.  Air borne  fungal diseases Includes Cryptococcosis,  Blastomycosis, Coccidioidomycosis,  Histoplasmosis and Aspergillosis. 

1.  Cryptococcosis

Causative agent is a yeast, Cryptococcus neoformans. It is a soil saprophyte and inhalation of soil particles containing the causative agent will cause the disease.  Most of the infections are asymptomatic, but sometimes the infection may cause pulmonary cryptococcosis, leading to mild pneumonitis or inflammation of the lungs. 

2.  Blastomycosis

Causative agent is Blastomyces dermatitis.  This is also a soil borne fungus liberated to air by environmental or human activities. Inhalation of these soil particles containing the fungus will cause infection. Blastomycosis is a type of chronic infection. Suppurative (pus forming) and granulomatous lesions are formed in any part of the body. 

3. Coccidioidomycosis

Causative agent is soil fungus, Coccidioides immitis. Arthrospores of this fungus are liberated from soil into air by environmental and human activities. Inhalation of spores through the dust particles will lead to infection. Respiratory infection by Coccidioides immitis may be inpparent, benign, severe or even fatal. In most cases, respiratory infection is asymptomatic and gives lifetime immunity. Usually infection leads to self limited influenza  fever called “Valley fever” or “Desert rheumatism”. However, less than 1% of individuals develop chronic disease called “Coccidioidal  granuloma”,  which is fatal.

4. Histoplasmosis

Causative agent is Histoplasma capsulatum. This fungus is seen on rotting trees and in soil. Inhalation of spores liberated in air causes infection. Most of the infections are asymptomatic, but sometimes the fungus can cause intracellular infection of the reticuloendothelial system. This infection results in lymphadenopathy, hepatosplenomegaly, fever and anaemia which is highly fatal. Some people develop pulmonary disease resembling tuberculosis or TB.

5. Aspergillosis

Aspergillosis is caused by Aspergillus fumigatus. Aspergillus is an omnipresent fungus seen in organic matter, in soil, decomposing plants and animals, household dust, building materials, in some food and water.  Aspergillus is an opportunistic pathogen, meaning it is not a pathogen under normal conditions,  but when the conditions are favourable, it will cause infection in human beings. Major route of infection is the inhalation of conidiospores liberated in air from various sources containing this fungus. Inhalation of this fungus causes two types of Pulmonary aspergillosis. First type is called Allergic aspergillosis in which allergic response and asthma attacks are the results. Second type is called Bronchopulmonary aspergillosis. In this case, due to type 1 and type 2 hypersensitivity reactions, Bronchitis (inflammation of bronchi) is developed.  Also fungal growth in lungs will produce fungus balls called ‘aspergilloma’. Aspergilloma consists of tangled mass of fungus hyphae growing in the lungs which may spread to other organs and tissues. 

Air borne viral diseases-Dr C R Meera

 A number of diseases are transmitted through air. Airborne diseases are divided into bacterial diseases, viral diseases and fungal diseases. Diseases that are transmitted through air and caused by viruses are known as Air Borne Viral Diseases. Common air borne viral diseases include common cold, influenza, measles, mumps and adenoviral diseases. Causative agent, disease caused and the mode of transmission through air are to be mentioned under airborne viral diseases. 

1. Common cold

Causative agent of common cold is Rhinovirus. Symptoms include running nose and sometimes respiratory discomfort. It is most frequent of all human infections. Source of infection is the droplets liberated from the nose and throat discharges of the infected patient. Inhalation of the bioaerosols from patients by the healthy individuals will lead to infection.

2. Influenza 

Causative agent of influenza is Orthomyxovirus. Symptoms include nasal discharge , headache, muscle pain, sore throat and general weakness. Infected patients will liberate bioaerosols into a air by activities like coughing, sneezing, talking etc., and  the inhalation of these bioaerosols by healthy individuals lead to the transmission of infection. 

3. Measles

Causative agent is Morbilli virus. It is the most common communicable disease of human beings,  mainly affecting children. Symptoms are fever, cough, cold and red blotchy skin rashes. Blotchy skin rashes are irregular red spots on the body. Source of infection is the respiratory tract secretion from the patient as aerosols. Inhalation of these bioaerosols  will lead to the infection in healthy individuals.  

4. Mumps

Mumps is caused by Mumps virus or Paramyxovirus. It is a communicable disease, also considered as a common childhood disease. Infection is characterized by painful swelling and tenderness of the parotid glands and other salivary glands. Parotid glands are the salivary glands located near the ears. Low grade fever, headache, weakness, fatigue and loss of appetite etc., are other associated symptoms. Source of infection is droplets containing infected saliva and inhalation of the droplets lead to infection. 

5. Adenoviral Diseases

Adenoviral diseases are caused by Adenoviruses.Adenoviruses are a group of viruses that can cause various respiratory & eye infections like conjunctivitis.  Rrespiratory infections like  common cold, bronchitis and pneumonia can be caused by Adenoviruses. Airborne adenoviral diseases are commonly acute febrile pharyngi, Acute respiratory disease and Adenovirus pneumonia. The transmission mode is through the bioaerosols. 

