MCB 251 Week 1: Aseptic Technique & Sterilization

• The lab work for MCB 251 / Week 1, covers several topics
• Aseptic Technique
• Pipetting
• Dilution Factors
• Spread Plates
• Streak Plates
• Centrifuges

Aseptic Technique

• Asepsis means to be free of microorgaisms
• Aseptic technique is a set of practices used to promote asepsis
• In microbiology, the main purpose of aseptic technique is to avoid contamination by undesirable organisms

Aseptic Technique: Sterilization

• Asepsis requires sterile tools and materials
• Fire kills things quickly but Heat often degrades enzymes in vegetative organisms.
• Fire is a low energy plasma that causes ionization and combustion of most carbon compounds.
• Liquid chemicals that sterilize include bleach, ethanol, formaldehyde, and hydrogen peroxide.
• Toxic sterilizing gasses include ethylene oxide, ozone, gaseous hydrogen peroxide, and nitrogen dioxide; they are oxidizing agents.
• Autoclaves use high-temperature steam to kills most things, including tough bacterial endospores.
• Bacterial endospores are especially hardy, and can withstand the heat of an autoclave, but not the heat and steam
• Ionizing radiation, such as gamma emissions from Cobalt-60, is used for sterilization. Sterile filtration uses a membrane with small (0.22 µm or less) pores to allow the passage of water and other non-viscous materials but denies passage to bacteria.
• Sterile filtration does not filter out many viruses.
• Some disposable tools, such as plastic serological pipettes, are pre-sterilized.

Aseptic Technique - Dos and Don'ts

• Tools must be sterilized properly before use
• Be careful not to touch the 'business end' of a tool to anything that is not the material/culture/organism to be handled.
• Sterility is not forever
• Don't reuse non-disposable tools without sterilizing them between uses
• Don't reuse disposable tools
• Don't set down sterile tools if they will be used.
• Don't allow tools to sit open to the air for long.

Pipetting

• Micropipettes and Pipettes are essential for moving precise amounts of liquid in benchtop biology
• Both micropipettes and pipettes types are air displacement pipettes.
• Fluid is moved using the displacement of air from the piston, creating a vacuum when the piston moves back from the spring.
• Fluid being pipetted should not come into contact with the pipette components.
• The volume indicated is 'to-deliver' (TD), meaning the amount delivered upon expulsion.

Micropipette Usage

• Soft Stop stops at normal resistance
• Hard stop is with extra pressure

Micropipette Usage - Caveats, Dos & Don'ts

• Move the plunger in a slow, controlled way; snapping it upward obliterates accuracy
• Snapping the plunger upward causes air to be pulled into the tip or fluid to be sprayed up the tip, wetting the barrel
• A wet barrel means the pipette must be cleaned and possibly calibrated if autoclaving is required.
• Keep the pipette at 90°
• Immerse the pipette about 5 mm into the fluid being transferred.
• Draw up and expel material three times before transferring materials.
• This allows evaporation of a small amount of solvent to humidify the gas in the pipette barrel and piston.
• Keep the pipette within 30 degrees of vertical to reduce gas drawn into the pipette and friction on the chamber wall.
• Storing pipettes on their sides is discouraged.

Centrifuges

• Centrifuges separate material by centripetal force and density.
• Solids that accumulate at the bottom are called the pellet, and the fluid at the top is the supernatant..
• Centrifuges can force liquid together, but are not useful for uniformly mixing different solutions, so use pipettes to mix/resuspend

Centrifuges - Dos & Don'ts

• Load tubes with equal volumes and balanced placement symmetrically about the rotor
• Remove internal lid (if present)
• Don't run unbalanced loads

Streak Plates

• To study microorganisms, it is necessary to isolate a pure culture of a particular organism
• A pure culture allows for the study of phenomena caused by a species of microorganism, free of interfering phenomena
• A streak plate permits scientists to separate one particular bacteria from the dizzying number that is found in many places in nature.
• Streaking technique isolates single colonies that allow the creation of a pure culture.
• Each colony is an isogenic, genetically identical descendant of the single cell laid down.
• Streaking involves spreading bacteria out into a thinner layer
• As a technician drags the loop or toothpick,some cells are lost, so single cells are deposited.

Streak Plate - How-to

• Follow this procedure
• Flame the platinum loop in a Bunsen burner and let it cool.
• Touch the loop to a colony/culture, and streak on one side of the plate (1).
• Flame and cool the loop.
• Gently move back and forth across the primary streak, covering 1/3 to 1/2 of the plate (2).
• Flame and cool the loop.
• Begin streaking in one half of the unstreaked portion of the plate, dragging across previous streaks in the previous sector (3).
• Flame the loop.
• Finish streaking the plate without touching the second streaked sector.
• Re-sterilizations and a small number of cells picked up on the loop should result in single colonies in step 4.
• After streaking, place plates lid-side-down on trays at back of the room.

Dilution Factors and Spread plates

• It is necessary to determine what thenumber of cells participate in a process or assay in many experiments
• An infection can be established by cells
• An easy way to count the number of cells in a liquid culture is dispensing a known amount to an agar plate, incubating the plate, and then counting the number of colonies.
• Each colony is the descendant of a single cell
• The number of colonies is the estimated number of cells.
• The number of cells is reported as colony forming units or cfu.
• Millions of cells in the beginning requires diluting the sample, and using smaller volumes.