How to plate bacteria

OCC Biology
16 Jan 201503:19
EducationalLearning
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TLDRThis instructional video demonstrates the proper technique for spreading a bacterial culture of E. coli on LB agar plates. Key steps include resuspending the E. coli in LB, applying it to the plate with a micropipette, and using a sterile glass spreader. The spreader is sanitized by flaming and cooled before gently distributing the culture without applying pressure to avoid damaging the plate. The process is repeated with careful sterilization between plates to ensure accurate bacterial growth and colony formation.

Takeaways
  • 🧫 Spread bacteria culture evenly on LB plates.
  • 📏 Measure and resuspend E. coli in LB.
  • 🛠️ Use a micro pipetter to apply E. coli to plates.
  • 🚮 Eject pipet tip into pipet tip waste.
  • 🔬 Sterilize the spreader before using it on the plate.
  • 🔥 Use ethanol to sterilize the spreader by catching it on fire.
  • ❄️ Cool down the spreader by touching it to the agar before spreading.
  • 🌡️ Gently spread the bacterial culture around the plate without pressing down.
  • ↔️ Tilt the plate and swipe back and forth to distribute evenly, avoiding pressure.
  • 🔄 Repeat the swiping process after turning the plate a quarter turn.
  • 🚫 Avoid talking during the process to prevent contamination.
  • 🗄️ Store plates agar side up in the incubator.
  • 🔄 Repeat the process for each additional plate.
Q & A

  • What is the first step in spreading bacteria culture on LB plates?

    -The first step is to take the measured quantity of E. coli resuspended in LB and apply it to the plates using a micro pipetter.

  • Why should the plate be closed after applying the E. coli suspension?

    -Closing the plate keeps it sterile, preventing contamination.

  • What should you do with the pipet tip after applying the E. coli suspension?

    -The pipet tip should be ejected into the pipet tip waste container.

  • How should the spreader be prepared before using it on the plate?

    -The spreader should be sterilized by soaking in ethanol and then catching it on fire to further sterilize it.

  • Why do you need to cool down the spreader before spreading the bacterial culture?

    -The spreader is warm after being sterilized by fire, so it needs to be cooled down to avoid killing the bacterial cells.

  • How should you spread the bacterial culture on the plate?

    -Gently spread the bacterial culture around the plate without pressing down, to avoid digging into the plate or breaking the spreader.

  • What should you avoid doing while spreading the bacterial culture?

    -Avoid applying pressure on the spreader and talking, as bacteria from your mouth might land on the plate.

  • How do you distribute the bacterial culture evenly after the initial spreading?

    -Tilt the plate, take the spreader at a 90° angle to the plate, and gently swipe it back and forth from top to bottom. Then, turn the plate a quarter turn and repeat the process.

  • How should the plates be stored after spreading the bacterial culture?

    -The plates should be stored with the agar side up and the cap down. They can be stacked and placed into the incubator.

  • What happens to the E. coli in the incubator?

    -The E. coli will begin to multiply, and each individual E. coli that can survive will form a colony, which is a mound of clone cells.

Outlines
00:00
🔬 Sterilization and Spreading Bacterial Culture

This paragraph outlines the process of spreading a bacterial culture evenly on LB plates. It begins with the preparation of a measured quantity of E. coli resuspended in LB, using a micropipette for application. The importance of maintaining sterility is emphasized, including the disposal of used pipette tips. The paragraph then details the sterilization of a glass spreader through flaming in ethanol, followed by cooling it by touching the augur of the plate. The spreading technique involves gentle motion without pressing down to avoid damaging the plate or spreader. The process of distributing the culture is described, including tilting the plate and using the spreader at a 90° angle to ensure even distribution. The paragraph concludes with advice on avoiding contamination from talking and the correct storage of plates before incubation.

