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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Can a simple disinfection process avoid infection and diseases?

Disinfection process

Disinfection process is one of the methods which is used to kill micro-organism. In this process, a chemical substance called as a disinfectant is applied on the surface of living and non-living objects to kill micro-flora. Disinfection process kills almost all micro-organisms except some resistant fungal spores. Compared to sterilization technique, disinfection process is less efficient. But in our day to day life, disinfection could be very helpful as well as very easy in application.

Disinfectants can be used in kitchens, bathrooms, floors, hand wash, food industry, dairy, brewery industry, hospitals, pharmaceutical industry, biological laboratories etc.

 Disinfectants

Disinfectants

Properties of Disinfectants

  • It should be non-corrosive, inexpensive and effective.
  • It should be broad spectrum and should not cause deterioration of the object to which it is applied.
  • It should not be harmful to human beings and animals.
  • It should have a pleasant odour.
  • It must be soluble in water and safe for regular use.
  • It should not cause any kind of pollution when disposed in environment.

Types of Disinfectants

1] Air disinfectant - Air disinfectant is used to disinfect or kill the micro-organisms which are suspended in air. Disinfection of air in hospitals, especially in operating theatres is very important to prevent spread of diseases. This type of disinfectant is used in aerosol form. This chemical should be safe to use as it spreads in the air and could be inhaled by people

Examples of chemicals used in air disinfectant are Propylene glycol, Triethylene glycol.

2] Alcohols – Generally, ethanol and isopropanol are used as disinfectant. Alcohol is inflammable but it can also evaporate quickly. So it is possible to use ethanol and isopropanol as a surface disinfectant.

  1. When high concentration mixture of ethanol and isopropanol is used, it can inhibit lipid envelop viruses.
  2. If 70% ethanol or isopropanol is diluted with distilled water, it can inhibit a wide range of micro-organisms.
  3. The efficiency of alcohol is increased when it is mixed with dodecanoic acid (wetting agent). This solution can inhibit broad spectrum of bacteria, viruses and fungal cells.

3] Phenol –Phenolic solutions are generally used as household disinfectants and antiseptics for cleaning floors, surfaces, mouth wash as well as in the form of disinfectant soaps. These phenolic solution may be corrosive in nature and cause some skin allergies on sensitive skins. They should be kept away from children.

Examples of some phenolic substances

  • Thymol – It is used as a broad spectrum disinfectant.
  • Chloroxylenol – It is used as a antiseptic as well as disinfectant.
  • Amylmetacresol – It is used in tablet form for throat disinfection.
  • O-phenyl phenol –It is an alternative used for phenol.

These Phenolic solutions are available in concentrated form. They should be first diluted with water and then used for disinfection of surfaces.

4] Oxidizing agents – Oxidising agents are used to oxidise cell membrane of micro-organism and cause lysis of cell.

Examples of some oxidising agent

  • Chloramines- It is used in drinking water treatment.
  • Chlorine-dioxide – It is used in disinfection of water by killing bacteria that are responsible for water born diseases.
  • Hydrogen peroxide- It is used for disinfection of a living tissue as well as hydrogen peroxide vapours are useful in disinfection of a room these vapours have some toxic effects to eyes and respiratory system so a special care should be taken during its application.
  • Sodium hypo chloride – It is used as a bleach it is a common household disinfectant used for cleaning toilets, washbasins and other surfaces.
  • Ozone – Ozone gas is used for disinfection of water, air and food etc.
  • Calcium hypo chloride- It is used for disinfection of swimming pool.

Disinfection process can help you to be free from infections and diseases.


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Super Bug Bacteria | A Global Threat

Super Bug Bacteria | A Global Threat

A Super bug can be defined as bacteria which are resistant to multiple drugs or antibiotics. These super bugs have become a global threat. Regular disease-causing bacteria have been getting tougher to kill and could get converted into a super bug. Such a super bug would remain unaffected even after treatment with a multiple range of antibiotic. In additional to this, these super bugs are spreading quickly and could become a major threat to world.

How Antibiotics fight micro-organisms?

Antibiotics are the chemical compounds that inhibit the growth of micro-organisms. Generally these antibiotics are antibacterial and anti-fungal in nature. Antibacterial agents are the substances that kill or inhibit the growth of bacteria as well as anti-fungal agents are the substances that kill fungus to cure a disease or infection. Usually some bacterial pathogens are resistant to many antibiotics. These pathogens are called as multi-drug resistant pathogens.

