UNIVERSITY OF KUALA LUMPUR (UNIKL)

MALAYSIAN INSTITUTE OF CHEMICAL AND BIO-ENGINEERING

TECHNOLOGY

Experiment 4: Bacterial Identification

Prepared by:
Nur Nabilah binti Ramlee
55211114146

Lecturer: DR. Leong Chean Ring

Submission Date: March 18, 2016

1.0 OBJECTIVE
1. To develop the laboratory skill in proper ways.
2. To apply the basic of aseptic technique.
3. To understand the biochemical testing and the types of biochemical reactions which
each organism undergoes for its identification.
4. To prepare the experiment using MacConkey agar, Starch hydrolysis test, Methyl red,
Voges Proskauer and citrate utilization test.
5. To distinguish between B. subtillis and E. coli.
6. To perform bacteria identification by using colony morphology on nutrient agar
techniques.

2.0 INTRODUCTION
Bacteria is the smallest and most common microorganism which are prokaryotes. It is
unicellular organisms that lack a nucleus. Bacteria can be identified by characteristics such as
shape, the chemical nature of their cell walls, the way they move and the way they obtain
energy.
Bacterial colonies can differ greatly in their morphologies. Therefore, difference
species of bacteria can be identified. Besides, staining provides valuable information as to
bacterial morphology, gram reaction and presence of such structures as capsules and
endospores. In identifying of bacteria, biochemical test were conducted. There were many
types of biochemical test. However, in this experiment, MacConkey agar, Starch hydrolysis
test, Methyl red, Voges Proskauer and citrate utilization test were conducted to identify the
bacteria.
MacConkey agar is the medium of both selective and differential which the crystal
violet and bile salts will inhibit the growth of gram-positive organisms. Then, the gramnegative bacteria have ability to ferment lactose and turn pink in colour.
Next, the aim for starch hydrolysis test is to check the ability of an organism to
produce hydrolytic enzymes or exoenzymes such as amylase that excreted out by organisms

to breakdown large substances into smaller ones. In the presence of starch, iodine will turns
into dark blue colour and gold colour around the growth.
Then, methyl red is the method that act as indicator dye because that turns red in
acidic solution. In microbiology, to identify bacteria that produce stable acids can be done by
methyl red in the methyl red test (MR test). This test determines whether the microbe
performs mixed acids fermentation when supplied glucose. Mixed acids fermentation results
in accumulation of a variety of acids and a significant drop in the pH of the medium. After
addition of methyl red, the culture medium will turns into red colour that indicates positive
result.
Voges Proskauer named after two microbiologists working at the beginning of the
20th century which is Daniel Wilhelm and Bernhard Proskauer. The method is to test the
presence of diacetyl, the oxidation product of acetoin in a bacterial growth. Positive results
represented by the presence of red colour while the yellow colour represent negative.
Lastly, citrate utilization test is to observe either microbe may use the compound
citrate as energy for growth and its main source of carbon. Simmon-citrate agar slants were
used in the medium that contains mineral salts, ammonium phosphate for its nitrogen source
and sodium citrate for carbon. The alkaline pH turns the pH indicator (bromthymol blue)
from green to blue that indicates the positive result.

3.0 SUMMARY
This experiment was conducted to perform bacteria identification by using colony
morphology on nutrient agar and biochemical method. There were five biochemical tests that
conducted in this experiment which are differential on MacConkey agar, starch hydrolysis
test, methyl red test, voges proskauer test and citrate utilisation test. Aseptic technique
applied during the experiment to prevent contamination of other living microorganisms on
samples. Firstly, for MacConkey agar test and starch hydrolysis test, the plate of the agar
have been divided into two zones (A and B) by draw a line using marker pen. After that, both
of the B. subtillis and E-coli were inoculated in the agar and the plates were incubated at
37C for 24 hours. Then, for MacConkey agar, the growth of both bacteria on the plate were
observed. In starch hydrolysis, after incubation, the iodine was poured on the plate evenly to
observe which bacteria shows positive result by production of a clear zone around the
bacterial growth. Then, for methyl red and voges proskauer, the colony transferred into the
glucose phosphate broth by inoculated wire loop and incubated at 37C for 24 hours. After
that, for methyl red, two drops of methyl red solution were added into the culture to observe
the colour changes while in voges proskauer, two drops of creatine solution and 1 ml of 4%
potassium hydroxide solution were added to observe the colour changes. Lastly, in citrate
utilization test, each of the colony were streaked by inoculated wire loop onto the slant of
Simmon-citrate medium and incubated for 37C for 24 hours. Then, observe the colour
change and growth of the microorganisms.

