principle of biochemcial test of gram negative bacteria in microbiology

1. Triple Sugar Iron test
.Principle: 
  Triple sugar iron agar test is used to determine whether gram negative bacilli utilize glucose and lactose or sucrose fermentatively and produce hydrogen sulfide (H2S).  It contains 10 parts of lactose: 10 parts of sucrose: 1 part of glucose and peptone.  Phenol red and ferrous sulphate serves as an indicator for acidification of medium and H2S production respectively.

Glucose is utilized first by a fermentative organism and the entire medium becomes acidic (yellow) in 8 to 12 hours. Butt remains acidic even after 18 to 24 hours incubation peroid because of the presence of organic acids resulting from the fermentation of glucose under anaerobic conditions in the butt of the tube.The slant reverts to alkaline state that is indicated by red color as the fermentation products gets oxidised to carbon dioxide (CO2) and water (H2O) and peptone in aerobic condition the slant undergoes oxidation releasing alkaline amines(Phenol red in alkaline pH turns red while in acidid pH turns yellow).

• If the organism ferments glucose but does not ferment lactose and/or sucrose, then the slant becomes red and butt remains yellow (K/A) within 18 to 24 hrs.
• If the organism in addition to glucose ferments lactose and/or sucrose, the fermentation product formed on the slant will more than neutralize the alkaline amines rendering the slant acidic (yellow) (A/A) provided the reaction is read within 18 to 24 hours.
• If the organism is non fermenter, instead of sugars, peptone is utilised as an alternate source of energy under aerobic condition on the slant which makes it alkaline indicated by the red color while there is no change in the color of the butt. K/NC

Reactions in TSI should not be read after 24 hours of incubation, because aerobic oxidation of fermentation products form lactose and/or sucrose will occur and the slant will eventually revert to alkaline state.

The formation of CO2 and H2 is indicated by the presence of bubbles or cracks in the medium or by the separation of the agar from sides or bottom of the tube. The production of H2S rqires an acidic condition and is indicated by blackening of the butt of the medium in the tube.

Procedure:

1. Touch a well isolated colony with a sterile straight wire.
2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and then streak the surface of the slant.
3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
4. Observe the reaction

Expected results:

1.Alkaline slant/no change in butt (K/NC) or Alkaline slant/Alkaline butt (K/K) = glucose, lactose and sucrose nonfermenter.(red/red)

2.Alkaline slant/acidic butt (K/A)- glucose fermentation only. (red/yellow)

3.Acidic slant/acidic butt (A/A)- glucose, lactose and/or sucrose fermenter. (yellow/yellow)

A black precipitate in the butt indicates production of H2S . H2S produced reacts with ferric salt to produce black precipitate of ferrous sulfide.

Bubbles or cracks in the tube indicate the production of CO2 or H2. Drawing of circle around butt indicates that gas is produced by glucose and sucrose or glucose and lactose and glucose, sucrose and lactose fermenter.

Examples: 

A/A = E. coli and Klebsiella

K/A = Salmonella and shigella

K/K = Pseudomonas

2. SULPHUR INDOLE MOTILITY TEST (SIM)

 >PRINCIPLE: 

  The medium contains ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide. Hydrogen sulfide production is detected when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with H₂ S gas.

Casein peptone, another component of SIM Medium, is rich in tryptophan. Organisms possessing the enzyme tryptophanase degrade tryptophan to indole. Indole is detected upon the addition of Kovacs Reagent  following incubation of the inoculated medium. Indole combines with p-dimethylaminobenzaldehyde and produces a red band at the top of the medium. A negative indole test produces no color change upon the addition of Kovacs Reagent.

The small amount of agar added to the medium provides a semi-solid structure allowing for the detection of bacterial motility. Motile organisms extend from the stab line and produce turbidity or cloudiness throughout the medium. Non-motile organisms grow only along the stab line and leave the surrounding medium clear.

SIM Medium also contains animal tissue which provides amino acids and nutrients necessary for bacterial growth.

INTERPRETATION OF RESULTS

A positive H ₂ S test is denoted by a blackening of the medium along the line of inoculation. A negative H ₂ S test is denoted by the absence of blackening.

A positive motility test is indicated by a diffuse zone of growth flaring from the line of inoculation.

A negative motility test is indicated by growth confined to the stab line.

A positive test for indole is denoted when a pink to red color band is formed at the top of the medium after addition of Kovacs Reagent. A yellow color denotes a negative indole test after addition of Kovacs Reagent.