Causes and distribution of a Super bug

These days, in many third world countries, antibiotics are easily available and people purchase and use these antibiotics without a doctor’s prescription. This over use of antibiotics for a longer period of time may result in development of drug resistant bacteria. These bacteria are not only resistant to a single drug but resistant to multiple drugs. Over use of this drug is not only responsible for rise of Super bug but there are many are reasons which are responsible for giving a rise to a Super bug. The reasons are as follows

  • Anti-bacterial soaps, detergents and liquid are easily available and used regularly around the world. These could lead to the development of stronger resistant bacteria.
  • The day to day research is carried out in pharmaceutical industry. The bacteria are tested against wide range of antibiotics to determine its resistance, sensitivity and minimum inhibitory concentration (MIC) due to continuous exposure, the sensitive bacteria also starts to develop a resistance against multiple drugs and then develops into a super bug.
  • Livestock animals are fed with food plus antibiotics to protect them from diseases. Antibiotics are given to livestock for three purposes to treat the infection, to improve the efficiency of food and promote growth of live stock. The antibiotics which are not suitable for human use are given to livestock. Due to this, the drugs get utilized in an animal body as well as due to continuous exposure the micro-flora present in animal body develop a resistant against these drugs. Further these resistant bacteria get transmitted to humans via consumption of animal products like meat, milk and eggs as well as by direct contact with the animal.
  • Various devices and tools are used in surgery. If these are not properly sterilized, it could lead to a super bug infection. Endoscope can result in infection of a CRE that is Carbapenem resistant enterobacteriaceae family super bug.
  • The multiple drug resistant bacteria get exposed to the environment through human beings, animal waste and pharmaceutical industry waste.
  • Further when such bacteria are exposed to the environment, they carry out multiplication and replication. The drug resistant genes of bacteria get multiplied and survive, and these multiplied cells get distributed in the environment via air, water, soil, human beings and medical devices used in hospital can result in super bug infection.
  • These distributed super bugs have an ability to spread a mutated gene that is highly drug resistant to other normal bacteria via gene transfer by multiplication (conjugation, transformation and transduction).
  • In this way a Super bug is developed and distributed.

Detection of a Super bug Infection

Generally when a patient is treated for any diseases, he or she is first exposed to first line antibiotics. These first line antibiotics are mild, safe, cheap and easily available and have no side-effects. The first line antibiotics are generally narrow spectrum antibiotics. If these narrow spectrum antibiotics do not work, then the patient is prescribed with second line broad spectrum antibiotics. The second line antibiotics inhibit a wide range of micro-flora. They are expensive, strong and show some side-effects on patient. Further if the patient still continues to have an infection, he or she is treated with a third line of high spectrum and high level antibiotics. Third level antibiotics are very strong and they show adverse side effects on patient.

If such a condition develops where these wide range of antibiotics do not succeed in curing the disease and a situation becomes critical then we can say a patient is infected by a super bug.

Examples of Super bugs

On the basis of type of antibiotic resistance, the Super bugs are divided into some types.

  1. NDM-1
    This super bug was reported in India. This super produces a enzyme called as New Delhi Metallo-Beta-Lactamase this enzyme destroys beta lactamase ring present in the structure of antibiotics. This super bug is resistant to all antibiotics that contains beta lactamase ring.
  2. Methicillin resistant Staphylococcus aureus (MRSA)
    This super bug is resistant to Methicillin antibiotic and causes wound infection.
  3. Escherichia coli
    It is also called as ESBL. This super bug is resistant to fluoroquinolane antibiotics and they cause blood poisoning as well as urinary tract infection.
  4. Clostridium difficile
    This super bug causes severe diarrhoea, inflammation of stomach and dehydration as well as this super bug produces 20 times more toxins than the normal strain. Clostridium difficile super bug is resistant to Clindamycin antibiotics.
  5. Streptococcus and Enterococcus
    Super bug causes pneumonia, otitis, meningitis, sinusitis and arthritis. It is resistant many antibiotics.
  6. Pseudomonas aeruginosa and Mycobacterium tuberculosis
    It is multi-drug resistant superbug.

Replacement for antibiotics to fight the Super bug problem?

Bacteriophages seem to be a solution. A bacteriophage is a virus that infects and replicates within bacteria.These can be designed to target a specific bacteria and kill it. These will replace antibiotics within this decade.


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