Triple Sugar-Iron Agar Test-Dr C R Meera

 

The Triple Sugar-Iron (TSI) agar test is designed to differentiate among the different groups or genera of the Enterobacteriaceae, which are all gram negative bacilli capable of fermenting glucose with the production of acid, and to distinguish the Enterobacteriaceae from other gram negative intestinal bacilli.  This differentiation is made on the basis of differences in carbohydrate fermentation patterns and hydrogen sulphide production by the various groups of intestinal organisms. Some bacteria liberate sulfur from sulfur containing aminoacids or other sulfur containing compounds.  The sulfur is used as final hydrogen acceptor leading to the formation of H₂S. 

Aim

To differentiate among members of the Enterobacteriaceae; and also between the Enterobacteriaceae and other groups of intestinal bacilli.

Principle

The TSI agar slants contain lactose (1%), sucrose (1%) and glucose (0.1%) to facilitate observation of carbohydrate utilization patterns. The acid-base indicator phenol red is also incorporated to detect carbohydrate fermentation that is indicated by a change in color of the medium from orange-red to yellow in the presence of acids.  The slant is inoculated by means of a stab and streak procedure.  Following incubation, the fermentative activities of the organisms are determined as follows:

1.      Alkaline slant (red) and acid butt (yellow) with or without gas production (breaks in the agar butt): Only glucose fermentation has occurred.  The organisms preferentially degrade glucose first.  Since this substrate is present in minimal concentration, the small amount of acid produced on the slant surface is oxidized rapidly.  The peptones in the medium are also used in the production of alkali.  In the butt the acid reaction is maintained because of reduced oxygen tension and slower growth of the organisms.

2.      Acid slant (yellow) and acid butt (yellow) with or without gas production: Lactose or sucrose fermentation has occurred.  Since these substances are present in higher concentrations, they serve as substrates for continued fermentative activities with maintenance of an acid reaction in both slant and butt.

3.      Alkaline slant (red) and alkaline butt (red) or no change (orange-red) butt: No carbohydrate fermentation has occurred.  Instead, peptones are catabolized under anaerobic or aerobic conditions, resulting in an alkaline pH due to production of ammonia.  If only aerobic degradation of peptones occurs, the alkaline reaction is evidenced only on the slant surface.  If there is aerobic and anaerobic utilization of peptone, the alkaline reaction is present on the slant and the butt.

To obtain accurate results, it is essential to observe the cultures within 18 to 24 hrs following incubation.  This will ensure that the carbohydrate substrates have not been depleted and that degradation of peptones yielding alkaline end products has not taken place. 

The TSI medium also contains sodium thiosulphate, a substrate for hydrogen sulphide (H₂S) production, and ferrous sulphate for detection of this colorless end product.  Following incubation, only cultures of organisms capable of producing H₂S will show an extensive blackening in the butt because of the precipitation of the insoluble ferrous sulphide. 

Requirements

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

Triple Sugar-Iron Agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

 1.      Using sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of a stab and streak inoculation. One tube kept as control.

2.      Incubate for 18-24 hrs at 37^oC.

3.    3.  Examine the color of both the butt and slant of all inoculated tubes and determine the type of reaction that has taken place (acid, alkaline or none) and the carbohydrate that has been fermented in each culture.

4.     4.  Examine all cultures for the presence or absence of blackening with in the medium and determine the ability of the organism to produce H₂S.

 Observations    

Bacillus               : Acidic slant, no color change in butt, no gas and no H₂S formation.                            

 Streptococcus     : Acidic slant, acidic butt, gas produced and no H₂S formation.                                     

 Staphylococcus   : Acidic slant, acidic butt, no gas and no H₂S formation.

E.coli                   :  Acidic slant, acidic butt, gas produced and no H₂S formation.                                   

 Pseudomonas     :  Alkaline slant, no color change in butt, no gas and no H₂S formation.

