Lab 1: Aseptic technique
1. Describe aseptic technique
Aseptic Technique is a method provided to prevent any contamination from anything on the surface.
2. Describe the steps to create and maintain a sterile work area
- Decontaminating the work surface with ethanol
- Light the Bunsen burner and adjust it to produce a roaring blue flame
- Place only necessary materials in the sterile work area and arrange them so the updraft that is creating the sterile field is not disturbed
- Minimize movements that can disturb the sterile field around the Bunsen burner
- Hold any lids and closures in the hand in the sterile field rather than placing them on the work surface
- Sterilize any equipment by holding it in the flame
- Use either a new disposable item (such as pipette tips) for each action or sterilize reusable equipment (such as glass spreaders) before and
after each action
3. Describe the purpose of Bunsen burners in a microbiology lab.
It creates a radial sterile field around it. Items can also be sterilized by passing them through the flame. This allows for the use of reusable equipment.
4. Be able to differentiate between slants, broth, and plate cultures.
Slant: Solid medium in a slanted test tube Broth: Liquid medium in a test tube. Plate Culture (Petri Dish): Solid medium in a flat petri dish
5. Describe how to set up liquid and solid cultures.
To create a liquid culture, a colony from an agar plate is added to a liquid medium using a culture loop, or a small volume of liquid is added using a
pipette. To create a solid culture, a colony is streaked across a petri dish containing a solid medium( aka agar plate) using a culture loop, or a small
volume of liquid culture is added to the plate and spread across the surface using a spreader. All equipment must be sterilized before and after each use.
6. Describe what is a control and its purpose.
To ensure a good aseptic technique is used, the best negative control is an uncultured medium. Since the medium and equipment should be sterile,
microbial growth in this control indicates contamination.
Lab 2: Biosafety
1. Describe what is laboratory containment.
Used to describe methods, practices, facilities, and equipment used to safely manage hazardous materials.
2. List the four types of biosafety levels and describe the characteristics of each.
Biosafety 1: basic teaching; research with good microbiological practice. No safety equipment required for use.
Biosafety 2: primary health and diagnostic service; research. Use good microbiological practice plus protective clothing with biosafety cabinets.
Biosafety 3: special diagnostic service; research. As level 2 but special clothing, controlled access, directional airflow and level 1 biosafety cabinet.
Biosafety level 4: Dangerous pathogen units as level 3 plus airlock entry, shower exit, special waste disposal, class 3 biosafety cabinet.
3. Explain how a Biosafety containment level III laboratory is constructed.
In a separate building or isolated in restricted areas with a dedicated supply and exhaust air with vacuum lines and decontamination systems.
4. What is the purpose of negative/positive pressure and how are they used?
Negative pressure flow is an isolation technique used to prevent air from flowing outside. The room will have a lower pressure than outside the room
because air flows from high to low pressure.
5. Explain how a BSL 3 culture is set up.
Same way as a normal lab however, more care is needed. No flip top tubes are used and the little finger technique is applied to open screw top lips
safely when pipetting.
Lab 3: Bacterial Isolation
1. Describe the importance of a single colony
The importance of a single colony is that they offer the opportunity to establish a pure culture, a generally identical bacterial population.
2. Describe how streak plate is used to isolate bacterial cultures.
1) Spreading a large # of cells over an agar plate in sequented manner until they are well separated.
2) Each streak is numbered/colored to indicate the order of application
3) After streaking, the plate is incubated, and cells start dividing, forming colonies.
3. Describe the steps involved in the streak plate technique
Same as above
4. Describe the importance of flaming the loop between quadrants in the streak plate technique.
To prevent cross-contamination
5. Describe the steps used to keep laboratory work sterile.
1) Lab doors/windows are kept closed to prevent air currents and surface microorganisms.
2) Wire loop/glass spreaders are sterilized before and after each use.
3) Lids are held when removed, not placed on bench.
4) Neck of bottle must be immediately heated using bunsen burner so that any air movement is outward
5) Bottles are opened for the least amount of time as possible, work performed close to bunsen burner.
6) Media and equipment are sterilized to prevent growth of unwated microorganisms.
6. Be able to identify between a good streak (isolation successful) and a bad streak plate (no isolation obtained)
If no colonies are present, the plate was contaminated. Colonies must be identifiable for a good streak.
EX 4: Bacterial Cell Structures
1. Be able to identify the shape and arrangement of cells (Shape: bacillus: rod shaped, coccus: sphere shaped,spirillum: spiral shaped;
Arrangement: Single 1, Diplo 2, Strepto filament, Staphylo cluster)
Cell Shapes: Cell Arrangements: Prokaryotic Eukaryotic:
Bacillus: Rod-shaped Single: One cell no nucleus nucleus
Coccus: Spherical Diplo: Pairs no membrane-bound membrane-bound
Spirillum: Spiral Strepto: Chains
Staphylo: Cluster
Cell Membrane: selectively permeable, keep things in the cell stable- homeostasis
Cytoplasm: surrounds all internal cell structures.
Cytoskeleton: a collection of fibers that will support the cell and its organelles and can play a role in cell movement.
Ribosomes: not membrane-bound, make protein. They can be free in the cytoplasm.
In Eukaryotes you can find these:
Nucleus: ‘cell headquarters’ holds genetic material such as DNA.
Nucleolus: where ribosomes can be produced
Endoplasmic Reticulum (ER): processing of molecules like protein folding and transporting the molecules. Smooth ER: detoxification, make some types of
lipids.
