Nitrate Reduction Test

Nitrate is a poly-atomic inorganic molecule and its molecular formula is NO3-  

Nitrate is reduced by micro-organism in two ways.

1. Assimilatory Nitrate reduction.

Assimilatory nitrate reduction mainly occurs during aerobic condition in absence of nitrogen.In this reduction process nitrate and ammonia are used for synthesis of organic nitro-compounds and amino-acids.In assimilatory nitrate reduction the nitrate do not play any role in energy production.

2. Dissimilatory nitrate reduction.

Dissimilatory nitrate reduction is carried out under anaerobic condition where bacteria use nitrate as a electron acceptor.

These assimilatory and dissimilatory nitrate reduction process is carried out nitrate reductase enzyme.This nitrate reductase enzyme converts nitrate to nitrite .The reaction is as follows

NO3- + NADPH + H+       Nitrate reductase           NO2+ NADP+ + H2O           

Further this nitrite is converted to ammonia by nitrite reductase and the ammonia formed is assimilated.The nitrate reductase test is performed by using two indicators and the reduction of nitrate to nitrite is detected by using two indicators and that are sulphanilic acid and α- napthylamine.

Aim 

Aim of the experiment is to carry out nitrate reduction test .

Requirement

  1. Peptone nitrate broth
  2. Test culture.
  3. Reagent A-Sulphanilic acid.
  4. Reagent B- α- napthylamine.
  5. Zinc dust.

Composition of Peptone nitrate broth

  • Peptone            -0.5 gm
  • KNO3                 -0.2 gm
  • Distill water    -100 ml
  • pH                       -7

Principle

In nature many micro-organism posses nitrate reductase enzyme  when this micro-organisms are exposed to nitrate as a sole source of energy have a ability to reduce nitrate to nitrite.Here in nitrate reductase test we expose these micro-organism to a media containing nitrate as a sole source of nitrogen and energy and further this nitrite is detected by using two reagents Sulphanilic acid and α- napthylamine.

Procedure

  1.  Take sterile peptone nitrate broth and  innoculated it with loopful of  test culture  and this innoculated broth is incubated for about 24 hours at about 37 ° C .
  2. After incubation 0.5 ml of reagent A that is  Sulphanilic acid is added and then reagent B α- napthylamine is added observe the test tube for development of red colour.
  3. Further if no red colour develops a pinch of zinc dust is added and mix properly and test tube is observed for development of red colour.

Flow chart of Nitrate reduction test procedure

 

Reagent A is Sulphanilic acid and Reagent B alpha napthylamine

Reagent A is Sulphanilic acid and Reagent B alpha napthylamine

 

Conclusion

  • If development of red colour is observed after addition of reagents then it indicates positive nitrate reduction test.
  • If after addition of both reagents there is no red colour formation then it may indicate negative nitrate reduction test or false result  because there is a possibility that the microflora present in the tube may reduce the nitrate to other byproducts like ammonia,molecular nitrogen,nitrous oxide,nitric oxide and as the test reagents detects only nitrates so it can give a false negative result so to clear this doubt a pinch of zinc dust is added and tube is observed for red colour development .Here zinc ions reduce nitrates to nitrites and we can observe development of red colour as zinc ions confirms presence of residual nitrates and this development of red colour indicates negative nitrate reduction test.

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Catalase Biochemical Test

Catalase Biochemical Test

Introduction

Catalase is a name of a enzyme.This enzyme is nearly present in all living plants and animals which are always exposed to oxygen.Catalase enzyme is also present in aerobic and facultative anaerobic micro-organisms.Generally this micro-organisms utilise oxygen and produce hydrogen peroxide and this hydrogen peroxide is toxic to the cells and it effects the cells enzyme system so to avoid this toxic effect micro-organism produces catalase enzyme. The role of catalase enzyme is to convert hydrogen peroxide molecule to water and oxygen and this reaction is a irreversible reaction.

2H2O2   is converted to     2H2O + O2

It is also one of the reason that anaerobic micro-organisms do not survive in presence of oxygen as hydrogen peroxide is produced and due to lack of catalase enzyme this hydrogen peroxide destroy cells enzyme system and the cell dies.

Why is it important to carry out Catalase test ?

