INTRODUCTION

Selective, differential and enrichment media are often used to isolate and identify
microorganism especially those from infectious diseases.

A selective medium is medium that encourages the growth of some microorganism but
suppress the growth of others. For example, antibiotics sulfadiazine and polymyxin sulfate
(SPS) are added to anaerobic cultures of Clostridium species, bacteria that is suspected of being
agent of food poisoning. A differential medium is medium that has component that causes an
observable change such as colour change, or pH change in the medium when certain
biochemical reaction occurs. These changes allow distinguishing a certain type of colony from
others growing on the same plate.

Many media, such as SPS and MacConkey agar, are both selective and differential. For
example, MacConkey agar contains crystal violet and bile salts, which inhibit growth of Gram-
positive bacteria while allowing growth of Gram-negative. MacConkey agar also contains
sugar lactose and pH indicator that turns colonies of lactose fermenters red and leaves colonies
of non-fermenters colourless and translucent (Black, 2008).

Bacteria are generally not readily identified by using microscopic appearance because
different species may have similar look. Other techniques, which characterized bacteria’s
cellular metabolism must be included to precisely identify a bacterium. Method that utilizes
microorganism’s metabolism is called biochemical tests where the fundamental chemical
characteristic such as nutrient requirement, products given during growth period, enzymes
presence and energy production mechanism can be determined (Talaro & Chess, 2008).

Growth on selective and differential media is based on its growth characteristics on

primary isolation media where the bacteria is subcultured onto specialized media designed to
measure one of many different biochemical reactions. The media used for microbial
identification can be selective, differential or both. For example, eosinmethylene blue (EMB)
is used widely as selective and differential medium for isolation and identification of enteric
bacteria (Talaro & Chess, 2008).

Differential media uses biochemical tests to measure the presence of absence of

enzymes involved in catabolism of a specific substrate or substrates. For example, fermentation
of sugar is measured by using pH indicator dyes that change colour when acidification occurs
(Madigan etc, 2012).
OBJECTIVES

To identify bacteria using biochemical testing

PROCEDURE

(A) Colony morphology on Nutrient Agar

1. The colony morphology of B. substilis and on nutrient agar were described using
below traits as reference.

(B) Growth on selective and differential MacConkey agar

1. B.substilis and E.coli were streak on McConkey agar.
2. The plates were incubated at 37℃ for 24 hours.
3. The growth of both bacteria on the plate were observed.

(C) Starch Hydrolysis Test

1. B. substilis and E.coli were streak on Starch agar.
2. The plated were incubated at 37℃ for 24 hours.
3. Enough iodine was poured over the surface of the starch plate to cover the entire plate.
The plate was rotated and tipped to spread the iodine. Positive result was observed
(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. The colony was transferred into the glucose phosphate broth using the inoculating
wire loop and incubated at 37℃ for 24 hours.
 2. After 24 hours, 2 drops of methyl red solution were added into the culture. The test
tubes were shaken gently, and the final colour change was observed. Red indicates
positive result.

(E) Voges Proskauer (2 test tubes containing 5 ml of phosphate broth + 0.5% (w/v) glucose)
1. Each of the colonies were transferred into the glucose phosphate broth using the
inoculating wire loop and incubated at 37℃ for 24 hours. Colour changes from
yellow to pink indicates positive reaction.
2. After 24 hours, 2 drops of creatine solution and 1 ml of 4% potassium hydroxide
solution were added. The mixture was shaken and examined after 1-4 hours.

(F) Citrate utilization test (2 Universal bottles containing Simmon-citrate agar slants)
1. Each of the colony was streaked onto the slant of Simmon-citrate medium using the
inoculating wire loop and incubated at 37℃ for 24 hours.
2. After 24 hours, the colour changes and the growth of the microorganism were
examined. The utilization of citrate is indicated by the colour change from green to
blue.
RESULT AND DISCUSSION

(A) Colony morphology in Nutrient Agar

Figure 1: B. substilis

(B) Growth on the selective and differential MacConkey Agar

Figure 2: E. coli Figure 3: B. substilis

(C) Starch Hydrolysis Test

Figure 4: E. coli before iodine test

Figure 5: B. substilis before iodine test
 Figure 6: E. coli after iodine test Figure 7: B. substiles after iodine test

(D) Methyl red

Figure 8: B. substilis (left) and E. coli (right)

(E) Voges Proskauer

Figure 9: E. coli (left) and B. substilis

Figure 10: B. substilis (left) and E. coli
(right) before putting creatine and KOH
(right) 1 hour after inserting creatin and
KOH

(G) Citrate Utilization test

Figure 11: E. coli (above) B. substilis (below)

Table 1: Result from Bacteria Identification

Test Sample Observation

Nutrient Agar B. substilis The characteristics of the
colonies are;
 Irregular form
 Convex elevation
 Undulated (wavy)
margin
MacDonkey B. substilis No growth of microorganism
E. coli Microorganism grown
Starch Hydrolysis B. substilis More clear zones around the
microorganism
E. coli No clear zone around the
microorganism
Methyl Red B. substilis The colour changes to
orangish
E. coli The colour changes to
reddish
Voges Proskauer B. substilis Less murky
E. coli Murkier
Citrate utilization B. substilis Slant agar’s colour changes
from green to blue
E. coli No changes from green
colour
 From Table 1, test using Nutrient Agar is to identify the morphology of the B. substilis
cultured. From the experiment, it was found out that the characteristics are irregular form,
convex elevation and margined with undulated shape.