1. Describe aseptic technique
Aseptic Technique is a method provided to prevent any contamination from anything on the surface.
2. Describe the steps to create and maintain a sterile work area
- Decontaminating the work surface with ethanol
- Light the Bunsen burner and adjust it to produce a roaring blue flame
- Place only necessary materials in the sterile work area and arrange them so the updraft that is creating the sterile field is not disturbed
- Minimize movements that can disturb the sterile field around the Bunsen burner
- Hold any lids and closures in the hand in the sterile field rather than placing them on the work surface
- Sterilize any equipment by holding it in the flame
- Use either a new disposable item (such as pipette tips) for each action or sterilize reusable equipment (such as glass spreaders) before and
after each action
3. Describe the purpose of Bunsen burners in a microbiology lab.
It creates a radial sterile field around it. Items can also be sterilized by passing them through the flame. This allows for the use of reusable equipment.
4. Be able to differentiate between slants, broth, and plate cultures.
Slant: Solid medium in a slanted test tube Broth: Liquid medium in a test tube. Plate Culture (Petri Dish): Solid medium in a flat petri dish
5. Describe how to set up liquid and solid cultures.
To create a liquid culture, a colony from an agar plate is added to a liquid medium using a culture loop, or a small volume of liquid is added using a
pipette. To create a solid culture, a colony is streaked across a petri dish containing a solid medium( aka agar plate) using a culture loop, or a small
volume of liquid culture is added to the plate and spread across the surface using a spreader. All equipment must be sterilized before and after each use.
6. Describe what is a control and its purpose.
To ensure a good aseptic technique is used, the best negative control is an uncultured medium. Since the medium and equipment should be sterile,
microbial growth in this control indicates contamination.
Lab 2: Biosafety
1. Describe what is laboratory containment.
Used to describe methods, practices, facilities, and equipment used to safely manage hazardous materials.
2. List the four types of biosafety levels and describe the characteristics of each.
Biosafety 1: basic teaching; research with good microbiological practice. No safety equipment required for use.
Biosafety 2: primary health and diagnostic service; research. Use good microbiological practice plus protective clothing with biosafety cabinets.
Biosafety 3: special diagnostic service; research. As level 2 but special clothing, controlled access, directional airflow and level 1 biosafety cabinet.
Biosafety level 4: Dangerous pathogen units as level 3 plus airlock entry, shower exit, special waste disposal, class 3 biosafety cabinet.
3. Explain how a Biosafety containment level III laboratory is constructed.
In a separate building or isolated in restricted areas with a dedicated supply and exhaust air with vacuum lines and decontamination systems.
4. What is the purpose of negative/positive pressure and how are they used?
Negative pressure flow is an isolation technique used to prevent air from flowing outside. The room will have a lower pressure than outside the room
because air flows from high to low pressure.
5. Explain how a BSL 3 culture is set up.
Same way as a normal lab however, more care is needed. No flip top tubes are used and the little finger technique is applied to open screw top lips
safely when pipetting.
Lab 3: Bacterial Isolation
1. Describe the importance of a single colony
The importance of a single colony is that they offer the opportunity to establish a pure culture, a generally identical bacterial population.
2. Describe how streak plate is used to isolate bacterial cultures.
1) Spreading a large # of cells over an agar plate in sequented manner until they are well separated.
2) Each streak is numbered/colored to indicate the order of application
3) After streaking, the plate is incubated, and cells start dividing, forming colonies.
3. Describe the steps involved in the streak plate technique
Same as above
4. Describe the importance of flaming the loop between quadrants in the streak plate technique.
To prevent cross-contamination
5. Describe the steps used to keep laboratory work sterile.
1) Lab doors/windows are kept closed to prevent air currents and surface microorganisms.
2) Wire loop/glass spreaders are sterilized before and after each use.
3) Lids are held when removed, not placed on bench.
4) Neck of bottle must be immediately heated using bunsen burner so that any air movement is outward
5) Bottles are opened for the least amount of time as possible, work performed close to bunsen burner.
6) Media and equipment are sterilized to prevent growth of unwated microorganisms.
6. Be able to identify between a good streak (isolation successful) and a bad streak plate (no isolation obtained)
If no colonies are present, the plate was contaminated. Colonies must be identifiable for a good streak.
EX 4: Bacterial Cell Structures
1. Be able to identify the shape and arrangement of cells (Shape: bacillus: rod shaped, coccus: sphere shaped,spirillum: spiral shaped;
Arrangement: Single 1, Diplo 2, Strepto filament, Staphylo cluster)
Cell Shapes: Cell Arrangements: Prokaryotic Eukaryotic:
Bacillus: Rod-shaped Single: One cell no nucleus nucleus
Coccus: Spherical Diplo: Pairs no membrane-bound membrane-bound
Spirillum: Spiral Strepto: Chains
Staphylo: Cluster
Cell Membrane: selectively permeable, keep things in the cell stable- homeostasis
Cytoplasm: surrounds all internal cell structures.
Cytoskeleton: a collection of fibers that will support the cell and its organelles and can play a role in cell movement.
Ribosomes: not membrane-bound, make protein. They can be free in the cytoplasm.
In Eukaryotes you can find these:
Nucleus: ‘cell headquarters’ holds genetic material such as DNA.
Nucleolus: where ribosomes can be produced
Endoplasmic Reticulum (ER): processing of molecules like protein folding and transporting the molecules. Smooth ER: detoxification, make some types of
lipids.
