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

Food Microbiology;Quality Standard : BS 5750

British Standard 5750:

A British Standard for Quality Assurance (BS 5750 'Quality Systems') was published in 1979. It contained a description of the controls which it prescribed were required to be instituted in order for a supplier to claim that it was a 'Quality Assured' Organisation.

In the same way as with the registration of products to a particular standard, an organisation could not be accredited to BS 5750 unless it had been inspected (and formally accredited) by an independent authority (such as the British Standards Institute) against the standard. In contrast to the Kite Mark (which is a method of product certification), BS 5750 is a form of company certification.

The standard specifies all those 'elements' of the management system which are seen to be critical to the quality of the final product and describes how these elements are to be controlled. 3 Although its initial adoption by industry was quite slow, a number of organisations have now implemented Quality Systems commensurate with the requirements of BS 5750, although its predominance in the engineering sector remains. In fact, BS 5750 has been increasingly criticised for its continued focus on the engineering / manufacturing environments - which a quick glance at the index of the standard will show.

In more recent years, in particular, a number of non-engineering and service sector organisations have recognised that the philosophy of Quality assurance is in fact applicable to every organisation and have sought a more broadly based guideline or standard. In an effort to accommodate the views of these other industry sectors, a number of QAS (Quality Assurance Schedules) have been produced to augment / amplify the standard.

Schedule no. 8 for example, is written specifically for the Service Sector industries and contains some additional requirements and guidance on the interpretation of the standard's requirements for these organisations. In 1987 the entire standard was revised and republished and its format was significantly amended.

The text is now identical with that of its equivalent International and European Standards - ISO 9000 and EN 29000. BS 5750 is due for another major renewal in 1996 and there is currently some interesting discussion underway as to its most desirable format and scope. It should be noted that the Nuclear Industry has for some time had its own Quality System Standard. In the UK this is BS 5882 ('Specification for a Total Quality Assurance Programme for Nuclear Power Plants'), which is similar in philosophy to BS 5750.

Ref : http://mt-archive.info/Aslib-1992-Wedlake.pdf

Oxidase Test

Objective:

•    To demonstrate the ability of bacteria that produces the enzyme cytochrome oxidase.
•   To distinguish the bacterial groups based on oxidase activity.

Principle:

            In aerobic respiration, oxidase enzyme plays a vital role in operation of electron transport chain. Cytochrome oxidase transfers the electrons from donor to molecular oxygen, during the oxidation of reduced cytochrome c to form water (or) hydrogen peroxide and oxidized cytochrome c.

            The ability of bacteria produce cytochrome oxidase enzyme can be determined by using oxidase disc impregnated with N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride (TMPD) reagent. This reducing dye become oxidized and turns into deep purple color from colorless by oxidase enzyme, the colored end product is called indophenol. Pseudomonas is the only species which gives positive reaction in enterobacteriaceae family.

Materials required:

- Bacterial cultures (Pseudomonas sp., and E.coli )

- Oxidase disc (Readymade)
(Preparation of Reagent:  1% solution of N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride (5g in 5ml in distilled water).  Disc preparation: Soak the sterile filter paper disc in few drops of reagents)

Procedure :
1.      Pick the 24 hours old bacterial cultures by platinum loop/plastic loop and smear over the oxidase    disc.
2.      After 10 min. observe the color change on disc.

Observation :
-   Deep purple color change of the disc with   Pseudomonas sp.,
-   No color change in the disc with E.coli

      

Result :
-  Pseudomonas sp., gives oxidase positive
- E.coli shows oxidase negative.

                             

       

Interpretation:

            Pseudomonas sp., gives positive result because it oxidizes the reagent N, N, N΄, N΄ tetramethyl p-phenylene diamine dihydrochloride and turns into deep purple color by oxidase enzyme. The purple colored compound known as indophenol. E.coli gives negative reaction because it doesn’t produces oxidase enzyme.

Oxidase positive organisms:

Pseudomonas sp., Vivrio chlorae, Nisseriae  sp., Camphylobacter sp., Alcalgenes sp., and Helicobacter sp.,

Precautions:

1.      The regent is to be prepared fresh.

2.      Nichrome wire is not to be used take culture. Trace of iron gives false positive result

3.      Should not take culture from selective medium

4.      Observe the result within the prescribed time.

Casein Hydrolysis Test

Objective:

 To demonstrate the ability of certain microorganisms to produce extracellular enzymes,    capable of degrading the milk protein casein.

