cell & molecular biology

BSBO102

Experiment 4

Aim of the Experiment

Sterility practices in cell and tissue culture.

Introduction:

Asectic technique

This protocol describes basic procedures for aseptic technique for the novice in cell culture technology. One basic concern for successful aseptic technique is personal hygiene. The human skin harbours a naturally occurring and vigorous population of bacterial and fungal inhabitants that shed microscopically and ubiquitously. Most unfortunately for cell culture work, cell culture media and incubation conditions provide ideal growth environments for these potential microbial contaminants. This procedure outlines steps to prevent introduction of human skin flora during aseptic culture manipulations. Every item that comes into contact with a culture must be sterile. This includes direct contact (e.g., a pipet used to transfer cells) as well as indirect contact (e.g., flasks or containers used to temporarily hold a sterile reagent prior to aliquoting the solution into sterile media). Single-use, sterile disposable plastic items such as test tubes, culture flasks, filters, and pipets are widely available and reliable alternatives to the laborious cleaning and sterilization methods needed for recycling equivalent glass items. However, make certain that sterility of plastic items distributed in multiunit packages is not compromised by inadequate storage conditions once the package has been opened.

Ideally, all aseptic work should be conducted in a laminar cabinet. However, work space preparation is essentially the same for working at the bench. Flame sterilization is used as a direct, localized means of decontamination in aseptic work at the open bench. It is most often used (1) to eliminate potential contaminants from the exposed openings of media bottles, culture flasks, or test tubes during transfers, (2) to sterilize small instruments such as forceps, or (3) to sterilize wire inoculating loops and needles before and after transfers. Where possible, flame sterilization should be minimized in laminar-flow environments as the turbulence generated by the flame can significantly disturb the sterile air stream.

Materials:

Antibacterial soap
70% ethanol or other appropriate disinfectant
95% ethanol
Clean, cuffed laboratory coats or gowns
Latex surgical gloves
Clean, quiet work area
Shallow discard pans containing disinfectant
Bunsen burner or pilot-activated burner.

Take personal precautions

1. Just prior to aseptic manipulations, tie long hair back behind head. Vigorously scrub hands and arms at least 2 min with an antibacterial soap. Superficial lathering is more prone to loosening than removing flaking skin and microbial contaminants. Loosely adhering skin flora easily dislodge and can potentially fall into sterile containers.

2. Gown appropriately. For non-hazardous sterile-fill applications, wear clean, cuffed laboratory coats and latex gloves. Greater stringencies may be necessary depending upon laboratory regulatory requirements. Work with potentially hazardous agents certainly mandates additional considerations for safety. Front-closing laboratory coats are not recommended for work with hazardous biological agents. Safety glasses should be worn by laboratory personnel when manipulating biological agents outside the confines of a biosafety cabinet.

3. Frequently disinfect gloved hands with 70% ethanol while doing aseptic work. Although the gloves may initially have been sterile when first worn, they will no doubt have contacted many nonsterile items while in use. Note that 70% ethanol may not be an appropriate agent for latex glove disinfection when working with cultures containing animal viruses, as studies have shown that ethanol increases latex permeability, reducing protection for the wearer in the event of exposure. In this case, quaternary ammonium compounds are more appropriate.

4. Dispose of gloves by autoclaving after use. Do not reuse. Bag and autoclave single-use laboratory coats after use. Bag, autoclave (if necessary), and wash other laboratory coats within the laboratory facility or send out for cleaning at a laundry certified for handling biologically contaminated linens. Never take laboratory clothing home for washing.

5. Thoroughly wash hands after removing protective gloves.

Prepare and maintain the work area

6. Perform all aseptic work in a clean work space, free from contaminating air currents and drafts. For optimal environmental control, work in a laminar-flow cabinet.

7. Clear the work space of all items extraneous to the aseptic operation being performed.

8. Wipe down the work surface before and after use with 70% ethanol or other appropriate disinfectant.

9. Wherever feasible, wipe down items with disinfectant as they are introduced into the clean work space. Arrange necessary items in the work space in a logical pattern from clean to dirty to avoid passing contaminated material (e.g., a pipet used to transfer cultures) over clean items (e.g., flasks of sterile media).

10. Immediately dispose of any small contaminated items into a discard pan.

11. When the aseptic task has been completed, promptly remove any larger contaminated items or other material meant for disposal (e.g., old culture material, spent media, waste containers) from the work space and place in designated bags or pans for autoclaving. Disinfect the work space as in step 8.

