Carbohydrate Fermentation Test

Objective:

To determine the ability of microorganism to ferment the given carbohydrates with acid or acid and gas end products. 

Principle:

Fermentation is the metabolic process to breaking down or catabolism of the sugars under anaerobic conditions. The result of this process is production of energy source ATP by substrate level phosphorylation, in the absence of aerobic respiration electron transport chain. Microorganisms utilize the carbohydrates by their enzymatic reactions and produce organic acids, acid with gas and alcohol. The types and proportion of the products depend on bacterial species and carbohydrates.

 Formation of acids in sugar broths can be detected by using the pH indicator phenol red which is red at neutral pH and changes to yellow at acidic conditions, slight amount of acid can cause color change. Gas formation can be detected by using an inverted ‘Durham’s tube’.

Materials required:

-24 Hours old bacterial cultures (E.coli. Pseudomonas sp. Klebsiella sp. and Salmonella sp.)

-Peptone broth. Nutrient broth with pH indicator (Phenol red)

-Test tubes and Durham tube

Procedure:

- Prepare and sterilize the tubes containing 5 ml of peptone broth/nutrient broth for each sugar lactose, sucrose and glucose with pH indicator.

- Carefully insert the Durham tube in the broth tube in inverted position without air bubble.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

-E.coli and Klebsiella sp., shows both yellow color changes in the medium and bubble formation inside Durham’s tubes in all the sugars.

-Salmonella sp. shows yellow color changes in the medium and gas bubble formation in glucose, only yellow color change in sucrose and no reaction in lactose sugar.

-Pseudomonas sp. shows yellow color change in glucose, no reaction in sucrose and lactose.

Interpretation:

If the medium changes colorless to yellow color and gas bubble formation in Durham’s tube that indicates acid production and gas production. In some cases the gas does not evolved during fermentation. If no changes in the medium it indicates the sugars not degraded by the organisms.

Carbohydrate Fermentation Medium Composition:

Peptone     10.0 g

Sodium chloride   5.0 g

Sugar    1 g

Phenol red   5 ml

Distilled water    1000ml

pH   - 7.00

Sugar fermentation by different bacterial species

Urease Test

Objective:

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

Principle:

 Urea is a nitrogen containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is highly soluble in water and is therefore an efficient way for the human body to discharge excess nitrogen. This excess urea is then taken out of the body through the kidneys as a component of urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea to ammonia and carbon dioxide.
 Many enteric bacteria have the ability to hydrolyze urea as part of their metabolism, members of the genus Proteus are considered rapid urease producers due their efficiency in carrying out this process. Therefore, this experiment is useful in distinguishing members of Proteus, a urinary tract pathogen, from other enterics based on their ability to rapidly hydrolyze urea. Many enterics can hydrolyze urea but only a few can degrade  it rapidly. These are commonly referred as "rapid urease-positive" organisms. Members of the genus Proteus have the ability to hydrolyze urea rapidly.

  

Urea Hydrolysis:  Urea is waste product excreted in urine by animals. Some enteric bacteria produce the enzyme urease, which splits the urea molecule into carbon dioxide and ammonia. 

Urease, which is produced by some micro organisms, is an enzyme that is especially helpful in the identification of Proteus vulgaris, although other organisms may produce urease, their action on the substrate urea tends to be slower than that seen with Proteus species. Therefore this test serves to rapidly distinguish members of this genus from other lactose non fermenting enteric microorganisms.Urea is unstable and is broken down at 15 psi or pressure. It cannot be added to the medium for autoclaving and is therefore filter sterilized and added to the medium after autoclaving.

 Materials Required:

24 hours broth cultures (Proteus sp. and E.coli). 

Urease both / Urease Slant

Procedure: 

Using a sterile technique, inoculate each experimental organism into its appropriately labeled tube by means of loop inoculation. Incubate cultures 24-48 hours at 37°C.

Urea Broth method: -Inoculate the urea broth with the inoculation loop containing the organism from the broth cultures.-Incubate for 24-48 hours at 37°C. –-Obtain the broths from the incubator and observe the colour.

Urea Slant method:-Inoculate the urea slant (slope) with the inoculation loop containing the organism from the broth cultures. Do not stab the butt.-Incubate for 24-48 hours at 37°C.-Obtain the broths from the incubator and observe the color.

Result: -Proteus sp. gives urease positive reaction  -E.coli gives urease negative reaction

 Interpretation: Proteus sp. produces urease enzyme to degrade the urea to release the ammonia, it leads to alkaline condition of the medium. The phenol red indicator in the medium turns red pink color in alkaline condition and gives positive result.  E.coli does not produces urease enzyme and giving negative reaction.

Urea Broth Composition:

Urea      20g

Dibasic Sodium phosphate    9.5 g

Monopotassium phosphate    9.1 g of

Phenol red      0.01 g

The pH is made to   6.8±0.2 at 25°c.

Distilled water   1000 ml

  

Urea Slant Composition:

Same as above composition, but add agar as one of the ingredient.