Principle:

           

            Casein is the principle protein of the milk. It is the large polymer and colloidal suspension which gives opaque whiteness to the milk.   Casein is too large to enter into the bacterial cell membrane. In order to utilize the casein, bacterial cells secrete proteolytic exoenzymes (caseinase and peptidase) outside of the cell that break down the protein into amino acids.  The amino acids can then be used by cells after crossing the cell membrane via transport proteins. Casein hydrolysis is tested by growing an organism on a skim milk agar plate (providing nutrients and the casein) and then checking the plates for hydrolysis.  Plates without any hydrolysis will be white from the casein, whereas those exhibiting hydrolysis will have zones of clearing around the growth.

Materials required:
 - Bacterial culture (Bacillus sp.,)
- Skim milk agar

Procedure:
1.      Inoculate the bacterial cultures on skim milk agar medium by streaking. 
2.      The plates are incubating at 37° C for 24 hrs. 
3.      Following incubation, observe the zone of hydrolysis around the line of growth.

Observation:
-   Zone of clearance around the colonies of Bacillus sp.,

      

Result : 
-  Bacillus sp., shows positive result in casein hydrolysis  

       

Interpretation:

            A positive reaction is indicate by the zone of hydrolysis around the colonies of Bacillus sp. This is due to breakdown of casein by caseinase and other enzymes. In negative reaction indicates absence of enzymes showing no zone of hydrolyzing.

Casein hydrolyzing organisms:

Bacillus subtilis., Pseudomonas sp.,

Skim Milk Agar Composition:

Skim milk powder    100 g

Peptone    5g

Agar       15 g

Distilled water 1000 ml

Starch Hydrolysis Test

Objective:

-To determine  the ability of certain microorganisms to produce extracellular enzyme  amylase that breaking down starch.

-To identify the starch hydrolyzing bacteria.

Principle:

           

            Starch is a high molecular weight branching polymer. Starch as such cannot be transported to the bacterial cell for energy generation due to its high molecular weight. Therefore starch hydrolyzing exoenzymes are released into the surrounding media which can degrade starch to its glucose monomeric units. These soluble low molecular weight glucose can pass into the cell can be utilized energy generation by microbes.

                                     

Starch agar is nutritive medium with starch. The presence and absence of starch in the medium can be detected by addition of iodine, as iodine reacts with starch to import blue black color.

Materials required:

Bacterial cultures (Bacillus sp., and  E.coli)

Starch agar medium

Procedure :
1.      Inoculate the bacterial cultures on starch agar medium by streaking.
2.      The plates are incubating at 37° C for 24 hrs.
3.      Following incubation, pour the iodine solution on medium and observe the clear zone around the area of growth.

Observation:
- Clear zone around the line of growth of Bacillus sp., 
 In E.coli Plate turns completely blue black color, without zone formation.

Result :
-  Bacillus sp  : Starch hydrolysis positive
- E. coli : Starch hydrolysis negative

Interpretation:

           

            The clear zone of hydrolysis around Bacillus sp., when flooded with iodine solution, indicates that organism produces extra cellular amylase enzyme that hydrolyse starch in the media. E.coli showed negative result  as it did not produce amaylase. Thus could not hydrolyse starch and hence when iodine added it produced blue black color.

  

Starch Hydrolysing organisms:

Bacillus subtilis, Bacillus megatarium

 

Clear zone around the the Bacillus sp.,

(Starch hydrolyzing bacteria)

Below the first plate is positive and the second one is negative

                              

Starch Agar Composition

Ingredients / Liter

                     

Beef extract                             3.0 g   

Starch, soluble                         10. g

Agar                                        12.0 g

pH ( at 25°C) 7.5


Iodine Solution Preparation:

Potassium iodide   2g

Iodine crystals  1g

Distilled water  300ml

Dissolve 2g of potassium iodide in 300 ml of water. Then add 1g of finely ground iodine crystals. Stir at room temperature until completely dissolved.