Duisburg-Essen University

Master Water Science

Practical Course Environmental
Microbiology
2015- Summer Semester

EXPERIMENT 3

ENRICHMENT AND ISOLATION OF

Bacillus subtilis
Group 17
Hellen Barinas- 3025424
Hellen0430@gmail.com
riskyanti_sutady@yahoo.co.id

Riskyanti Lanyumba-3023859

ABSTRACT

During this experiment the enrichment and isolation of Basillus

subtilis

was

performed.

The

characteristics

of

each

colony

encountered during different days of practice were analyzed. In the

end, we managed to obtain a pure culture of Bacillus.

1 Introduction
Bacterial populations in different crops like potatoes and rice are not
distributed randomly. Factors such as the composition of the soil,
organic matter, pH, water and oxygen availability play an important
role. One of these bacteria are Bacillus subtillis bacteria which are
soil-dwelling often present in the rhizosphere.
This genus of Gram positive bacteria have the advantage of having
several mechanisms to ensure their survival in unfavorable physical
conditions, under these conditions Bacillus spp. begins a series of
responses; if these responses fail to remain in a vegetative state
sporulation (Petersohn et al., 2001) is induced. The ability of Bacillus
species to form highly resistant endospores gives them a significant
competitive advantage in an environment such as soil (C., 1998).

Likewise, it produces endospores which are heat-resistant and also

resists harmful physical factors radiation as drying and disinfecting
acids

chemicals,

produce

extracellular

enzymes

Absorbent

decompose polysaccharides, nucleic acids allowing the organism use

these products as carbon source and electrons, producing antibiotics
such as bacitricina, polymyxin, gramicidin and circulina, fermented
casein and starch, lives within the limits of 55-70 C. Bacillus spp.
must also adapt to sudden changes in temperature, for this feature
thermal shock inducible genes including chaperone proteins and
proteases (Petersohn et al., 2001).
The aim of this study was to create and maintain an enrichment
culture from a soil sample. In addition, a starch decomposing strain of
Bacillus subtillis was isolate from this enrichment culture.

2 Material and procedure

2.1 Enrichment culture
To create an enrichment culture potato pieces about 1 cm 3 were used.
These were placed in an Erlenmeyer with a small amount of water,
enough to cover the pieces of potato. Then, the flask was inoculated
with a small amount of soil. In order to remove all vegetative cells of
this sample it was pasteurized at 100 C for 10 minutes. After that,
the water was decanted and the enrichment culture was incubated at
room temperature for one week under the fume hood.
Then, the enrichment culture was analyzed by hanging-drop method
and Safranin staining.

2.2 Pure culture

In order to have a good selective medium for Bacillus subtillis was
prepared starch-pepton agar which serves as a source of salt and
carbon. Using the `13 steak procedure 2 agar plates were inoculated.
Then, incubated for 2 days at 30 C.

The following two practice the purity of the colonies was analyzed by
microscopic methods used before. Subsequently, two other plates
were inoculated using again the method `13 steak.

3 Result
In the second week the enrichment culture was examined both macro
and microscopically. In the macroscopic analysis it was found that the
colonies grown on the surface of the potatoes were a bit crease,
rough, dry in the center but wet around. The colour in some areas was
white (around) and other coffee (especially in the center).
Microscopic examination allowed to demonstrate the difference
between the colony of cells young and old. In the colony of young
cells they isolated some long and short chains of Bacillus bacteria
were observed. Limit amount of spores were observed in this sample
(figure 1). Furthermore, the old cells evidenced a reduce number of
spores, and the presence of some long chains of Bacillus (Figure 2).

Figure 1 Enrichment culture Bacillus subtilis, younger colony

Figure 2 Enrichment culture Bacillus subtilis, older colony

Safraning staining analysis was done for both colonies. Results are
shown in Figures 3 and 4.

Figure 3 Staining enrichment culture Bacillus subtilis, younger colony

Figure 4 Staining enrichment culture Bacillus subtilis, older colony

In the third and fourth week analysis of the outcome of the

proceedings "13 streak" was held. In the third week a parallel growth
between the first six lines are presented. The growth trend was
decreasing between line 7 and 9. Finally a colony isolated on line 11
was found.
In the fourth week, a decrease was observed in the growth of the
colonies. In this case only prominent growth was presented in lines
2,3, 5 and 6. The isolated colony is present on line 8. (Figures 5 and 6)

Figure 5 `13 Steak Procedure week 3

Figure 6 `13 Steak Procedureweek 4

The macroscopic characteristic of both single colony is show in the

table 1.
Table 1 Macroscopic characteristics of the single colonies.

THIRD WEEK

FOURTH WEEK

Small- medium
Dry

Small- medium
Damp

COLOUR
TRANSPARENCY

white
Matt

white
Matt

SHAPE
MARGIN (EDGE)

Round
Entire

Irregular
undulate

flat

convex

Rough

Mucoid

SIZE
CONSISTENCE

ELEVATION
TEXTURE

Microscopic analysis of each colony obtained in different weeks were

performed. Figures 7 shows the results obtained in the analysis of the
colony of the week 3. It may show that there are numerous bacteria
Bacillus. However, there were not evidence of the long chains that
were observed previously. In this case, only pairs and single were
observed. Additionally, the size of bacteria was significantly reduced
and can not be distinguished easily from the spores. mobility initially
observed in bacteria, is not evidenced at this stage. In the process of
stained bacteria and spores could be observed. However, their size
also decreased compared to the first analysis.

