Sugar fermentation tests, Cetrimide agar medium, Hugh Leifson medium

Combined presentation on:-
1. Sugar fermentation tests
2. Cetrimide agar
3. Hugh Leifson medium
Covering principle, preparation of the medium, test protocol, result interpretations, test conclusions.

1. Sugar Fermentation Mr. Shivam Kumar Sriwas MSc Biotechnology TESTS MEDIA

2. We Will Talk About What is sugar fermentation? How to test ? Media used to test Hugh Leifson Medium & Cetrimide Agar: Principle, Composition, Preparation, Uses, Quality control.

3. What is Sugar fermentation? Sugars serve as energy sources when broken down by bacteria and other cells. Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which sugars are broken down for energy production.

4. Tests We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced: Acid end products. Acid and gas end products .

5. Tests The sugar utilization tests are designed to detect the change in pH which would occur if fermentation of the given sugar occurred . Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow . If the bacteria do not ferment the sugar then the media remains red . If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it.

6. Phenol Red Carbohydrate Broth is commonly used in carbohydrate fermentation test. The carbohydrate source can varies based on your test requirements. Common broth media are: Phenol Red Glucose Broth Phenol Red Lactose Broth Phenol Red Maltose Broth Phenol Red Mannitol Broth Phenol Red Sucrose Broth Test procedure

7. Composition of the media   Peptone: 10 g Sodium Chloride ( NaCl ): 5 g Beef extract (optional): 1 g Phenol red (7.2 ml of 0.25% phenol red solution): 0.018 g Carbohydrate source: 10 g

8. Composition of the media   Importance and role of different pH Indicators

9. Preparation of  the media   Prepare broth media by mixing all ingredients in 1000 mL of distilled and heating gently to dissolve it. Fill test tubes with 4-5 ml of phenol red carbohydrate broth. Insert a Durham tube to detect gas production. Autoclave the prepared test media (at 121°C for 15 minutes) to sterilize. The sterilization process will also drive the broth into the inverted Durham tube .

10. Interpretation of the results 

11. Interpretation of the results 

12. Hugh Leifson Medium

13. Hugh Leifson Medium The oxidative-fermentative (OF) test was developed by Hugh and Leifson in 1953 . They developed OF media to differentiate between oxidative bacteria ( that produces acid from carbohydrates under aerobic condition only ) and fermentative bacteria ( that produces acid both under aerobic and anaerobic conditions ).

14. Hugh Leifson Medium

15. Hugh Leifson Medium Preparation Suspend 20.33 grams in 1000 ml distilled water. Digest to dissolve the medium completely. Dispense into test tubes in duplicate for aerobic and anaerobic fermentation. Sterilize by autoclaving at 10 lbs pressure (115°C) for 20 minutes. Cool the tubed medium in an upright position

16. Hugh Leifson Medium Principle The oxidative-fermentative test determines if certain gram-negative rods metabolize glucose by fermentation or aerobic respiration ( oxidatively ). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will  turn the bromthymol blue indicator in OF media from green to yellow  in the presence or absence of oxygen .

17. Hugh Leifson Medium Principle

18. Hugh Leifson Medium Uses: OF Test is used to determine if gram-negative bacteria metabolize carbohydrates oxidatively , by fermentation, or are nonsacchrolytic (have no ability to use the carbohydrate in the media ). Limitations : This medium is general purpose medium and may not support the growth of fastidious organisms.