 Result

 Bacillus                :A/ NC                                                                                                                         

 Streptococcus     :   A/A, G                                                                                                                          

 Staphylococcus   :   A/A

 E.coli                    :  A/A, G

 Pseudomonas      :  K/NC

Table 1. Result interpretation of Triple Sugar-Iron Agar test

Results (slant/butt)	Symbol	Interpretation
Red/yellow	K/A	Glucose fermentation only; Peptone catabolized
Yellow/yellow	A/A	Glucose and lactose and/or sucrose fermentation
Red/red	K/K	No fermentation; Peptone catabolized
Red/no color change	K/NC	No fermentation; Peptone used aerobically
Yellow/yellow with bubbles	A/A,G	Glucose and lactose and/or sucrose fermentation; Gas produced
Red/yellow with bubbles	K/A,G	Glucose fermentation only; Gas produced
Red/yellow with bubbles and black precipitate	K/A,G, H2S	Glucose fermentation only; Gas produced; H2S produced
Red/yellow with black precipitate	K/A, H2S	Glucose fermentation only; H2S produced
Yellow/yellow with black precipitate	A/A, H2S	Glucose and lactose and/or sucrose fermentation; H2S produced
No change/no change	NC/NC	No fermentation
A=acid production; K=alkaline reaction; G=gas production; H2S=sulfur reduction

 §  Triple Sugar-Iron Agar slants

Beef extract                                        3.0 g

Yeast extract                                       3.0 g

Peptone                                               15.0 g

Proteose peptone                                 5.0 g

Lactose                                                10.0 g

Saccharose                                          10.0 g

Dextrose                                              1.0 g

Ferrous sulfate                                    0.2 g

Sodium chloride                                  5.0 g

Sodium thiosulphate                           0.3 g

Phenol red                                           0.024 g

Agar                                                    12.0 g

Distilled water                                    1 litre

pH                                                       7.4

 

 

Nitrate reduction test- Dr C R Meera

 

Nitrates serve as source of nitrogen for many bacteria.  They can also act as final electron acceptor.  Many organisms can be differentiated and identified by their capacity to reduce nitrates to nitrites.  Most of the enterobacteriaceae reduce nitrates.  This character is useful for the identication of the species in Neisseria, Haemophilus and Branhamella.  Some Pseudomonas and nonfermenters reduce nitrate to nitrite and further down to ammonia or to molecular nitrogen.

The enzyme nitrate reductase possessed by organisms reduces nitrates to nitrites. The reduction of nitrates by some aerobic and facultative anaerobic microorganisms occurs in the absence of molecular oxygen, an anaerobic process.  In these organisms anaerobic respiration is an oxidative process whereby the cell uses inorganic substances such as nitrates (NO₃^-) or sulfates (SO₄ ^2-) to supply oxygen that is subsequently utilized as a final hydrogen acceptor during energy formation.  The biochemical transformation may be visualized as follows:

NO₃^-  +  2H⁺  +  2e^{-        Nitrate Reductase}    NO₂^- + H₂O

Nitrate     Hydrogen    Electrons                                                   Nitrite        Water

Some organisms possess the enzymatic capacity to act further on nitrites to reduce them to ammonia (NH₃⁺) or molecular nitrogen (N₂) and this process is called denitrification.  The reaction may be described as follows:

 NO₂^-    ------->         NH₃⁺

Nitrite                                 Ammonia     

 Or

 2NO₃^-   +  12H⁺  +  10e^{-   -------->} N₂   +  6 H₂O

Nitrate           Hydrogen      Electrons                  Molecular nitrogen and water

 Aim

To determine the ability of some microorganisms to reduce nitrates (NO₃^- ) to nitrites (NO₂^-) or beyond the nitrite stage.

Principle

Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium.  The medium is basically a nutrient broth supplemented with 0.1% potassium nitrate (KNO₃) as the nitrate substrate.  In addition, the medium is made into a semisolid by the addition of 0.1% agar.  Following incubation of the cultures, an organism’s ability to reduce nitrates to nitrites is determined by the addition of two reagents: Solution A, which is sulfanilic acid, followed by Solution B, which is α- naphthylamine.  Nitrites in acid environment immediately produce a cherry red coloration due to the formation of a red diazomium dye, p- sulfo benzene-azo-alphanaphthylamine.