The purpose of carrying out a catalase test is to detect production of catalase enzyme by bacteria. Secondly catalase test help us to determine whether the micro-organisms are aerobic,anaerobic or obligate anaerobic in nature and due to this we can differentiate the organisms in catalase positive or catalase negative group. This differentiation helps us in identification and classification of bacteria.

How is the Catalase test performed ?

The Catalase test can be performed by using two methods.

  1.  Test tube method.
  2. Slide method.

1.Test tube method.

In test tube method the catalase test is performed in sterile test tube.

Aim

The aim of the experiment is to carry out catalase test by tube method.

Requirement

  1. Bacterial culture
  2. Sterile test tube.
  3. Dropper.
  4. Wireloop.
  5. Hydrogen peroxide.

Procedure

  1. A sterile test tube is taken and with the help of a dropper 5 to 6 drops of hydrogen peroxide are added in the all this procedure is carried out under sterile conditions.
  2. Further with the help of a sterile nichrome wireloop a colony of a test culture is picked up and inserted in hydrogen peroxide containing test tube.
  3. The test tube is observed for production of effervescence that is bubble formation this reaction occurs immediately after addition of culture.

Observation

 Positive catalase-test

Positive catalase-test

neg-catalese-test

Negative catalase test

 Conclusion

If effervescence is observed, test culture is catalase positive and if their are no effervescence test culture shows negative catalase test.

2. Slide method

Aim

The aim of the experiment is to carry out Catalase test by Slide method.

Requirement

  1. Bacterial culture.
  2. Clean grease free slide.
  3. Wireloop
  4. Hydrogen peroxide.

Procedure

  1. Take a clean grease free slide and on it take a drop of sterile water and with the help of a sterile nichrome wireloop pick a colony of a test culture.
  2. Further with the with the help of a dropper add 3 to 4 drops of hydrogen peroxide all this procedure should be carried in sterile condition.
  3. After addition of hydrogen peroxide the slide is observed for formation of effervescence that is bubbles.

Conclusion

If effervescence are observed within 20 second then catalase test is positive and if effervescence are not observed then catalase test is negative.

List of some Catalase positive micro-organism.

  • Pseudomonas aeruginos
  • Mycobacterium tubercule
  • Nisseria gonorrhea
  • Helicobacter pylori
  • Candida
  • Staphylococcus aureus
  • E.coli

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How is IMViC Biochemical test performed ?

How is IMViC Biochemical test performed ?

In IMViC test there are four biochemical tests and this test are carried out individually. IMViC test is carried out to identify members of Enterobacteriaceae family.

Each letter of IMViC test stand for individual test

  1. I – Indole production test.
  2. M – Methyl red test.
  3. V- Voges- Proskaver test.
  4. C – Citrate utilization.
  5. i – Lower case i is used for ease of pronunciation.

We will go through these tests one by one.

Indole production test.

Aim

The aim of this test is to determine the capability of a bacterial culture to produce Indole.

Requirements

  • Bacterial culture
  • Tryptophan broth
  • Kovac’s reagent or Ehrlich’s reagent.
  • Xylene

Composition of media and reagents

1. Tryptophan broth medium

Tryptone         – 1 gm

Distilled water- 100 ml

2. Kovac’s reagent

p-dimethylaminobenzaldehde -15 gms

Isoamly alcohol                           – 150 ml

HCL                                                – 75 ml

3. Ehrlich’s reagent

p-dimethylaminobenzaldehde  – 1 gm

95 % ethanol                                – 95 ml

Concentrated HCL                        -20 ml

 

Procedure

  1. Tryptone broth is prepared, distributed in test tube and sterilized by autoclaving.
  2.  The sterile tryptone broth tubes are inoculated with loopful of suspension and incubated at 37° C for 24 hours.
  3. After incubation add 3-4 drops of xylene in tubes and shake vigorously and keep the tubes still so that two layers get separated.
  4. After separation of two layers add 1 ml of Kovac’s reagent or Ehrlich’s reagent and tubes are observed for formation of pink colour ring.

