Isolation of Environmental Microorganisms

Created: Sep 2021


The aim of this experiment is the preparation of liquid and solid Lysogenic Broth (LB) culture mediums, Wx inorganic salt agar medium containing 3 different types of carbon source compound (glucose, succinic acid, vanillic acid) and saline solution. The prepared mediums and solutions will be used in this and following experiments.

Another objective is to examine the growth of microorganisms from the environmental sample (e.g. soil) on culture mediums of different composition and to isolate a single microorganism colony by transplanting it to a LB medium plate.

Materials and Method

Experiment preparation: Culture Medium and Reagent Preparation

Growth medium:

• Liquid LD culture medium: 6 L-shaped tubes (L-tubes)

• Agar LD culture medium: 20 plates

• Wx inorganic salt agar medium (5mM of 1 type of carbon source compound): 10 plates (20 ml per plate)

Experimental Procedure:

a) LB Culture Medium

Measure 5.0 g of Bacto- tryptone, 2.5g of Bacto-yeast extract and 2.5 g of NaCl into a 500 ml conical flask and dissolve in 500 ml of distilled water using a magnetic stirrer.

i. Liquid LB Culture Medium

Pour the dissolved LB medium into 6 L-shaped tubes (10ml per tube). Insert the plugs into the L-tubes. Cover the test tube rack with 2 layers of foil.

ii. Agar LB Culture Medium

Place 6.6 g of agar into the conical flask with the remaining LB medium (no need to dissolve). Cover the mouth of the flask with 2 layers of aluminium foil, leaving the stir bar inside of the medium.

b) Wx inorganic salt agar medium

Place 3.0 g of agar and 200ml of distilled water into a 300ml conical flask (no need to dissolve). Cover with 2 layers of aluminium foil. Carbon source compound will be distributed after autoclave treatment.

c) Saline solution (100 ml)

Dissolve 0.9 g of NaCl in 100 ml of distilled water, inside of a 100 ml reagent bottle, creating a 0.9% (w/v) saline solution. Cover with 2 layers of aluminium foil.

d) Culture medium and saline solution sterilization

Place the culture medium and saline solution in the autoclave for 15 minutes at 120ºC (high pressure steam sterilization).

e) Treatment after autoclave processing

Keep the culture medium in L-tubes and saline solution at room temperature. Keep stirring the medium in 500 ml conical flask to make it cool down, distributing the Wx inorganic salt agar medium to plates in the meantime.

f) Creating the Wx inorganic salt agar medium containing 5mM carbon source (200 ml)

Place the flask from b) inside of a clean bench, add 20 ml of Solution I, 2 ml of Solution II and 2 ml of 500mM glucose or succinic acid (コハク酸) or vanillic acid (depending on the group). Shake the flask gently. Pour the solution from the flask into 10 plates (about 20 ml per plate) and let it solidify. Distribute the plates between groups, so that every person has 3 plates containing 3 different carbon sources.

g) Creating the agar LD culture medium

Place the 500 ml conical flask inside of the clean bench. Distribute the solution into 20 plates.

Microorganisms Isolation

Equipment and Reagents:

• Environmental sample (soil etc.)

• Agar LB culture medium, 2 plates per person

• Wx inorganic salt agar medium containing 5mM glucose, succinic acid and vanillic acid, 1 plate of each carbon source

• 0.9% saline solution

a) Environmental sample collection

Collect a sample of soil, water or plant into a 15 ml tube so that it occupies about 1/3 of its volume.

b) Sample cultivation

i. Preparation for the application of the sample to the culture medium

Working inside of the clean bench, pour the saline solution into the 15 ml tube so that 2/3 of its volume is occupied. Shake strongly and suspend the sample using a vortex mixer and leave for 1 minute, allowing it to precipitate. In case of a soil sample, pour 1 ml of the supernatant into a 1.5 ml microtube. Place 900 μl of saline and 100 μl of the supernatant solution in another microtube, diluting it 10 times.

ii. Application of the diluted sample

Place 100 μl of the diluted solution in the middle of an LB medium plate. Submerge a cell spreader in ethanol, then put it in a flame to sterilize it. Dissipate the heat on the edge of the LB medium plate, then spread the diluted sample on the plate.