Cultures not producing a color change suggest one of two possibilities: (1) nitrates were not reduced by the organism, or (2) the organism possessed such potent nitrate reductase enzymes that nitrates were rapidly reduced beyond nitrites to ammonia or even molecular nitrogen.  To determine whether or not nitrates were reduced past the nitrite stage, a small amount of zinc powder is added to the basically colorless cultures already containing Solutions A and B.  Zinc reduces nitrates to nitrites.  The development of red color therefore verifies that nitrates were not reduced to nitrites by the organism.  If nitrates were not reduced, a negative nitrate reduction reaction has occurred.  If the addition of zinc does not produce a color change, the nitrates in the medium were reduced beyond nitrites to ammonia or nitrogen gas.  This is a positive reaction.

Requirements

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

Nitrate broth, Solution A (sulfanilic acid), Solution B (α- naphthylamine), zinc powder, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  Using sterile technique, inoculate each experimental organism into its appropriately labeled tubes by means of loop inoculation. One uninoculated tube kept as control.

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

3. Add 5 drops of Solution A and then 5 drops of solution B to all nitrate broth cultures and observe for the red color development.

4. Add a minute quantity of zinc to the cultures in which no red color developed.  Observe for the color change to red.

 

Observations

Bacillus, Staphylococcus and E.coli produced cherry red coloration immediately after adding Solutions A and B, indicating that they are nitrate positive.  Pseudomonas and Streptococcus produced no cherry red coloration on addition of Solutions A and B.  Addition of zinc powder to these tubes produced a red color in Streptococcus inoculated ones which indicates a negative nitrate reduction result. No colour formation in the tube inoculated with Pseudomonas indicated the conversion of nitrate beyond nitrites to ammonia or nitrogen gas.

Result

Bacillus, Staphylococcus, E.coli and Pseudomonas are nitrate test positive whereas Streptococcus is nitrate negative.

 

§         Nitrate broth

Peptone                                               5.0 g

Beef extract                                        3.0 g

Potassium nitrate                                 5.0 g

Distilled water                                    1 litre

pH                                                       7.2

Citrate utilization test-Dr C R Meera

Aim

To demonstrate the ability of microorganisms to utilize citrate as the sole source of carbon.

Principle

In the absence of fermentable glucose or lactose, some microorganisms are capable of using citrate as a carbon source for their energy.  This ability depends on the presence of a citrate permease that facilitates the transport of citrate in the cell.  Citrate is the first major intermediate in the Kreb’s cycle and is produced by the condensation of active acetyl with oxaloacetic acid.  Citrate is acted on by the enzyme citrase, which produces oxaloacetic acid and acetate.  These products are then enzymatically converted to pyruvic acid and carbon dioxide.  During this reaction the medium becomes alkaline- the carbon dioxide that is generated combines with sodium and water to form sodium carbonate, an alkaline product.  The presence of sodium carbonate changes the Bromothymol blue indicator incorporated into the medium from green to deep Prussian blue.  Bromothymol blue is green when acidic (pH 6.8 and below) and blue when alkaline (pH 7.6 and higher). 

Following incubation, citrate positive cultures are identified by the presence of growth on the surface of the slant, which is accompanied by blue coloration.  Citrate negative cultures will show no growth and the medium will remain green.

Requirements

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

Simmons citrate agar slants, Bunsen burner, inoculating needle, glass marking pencil etc.

 Procedure

1.  Aseptically 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

In  Streptococcus, Bacillus, Staphylococcus and Pseudomonas inoculated slants, growth was visible on the surface and the medium color changed to blue.  In E.coli inoculated slants there was no growth as well as change in color of the medium.

Result

Among the given cultures, Streptococcus, Bacillus, Staphylococcus and Pseudomonas sp. are Citrate positive whereas and E.coli is Citrate negative.