Hydrogen Sulphide (H2S) Production Test

Objective:

            To determine the ability of microorganism reduces the sulphur containing compounds to hydrogen sulphide during metabolism.

Principle:

            Some bacteria utilize sulphur containing amino acids such as cysteine from the proteins by enzyme desulferase; it loses the sulfur atom then reduced by addition of hydrogen atom from water to form hydrogen sulphide.  This organism also reduces the inorganic sulphur compounds such as sodium thio sulfate, sulfate and sulfite to gaseous hydrogen sulphide.

            The SIM (Silfide, Indole, and Motility) agar contains the peptone a source of cysteine and sodium thiosulfate as sulfur substrates, ferrous ammonium sulfate which behaves as the H₂S indicator. If H₂S produced it combines with ferrous ammonium sulfate, forming insoluble black ferrous sulfide precipitate.   

Materials required:

24 Hours old bacterial cultures (E.coli. Proteus sp., Klebsiella sp. and Salmonella sp.)

SIM agar medium

Procedure:

- Prepare and sterilize the SIM agar deep tubes, adjust the pH to 7.4

- Inoculate the bacterial cultures by straight stabbing to a depth of 2-3 cm.

- Incubate the tubes at 37°C for 24 - 48 hours

Result:

Salmonella sp. and Proteus sp. gives positive result.

E.coli and Klebsiella sp. gives negative result.

Interpretation:

Salmonella sp. and Proteus sp.  shows blackening color due to the production of hydrogen sulphide and gives positive reaction. E.coli and Klebsiella sp. are not showing blackening color.

SIM Agar Medium Composition:

Beef extract    3.0 g

Peptone  30.0 g

Ferrous ammonium sulphate   0.2 g

Sodium thiosulphate 0.025 g

Agar   3.0g

Final pH ( at 25°C) 7.3±0.2

Distilled water    1000ml

Triple Sugar Iron (TSI) Test

Objective:

            To differentiate the enterobacteriaceae members according to their ability to ferment lactose, sucrose and glucose sugars and production of the hydrogen sulphide.

Principle:

            The fermentation of sugars will help to distinguish enteric bacteria from other gram negative bacilli. The TSI agar contains 1% concentrations of lactose and sucrose, 0.1 % of glucose. The phenol red is an acid base indicator is incorporated in this medium to detect acid production from carbohydrate fermentation.

            Acidification of medium is caused by intestinal organisms to attacking the sugars and it changes the phenol red to yellow color. This medium also contains sodium thio sulfate, the organisms reduces the sulfur to form hydrogen suphide gas and it is react with ferrous sulfate which is present in the medium to give black precipitation.

-0.1% Glucose: If only glucose is fermented, only enough acid is produced to turn the butt yellow.  The slant will remain red.

-1.0% lactose/1.0% sucrose:  a large amount of acid turns both butt and slant yellow, thus indicating the ability of the culture to ferment either lactose or sucrose.

-Sodium thio sulfate : Substrate for Production of Hydrogen sulphide (H₂S)

-Iron: Ferrous sulfate: Indicator of H₂S formation

-Phenol red: Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions).

Materials required:

24 Hours old bacterial cultures (E.coli ., Proteus sp. and Klebsiella sp.)

Triple sugar iron agar

Procedure:

- Prepare TSI medium and adjust the pH to 7.4

- Sterilize the medium and pour into sterile test tubes and make slants

- Inoculate TSI agar slants by first stabbing in center of the medium and streaking on the surface of the slant

- Incubate the tubes at 37°C for 18 - 24 hours

Result:

-E.coli and Klebsiella sp., - shows yellow (acid) slant/ acid (yellow) butt with gas production.

-Proteus sp., shows acid butt, alkaline (red) slant with hydrogen sulphide (black precipitation) and gas production.

E.coli  & Klebsiella sp.             -   A/A with gas

Proteus sp.,                                -  K/A with H2S production

Interpretation:

From the above results klebsiella sp. and E.coli both are able to ferment all the sugars present in the TSI agar and produces acid and gas production indicate by bubble formation. Proteus sp. is ferment the glucose only, doesn’t ferment the remaining sugars, this organism also reduced the sulfur to hydrogen sulphide gas and it is observed as blackening precipitation.

Composition of TSI Agar

Beef extract   3.0 g

Yeast extract  3.0 g,

Peptone     15 g,

Protease peptone  5 g,

Lactose 10.0 g

Sucrose 10.0 g

Glucose 1.0g

Ferrous sulphate   0.2 g

Sodium chloride   5.0 g

Sodium thiosulphate   0.3 g,

Phenol red    0.024 g

Agar   12 g

Distilled water    1000ml

some examples of Triple Sugar Iron (TSI) Agar Reactions: 

Name of the organisms	Slant	Butt	Gas	H2S
Escherichia, Klebsiella, Enterobacter	Acid (A)	Acid (A)	Pos (+)	Neg (-)
Shigella, Serratia	Alkaline (K)	Acid (A)	Neg (-)	Neg (- )
Salmonella, Proteus	Alkaline (K)	Acid (A)	Pos (+)	Pos (+)
Pseudomonas	Alkaline (K)	Alkaline (K)	Neg (-)	Neg (-)

Biosynthesis of Glutamate family amino acids

Biosynthesis of Aromatic Amino Acids

Gelatin Hydrolysis Test

Objective:

            To determine the ability of bacteria that produces extracellular hydrolytic enzyme Gelatianse that degrade gelatin.

