1. The document discusses sugar fermentation, how to test for it using media like phenol red carbohydrate broth, and the interpretation of results. 2. Hugh Leifson medium and Cetrimide agar are described as media used to differentiate bacterial metabolism and isolate Pseudomonas aeruginosa, respectively. 3. Key components, principles, preparation, and expected results are outlined for both Hugh Leifson medium and Cetrimide agar tests.

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:
1. Acid end products.
2. 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 35oC 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 Pseudomonasspecies
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

33. 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

34. 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