4.0 PROCEDURE
(A)

Colony morphology on Nutrient Agar

1.

Described the colony morphology of B. subtilis and on nutrient agar.

B)

Growth on the selective and differential MacConkey Agar

1. Took one plate of MacConkey agar and a line drawn used marker pen to separate it
into two zones: A and B.
2. Inoculated B. subtillis on zone A and E. coli on zone B.
3. Incubated the plates at 37 C for 24 hours.
4. The growth of both bacteria were observed on the plate.

(C)

Starch Hydrolysis Test

1. Took one plate of starch agar and a line drawn used marker pen to separate it into two
zones: A and B.
2. Inoculated B. subtillis on zone A and E. coli on zone B.
3. Incubated the plates at 37 C for 24 hours.
4. Poured enough iodine over the surface of the starch plate to cover the entire plate.
Rotated and tipped the plate to spread the iodine.
5. Observed which bacteria shows positive result (shown by production of a clear zone
around the bacteria growth.

(D)

Methyl red (2 test tubes containing 5 ml of phosphate broth + 0.5% (w/v)

glucose)
1. Used the inoculating wire loop, the colony was transferred into the glucose phosphate
broth and incubated at 37 C for 24 h.
2. After 24 h, 2 drops of methyl red solution added into the culture. Shacked gently and
observed the final colour change. Red indicates positive result.

(E)

Voges Proskauer (2 test tubes containing 5 ml of phosphate broth + 0.5% (w/v)

glucose)
1. Used the inoculating wire loop, transferred each of the colonies into the glucose
phosphate broth and incubate at 37 C for 24 h.
2. After 24 h, 2 drops (about 0.5 ml) of creatine solution and 1 ml of 4% potassium
hydroxide solution were added. Shacked the mixture and examined after 1-4 h. A
positive reaction was indicated by a change of colour from yellow to pink.

(F)

Citrate utilization test (2 Universal bottles containing Simmon- citrate agar

slants)
1. Used the inoculating wire loop, streaked each of the colony onto the slant of Simmon-

citrate medium and incubated at 37 C for 24 h.

2. After 24 h, examined the colour change and growth of the microorganisms.

Utilization of citrate was indicated by the colour changes from green to blue.

5.0 RESULTS
A) Growth of the selective and differential MacConkey agar

Zone A

Microorganism

(B)

Zone B

Changes of colour

Reaction

Fermentation of lactose

B. subtillis (zone A)

No changes

(-)

Absence

E. Coli (zone B)

Pinky-red

(+)

Presence

Starch Hydrolysis Test

Zone B

Zone A

Microorganisms

Growth

Colour of medium
around colonies after
addition of iodine

Starch hydrolysis

B. subtillis (zone A)

(+)

Light yellow

(+) = yes

E. Coli (zone B)

(+)

Colourless

(-) = no

(C) Methyl red (2 test tubes containing 5 ml of phosphate broth + 0.5% (w/v) glucose)

Sample 1

Microorganism

Sample 2

Colour changes

Reaction

B. subtillis (Sample 1)

Remains yellow

(-)

E. coli (Sample 2)

Reddish

(+)

(D) Voges Proskauer (2 test tubes containing 5 ml of phosphate broth + 0.5% (w/v) glucose)

Sample 2

Sample 1

Microorganism

Colour change

Test

B. subtillis (Sample 1)

Remained yellow

(-)

E. coli (Sample 2)

Remained yellow

(-)

(E) Citrate utilization test (2 Universal bottles containing Simmon- citrate agar slants)