Flame sterilize the opening of a vessel

12. For a right-handed person, hold the vessel in the left hand at !45° angle (or as much as possible without spilling contents) and gently remove its closure. Do not permit any part of the closure that directly comes in contact with the contents of the vessel to touch any contaminating object (e.g., hands or work bench). Ideally, and with practice, one should be able to hold the closure in the crook of the little finger of the right hand while still being able to manipulate an inoculating loop or pipettor with the other fingers of the hand. Holding the vessel off the vertical while opening will prevent any airborne particulates from entering the container.

13. Slowly pass the opening of the vessel over the top of (rather than through) a Bunsen burner flame to burn off any contaminating matter. Be careful when flaming containers of infectious material. Any liquid lodged in the threads of a screw cap container will spatter as it is heated. Aerosols thus formed may actually disseminate entrapped biological agents before the heat of the flame is hot enough to inactivate them.

14. While still holding the vessel at a slant, use a sterile pipet and pipettor to slowly add or remove aliquots to avoid aerosol formation.

15. Flame-sterilize again as in step 13, allow the container to cool slightly, and carefully recap the vessel.

Flame sterilize small hand instruments

16. Dip critical areas of the instrument (i.e., those that come into contact with the material of concern) in 95% ethanol. Make certain that the alcohol is in a container heavy enough to support the instrument without tipping over.

CAUTION: 95% ethanol is flammable; keep the container at a safe distance from any open flame.

17. Remove the instrument from the alcohol, being careful not to touch the disinfected parts of the instrument. Allow excess ethanol to drain off into the container.

18. Pass the alcohol-treated part of the instrument through the flame of a Bunsen burner and allow residual alcohol to burn off.

19. Do not let the sterilized portion of the instrument contact any nonsterile material before use. Let the heated part of the instrument cool for !10 sec before use.

20. After use, return the instrument to the alcohol disinfectant until needed again. Flame sterilize inoculating loops and needles

21. Hold the inoculating wire by its handle and begin in the canter of the wire to slowly heat the wire with the flame of a Bunsen burner. Proceed back and forth across the wire’s full length until it glows orange.

22. While still holding the handle, allow the inoculating wire to cool back to room temperature (!10 sec) before attempting any transfer of material. If transfers are made while the inoculating wire is hot, cells will be killed by the hot wire, and aerosols created from spattering material can disperse biological material throughout the work space.

23. After the transfer is made, reheat the inoculating wire as in step 21 to destroy any remaining biological material. Let cool to room temperature before putting aside for next use.

USE OF THE HORIZONTAL LAMINAR-FLOW CLEAN BENCH

Laminar-flow cabinets (hoods) are physical containment devices that act as primary barriers either to protect the material being manipulated within the hood from worker generated or environmental sources of contamination, or to protect the laboratory worker and laboratory environment from exposure to infectious or other hazardous materials that are present within the hood. Cell culture applications utilize two types of laminar-flow hoods: (a) the horizontal-flow clean bench (described here) and (b) the biological safety cabinet (see Alternate Protocol). Both types of hoods use a high-efficiency particulate air (HEPA) filter and blowers that generate a nonmixing stream of air.

The horizontal laminar-flow clean bench is used to provide a near-sterile environment for the clean (i.e., noncontaminating) handling of non-hazardous material such as sterile media or equipment. Because the air stream pattern directs the flow of air within the hood directly back to the hood operator and the room (Fig. 1.3.1), horizontal flow hoods are never to be used with infectious agents or toxic chemicals.

Materials:

70% ethanol or other disinfectant
Horizontal laminar-flow hood, certified for use
Swabs (e.g., cheesecloth, paper towels)
Pilot light–activated Bunsen burner.

1. Completely clear the bench of the laminar-flow hood and disinfect the bench working surface and the left and right sides of the hood with 70% ethanol or other disinfectant. Do not spray the back (gridded) wall where the HEPA filter is housed. Resist the urge to leave frequently used items (e.g., pipet canisters or a bag of disposable plastic tissue culture flasks) in the hood between uses. Their presence makes thorough disinfection of the work space difficult.