Mechanism

Indole is aromatic heterocyclic organic compound.Indole can be produce by some members of Enterobacteriaceae family by hydrolysis of tryptophan.Indole production is a protein utilization test. Here we are going to use a protein rich that is tryptophan rich broth. This media contains tryptophan as a source of protein. The bacterial cells that are able to produce trytophanase enzyme hydrolyse and deaminate tryptophan into Indole, pyruvate and ammonia. Further these degraded products are treated with Kovac’s reagent or Ehrlich’s reagent in presence of heat. If the degraded products in the tube contain Indole then this Indole reacts with Kovac’s reagent or Ehrlich’s reagent and form pink colour rose Indole complex. In this way Indole production is detected.

[Note – Indole is soluble in organic compound so it is recommended to add xylene/chloroform/ether by adding this substance Indole get extracted from the whole medium and forms a separate layer on the surface of medium. So that Kovac’s reagent or Ehrlich’s reagent can easily react with the indole and form a pink colour ring.

Result – If there is formation of pink colour ring then the Indole production test is positive and if there is no ring formation then the test is negative.

Methyl Red Test {MR test}

Aim

To carry out Methyl-red test to determine fermentation capacity of given bacterial culture.

Requirement

  • Glucose phosphate broth
  • Methyl-red indicator
  • Test culture.

Composition of media

Glucose phosphate broth

  1. Glucose  – 0.5 gms
  2. K2HPO4 -  0.5 gms
  3. Peptone – 0.5 gms
  4. Distill water – 100 ml
  5. pH – 7

Procedure

  1. Glucose phosphate broth is prepared and distributed in test tubes this test tubes are further sterilized by autoclaving.
  2. The sterile test tubes are inoculated with test culture and incubated at 37 ° C for 24 hours.
  3. After incubation the five drops of methyl-red indicator is added to the medium and the tubes are observed for development of red colour.

Mechanism

As we know we carry out IMViC test for determination of members of Enterobacteriaceae family. The micro-organisms from Enterobacteriaceae family carry out fermentation pathway they metabolise glucose to pyruvic acid  to formic acid and this formic acid formation is called formic acid fermentation process.

In general there are two types of formic acid fermentation pathway and they are:-

  1. Mixed acid fermentation- This type of fermentation is carried out in E.coli, Salmonella and Proteus.In this fermentation ethanol and mixture of various acid like succinic acid, formic acid, acetic acid and lactic acid are formed.
  2. 2.       Butanediol fermentation – This fermentation is carried out in Bacillus, Enterobacteriacea, Serratia  . Here pyruvate is converted to acetyl methyl carbonyl and further from this Butanediol is formed. This fermentation mainly produces neutral products. 

Now when we compare this two fermentations process, mixed acid fermentation produces more acidic products and it acidifies the incubation media. Here only mixed acid fermentation produces sufficient amount of acid which can be detected by methyl red indicator.

As here we use glucose phosphate broth as incubation medium this medium is highly buffered and small amount of acid is produced the media can resist change in pH. So here we use Methyl red the pH range of methyl red is at 4.4 ph it show red colour and where as at 6.2 pH it shows yellow colour. So after incubation the pH indicator methyl red and medium is observed for change in colour of medium.

If acid is produced in medium and after addition of methyl red the medium shows red colour then the Methyl red test is positive.

The Methyl red test is shown positive in case of mixed acid fermentation as complex mixture of acid is produced.

Result

If there a stable red colour is developed in the media after addition of Methyl red indicator then the M-R Test is positive.

Voges-Proskauer (V-P) Test

Aim

To carry out Voges-Proskauer test determine the capacity of bacterial culture to carry out fermentation.

 Requirement

  • Glucose phosphate broth
  • 5 % alcoholic alpha-naphthol
  • 40 % KOH solution
  • Test culture

Procedure

  1. Glucose phosphate broth is prepared and distributed in test tubes these test tubes are further sterilized by autoclaving.
  2. The sterile test tubes are inoculated with test culture and incubated at 37 ° C for 24 hours.
  3. After incubation 0.6 ml of alpha naphthol and 0.2 ml of KOH solution per ml of culture broth media is added.
  4. Further after addition of these two reagents the culture tubes are shaken properly and kept in slanting position to increase aeration. Keep these tubes in slanting position for about one hour and then results are noted down.