Sample 2

Sample 1

Microorganism

Colour change

Result / Growth

B. subtillis (Sample 1)

Slightly blue

(+) / yes

E. coli (Sample 2)

Remained green

(-) / yes

6.0 DISCUSSION
The objective of this experiment is to distinguish between different types of bacteria
which able to grow on the different sample by five biochemical method and morphology
method. Besides, to understand the biochemical testing and the types of biochemical reactions
which each organism undergoes for its identifications. The two types of bacteria were used in
this experiment which are B. subtillis and E. coli. Firstly, the morphology of the bacteria must
be observed in order to differentiate them. E. coli have circular shape, raised in elevation,
entire margin, smooth surfaces, and shiny. Besides, B. subtillis is the gram positive,
sporeforming rods produce colonies which are dry, flat, and irregular, with lobate margins.
Then, MacConkey agar is the selective media because of it contain bile salts and
crystal violet dye that will inhibit gram positive bacteria and allowed the selection and
isolation of gram-negative bacteria. The bacteria that have ability to ferment lactose will turn
the colour from red to pink because when lactose present in medium, the bacteria will produce
acid and lower the pH. However, the bacteria that cannot utilize lactose will peptone and
forms ammonia and results in higher pH and the formation of colourless colony in the
medium. In this experiment, it were observed that E. coli turn to pinky-red colour which
showed that it was able to ferment lactose while B. subtillis have no change in colour which
showed that it cannot utilize lactose.
Next, starch hydrolysis test was conducted to check the ability of an organism to
produce hydrolytic enzymes to breakdown large substances into smaller one. Besides, the
purpose is to see either the microbe able to use starch as a source of carbon and energy for
growth. Both bacterium were streaked on the agar and were incubated for 24 hours and the
presence of starch was detected by added the iodine reagent. It were observed that B. subtillis
had presence of starch due to formation of light yellow colour of medium around colonies
after addition of iodine over the surface of the medium. However, the E. coli showed negative
results in starch hydrolysis.
Methyl red is the method used to determine either the microbe performs mixed acids
fermentation when supplied glucose. After incubation for 24 hours, five drops of methyl red
solution were added into the culture. Methyl red acted as indicator dye because of its
characteristics that turns red in acidic solution. Theoretically, the observed will showed that
yellow at neutral pH and turn red at pH under 4.0. Mixed acids fermentation results in a red
colour change. It were observed that E. coli showed positive result by change the colour to

reddish while B. subtillis showed negative result. This proved that E. coli can undergo mixed
acid fermentation because able to produce sufficient acidic end products that lower the pH
while B. subtillis cannot undergo the mixed acid fermentation because of produced insufficient
acidic end products.
Voges proskauer is the method to detect organisms that able to utilize the butylene glycol
pathway and produced acetone. After broken down of glucose, it will react with creatine solution
and potassium hydroxide that results turn the colour to red which is a positive result. After
incubated for 24 hours, five drops of creatine solution which act as catalyst and 1 ml of 4%
potassium hydroxide were added into the cultures. Theoretically, E. coli will shows negative
results which remain yellow while B. subtillis will show positive results which change colour
from yellow to pink. However, in this experiment, there were no changes in colour for both
cultures. This is due to some error while preparing the reagents or the loop is too hot when
transfer the colony into the broth that might kills all the microorganism inside.
Lastly, citrate utilization test is to observe either microbe can use the compound citrate as
its sole source of carbon and energy for growth. The experiment used simmon-cirate agar slants
as the medium. The agar contained mineral salts, sodium citrate as the carbon source and
ammonium phosphate as nitrogen source. The formation of ammonia from ammonium phosphate
will raise the pH of the agar and results of alkaline condition that will change the indicator from
green to blue. It will grow on Simmons citrate agar if the microbe can use citrate as carbon and
energy and cause the pH of the medium increased, and a pH indicator known as bromothylmol
blue will change in colour to blue in alkaline condition. After incubated for 24 hours, the colour
changes and growth were observed. In this experiment, it showed that E. coli have negative
results because the agar remained green in colour. This proved that it cannot utilize citrate as a
carbon source. Next, B. subtillis have positive result because it change from green to slightly
blue. This proved that it was able to utilize the citrate as carbon source.
As the conclusion, the results obtained may be slightly different from the theory. This is
due to some error that occurred during the experiment. Firstly, proper aseptic technique did not
apply during the experiment. Second, the wire loop might be too hot when transfer the colony
into the cultures. Thirdly, there was error in prepared dilution. Lastly, the wrong streaking
technique.