Components of Manuscript/ Research Paper (Part III)- Results, Discussion, Conclusion, Acknowledgement & References

 

1.   Results

Results forms the core or data of the manuscript. This section has two components. Initially the overall description of experiments have to be provided without repeating the minor details of experiments as explained in Materials & Methods part. Second part is the presentation of findings or data of the analysis in past tense. In this part, first the   main findings of the study in order, relating them in turn to the hypotheses and methods used are presented.  Followed by stating the subsidiary findings, relating them in turn to the hypotheses and methods used. Negative results should be also stated.

If one or only a few determinations are to be presented, it can be included in the descriptive part of results. If repetitive determinations are made, data have to be tabulated and presented as graphs and tables. Figures could be added if required to explain the results. Do not verbose in citing graphs, figures and tables. For eg: “It is clearly shown in Table 1 that Penicillin could inhibit the growth of N.gonorrhoea”- Wrong usage.

“Penicillin inhibited the growth of N.gonorrhoea (Table 1)”- Correct usage.

Statistical analysis of results have to be also performed.

Result section should be short and without verbiage. Results should be stated clearly and simply because this part includes the new knowledge you are contributing to the world. Results should be presented with crystal clarity because the stand or fall of the whole paper is decided on the basis of result section.

2.             Discussion 

Discussion part is the hardest section to write. Discussion sections are long and verbose.

   ·       Restate the findings and accomplishments. State principles, relationships and generalisations shown by the results.

·       Evaluate how the results agree with the previous findings – do

they contradict, qualify, agree or go beyond them?

·       Offer an interpretation/explanation of these results and ward off

counter-claims.

·       List potential limitations to the study.

·       State the theoretical implications as well as potential applications and recommend further research.

3.             Conclusion

Summarise the major findings in simple and effective manner. Should be a short brief paragraph. In some journals, it forms the last part of Result & Discussion section. In some, it comes under separate head.

  4.             Acknowledgements

It is courteous to thank sources of financial support (funding agencies), and colleagues and referees for their help during work as well as in improving articles.

·     Financial (recognition of extramural or internal funding);

·     Instrumental/technical (providing access to tools, technologies, facilities, and also furnishing technical expertise, such as statistical analysis);

·     Conceptual (source of inspiration, idea generation, critical insight, intellectual guidance, assistance of referees etc.);

·     Editorial (providing advice on manuscript preparation, submission, bibliographic assistance etc.)

·     Moral (recognising the support of family, friends etc.).

Eg: Portions of this research were funded by a grant from the National

Institutes of Health (MH53291). We would like to thank Greg Hixon, Amy Kaderka and Girish Tembe for their assistance on this project and

Amie Green, Timothy Loving, Mathew Newman, William Swann, and Simine Vazire for their helpful comments on an earlier draft of this article.

(Slatcher and Pennebaker, 2006, p. 663)

5.             References

References serve many purposes in a scientific manuscript. References should include details like author name, year of publication, topic of paper, Journal name, volume, issue, page number etc.

They:

• Establish where ideas came from
• Give evidence for claims
• Connect readers to other research
• Provide a context for your work
• Show that there is interest in this field of research

Many different styles of referencing have developed over the years. Journals considerably vary in the style of reference section. Currently there are four main styles.

1.       The APA style. This system is also known as the Harvard or, more colloquially, as the ‘name (date)’ system. This is because an author’s surname in the text is followed by the date of the publication in brackets.

Eg: Zammuner, V. L. (1995). Individual and co-operative computer writing and revising: Who gets the best results? Learning and Instruction, 5(2), 101–24.

 

2.     The Modern Languages Association (MLA) style. In this version the authors’ surnames (with or without the dates) appear in the text and the first author’s surname comes first in the reference list.

 

Eg: Speck, Bruce W., Teresa R. Johnson, Catherine Dice, and Leon B. Heaton. Collaborative Writing: An Annotated Bibliography. Westport, Connecticut: Greenwood Press, 1999. 

 

3.      The Institute of Electronic and Electrical Engineers (IEEE) style. Here, the authors in the text are numbered in order of their appearance in the text, sometimes without their names, and the numbers are enclosed in square brackets. The reference list is then numbered sequentially.

    Eg: [1] V. L. Zammuner, ‘Individual and co-operative computer writing and revising: Who           gets the best results?’ Learning and Instruction, vol. 5, no.2, pp. 101–24, 1995.

 

4.   The Vancouver style- popular in medical journals. the authors are numbered in the text in order of their appearance, and the numbers are enclosed in square brackets. The reference list is numbered sequentially, but the authors are listed surnames first, followed by their initials. Again the dates of publications are given after journal titles, or at the ends of the references for books etc

 

Eg:

1.   Speck BWM, Johnson TR, Dice CP, Heaton LB. Collaborative    writing: an annotated bibliography. Westport, CT: Greenwood Press, 1999.

References:

1.             James Hartely, 2008. Academic writing and publishing-A practical handbook (ISBN 0-203-92798-2), Routledge Taylor & Francis Group, New York

2.             Robert A Day and Barbara Gastel, 2012. How to write and publish a scientific paper (ISBN 978-1-107-67074-7), Cambridge University press, UK.