For the second test, using MacConkey agar, both B. substilis and E. coli were streaked
on the same media. The result shows that only E. coli was able to grow on the media while no
growth of B. substilis on the plate. In addition, the colour of the E. coli changes from red to
colourless. This proves that E. coli is a Gram-negative and non-fermenters bacterium. This is
because MacConkey agar is a selective media where it allows only Gram-negative to grow and
it also a differential media where the non-fermenter bacteria changes colour from red to
colourless (Black, 2008). MacConkey agar contains bile salts and crystal violet dye to inhibit
Gram-positive bacteria and neutral red pH dye which turns pink of the bacteria is a fermenting
lactose (MacConkey, 1905). The lactose fermenters will produce acid which lowers the pH of
the agar below 6.8 and results in the pink colonies appearance. Bile salts precipitate in the
immediate neighbourhood of the colony, causing the medium surrounding the colony to
become hazy (MacConkey, 1908).

For the third test, Starch Hydrolysis test, both B. substilis and E. coli were streaked on
starch agar. The result of the experiment showed that plate with B. substilis has clear zones
around the microorganism while area around the E. coli has no clear zone. From the
observation, iodine changes colour from brownish yellow to blackish blue in the presence of
starch. The plate with E. coli turned completely black because all the starch was still present.
While for plate with B. substilis, clear zone around the growth of the microorganism indicates
that the starch has been removed in the area around the bacterial inoculum. This is because B.
substilis produces the exoenzyme, enzyme amylase which hydrolyses starch in the agar. In the
bacterial species produces and releases amylase, starch hydrolysis in the media will occur (Bird
& Hopkins, 1954).

For the fourth test, Methyl Red test, both B. substilis and E. coli were dipped in glucose
phosphate broth. The result from this experiment , as in Figure 8, showed that the colour of
solution contained B. substilis was more orangish while the one with E. coli was more reddish.
From this observation, E. coli has lower pH because in methyl red test, when the colour changes
to red is a positive test in which the pH was lowered to below 4.2. While when the methyl red
is yellow, it is negative test where the pH is more than 6.2 (Kirner & Clarke, 1941). Since the
colour of B. substilis is orangish, the pH value was between 4 and 6. If the pH is lower, it means
that the bacteria produces more acid during growth compared to the other. In this case, E. coli
produced more acid than B. substilis.

The fifth test, Voges Proskauer, both B. substilis and E. coli were dipped into glucose
phosphate broth. After 24 hours of incubation, creatine and potassium hydroxide were added.
As shown in Figure 10, the mixture with E. coli is murkier than that of B. substilis.
Theoretically, positive test is when red colour was developed after 15 minutes indicating the
presence of diacetyl, the oxidation product of acetoin (or acetylmethyl carbinol); product of
glucose fermentation. In this experiment, both B. substilis and E. coli does not change into red
indicating that both do not produce acetoin.

Lastly, in citrate utilization test, both B. substilis and E. coli were streaked onto slant of
Simmon-citrate medium. From Figure 11, B. substilis changes colour from green to blue while
E. coli remained in green colour. B. substilis turns the bromthymol blue indicator in the medium
from green to blue as the pH shift above 7.6 (Tille, 2014). This is because B. substilis
metabolized the citrate in the media provided and broken down the ammonium salts to
ammonia which increased the alkalinity of the medium. As citrate agar is to test the ability of
an organism to utilize citrate as a source of energy, where citrate as the sole source of carbon
source and inorganic ammonium salts as the sole source of nitrogen, in this experiment, B.
substilis was proven as an organism that can do so while on the other hand, E. coli were not
able to utilize citrate as energy source upon growth.

CONCLUSION

As a conclusion, as discussed in the previous section, the objectives of this experiment

were successfully achieved as biochemical metabolism of the bacteria tested were able to be
identified using several different media. Firstly, the morphology of a bacteria can be added into
consideration when identifying bacteria. In addition, from the tests it can be assumed that B.
substilis is a Gram-positive bacterium while E. coli Is a Gram-negative bacterium from
MacConkey Test. Next, B. substilis also produces exoenzyme which hydrolysed the starch
while E. coli does not from Starch Hydrolysis Test. In addition, E. coli produced more acid
than B. substilis as the methyl red colour of that from E. coli in methyl red is red. Other than
that, when tested with Voges Proskauer, B. substilis and E. coli both proven to be none acetoin
producing bacteria. Lastly, B. substilis is a bacterium that can use citrate as a source of energy
while E. coli cannot as shown in Citrate Utilization test.
REFERENCES

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MacConkey, A. T. (1908). Bile Salt Media and their advantages in some Bacteriological
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Bird, R. & Hopkins. R. H. (1954). The action of some alpha-amylases on amylase. Biochem.
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Clarke, H. T. & Kirner, W. R. (1941). "Methyl Red". Organic Syntheses.; Collective Volume,
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