7.0 TUTORIAL
QUESTION:
You have isolated an amylase-producing bacterium from the soil sample collected from oil palm
plantation in Alor Gajah, Melaka. Propose a few alternatives to identify the bacterial strain.
ANSWER:
1. Serological testing can differentiate not only among microbial species, but also among
strains within species. Strains with different antigens are called serotypes, serovars or
biovars. It will identify unknown bacterium. Samples unknown bacterium placed in a
drop of saline on each several slides. Different known antiserum added to each sample.
Antiserum is antibodies that identify many organisms. Bacteria will agglutinate (clump)
when mixed with antibodies that were produced in response to that species of bacterium.
2. Phage typing is a test for determining which phage a bacterium is susceptible to.
Bacterophages (phages) are bacterial viruses and that they usually cause lysis of the
bacterial cells they infect. They are highly specialized, in that they usually infect only
members of a particular species, or even particular strains within a species. One bacterial
strain might be susceptible to two different phages, whereas another strain of the same
species might be susceptible to those two phages plus a third phage.
3. DNA fingerprinting is modern biochemical method to determine the entire sequence of
bases in an organisms DNA. This method is currently impractical for laboratory
identification because of the great amount of time required. However, the use of
restriction enzymes enables researchers to compare the base sequences of different
organisms. Restriction enzymes cut a molecule of DNA everywhere a specific base
sequences occurs, producing restriction fragments.

8.0 CONCLUSION AND RECOMMENDATION

As the conclusion, five biochemical method were test in this experiment which are
growth on the selective and differential MacConkey agar, starch hydrolysis test, methyl red,
voges proskauer test and citrate utilization test. The microorganisms that used in this experiment
were B. subtillis and E. coli. In the experiment of growth on the selective and differential
MacConkey agar, E. coli turn to pinky-red means present of fermentation of lactose while B.
subtillis remained in red colour that showed negative results. Next, for the starch hydrolysis test,
both B. subtilis and E.coli have growth. However, B. subtillis showed positive result by light
yellow colour of medium around colonies after addition of iodine while E. coli present negative
result in starch hydrolysis. The experiment of methyl red test, E-coli showed positive result by
turn into reddish colour while B. subtillis remained yellow colour. Then, in the voges proskauer
test, there were no changed in colour in both E coli and B. subtillis. This happened because of the
solvent may be contaminated by the wrong preparation technique. Lastly, in citrate utilization
test on the Simmons slant agar. The B. subtillis showed positive result by turn into slightly blue
colour and had growth on the surface of the agar while E. coli showed negative result by
remained in green colour. Lastly, this experiment need some recommendation for improvement.
Firstly, apply the proper aseptic techniques to avoid the contaminations on sample. Second, when
inoculate the sample with wire loop, always make sure that the loop is not too hot because it
might kill all the microorganisms inside. Lastly, the proper streaking technique should be apply
in order to get the true results of growth of the colony on the medium.

9.0 REFERENCES
1. G. J., B. R., C. L. (2007). MICROBIOLOGY (Ninth ed.). United States: Pearson
2. Identifying and distinguishing bacterial strains using Real Time PCR and Microarrays.
(n.d.). Retrieved March 17, 2016, from
http://www.premierbiosoft.com/tech_notes/bac-id.html
3. Methyl Red (MR) test: Principle, procedure and results - microbeonline. (2014).
Retrieved March 17, 2016, from
http://microbeonline.com/methyl-red-mr-test-principle-procedure-results/
4. Voges-Proskauer Test. (n.d.). Retrieved March 17, 2016, from
http://www.vumicro.com/vumie/help/vumicro/Voges_Proskauer_Test.htm
5. Starch Hydrolysis Test. (n.d.). Retrieved March 17, 2016, from
http://www.vumicro.com/vumie/help/vumicro/Starch_Hydrolysis_Test.htm
6. Janda, J. M., & Abbott, S. L. (n.d.). Bacterial Identification for Publication: When Is
Enough Enough? Retrieved March 17, 2016, from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130684/