PRAK. KE-1B. ISOLASI RHIZOBIUM - BIOTEK PERTANIAN (Lab. Biotan Faperta Unpad)

Summary

In this practicum, the process of isolating rhizobium from root nodules of soybean plants is demonstrated. The procedure begins with preparing selective media, followed by sterilizing root nodules with HgCl2 and alcohol. After the sterilization, the nodules are crushed to extract liquid, which is then used for rhizobium isolation. The isolation involves streaking the liquid onto agar plates in a zigzag pattern and incubating the cultures at 30°C. The colonies that grow are further purified through repeated streaking to obtain pure rhizobium isolates. The entire process is explained in detail to ensure successful isolation for further study.

Takeaways

• 😀 Prepare selective media for rhizobium by heating yema media, pouring into a sterile Petri dish, and allowing it to cool.
• 😀 Use root nodules from legume plants, specifically soybean plants, for rhizobium isolation.
• 😀 Sterilize the root nodules by first washing them with running water and then using 0.1% HgCl2 for 1 minute.
• 😀 After sterilizing with HgCl2, soak the nodules in 70% alcohol for 1 minute for a second sterilization.
• 😀 If any alcohol or HgCl2 residue remains, soak the nodules in sterile distilled water for 3 minutes.
• 😀 Split the sterilized root nodules using tweezers and crush the agar ball to release the liquid needed for rhizobium isolation.
• 😀 Use a sterile Ose needle to streak the liquid in a zigzag pattern across the media in quadrant 1, then move to quadrant 2 and 3 as instructed.
• 😀 After incubation, select separate colonies for further purification using the same zigzag streaking method.
• 😀 Sterilize the Ose needle between each streaking to prevent contamination.
• 😀 Label the plates and seal them with plastic wrap before incubating at 30°C for 3-5 days to isolate pure rhizobium colonies.

Q & A

What is the first step in isolating rhizobium from root nodules?

The first step is to prepare selective media for rhizobium, such as heating the yema media and pouring it into a sterile Petri dish, then waiting for it to freeze and cool.

What type of plants are used to collect root nodules for this practicum?

Legume plants, specifically soybean plants, are used to collect root nodules for this practicum.

How are the root nodules sterilized before use?

The root nodules are sterilized by washing them with running water, then soaking them in 0.1% HgCl2 for 1 minute, followed by soaking them in 70% alcohol for 1 minute. Any remaining alcohol or HgCl2 is removed by soaking the nodules in sterile distilled water for 3 minutes.

What should you do if there is remaining alcohol or HgCl2 on the root nodules?

You should soak the root nodules in sterile distilled water for 3 minutes to remove any remaining alcohol or HgCl2.

How are the root nodules prepared for rhizobium isolation?

The root nodules are split using tweezers in a sterile Petri dish, and the agar ball is crushed to release the liquid, which will be used for rhizobium isolation.

What is the method for inoculating the media with rhizobium?

The liquid from the crushed root nodules is taken on an ose, and a zigzag streak is made in quadrant 1. The streak continues into quadrant 2, and then quadrant 3. The ose is sterilized between each streaking.

What is the purpose of sterilizing the ose needle each time it is used?

Sterilizing the ose needle between streaking helps to prevent contamination and ensures that only isolated rhizobium colonies grow on the media.

What happens after the streaking process is completed?

After streaking, the Petri dish is labeled and wrapped with plastic wrap, then incubated at 30°C. This allows rhizobium colonies to grow.

How long should the Petri dish with rhizobium isolates be incubated?

The Petri dish should be incubated for 3-5 days at a temperature of 30°C.

What are the visible results after incubation?

After incubation, colonies will be visible on the media, separated from each other. The largest colonies can be identified, which are the isolated rhizobium.