19. Hugh Leifson Medium Quality Control Appearance Light yellow to bluish green homogeneous free flowing powder. Gelling Semisolid,comparable with 0.2% Agar gel. Colour and Clarity of prepared medium Greenish blue coloured, clear to slightly opalescent gel forms in tubes as butts pH 6.90-7.30

20. Hugh Leifson Medium Procedure Inoculate two tubes of OF test medium with the test organism using a straight wire by stabbing “half way to the bottom” of the tube . Cover one tube of each pair with 1 cm layer of  sterile mineral oil or liquid paraffin , leaving the other tube open to the air . Incubate both tubes at 35 o C for 48 hours

21. Hugh Leifson Medium Interpretation Fermentative result:   Acid production on both (open and covered) tubes. The acid produced changes the pH indicator, bromthymol blue, from green to yellow. e.g.  Escherichia coli  Oxidative result : Acid production in the open tube (aerobic) and not the oil-covered tube (anaerobic) indicates an oxidative result.  Nonfermenting bacteria that metabolize glucose via oxidative metabolism give an oxidative result. e.g.  Pseudomonas aeruginosa  Non saccharolytic (Negative OF result):   Nonsacchrolytic bacteria give a negative OF result. The negative result is indicated by no color change in the oil-covered tube and in some cases an increase in pH (pH 7.6) changing the bromthymol blue from green to blue in the top of the open tube. The increase in pH is due to amine production by bacteria that break down the peptone (protein) in the medium. e.g. Alcaligenes faecalis

22. Hugh Leifson Medium Interpret Open (Aerobic) Tube Covered (Anaerobic) Tube Metabolism Acid (Yellow) Alkaline (Green) Oxidative Acid (Yellow) Acid (Yellow) Fermentative Alkaline (Green) Alkaline (Green) Non saccharolytic (glucose not metabolised) Following are the reaction patterns:

23. Cetrimide Agar

24. Cetrimide Agar Pseudosel Agar or Cetrimide Agar is used for the selective isolation and presumptive identification of   Pseudomonas aeruginosa   . The medium was first developed by Lowburry and is a modification of Tech Agar (developed by King et al.) with addition of 0.1% cetrimide ( cetyl trimethyl ammonium bromide) for the selective inhibition of organisms other than  Pseudomonas aeruginosa .

25. Cetrimide Agar

26. Cetrimide Agar Glycerol acts as the carbon source. Magnesium chloride and Potassium chloride enhance the production of pyocyanin and fluorescein. Agar is the solidifying agent. Glyceol is supplemented as a source of carbon. Cetrimide is the selective agent.It is a toxic substance that inhibits the growth of many bacteria.

27. Cetrimide Agar Principle Cetrimide is a quaternary ammonium salt, which acts as a cationic detergent when comes in contact with bacterial cell, causes the release of nitrogen and phosphorous which in turn has denaturing effects  on membrane proteins of bacterial cell. It exhibits inhibitory actions on a wide variety of microorganisms including Pseudomonas species other than  Pseudomonas aeruginosa .

28. Cetrimide Agar Principle Pseudomonas aeruginosa  produces a number of water soluble pigments, including the  yellow-green or yellow-brown fluorescent pigment pyoverdin (fluorescein) .When pyoverdin combines with the  blue water -soluble pigment pyocyanin , the bright green color characteristic of  Pseudomonas aeruginosa  is created.

29. Cetrimide Agar Principle

30. Cetrimide Agar Preparation Suspend 45.3 g of the medium and 10 ml of glycerol in one liter of purified water . Digest with frequent agitation and boil for one minute to completely dissolve the medium . Autoclave at 121°C for 15 minutes . Mix well and pour into sterile Petri plates.

31. Cetrimide Agar Expected Results Positive : Growth of organism is seen in the slant (see the image above ) Negative: No growth

32. Q & A Is Hugh Leifson medium differential ? YES Other name for Cetrimide agar Pseudosel Agar Which organism’s isolation is perfomed using Pseudosel agar ? Pseudomonas aeruginosa What is fullform of ATCC ? the American Type Culture Collection

33. Reference Used Cetrimide Agar: Composition, Principle, Preparation and Uses -By Nisha Rijal https:// microbeonline.com/cetrimide-agar-composition-principle-preparation-uses/ ppt : Biochemical test of bacteria ~ By K.P . Senthil Kumar.,M. Sc ., M.Phil.,ADAB ., HiMedia Laboratiories : Technical Data- Cetrimide agar, Hugh Leifson Medium www.microbiologyinpictures.com