Mindmap
Keywords
💡Bacteria Culture
A bacteria culture refers to a controlled environment where bacteria are grown and maintained, typically in a laboratory setting. In the video, the bacteria culture is a critical component as it is spread on LB plates to study bacterial growth. The script mentions 'your measured quantity of E. coli that you have, resuspended in LB,' which is a process of mixing bacteria with a growth medium known as Luria-Bertani (LB) broth.
💡LB Plates
LB plates are Petri dishes containing Luria-Bertani agar, a medium used for the growth of various types of bacteria. They are essential in microbiology for isolating and growing bacteria. The script describes the process of spreading a bacteria culture on these plates to ensure even distribution for further analysis, as in 'spread your bacteria culture, evenly on your LB plates.'
💡Micro Pipetter
A micro pipetter is a laboratory instrument used to transfer small, measured volumes of liquid. It is crucial for maintaining sterility and accuracy in the process. The script refers to using a micro pipetter to 'apply it to your plates,' indicating its use for transferring the bacteria culture onto the LB plates.
💡Sterile
Sterile refers to the absence of living organisms, such as bacteria, which is critical in microbiology to prevent contamination. The script emphasizes the importance of sterility, as seen in 'the spreader needs to be sterile before it goes on the plate' and the use of ethanol and fire to sterilize the spreader.
💡Spreader
A spreader is a tool used to evenly distribute a sample, like a bacteria culture, across a surface, such as an agar plate. The script describes the sterilization process of the spreader 'soaking in ethanol' and 'catching it on fire' to ensure it is sterile before use.
💡Ethanol
Ethanol is an alcohol used as a solvent and disinfectant. In the script, it is mentioned as a substance in which the spreader is soaked to maintain sterility. The flammability of ethanol is also highlighted when the spreader is set on fire for further sterilization.
💡Auger
An auger is a tool or mechanical device used to move or lift bulk materials, such as the agar in an LB plate. In the script, the auger is used to cool down the sterilized spreader before it touches the bacterial culture, as mentioned in 'I'm going to open the plate and just gently touch to the auger, to cool it down a little bit.'
💡Colony
A colony is a visible mass of microorganisms, usually bacteria, that have grown from a single bacterium on a solid medium. The script explains that each individual E. coli will 'form a mound of clone cells which we call a colony,' indicating the growth outcome of the spread bacteria on the LB plates.
💡Incubator
An incubator is a controlled environment used to grow and maintain biological specimens at a constant temperature and humidity. The script mentions placing the plates into an incubator 'where the E. coli will begin to multiply,' showing the role of the incubator in promoting bacterial growth.
💡Sterility Maintenance
Sterility maintenance refers to the practices and procedures used to keep a microbiological sample free from contamination. The script provides several examples, such as using a micro pipetter and sterilizing the spreader, to illustrate the importance of maintaining sterility throughout the process.
💡Contamination
Contamination in microbiology refers to the unwanted presence of foreign organisms or substances that can affect the purity of a sample. The script warns against talking while spreading the culture to avoid 'bacteria from your mouth might land on your plate,' which would be a form of contamination.
Highlights

Demonstration of spreading bacteria culture evenly on LB plates.

Using a micro pipetter to apply resuspended E. coli to the plates.

Maintaining sterility by closing the pipette tip.

Sterilizing the spreader with ethanol before use.

Safety precautions with flammable ethanol when sterilizing the spreader.

Cooling the spreader by touching the augur to avoid damaging the cells.

Gentle spreading technique to avoid pressing down on the plate.

Distributing the culture by tilting the plate and using a 90° angle swipe.

Avoiding pressure to prevent damage to the plate and spreader.

Minimizing contamination by not talking during the spreading process.

Sequential spreading method by turning the plate and repeating the swipes.

Storing plates with augur side up and cap down for incubation.

E. coli multiplication and formation of mound-like colonies.

Repeating the process for each new plate with sterilization and cooling.

Importance of sterility and technique in bacterial culture spreading.

Final storage and incubation setup for optimal bacterial growth.

Transcripts

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Related Tags
Bacterial CultureLab TechniqueSpreading MethodE. coliLB PlatesSterilizationMicrobiologyLab SafetyDemonstrationAseptic Technique