Principle:

            Gelatin is a protein obtained from animal protein collagen, a major component of connective tissues and tendons of humans and animals. It is an incomplete protein with lacking of essential amino acid tryptophan. Gelatin maintains its gel properties below 25 ͦ C and exists as a solid in nature. At temperature above 25 ͦ C, gelatin is a liquid.

            Some microorganisms capable of producing proteolytic extracellular enzyme gelatinase which hydrolysis this gelatin into amino acids. After this degradation, it will not return to gel characteristics even at very low temperature 4 ͦ C.

Materials required:

24 Hours old bacterial cultures (Bacillus sp. and E.coli )

Nutrient gelatin

 Procedure:

-Prepare sterilized nutrient gelatin in test tubes and allow to solidify.

-Inoculate the organisms into gelatin tubes by stabbing

-Incubate the tubes at 37 ͦ C for 24 hours (May require upto 14 days to liquefy the gelatin.

-Following incubation, place the tubes in refrigerator at 4 ͦ C for 30 min.

-Observe the liquefaction (Liquid formation) in tubes

  (If bottom is resolidifies, slant the tubes and notice the surface of the medium is liquid or not)

Result:

Bacillus sp., is Gelatin hydrolysis positive (Gelatin liquefied)

E.coli is Gelatin hydrolysis positive (non liquefied)

Interpretation:

Bacillus sp. shows positive result as the medium remained liquefied after refrigeration. This result indicates that the organism produce gelatinase enzyme. E.coli give negative result as the medium remains solid after refrigeration.

           

Gelatin Hydrolysis Positive Organisms:

Bacillus sp.

Clostridium perfringens

Proteus vulgaris

Staphylococcus aureus

Gelatin Hydrolysis Negative Organisms:

E.coli

Staphylococcus epidermidis

Enterobacter aerogenes

Nutrient Gelatin Composition:

Peptone                      -              5 g

Beef extract               -               3 g

Gelatin                       -           120 g

Distilled water           -           1000ml

Catalase Test

Objective:

            To determine the ability of bacteria that produces Catalase enzyme which degrades the hydrogen peroxide.

 Principle:

           

            The enzyme Catalase produced by several bacteria, which is breakdown the hydrogen peroxide and releases oxygen and water. Organisms are producing O₂ products like superoxide and hydrogen peroxide during cell respiration, accumulation of hydrogen peroxide and superoxide leads to the destruction of cell constituents and death of the organisms. The superoxide dismutase enzyme, which catalyzes the destruction of toxic superoxide and Catalase enzyme which catalyzes the degradation of hydrogen peroxide. Obligate aerobes and certain anaerobes contain these types of enzymes, but most of the strict anaerobes lack of above enzymes and therefore they cannot tolerate oxygen.

              

Catalase production can be determined by addition of the substrate H₂O₂ on bacterial culture, if bacteria produce catalase enzyme the above said chemical reactions liberate oxygen gas and producing bubbles, it indicates the presence of catalase. This test also useful for differentiate the morphological similar organisms like Enterococcus (Catalse negative) and Staphylococcus (Catalase positive).

           

Materials required:

- 24 Hours old bacterial cultures (Staphylococcus  and Streptococcus )

- 3 % Hydrogen Peroxide (H₂O₂)
- Glass slide/ test tube

- Inoculation loop/ glass rod

Procedure:

1.      Slide Method:
   - Pure growth of the organisms will transfer the clean slide by using inoculation loop or glass rod.
  -   Immediately add a drop of 3% hydrogen peroxide on bacterial culture.
    -  Observe the bubble formation (Effervescence).

2. Tube Test:
-   Take one ml of 3 % hydrogen peroxide in test tube.

-   Small amount of bacterial culture introduce into the solution

-   Immediately observe the effervescence.

Result:

 Staphylococcus  is shows catalase positive with bubble formation

 Streptococcus is catalase negative with no bubble formation

                                                                            a)
                                                    
                                                                  b)

              

Interpretation:

           In aerobic organisms, during aerobic respiration, oxygen serves as hydrogen acceptor and hydrogen peroxide is formed in the cell. High concentration of H₂O₂  is formed which is toxic to cell.  Staphylococcus posses the catalase enzyme that converts hydrogen peroxide into oxygen and water. Streptococcus doesn’t releases catalase enzyme and this organism doesn’t convert

H₂O₂ into oxygen.

Catalase Positive Organisms:

Staphylococcus,           

E.coli,           

Pseudomonas aeroginosa,         

Klebsiella,

Salmonella,         

 Shiegella,

Proteus,

 Enterobacter,

 Citrobacter.

Catalase Negative Organisms:

Streptococcus

Most of the anaerobic organisms