Dilute the sample 10 times again (100 times dilution) and apply and spread 100 μl of the liquid per each of the 3 Wx inorganic salt agar medium plates.

iii. Cultivation

Cover the edges of the plates with parafilm and store them in an incubator at 37ºC for several days.

c) Observation and transplantation of the colonies

i. Observation

Observe and record the microbial growth on each plate. In order to distinguish different species of microorganisms, record characteristics of the colonies such as shape, colour, size, presence/ absence of gloss and contour. Try to determine the plate with the greatest diversity of species and whether there are different species developing on different culture medium plates.

ii. Transplantation

Decide which colony to transplant onto the LB culture medium. Place the inoculation loop in flame to sterilize it. Dissipate the heat on the edge of an LB medium plate. Carefully collect the colony to transplant with the inoculation loop and make streak 1 on the plate as shown in Figure 1. Make streak 2 and 3, sterilizing the inoculation loop each time and starting the streak from the endpoint of the previous streak.

d) Screening Evaluation

After several days, perform the screening of the colony that developed on the plate. Assess whether the isolation of a single colony has been successful.

Streak 2 starts from the end of streak 1, and streak 3 starts from the end of streak 2

Figure 1. Order of streaks during transplantation

Results and Discussion

In order to examine the growth of microorganisms from the environmental sample (e.g. soil) on culture mediums of different composition, a soil sample was collected outside the university building. The microorganisms from the sample were suspended in a solution and diluted, then placed on 4 plates: one LB medium plate and Wx inorganic salt agar medium plates containing glucose, succinic acid and vanillic acid (one plate per each carbon source). The plates were then incubated. After several days, colonies shown in Figure 2 have grown on the plates.

Circular microorganism colonies formed on 4 plates containing different mediums

Figure 2. Microorganism colonies on culture mediums of different constitution

The LB plate had a strong, pungent smell, which was created not only by the microorganisms, but also the medium itself. Glucose plate had a weak, slightly unpleasant smell. Succinic acid plate had a strong, moist smell of fresh soil. Vanillic acid plate smelled similar to the white powder cold medicine commonly used in Japan.

Each plate had a specific set of microorganisms present. The number of different colonies were classified depending on the characteristics. The results of the observations for each medium plate are summarised in the following four tables.

Table 1. Characteristics of the colonies on the LB medium plate

Table 2. Characteristics of the colonies on the Glucose Wx inorganic salt medium plate

Table 3. Characteristics of the colonies on the Succinic acid Wx inorganic salt medium plate

Table 4. Characteristics of the colonies on the Vanillic acid Wx inorganic salt medium plate

As can be seen from the tables and Figure 2, every medium plate has its own specific set of microorganisms, although there is a possibility that there are some species uniform across different mediums.

It is possible that colonies no 1 in glucose and succinic acid plates (Table 2 and 3) are in fact the same species. It may also be the same species as colony no 1 from LB plate (Table 1), although due to the slightly yellow colour of the LB medium it is difficult to determine. The tiny white dots of colony no 2 in glucose, succinic acid and valinnic acid plates can also represent the same species, although the dot size differs slightly. The species present in more than one plate are able to break down and feed on multiple types of compounds, so they are able to survive in a wider range of environments.

Only LB plate had the fuzzy contoured large colony no 4, which covered almost the whole plate in pale white haze. Translucent colony no 2 also appeared only on LD plate. LD culture medium’s composition differs greatly from that of the Wx inorganic salt culture medium, which may explain why LD plate has 2 species absent on other plates.

Moreover, succinic acid plate was the only one to have colony no 3, which resembled thin little threads in a star-like shape. It is likely that that particular species is only able to break down the succinic acid, and possibly other similar dicarboxylic acids, but is unable to process glucose or aromatic compounds such as vanillic acid.

The plates with the largest number of species present are the LB medium plate and Glucose Wx inorganic salt medium plate (4 species present), which means that those 2 plates have the highest diversity. This suggests that more microorganisms in the soil sample collected are able to break down the compounds present in LB and Glucose Wx plates, compared to other medium plates.

However, there are several sources of error in this experiment. It is possible that some of the species were not noticed during the observation of the plates, possibly because of their small size or translucent colour. Moreover, the samples could have been contaminated over the course of the experiment, introducing microorganisms that were not present in the soil sample, which affects the accuracy of the experimental results.

In the next part of the experiment, colony no 1 from LB culture medium plate has been transplanted onto a fresh LB plate and cultivated. The result of the cultivation can be seen in Figure 3.

Although the aim of the experiment was to isolate one species of microorganism (single colony), Figure 3 shows 2 distinct species, with the majority of the microorganisms being probably colony no 4 from Table 1. The colony that was initially chosen for transplantation is visible as a single white spot at the site of the second streak, on the right of the picture.

Since there are 2 species present, the single colony isolation has been unsuccessful. For more satisfactory results, it could have been better to transplant a colony from a more isolated part of the LB plate. Alternatively, the chosen colony from the plate in Figure 3 could be once again transplanted, which could lead to a successful isolation.

Two types of microorganisms instead of one grew on the plate

Figure 3. Single colony transplantation results