Infectious Hazards of Bacteriological Techniques (1950s?)

In many laboratories where infectious diseases are studied, there are persons who contract an infection from the microorganisms under investigation. An accident such as this may result in infection. But 80% of all reported illnesses are not attributed to any known accident. This led investigators to suspect that ordinary laboratory techniques performed without mishap provide the source of these infections. A great deal of investigation both in this country and abroad has been conducted on this problem. The purpose of this film is to review some microbiological techniques that have been studied and to suggest methods to improve safety in biological laboratories. The usual methods of studying laboratory hazards are air sampling using suitable air sampling devices and cotton swabbing to determine the extent of surface contamination. This civ type air sampler is highly efficient in trapping on augur media a majority of the airborne particles passing through it. Seratia indica, a red pigmented bacteria, is used as a tracer organism in many tests. The inoculating loop is one of the most frequently used of all laboratory tools, but it can become a dangerous weapon. When it is heated and used immediately to pick bacterial colonies from an augur plate, bacteria in the form of an aerosol are released into the air. When the heated loop is immersed in a flask of broth culture, it creates a disturbance violent enough to cause clumps of bacteria to escape from the flask in the resultant aerosol. Allowing the loop to cool before immersion into the broth or before touching the augur will prevent the creation of an aerosol. Often when a loop full of culture is withdrawn from a flask or test tube, the loop will accidentally touch the edge of the flask, break the culture film, and set up vibrations in the needle, releasing airborne bacterial particles. This hazard can be greatly reduced if care is taken when removing the loop from the flask. Using the inoculating loop to streak smooth augur plates with liquid culture will produce airborne colonies. A rough augur plate will cause the loop to vibrate and stick, producing larger numbers of colonies. Aerosol formation can be greatly reduced by substituting a sterile bent glass rod for the loop when streaking plates with liquid inocula. As with the loop, however, augur plates with rough surfaces result in an increase in the number of organisms aerosolized. The extent of aerosol contamination is indicated by this table showing the number of organisms recovered. A common use of the hypodermic syringe is to inoculate animals with infectious material. The first step in this procedure is the removal of the cotton plug from the test tube. This does not result in aerosol formation as long as the tube is not shaken. When the tube is agitated, bubbles form on the surface of the fluid. And when the stopper is removed, organisms escape into the air. Carefully drawing the inoculum into the syringe creates no hazard. However, if a portion of the fluid in the syringe is expelled against the inside wall of a test tube, an aerosol is created. Slowly emptying the syringe with the needle point below the surface of the liquid in the tube greatly reduces the chance of producing an aerosol. Sometimes a rubber stopper vaccine bottle is substituted for the cotton plugged test tube in an attempt to avoid the hazard involved in filling a syringe from an open test tube. However, when the needle is withdrawn from the rubber stopper, it frequently will vibrate and create an aerosol. Surrounding the needle and rubber stopper with an alcohol soaked cotton pledge affords good protection in this operation. An aerosol is seldom produced if excess fluid and air bubbles in the syringe are expelled into a wad of cotton soaked in alcohol. However, this procedure deposits viable organisms on the fingers of the technician. Following withdrawal of the needle from the injection site, some of the inoculum may leak out. The use of a good skin disinfectant significantly reduces the possibility of contamination to the technician. Repeated use of a syringe soon causes the distal end of the plunger to become contaminated. This in turn contaminates the fingers of the technician as shown by the use of a dye. A safety syringe with the rear portion of the plunger ground to eliminate capillary action minimizes contamination of the distal end of the plunger and protects the fingers. The use of this type of syringe can result in an accidental spray of the contents into the air or the needle may be forced from the syringe. This danger may be minimized by using only needle locking syringes as shown here. Therefore, in syringe operations, rubber gloves should be worn when handling infectious organisms. After using a syringe, place it in a disinfectant without removing the needle or emptying its contents. Using a pipet to transfer an inoculum to a tube or flask can be dangerous. Allowing the inoculum to drop or flow in a stream will agitate the surface of the broth, releasing an aerosol. When the inoculum is allowed to flow slowly down the side of the tube or flask, no agitation occurs and the aerosol is greatly reduced. Blowing the last drop from a pipet, as commonly practiced in placing an inoculum in a petri dish, may form bubbles on the tip of the pipet, which burst and create an aerosol. Fewer organisms escape into the air when the last drop is not blown, but allowed to drain. In this way, bubbles will not form on the tip of the pipet. Mixing a culture by alternately sucking and blowing with the pipet produces violent disturbance in the liquid. Such a hazard is greatly reduced by allowing the fluid to flow slowly down the inside of the tube or flask. Mouth pipetting frequently results in aspiration of bacterial suspension into the mouth. This pipet doesn't have a cotton plug in the mouthpiece. Use of a non-absorbent cotton plug in the proximal end of the pipet should be mandatory. Even though a cotton plug is used, finger contamination of the end of the pipet may cause oral contamination. Therefore, mouth pipetting of infectious or toxic fluids should be discontinued. Always use a pipeter such as this bulb type or this tube type. This is another bulb type, the pro pipeter. It can be