PROCEDURE

Specimen Collection: Specimen collection is not applicable since this medium is not intended for primary isolation from clinical specimens. As a general rule, infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection.

Method of Use:

1. Using isolated colonies from an 18-24 hour culture on solid media, inoculate the SIM Medium by stabbing the center of the medium to a depth of 1/2 inch.

2. Incubate the inoculated medium aerobically at 35ºC. for 18-24 hours.

3. Observe for H₂ S production and motility.

4. Once H₂ S and motility reaction have been read and recorded, apply three drops of Kovacs Reagent  to the surface of the medium.

5. Observe for the development of a pink to red color.

Indole positive organisms: Most strains of E.coli, Vibrio cholerae, P. vulgaris, M. morganii and Providenica are indole positive.

Indole Negative organisms: Klebsiella pneumoniae, Shigella sp., Salmonella sps.

Point to remember: Indole test can also aid in species differentiation.

1. Klebsiella species:  Klebs‪iella oxytoca is indole positive whereas Klebsiella pneumoniae is indole negative.
2. Citrobacter species: Citrobacter Koseri is indole positive where as Citrobacter freundii is indole negative
3. Proteus species: Proteus Vulgaris is indole positive whereas Proteus mirabilis is indole negative 

I used to remember above mentioned information with the help of MNEOMONIC,  which can be useful for you.

For this remember the phrase “OK VIP”

Where: O means: Oxytica (Klebsiella oxytoca), K means (koseri-i.e. Citrobacter koseri), V means vulgaris (Proteus vulgaris) and IP means Indole Positive.

H2S positive : Salmonella (except paratyphi A) 

H2S negative: Shigella, E coli, Klebsiella, Pseudomonas

Motility positive: Salmonella, E coli, Pseudomonas aeruginosa, 

Motility negative: Shigella, Klebsiella

Note: Hydrogen sulfide is produced if the sodium thiosulfate is reduced by the bacterial strain.  This happens when the strain either degrades the amino acid cysteine during protein degradation, or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen.  In either case H2S is produced (hydrogen sulfide) which reacts with the iron compound to form the black precipitate of ferric sulfide.  And The semi-solid medium (about 0.4% agar) allows for the testing of the motility of a strain.  The agar is at a concentration to allow the bacteria to move through a gel.

3. CITRATE UTILIZATION TEST

Media used: Simmon's Citrate Agar , (Koser's Citrate media)

Principle: 

When an organic acid such as citrate (remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced ultimately.  In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source.

Utilization of exogenous citrate requires the presence of citrate transport proteins (permeases). Upon uptake by the cell, citrate is cleaved by citrate lyase to oxaloacetate and acetate. The oxaloacetate is then metabolized to pyruvate and CO₂.

Citrate = oxaloacetate + acetate

oxalacetate = pyruvate + CO₂

Citrate Utilization Test

Further metabolic breakdown is dependent upon the pH of the medium.

A. Under alkaline conditions, pyruvate is metabolized to acetate and formate.
pyruvate = acetate + formate

B. At pH 7.0 and below, lactate and acetoin are also produced.
pyruvate = acetate + lactate + CO₂pyruvate = acetoin + CO₂The carbon dioxide that is released will subsequently react with water and the sodium ion in the medium to produce sodium carbonate, an alkaline compound that will raise the pH.  In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source.

When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue above pH 7.6.

Growth usually results in the bromothymol blue indicator, turning from green to blue. The bromothymol blue pH indicator is a deep forest green at neutral pH.  With an increase in medium pH to above 7.6, bromothymol blue changes to blue

Procedure of Citrate Utilization Test

1. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony.
2. Incubate aerobically at 35 to 37 C for up to 4-7 days.
3. Observe a color change from green to blue along the slant.

Examples:

Citrate positive organisms: 

• Klebsiella pneumoniae.
• Enterobacter species (minority of strains gives negative result)
• Citrobacter freundii.
• Salmonella other than Typhi and Paratyphi A.
• Serratia marcescens.
• Proteus mirabilis (minority of strains gives negative result)
• Providencia.

Citrate negative: E. coli, Salmonella, Shigella

4. UREASE TEST

Media: Christensen's Urea Agar

Bacteria may produce Urease enzyme. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours.

Weakly positive organisms may take several days, and negative organisms produce no color change or yellow as a result of acid production.

Urease positive: Klebsiella, Proteus, H. pylori, 

Urease negative : E. coli, Salmonella, Shigella