enrichment and isolation of bacillus subtilis
DESCRIPTION
ENRICHMENT AND ISOLATION OF Bacillus subtilisTRANSCRIPT
EXPERIMENT 3
ENRICHMENT AND ISOLATION OF Bacillus subtilisGroup 17
Hellen Barinas- 3025424 Riskyanti Lanyumba-3023859 [email protected] [email protected]
ABSTRACTDuring 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
Duisburg-Essen UniversityMaster Water Science
Practical Course Environmental Microbiology2015- Summer Semester
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 procedure2.1 Enrichment cultureTo create an enrichment culture potato pieces about 1 cm3 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 cultureIn 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 ºC.
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 Procedure´week 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
SIZE Small- medium Small- mediumCONSISTENCE Dry Damp
COLOUR white whiteTRANSPARENCY Matt Matt
SHAPE Round IrregularMARGIN (EDGE) Entire undulate
ELEVATION flat convex
TEXTURE Rough Mucoid
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.
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Torrez, F. J. (s.f.). Aislamiento y taxonomia de bacterias del género Bacillus recolectadas en suelos de un bosque de Pinus radiata y una pradera permanente en distintas epocas de muestreo. Valdivia, Chile.
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