Isolation Lab Report

Paper details:

Instructions

1. Complete the virtual lab in MH Connect.

2 Write your lab report in essay format. Your report must include the following in 1-2 pages:

Introduction
Hypothesis
Methods
Results
Conclusion
Methods
Phase 1: Mechanism of Quadrant Streak Plating for Isolation
Watch the video to see a quadrant streak plating procedure
Phase 2: Quadrant streaking basics
Select your answer to the first question
Select your answer to the second question
Phase 3: Preparing initial inoculum of quadrant streak plating
Label agar plate with marker
Turn on Bunsen burner
Sterilize loop by placing into flame of Bunsen burner
Pick up bacterial culture tube. Remove cap. Briefly heat mouth of tube in Bunsen burner flame to reduce contamination
Insert loop into tube to acquire bacterial sample
Briefly heat mouth of tube, replace cap, and return tube to rack
Use loop to streak bacterial sample onto one quadrant of plate
Sterilize loop using Bunsen burner
Phase 4: Isolation phases of quadrant streak plating
Select red arrow to turn agar plate to streak next quadrant
Sterilize inoculating loop
Streak bacterial sample from first quadrant onto second quadrant of plate
Turn agar plate
Sterilize inoculating loop
Streak bacterial sample from second quadrant onto third quadrant of plate
Turn agar plate
Sterilize inoculating loop
Streak bacterial sample from third quadrant onto final quadrant of plate
Sterilize loop using Bunsen burner
Select Incubate to incubate plate for 24 hours at 37°C
Observe your quadrant streak plate results for isolated colony formation. Record plate image, results, and interpretation in Lab Data
Phase 5: Apply what you have learned
Select your answer to the first question
Select your answer to the second question
Phase 6: Save Lab Data
Relevant Lab Data is available to be saved for personal reference. Data will be available if you return to this laboratory simulation



Key Concepts
The study of bacteria requires an ability to safely grow and work with them in a laboratory environment.
When sampling from environmental conditions, patient samples, or sites of growth, there are varying levels and types of microbes present.
Isolation techniques allow you to select out a specific bacterial organism from a source containing complex mixtures or combinations of different microorganisms.
Bacterial isolation requires the use of special laboratory techniques to obtain separated bacterial colonies, each derived from one bacterial cell (known as a colony forming unit).
Quadrant streak plate isolation techniques isolate one bacterial strain from a mixture of bacteria and are helpful in determining whether a bacterial culture may be pure.
The quadrant streak plate technique is a common microbiology isolation procedure for bacteria in culture, but does have two noted drawbacks. First, it can be difficult to master the technique, requiring practice to achieve consistent and acceptable results. Secondly, you cannot accurately count bacteria from a quadrant streak plate.
The purpose and methods of microbial isolation are important to the bacterial identification process and help us understand bacterial growth patterns.
Because the goals of the isolation simulations are to examine live organisms of interest, aseptic technique should be followed as closely as possible to prevent the introduction of unwanted or harmful organisms to a user or into an environment, culture, or agar sample.
Overview
The goal of this simulation is to take a sample of bacteria from a broth culture and streak the surface of a sterile agar plate using the quadrant streak method for isolation.
The quadrant streak plate technique can be described as having four key steps:
Use aseptic technique to transfer a bacterial sample to an agar plate.
Streak the bacterial sample across one quadrant (approximately ¼ of the agar surface) in a back-and-forth motion.
Re-sterilize the loop and then drag the loop through the previously inoculated quadrant to pick up a small fraction of sample to the next quadrant surface. Repeat until four quadrants are completed in the same manner.
Incubate the agar plate for 24 hours to grow isolated bacterial colonies.
A successful streak plate provides isolated colonies on the top of the agar by drag-diluting the colony forming units (CFUs) out across 4 sections of the plate.
During the steps of the quadrant streak plate method, the bacterial culture is only added to the plate one time. Each subsequent quadrant is a dilution of the initial inoculum and shows less bacterial growth than the previous quadrant area. Typically, the last dilution quadrant shows the isolated colonies.
Note the pattern used to create a quadrant streak across the agar surface. Pay special attention in each image to the ‘Start’ position. Make sure that during your quadrant streak, you only drag into the edge of the previous section with only 1-2 passages. This will prevent you from tracking too many organisms at higher concentrations into the next dilution of the quadrants.
Quadrant streak patterns
This is an example of a correctly performed quadrant streak plate. Notice the single, isolated colonies in the last quadrant.
Quadrant streak plate
Before you begin
In this simulation, you will be provided with a liquid bacterial culture sample, an agar plate for the quadrant streak isolation, and the necessary materials commonly used to transfer bacteria.
Agar plates are made by pouring hot, sterile agar into an empty, sterile petri dish and allowing it to cool. Often, condensation will form on the lid. Condensation could fall down onto the agar surface causing contamination or dispersion of any bacterial colonies. To prevent this, plates are always incubated upside down.
Plates should always be labeled when inoculated, ensuring proper tracking of samples in case of accidental exposures during experiments. Agar plates should be labeled on the bottom (agar-containing side) in case the lid becomes separated from the actual agar culture.
In many isolation labs, you will be working to transfer bacteria between multiple sets of equipment simultaneously (e.g., plates that are sterile and tubes that have been previously inoculated with bacteria). Many times, you may even be holding and working with several tubes or plates in the same hand! Make sure to keep focus as you transfer organisms between locations and be observant and systematic in your approach. Correct labeling and layout before starting experiments can be a huge help!
When performing a quadrant streak plate, your plate can rest on the surface of the lab bench as you streak while lifting the lid (known as the “clamshell” method) or you can invert the lid onto the benchtop and pick up the agar side of the plate as you work.
Clamshell method
Most bacteria grow well between 20–40°C and are commonly incubated at 37°C (human body temperature).
Make sure you are familiar with the following terms:
+ Agar media
+ Agar plate
+ Agar slant
+ Aseptic technique
+ Colony
+ Colony Forming Units (CFUs)
+ Culture
+ Heavy (or dominant) growth
+ Incubate
+ Inoculating loop

+ Inoculum
+ Intermediate (or moderate) growth
+ Isolation
+ Liquid broth culture
+ Petri dish
+ Pure culture
+ Quadrant
+ Scant (or scanty) growth
+ Sterile
+ Streak (or streaking)
+ Vegetative cell
Remember that while this is a safe simulation, you should practice good laboratory hygiene protocols when translating this learning to the benchtop. This includes following aseptic technique procedures, proper handwashing, and cleaning of benchtop surfaces as you complete similar exercises in the lab setting.

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