§        Simmons citrate agar

Ammonium dihydrogen phosphate    1.0 g

Dipotassium phosphate                       1.0 g

Sodium chloride                                  5.0 g

Sodium citrate                                     2.0 g

Magnesium sulphate                           0.2 g

Agar                                                    15.0 g

Bromothymol blue                              0.08 g             

Distilled water                                    1 litre

pH                                                       6.9

 

History of Microbiology; Theory of Spontaneous Generation Vs Biogenesis- Dr C R Meera

 

Microorganisms are closely related to our day today life. Many of the microorganisms are beneficial to mankind whereas others are detrimental. Beneficial effects of microbes are numerous. Microbes are involved in fermentation of idli batter, making of curd, bread, yogurt, cheese, wine and such so many food products. They are also involved in the production of many antibiotics, antiviral agents (interferons) etc. Microbes are the primary decomposers on earth playing a major role in the biogeocycling of organic compounds. At the same time harmful microbes cause many diseases, spoilage food and deterioration of materials like iron pipes, wood pilings etc.      

In Greek “Mikros” means “too small to be seen with the naked eyes”, “bio” means “life” and “ology” means “study of”. Microbiology is the study of living organisms that cannot be seen with our naked eyes or it is the study of “microorganisms”. Microorganisms include bacteria, protozoans, algae, parasites, fungi (Yeasts and, molds) and viruses.

Study of different groups of microorganisms are named as follows:

1. Bacteria                                               - Bacteriology

2. Protozoans                                          - Protozoology

3. Algae                                                   - Phycology

4. Parasites                                              - Parasitology

5. Yeasts and Molds (Fungi)                  - Mycology

6. Viruses                                                - Virology

In ancient times, it was believed that diseases are punishment for an individual's crime, or they are due to evil spirit or poisonous vapours from rotting organic matter. Existence of microorganisms was not known at that time.  First proponents of the idea that invisible organisms caused diseases, were Lucretius (B.C.) and Girolamo Fracastoro (1546).

Earliest microscopic observations were made by Francesco Stelluti and Robert Hooke. Francesco Stelluti, an Italian naturalist made first microscopic observations on bees and weevils, by using the microscope probably supplied by Galileo. Later microscopic observations and studies were done by Robert Hooke. Micrographia is a historically significant book by Robert Hooke about his observations through various lenses. It was the first book to illustrate insects, plants etc. as seen through microscopes. Published in January 1665, the first major publication of the Royal Society, London, it became the first scientific best-seller, inspiring a wide public interest in the new science of microscopy.

Theory of Spontaneous generation (Abiogenesis) Vs Theory of Biogenesis

Earlier in 17^th Century, it was believed that living creatures could arise from non-living matter. For instance, it was hypothesized that certain forms such as fleas could arise from inanimate matter such as dust, or maggots could arise from dead flesh or mud. Means, living things can originate from non- living things and this theory was called the “Theory of spontaneous generation” or “Theory of Abiogenesis”. This theory was put forward by the Greek Philosopher Anaximander.  Greek Philosopher Aristotle also supported the theory of spontaneous generation and concluded that even some invertebrates could arise from non-living matter by spontaneous generation.

The theory of spontaneous generation was first challenged by Italian physician Francesco  Redi. His experiments on decaying meat to disprove the theory of abiogenesis is known as Redi’s experiment. To test the hypothesis, Francesco Redi placed fresh meat in open containers [left]. As expected, the rotting meat attracted flies, and the meat was soon swarming with maggots (larvae of flies), which hatched into flies. Then he covered a jar with air tight material, so that flies could not get in [middle] and no maggots were produced from meat. To answer the objection that the air tight cover might have cut off fresh air necessary for spontaneous generation, Redi covered the third jar with porous gauze [right] instead of an air-tight cover. Flies were attracted to the smell of the rotting meat, clustered on the gauze, which was soon swarming with maggots, but the meat itself remained free of maggots. Thus he proved that flies are necessary to produce flies: they do not arise spontaneously from rotting meat. Life originate only from living forms and is known as the “Theory of Biogenesis”.