Figure 7 Pure culture week 3

Figure 8 Staining pure culture week 3

Colony obtained from the second inoculation (4 week) by the method

`13 streak was analyzed by the method hangning drop and Safranin
staining, the results can be seen in Figures 9 and 10.

Figure 9 Pure culture week 4

Figure 10 Staining pure culture week 4

Figure 11 Cell with and without spores modified form

4 Discussion
In order to obtain only bacteria of the genus Bacillus subtilis sample
pasteurized at 100 C. due to the resistance of these bacteria at high

temperature conditions it can be said that the majority of organisms

present in the enrichment culture belong to this genre.
The enrichment culture analysis allowed to observe the cells young
and old cells. Young cells showed a slight mobility in the hanging-drop
analysis. This mobility was also observed in the analysis by staining.
Long

chains

were

observed

accompanied

by

limited

spore

development that has shown consistent with the literature (Torrez),

because no sporulation occurs when cells are actively growing. In the
old cells was found not move, and a detailed analysis a small number
of spores were found. This is probably because at this stage the cells
still had nutrients like carbon or nitrogen from the soil. In the later
stages increasing of these was observed.

By the method "13 steak" the successful isolation of a colony in each

of the weeks was achieved. This was due to the use of the culture
medium (peptone-agar Starch) indicated as appropriate inoculation
the plates.

In analyzing the isolation of pure colonies, it can be seen couples and

single Bacillus. By performing a detailed observation can be seen
sporulation in Bacillus. We recommend using a microscope best
resolution for the best analysis of the colony. Using the method of
staining was also observed a large number of Bacillus and spores.
Which it is difficult to observe in the pictures due to camera
resolution.

5 Questions
5.1 WHY CAN A LOT OF ENDOSPORES BE FOUND IN THE
CENTRE OF A COLONY, BUT ONLY A FEW OR NONE AT THE
BORDER?
Spores growth in specific condition for example nutrient limitation,
stagnation of growth due to high cell numbers and therefore due to
quorum sensing are inducing factors for sporulation. The cells in the
center of the colonies are exposed to more unfavorable conditions
that the cells that are around. Likewise, being greater the number of
cells in the center, the amount of nutrients is depleted rapidly, which
quickly activates the process of sporulation. The cells at the border of
the colonies have space to grow and a nutrient rich surrounding.
However, they are able to also generate spores.

5.2 WHICH CONDITIONS LEAD TO A LOSS OF SPORE BUILDING

ABILITY?
Decrease of concentration of metabolites, or dehydration. This is
because release of the mature endospore is caused by auto-lysis of
the mother cells. Then the drop of concentration metabolites can lead
a loss is spore building.

5.3 YOU HAVE THE TASK TO CREATE A STOCK CULTURE OF A

CERTAIN STRAIN WITH KNOWN PROPERTIES OUT OF A SOIL
SAMPLE. DESCRIBE HOW YOU WOULD SOLVE THE
PROBLEM?
If you obtain a certain strain out of a soil sample, you have to apply
selection steps to the sample, regarding to the abilities of your strain.
It is necessary to supply all the necessary nutrients. This is because
all organisms need different nutritional supplements and different
amount, giving priority to the macronutrients. The conditions have to
be chosen appropriate. Conditions to select microorganisms from
each other are for instance pH, aerobic conditions, temperature,
pressure and selective media. After every selection, a subculture
should be obtained and exposed to a different kind of stress.
Combining and repetition of some steps can maximize the effect. The
pure culture in the end should be obtained by the 13 streak method.

6 References
C., S. (1998). Bacterial sporulation: A question of commitment?. . Current
Biology. 8, 45-48.
Ehrlich, L. (1996). Geomicrobiology. New York: Marcel Dekker.
M. Madigan, J. M. (2015). Brock Biology of Microorganism. Pearson Education
Limited.

Laboratory Script, Environmental Microbiology Practical, Summer

semester 2015
Starr, M. S. (1981). The prokaryotes: A Handbook on Habitats, isolation and
identification. Springer-Verlag.
Petersohn A., B. M. (2001). Global Analysis of the General Stress Response
of Bacillus subtilis. Journal of Bacteriology. Journal of Bacteriology,
5617-5631.
Torrez, F. J. (s.f.). Aislamiento y taxonomia de bacterias del gnero Bacillus
recolectadas en suelos de un bosque de Pinus radiata y una pradera
permanente en distintas epocas de muestreo. Valdivia, Chile.

Petersohn A., Brigulla M., Haas S., Hoheisel J., Lker U. & Hecker M. 2001.
Global Analysis of the General Stress Response of Bacillus subtilis. Journal of
Bacteriology. 183: 56175631.
Stephens C. 1998. Bacterial sporulation: A question of commitment?.
Current Biology. 8: 45-48.