EXPERIMENT -7

AIM: DETERMINATION OF METABOLIC ACTIVITIES OF BACTERIA THROUGH BIOCHEMICAL TESTS

INTRODUCTION:

The current storage procedures for bacteria are insufficient, as many bacterial species are
indistinguishable under the microscope. Despite appearing similar, each species has unique genetic
composition and enzymes, enabling specific biochemical activities. Biochemical assays are used to
distinguish between species based on factors like protein and fat metabolism, carbohydrate metabolism,
enzyme production, and chemical utilization abilities.

OBJECTIVES:
- To Explain the importance of biochemical tests;
- To Describe how to perform the biochemical tests in the laboratory.

PRINCIPLE:

Biochemical tests identify bacteria by analyzing their biochemical activity for different compounds.
Reactions may vary based on factors like incubation period, temperature, and bacteria inoculum.
Different bacterial species may produce different results, so media selection is crucial. pH indicators,
such as phenol red, bromocresol purple, and bromothymol blue, are used to measure pH shifts in
metabolic assays.
A. Carbohydrate Fermentation
Fermentation is a metabolic process used by certain bacteria to break down glucose in the absence of
oxygen. It involves reaction of glycolysis (the breakdown of a single molecule of glucose into two
molecules
of pyruvate) as well as other events that result in a range of end products (acids, alcohols, gases). The
end products are characteristics of individual bacterial species.
B. Gelatin Hydrolysis
C. Starch Hydrolysis
D. Casein Hydrolysis
E. Urea Hydrolysis
F. Citrate Utilization
G. Catalase Activity
H. Tryptophan Hydrolysis:
Respiration

REQUIREMENT:
a) For carbohydrate fermentation test: 1-1 tube of the Lactose + phenol red, Sucrose + phenol red,
Glucose + phenol red and bacterial culture of Proteus vulgaris, Escherichia coli, Bacillis subtilis
b) Gelatin Hydrolysis: 1 nutrient gelatin deep and bacterial culture of Staphylococcus aureus, Serratia
marcescens, Streptococcus faecalis, or Bacillis subtilis
c) Starch Hydrolysis: 1 starch agar plate and bacterial culture of Proteus vulgaris, Escherichia coli, Bacillus
subtilis
d) Casein Hydrolysis: 1 milk agar plate and bacterial culture of Bacillus subtilis and Enterobacter
aerogenes
e) Urea Hydrolysis: 1 urea slant and bacterial culture of Escherichia coli or Proteus vulgaris
f) Citrate Utilization: 1 Simmon’s citrate slant and bacterial culture of Escherichia coli or Serratia
marcescens
g) Catalase Activity: 1 TSA slant and bacterial culture of Staphylococcus aureus or Streptococcus faecalis
h) Tryptophan Hydrolysis: 1 tryptone broth (medium that contains tryptophan) and bacterial culture of
Escherichia coli or Enterobacter
aerogenes
i) Forceps
j) Incubators
k) Microscope and light source
l) Alcohol
m) Sterile water
Personal protective equipment
i) Gloves
ii) Apron
iii) Mask
iv) Shoe cover
v) Head cover

PROCEDURE:

