[생물공학실험]Inoculation and Culture for Microbes (Streaking and Spreading)

Inoculation and Culture for Microbes

Inoculation and culture for Microbes for streaking and spreading is the basest technology when experimenting with microbes. Microorganisms always exist as different kinds of mixtures in nature. To pick out and study just one type of microorganism in microbiology, therefore, one type of microorganism has to be sorted by using a streaking method. The stacking method is the most basic experiment in microbiology experiments. This is also a very common technique to use after gene recombination to pick up a single colon in mixed microbes. When you need to know the concentration of microorganisms in a certain liquid, a spreading method is a very good method. The spreading method and streaking are very useful method in biology experiments, and we’re going to cover them in this experiment.

 

미생물은 자연에서 항상 다른 종류의 혼합체로 존재합니다. 그래서 미생물학에서 단 한 종류의 미생물을 골라내어 연구하기 위해, 한 종류의 미생물이 선택되어야 합니다. 이러한 이유로, streaking 방법은 미생물학 실험에서 가장 기초가 되는 실험입니다. 이 방법은 유전자 재조합 이후에 단일 콜론을 픽업 하기 위해 사용하는 방법으로, 매우 흔한 기술입니다. 또한, 어떤 액체에 있는 미생물의 농도를 알아야 할 때, 확산 방법은 매우 좋은 방법입니다. spreading 방법 또한 생물학 실험에서 매우 유용한 방법이며, 이번 실험에서 다룹니다.

 

 

 

Experiment reagents and equipment

Escherichia coli BL21 (E. coli), sterile Distilled water

Incubator, Petri dish containing LB media (previous experiment), Micro-pipette, Pipette tip, Micro-centrifuge tube, Inoculating loop, Spreading rod, Alcohol lamp 

 

https://kielian-story.tistory.com/74

[생물공학 실험] E. Coli Culture, LB Media Preparation And Sterilization

 

 

 

Streaking Method

1. Sterilize an inoculating loop by placing it at an angle over a flame. Make sure that the entire loop turns orange. (Before the experiment, Culture media (LB) preparation and sterilization; https://kielian0116.com/?p=307)
2. Cool the inoculating loop by stabbing it at the agar.
3. Pick bacteria and streak it at the top end of the agar plate moving in a zig-zag horizontal pattern until 1/3 of the plate is covered.
4. Sterilize the loop again in the flame and cool it using the procedure in steps a and b. 
   e. Spread the bacteria from the first streak into a second area using the same motion in step c. Repeat it 2 times more.
5. Put the plates UPSIDE-DOWN in the incubator overnight at 37 ℃.

 

Spreading Method

1. Put the appropriate amount of bacterial suspension on the center of the plate using a sterile pipette. 
2. Dip a glass spreading rod into a 70% alcohol solution and then pass it quickly through a flame.
3. When all the alcohol has burned off and the rod has air-cooled, cool it by stabbing it at the agar.
4. Touch the rod to the cell suspension and drag it back and forth across the agar. Rotate the plate several times and continue to spread until you feel the agar is rough and dry.
5. Put the plates UPSIDE-DOWN in the incubator overnight at 37 ℃.

 

 

Streaking method result

  A picture is the result of streaking. This experiment was successful because you can see some single colons in the 3rd and 4th stretching. The single colon’s microbes have the same genes. If it’s worth researching, you can use microorganisms in a single colon for research.

 

사진은 줄무늬의 결과입니다. 이 실험은 성공적이었습니다. 3번째 streaking 과 4번째 streaking에서 몇몇 단일 콜론을 확인할 수 있습니다. 이렇게 생성된 단일 콜론은 동일한 유전체를 갖은 미생물들 입니다. 만일 연구의 가치가 있다면, 단일 콜론에서 미생물을 inoculation하여 연구에 사용할 수 있습니다.

 

Spreading method result

 The results were that a spreading experiment was performed from one unknown concentration of E. coli to dilute from 1/1000(10^-3) to 1/100000000(10^-8) with Distilled water. The result of 10^-6 dilution had a problem from caking one spot. Therefore, the number of clusters at this point could only be guessed. The original average concentration (expect ∞ concentrations) is (9.32 * 10^7 + 2.65 * 10^8 + 1.7*10^8 + 2.3*10^9)/4 (CFU/ml) = 7.07*10^8 (CFU/ml) but, 10^-8’s result should be lower than 10^-7’s result, so 2.3 *10^10 is too high number. Therefore, I think the results calculated during the experiment may have been a slight error.

“9.32 * 10^7 + 2.65 * 10^8 + 1.7*10^8 / 3 (CFU/ml) = 1.76 * 10^8 (CFU/ml) might be more reliably estimated at the original concentration.“

 

미지의 대장균 1농도에서 증류수로 1/1000(10^-3)에서 1/1000000(10^-8)까지 희석하여 확산 실험을 수행한 결과 사진 입니다. 10^-6 희석 결과에서 한 군데에서 문제가 발생했습니다. 따라서 이 지점 군집 수는 추측할 수 밖에 없었습니다. 원래 평균 농도는 (9.32 * 10^7 + 2.65 * 10^8 + 1.7*10^8 + 2.3*10^9)/4 (CFU/ml) = 7.07*10^8 (CFU/ml)이지만, 10-8의 결과는 10^-7의 결과보다 낮아야 하므로 2.3^10의 결과 값이 너무 높습니다. 따라서 실험 중 계산된 결과는 약간의 오차가 있었을 것으로 생각합니다.

 

9.32 * 10^7 + 2.65 * 10^8 + 1.7 * 10^8 / 3 (CFU/ml) = 1.76 * 10^8 (CFU/ml)은 원래 농도로 더 확실하게 추측될 것입니다.

 

 

 

(학사때 써논 건데 혹시나 필요하신 분들을 위한 정보입니다. 학사때 쓴거라 좀 허접함 주의)