Antony van Leeuwenhoek, a Dutch businessman and scientist is known as the “Father of Microbiology”. He was a cloth merchant. While running his draper shop, Van Leeuwenhoek wanted to see the quality of the thread better, by using the magnifying lenses. He developed an interest in lens making and it became his hobby. Leeuwenhoek made over 500 magnifying lenses and at least 25 single-lens microscopes during his lifetime. He started to observe drops of liquid like blood or pond-water etc., solid samples like as plant material or animal muscles etc.

His microscopes were made of silver or copper frames, holding hand-made lenses. The single-lens microscopes of Van Leeuwenhoek were relatively small devices, the largest being about 5 cm long. They were used by placing the lens very close in front of the eye, while looking in the direction of the sun. The other side of the microscope had a pin, where the sample was attached in order to stay close to the lens. There were also three screws to move the pin and the sample. He had made around 500 lenses and 25 single lens microscopes in his life time. But throughout his life the aspects of microscope construction and making of lenses, he kept as a secret.

The first microorganisms he observed under microscope were protozoans. He was also the first to record microscopic observations of muscle fibers, bacteria, spermatozoa and blood flow in small blood vessels. Van Leeuwenhoek did not write books, but sent letters to the Royal Society in London. He had also made drawings of moving “animalcules”. The letters were published in the Royal Society's journal Philosophical Transactions of the Royal Society. His observations laid the foundations for the sciences of bacteriology and protozoology and paved way to the development of new branch of science, Microbiology.

Leeuwenhoek’s discovery of microbes renewed the controversy between theory of Abiogenesis and Biogenesis. In 1748, another scientist John Needham challenged Redi’s findings and supported spontaneous generation of microbes by showing that even after boiling mutton broth and pouring into sealed containers, showed the growth of microbes. In this experiment he placed the broth in a bottle, heated the bottle to kill anything inside and then sealed it. Days later, he reported the presence of life in the broth as indicated by the development of turbidity and said that life had been created from nonlife. Actually, he did not heat it long enough to kill all the living forms in it.

In 1776, Lazzaro Spallanzani, an Italian scientist, again challenged the theory of spontaneous generation. Lazzaro Spallanzani, reviewed both Redi's and Needham's data and experimental design and concluded that perhaps Needham's heating of the bottle did not kill all living forms present in the broth. He constructed his own experiment by placing broth in each of two separate bottles. He boiled the broth in both bottles, then sealed one bottle tightly and left the other open. Days later, the unsealed bottle was teeming with small living things and the sealed bottle showed no signs of life. This experiment certainly excluded spontaneous generation as a viable theory.

In 1861, Louis Pasteur, Famous French scientist rigorously disproved the theory of spontaneous generation through his “Swan Neck Experiment”.  He designed   S-curve necked flasks (Swan necked or Goose necked flask) that were oriented downwards, so gravity would prevent access by airborne foreign materials. He placed nutrient-enriched broth in one of the goose-necked bottles, boiled the broth inside and left for few days. No life was observed in the jar. He then broke off the top of the bottle, exposing it more directly to the air, and noted life-forms in the broth within days. He noted that as long as dust and other airborne particles were trapped in the S-shaped neck of the bottle, no growth was observed in the broth. He reasoned that the contamination came from life-forms in the air and that only caused growth in broth. Hence he concluded that life originate only from living forms. Pasteur is known as the “Father of Modern Microbiology” and has made a number of significant of contributions to the field of Microbiology.

Louis Pasteur

Pasteur's Swan neck experiment

The mid 1800s to the early 1900s saw the rise of first microbiologists and is called the “Golden age of Microbiology”. During the golden age, many pathogens were identified, vaccines were developed, methodologies were perfected and foundations were established that support today’s modern research. These experiments and developments in this field led to the development of Microbiology as a new branch of science.

 

Youtube link: https://youtu.be/u7r94mig8sU