2. Turn the hood blower and lights on and let the air circulate within the hood 10 min before use.

3. Place items needed for the specific procedure into the hood, wiping each item with 70% ethanol or other disinfectant just before introducing it into the laminar environment. Do not overcrowd the work space. For horizontal laminar-flow effectiveness, maintain a clear path between the work area and the back wall of the cabinet where the HEPA filter is located.

4. Wash hands well before working in the hood and wear a clean laboratory coat and surgical gloves to further protect the work from shedding of skin flora that can contaminant any product.

5. While working in the hood, perform all work at least 4 in. back from the front opening, and avoid rapid movements that might disrupt the laminar air flow. Avoid moving materials or hands in and out of the cabinet as much as possible.

6. If flame sterilization is needed in the hood for a particular application, use a burner that can be activated by a pilot light when needed, rather than one that burns constantly. The open flame of a Bunsen burner causes turbulence that disrupts the unidirectional laminar air flow.

7. When work is completed, remove all material from the laminar work bench, clean any spills, and disinfect the bench working surface by wiping with 70% ethanol or other disinfectant.

8. Turn off hood blower and lights.

USE OF THE VERTICAL LAMINAR-FLOW BIOSAFETY CABINET

Biological safety cabinets provide a clean, safe environment for both the worker and the product. The Class II, Type A biosafety cabinet (Fig. 1.3.2) is frequently encountered in cell culture laboratories, and this protocol describes the use of this type of barrier device. The Class IIA biosafety cabinet is suitable for work with low- to moderate-risk biological agents in the absence of toxic or radioactive chemicals.

Materials:

Class II, Type A Biosafety Cabinet (BSC), certified for use

Pilot light–activated Bunsen burner or electronic

incinerator (e.g., Bacti-Cinerator III, VWR)

Closed-front laboratory gowns (for personnel working with biological agents)

1. Turn the hood blower on and verify air flow by feeling (by hand) the current near the front grill of the work surface. Turn the germicidal UV light off if it is on. Turn the fluorescent light on. Before use, the cabinet should already be empty and clean from prior activity. The view window should be lowered to the proper operating height (normally 8 in.) or as specified by the cabinet manufacturer. UV light is effective only for decontaminating clean, solid surfaces with which it comes in contact. It is not effective in decontaminating the cabinet air flow. UV light is not effective against bacterial spores. UV germicidal light tubes should be replaced frequently (at least every 6 months for biosafety cabinets in use on a daily basis) to assure that they are emitting light at 254 nm and at an intensity appropriate for decontamination.

CAUTION: UV light is harmful to the eyes. Laboratory personnel should not be near the cabinet or looking at the UV light when it is in use.

2. Wash and gown as required for the operation.

3. Wipe down the entire interior cabinet work surface area with 70% ethanol or other appropriate disinfectant.

4. Let blower run for 10 min to filter the cabinet air of any particulates.

5. Raise the front view window as needed to bring necessary items into the cabinet. Wipe each item with 70% ethanol or other disinfectant as it is placed in the cabinet. Do not crowd the work space and make sure no air vents are blocked by supplies or equipment. Do not position material so that it obscures any of the air vents at the front edges of the laminar hood. One frequent source of air flow restriction in biosafety cabinets is “lost” paper towels that have been drawn into the air ducts at the back of the work surface.

6. Organize the work surface for a clean-to-dirty work flow. Place clean pipets, flasks, and sterile media bottles at one side of the cabinet; place discard pans, spent cultures, and other wastes on the other side.

7. Return the view window to the 8-in. operating level. Wait !10 min for the blowers to filter the disturbed cabinet air before starting work.

8. While working, keep all material and perform work "4 in. back from the front opening of the cabinet, and minimize rapid movements or activity. Keep the view window opening as close to 8 in. as allows reasonable access to the work surface and equipment. These precautions assure that any drafts caused by arm movements will not disrupt air flow or churn room air currents into the clean work area.

9. If direct flame sterilization of items within the cabinet is necessary, use an electric burner or pilot light–activated flame burner located at the back of the work space. A constant open flame in the cabinet can disturb the laminar air flow.

10. At the end of the procedure, enclose all contaminated materials. Clean the cabinet work surface with 70% ethanol or other disinfectant, being especially careful to wipe any spills of culture suspensions or media that can serve as future contamination points. Clear all material from the cabinet.

11. Let the blower run for ≥10 min with no activity to remove any aerosols that were generated. During this period, turn off the fluorescent light and turn on the germicidal UV light. Allow the UV light to operate ≥30 min.