A. Carbohydrate Fermentation
• Taken 1 tube of each of the broths:
• Lactose + phenol red, Sucrose + phenol red, and Glucose + phenol red
• Proteus vulgaris, Escherichia coli, Bacillus subtilis, or Streptococcus faecalis ( 1 of any bacteria).
• Inoculated the three types of fermentation broth with selected bacteria. Made a note of any little
bubbles in the Durham tubes before inoculating the broths so that they are not misinterpreted as
signs of gas generation during fermentation.
B. Gelatin Hydrolysis
• taken 1 nutrient gelatin deep
• Used 1 of the following bacteria: Staphylococcus aureus, Serratia marcescens, Streptococcus faecalis,
or Bacillis subtilis
• To obtain some bacteria from stock culture, use an inoculation needle (not a loop).
• Inoculate the gelatin deep by using the inoculating needle to stab ~3/4 of the way down into the
nutrient gelatin and then drawing the needle straight back up.
C. Starch Hydrolysis
• Take 1 starch agar plate
• Use all of the following bacteria: Proteus vulgaris, Escherichia coli, Bacillus subtilis
• Divide starch agar plate into 4 areas using a wax pencil or a sharpie marker.
• Make sure that the draw will be on the bottom of the petri dish (the part that contains the agar) rather
than on the lid.
• to indicate which bacterium will be inoculated into each area should be properly labeled
• One area is left as a negative control. Using a loop, do spot inoculations of each bacterial species in the
areas. A spot inoculation is used to ensure that a large number of bacteria will grow at a single
location and produce a concentrated amount of amylase.
D. Casein Hydrolysis
• take 1 milk agar plate
• Use both bacteria, Bacillus subtilis and Enterobacter aerogenes
• Divide milk agar plate into 3 areas using a wax pencil or a sharpie marker.
• Label the plate to indicate which bacterium will be inoculated into each area.
• One area is left as a negative control.
• Using a loop, do spot inoculations of each bacterial species in the areas, just as you did on the starch
agar plate.
E. Urea Hydrolysis
• Take 1 urea slant
• Use one of the bacteria, Escherichia coli or Proteus vulgaris
• To inoculate the urea slant, use a loop to obtain some bacteria from the culture, and then carefully
streak the surface of the slant.
• Do not stab down into the slant’s interior. Replace the cap on the slant, but leave the cap loose.
• Wrap in the tube in aluminum foil.
• To inoculate the urea slant, use a loop to obtain some bacteria from the culture, and then carefully
streak the surface of the slant.
• Do not stab down into the slant’s interior. Replace the cap on the slant,but leave the cap somewhat
loose.
• Wrap in the tube in aluminum foil.
F. Citrate Utilization
• Take 1 Simmon’s citrate slant
• Use one of the bacteria, Escherichia coli or Serratia marcescens
• Using an inoculation needle (not a loop), inoculate the Simmon’s citrate by first stabbing the needle
into the butt of the agar slant and then streaking the surface of the slant before you pull the needle out
of the tube.
G. Catalase Activity
• Take 1 TSA slant
• Use one of the bacteria, Staphylococcus aureus or Streptococcus
faecalis
• Using a loop, inoculate the surface of the TSA slant. Make sure to use a heavy inoculum.
H. Tryptophan Hydrolysis
• Take 1 tryptone broth (Note: this medium contains tryptophan)
• Use one of the bacteria, Escherichia coli or Enterobacter aerogenes
• Inoculate the medium with any chosen bacteria.• Take 1 Simmon’s citrate slant
• Use one of the bacteria, Escherichia coli or Serratia marcescens
• Using an inoculation needle (not a loop), inoculate the Simmon’s citrate by first stabbing the needle
into the butt of the agar slant and then streaking the surface of the slant before you pull the needle out
of the tube.
G. Catalase Activity
• Take 1 TSA slant
• Use one of the bacteria, Staphylococcus aureus or Streptococcus faecalis
• Using a loop, inoculate the surface of the TSA slant. Make sure to use a heavy inoculum.
H. Tryptophan Hydrolysis
• Take 1 tryptone broth (Note: this medium contains tryptophan)
• Use one of the bacteria, Escherichia coli or Enterobacter aerogenes
• Inoculate the medium with any chosen bacteria.
RESULT:
PRECAUTIONS:

1) Always use the sterile glassware.

2) Before processing the samples in laminar flow bench, sterile the surface to remove the contamination.

3) Always on the UV light before and after processing the samples in laminar flow bench.

4) Always wear the lab coat, mask and gloves before start the analysis.

5) Always use the 70% alcohol for disinfect the working surface and after complete dry the surface turn
on the burner.

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On white side of the page –

OBSERVATIONS

A. Carbohydrate Fermentation: look at the results of the carbohydrate fermentation test.