EXPERIMENT 3

ENRICHMENT AND ISOLATION OF Bacillus subtilisGroup 17

Hellen Barinas- 3025424 Riskyanti Lanyumba-3023859 Hellen0430@gmail.com riskyanti_sutady@yahoo.co.id

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.

6 ReferencesC., 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 género 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: 5617–5631.

Stephens C. 1998. Bacterial sporulation: A question of commitment?. Current Biology. 8: 45-48.