Mechanism

As we know there are two types of fermentation carried out by members of Enterobacteriaceae family( Details are mention above in M-R test) In methyl red test we detected the mixed acid fermentation and here in Voges-Proskauer test we are going to determine butanediol fermentation by detecting product acetoin.

This acetoin is a precursor of butanediol and this acetoin is produced during butanediol fermentation. Here in V-P test after fermentation the medium is treated with alpha-naphthol and KOH solution and due to addition of this acetoin is oxidised to diacetyl .Now this diacetyl react with the peptone present in the broth and produce pink colour.

[ Peptone is present in a culture broth and this peptone contains guanidine nucleus of arginine and diacetyl reacts with guanidine nucleus of arginin and gives pink colour to the medium.]

Thus formation of pink colour indicates presence of acetoin and conforms Butanediol fermentation and indicates V-P test as positive.

Citrate Utilization Test

Aim

To carry out citrate utilization test to determine the ability of bacterial cell to utilize citrate.

Requirement

  • Simmons’s citrate agar
  • Test culture.

Composition of Simmons’s citrate agar

  1. Sodium citrate                                    –0.2 gm
  2. Magnesium sulphate                         –0.02 gm
  3. Sodium chloride                                  –0.5 gm
  4. Ammonium dihydrogen phosphate – 0.1 gm
  5. Dipotassium phosphate                      – 0.1 gm
  6. Bromothymol blue                               – 0.008 gm
  7. Agar-agar                                               – 3 gm
  8. Distill water                                            – 100 ml
  9. pH                                                             – 7

Procedure

  1. The Simmons’s agar is prepared according to the composition given above, sterilized and after sterilization the slants of this media are prepared.
  2. Further this slant are streaked heavily on the slant and incubated for 24 hours at about 37°C
  3. After incubation the slant are observed for change in colour and the results are recorded.

Mechanism

The members of Enterobacteriaceae family have the ability to obtain energy and carbon by utilization of citrate. This is an important characteristic to identify the members of Enterobacteriaceae family. For carrying out citrate utilization test the test culture should be exposed to medium which contains citrate as a sole source of carbon and energy .If the bacterial cells are able to produce enzyme citrate permase can utilize citrate because this enzyme can facilitate transport of citrate into bacteria then a bacterial cell can utilize citrate. For carrying out this test we need citrate agar that contains, ammonium phosphate as a source of nitrogen, Sodium citrate as source of carbon and bromothymol blue as a pH indicator .Bromothymol blue is yellow at acidic pH and blue at alkaline  pH  .

In citrate utilization test we use agar slant because for this test oxygen is required and after oxidation of citrate CO2 is liberated. Now this CO2 reacts with the sodium and water present in the medium and form sodium carbonate and this sodium carbonate is a alkaline products other hand the bacteria utilize ammonium citrate release nitrogen with production of ammonia and convert it to ammonium hydro-oxide and this is the other alkaline product. Now this alkaline products increases the pH of medium to alkaline side and thus colour of the medium changes to blue colour. Hence change in colour of medium to blue indicates positive citrate utilization test.

Result

Formation of a deep blue colour indicates positive citrate utilization test and the test culture has a ability to utilize citrate.

 

 

 

 


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Carbohydrate Fermentation Test

Carbohydrate Fermentation Test

Whenever a new micro-organism is discovered it becomes necessary to identify and catalogue it. For identification of this new microbe, it is first isolated and microscopically examined, and then further biochemically tested. Once these steps are carried out, we can identify this micro-organism. Biochemical tests are essential for the identification and classification of this micro-organism. Hence it is important to understand various biochemical tests and their functioning.

There are various biochemical tests and we will go through these tests one by one in my upcoming post. The list of biochemical test that play an important role in microbiology are…

  1. Carbohydrate fermentation test.
  2. Sugar Mannitol fermentation test.
  3. IMViC test.
  4. Catalase
  5. Oxidase
  6. Huge and Leifson’s test
  7. Indole Production test
  8. Hydrogen sulphide production test
  9. Decarboxylation test
  10. Phenylalanine Deaminase test
  11. Urea Hydrolysis test
  12. Nitrate Reduction test
  13. Ammonia Production test
  14. Starch Hydrolysis test
  15. Casein Hydrolysis test
  16. Gelatine Hydrolysis test
  17. Lipid Hydrolysis test
  18. Dehydrogenase test
  19. Coagulase test
  20. Hemolysis production
  21. Triple Sugar Iron Agar test
  22. Litmus test
  23. Lysin Decarboxylation test

In this article, we are going to have a look on Carbohydrate fermentation test.

Carbohydrate 

Carbohydrates are biological molecules which are made up of carbon, hydrogen and oxygen molecules. There are four types of carbohydrates and they are shown in the following figure.

Carbohydrates and its Types

Carbohydrates and its Types

Carbohydrates and its types

  • Monosaccharides
    Monosaccharides are simple molecule of sugar. It is water-soluble and crystalline in nature. Examples are glucose, fructose, glyceraldehyde and galactose.
  • Diasaccharides
    Diasaccharides is a simple carbohydrate formed when two monosaccharide molecules are joined together and a water molecule is removed. Examples are lactose,sucrose and maltose.
  • Oligossacharides
    Oligosaccharide is a polymer containing small number of monosaccharide.monosaccharide. Examples are fructo-oligosaccharide, galacto-oligosaccharide and mannan.
  • Polysaccharide
    Polysaccharide are complex carbohydrate containing long chain of monosaccharides.Examples are starch,glycogen,cellulose and chitin.

Fermentation

Fermentation is a metabolic process in which bacteria or yeast convert substrate into a product like acid, gas or alcohol. The product formed in fermentation process is of economic value.

Carbohydrate fermentation tests demonstrate fermentation of sugars like glucose, lactose or sucrose. The fermentation is noted by acid and gas production by bacterial cells.

Carbohydrate fermentation test

Aim

To determine fermentation of sugars (Glucose, Lactose and Sucrose) by bacterial cells.

Requirements

1.Nutrient broth medium

a) Glucose broth medium

b) Sucrose broth medium

c) Lactose broth medium

2. Indicator – Phenol red or Andrade’s indicator.

3. Durham’s tube

4. Bacterial cell culture

Composition of Sugar Broth Media

  1. Peptone                                                 -1 gm
  2. Meat extract                                       -0.3 gm
  3. NaCl                                                       -0.5 gm
  4. Distill water                                         – 100 ml
  5. Indicator                                              -0.008 gm
  6. Sugar(glucose/lactose/sucrose) -0.5 gm

Procedure

  1. Take three different tubes containing three different types of sugar broth and invert a Durham’s tube in it and screw cap the tubes.
  2. Further the three sugar broth tubes are sterilized by autoclaving.
  3. After sterilization, the tubes are cooled down to room temperature and inoculated with a cell suspension in aseptic condition.
  4. The tubes are incubated at 37°C for 24 hours.
  5. After incubation, the tubes are examined for acid and gas production and results are noted down.

Mechanism

  1. The three sterile sugar broth tubes are inoculated with bacterial suspension and incubated for 24 hours. During incubation the bacterial cells may or may not utilise sugar.
  2. If the cells utilize sugar, there is acid and gas production and this change is observed by change in colour of broth.
  3.  First of all, in sugar broth phenol, red indicator is added this pH sensitive indicator is added for determination of acid production. Initially the broth tubes are red in colour and after incubation, if there is acid production, the colour of broth changes from red to yellow. If there is no change in colour of broth, then there is no acid production.
  4. Secondly in sugar broth, a Durham’s tube is inverted to determine gas production. After incubation, if a gas bubble is observed then there is gas production.

Note

Phenol red is a pH sensitive indicator its pH range is at acidic pH it is yellow in colour and at alkaline pH it is red in colour.

Observation

There are three possibilities and they are after incubation

  1. If red colour of broth is observed then there is no fermentation.
  2. If yellow colour of broth is observed then there is acid formation.
  3. If yellow colour of a broth plus a gas accumulation in Durham’s tube is observed then there is acid plus gas formation.

Observation Diagram I

Observation Diagram II

Observation Diagram III

 Result

  • If there is no  change in colour of a broth then the test is negative.
  • If yellow colour of a broth is observed, fermentation is carried out resulting in acid production and it is indicated by + sign
  • If yellow colour of a broth along with gas bubble accumulation is observed then fermentation is carried out resulting in acid and gas production.

 


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