used accurately during all manipulations involving a pipet. There are many other types available. Sometimes during pipetting, one or more drops of culture may accidentally escape and fall upon the tabletop or floor. The amount of aerosol formed from a given volume spilled varies with the type of surface upon which it falls. This table shows the relative amounts of aerosol formed when drops of culture were allowed to fall upon various types of surfaces from a height of 1 ft. By far the best protection against falling drops is a towel soaked with 5% phenol or some other suitable disinfectant. The towel should be autoclaved after use. Used pipets should be placed in a suitable container filled with a disinfectant solution and autoclave before being cleaned. The preservation of bacterial cultures by sealing the organisms in glass tubes after drying under vacuum has become standard practice in many laboratories. Although there are wide variations in the types of apparatus, all offer ample opportunity for the escape of infectious particles during some stage of operation such as transfer of the culture to the liophalizing tube. or from an improperly clamped hose attachment. Swabbing the inside of the hoses and vacuum arms after use reveal organisms too numerous to count. The liophalizing apparatus should be sterilized after it has been used with highly infectious organisms. Since it is not feasible to sterilize the vacuum pump, a cotton filter should be inserted between the condenser and pump to prevent organisms from reaching the pump. Working with liophalized material may create infectious aerosol if good sound procedures are not followed. When the vial is broken, an appreciable aerosol may be liberated. Enclosing the breaking point of the vial in alcohol soaked cotton will prevent the escape of airborne particles. Shaking the contents of the vial into a flask or tube of broth produces a large aerosol. Reconstituting the dried material and transferring it into a tube of broth can also create a dangerous situation. Removing the dried mass with a cool wire loop is the least hazardous method. Accidental breakage of a vial containing liophalized culture will create a considerable aerosol which tends to persist. Organisms have been recovered from the air as long as 1 hour after breakage. It is recommended that all work with infectious liophalized products of a highly infectious nature be done in a ventilated cabinet. For many years, the high-speed blender or mixer has been recognized as a source of danger to those who use it with infectious materials. The conventional blender can liberate infectious aerosols in several ways. Aerosols may escape from under the lid of the blender, shown here by high-speed photography. through leaks in a worn bearing and when the lid is removed at the end of the blending operation. Here is one of the several types of safety blenders which have been developed. It has a screw top with a heavy rubber gasket. The drive shaft extends through a double housing to a bearing at the top of the bowl. The bowl is emptied by a siphon with a petcock connection or through a petcock drain. Because of the hazard existing for some time after blending is completed, it is best to empty the bowl through a closed system, either by siphoning or draining. But removing the connection to the siphon or drain can release a dangerous aerosol. Finger swabbing after each procedure of disconnecting the siphon shows that the operation may also contaminate the fingers. An alcohol soaked pledgeet placed around the point of disconnection eliminates contamination of air and hands. The hazards connected with the centrifuging of infectious or potentially infectious materials deserve careful consideration. If a tube breaks in the carrier cup, but the fluid remains in the cup, an aerosol is produced. A much larger aerosol will result when a tube breaks and releases culture into the centrifuge bowl. The hazard to the technician working at various distances from a centrifuge was demonstrated when bacterial air samplers were placed 6 in above the lid of the centrifuge, 2 ft from the centrifuge, 5 ft from the centrifuge, and 10 ft from the centrifuge. The results of this experiment are shown in this table. Avoidance of accidents begins with careful selection of uncracked heavyduty thickwalled centrifuge tubes. Carrier cup should be thoroughly inspected for irregularities. Each cup should be carefully balanced with a non-corrosive germicidal solution. Safety cups are available that hold test tubes, centrifuge tubes, and bottles up to 250 ml. The top of the cup is threaded and has a rubber O-ring gasket which prevents the escape of aerosol in the event of breakage during the centrifuging operation. Safety trunion cups are also available for use in an angle head centrifuge. Petri dishes containing cultures may be accidentally dropped in any laboratory. Such an accident can create a serious aerosol hazard to laboratory personnel. Glass petri dishes will usually shatter, disseminating large numbers of organisms. Plastic plates will not break, thereby reducing the number of organisms disseminated. This chart shows the number of organisms recovered from dropped glass plates as compared to dropped plastic plates. Potentially hazardous operations, no matter how carefully they are performed, should be carried out in a ventilated bacteriological safety cabinet. Several types of ventilated bacteriological cabinets are available. Portable cabinets of flexible plastic sheeting, conventional cabinets and modular cabinet systems. These modular systems are composed of several cabinets joined together with built-in incubators, refrigerators, and other laboratory equipment. The contaminated air in the cabinet is exhausted through a suitable filter for your own safety as well as that of your fellow worker. Remember, when working with highly infectious material, operations such as blending, [Music] mixing, opening liophalized cultures, centrifuging, opening centrifuge safety cups and pipetting. should be carried out in a ventilated cabinet. The ventilated safety cabinet is still the most important single piece of equipment in preventing laboratory infections. Of course, it cannot substitute for good training by a competent supervisor who accepts his responsibility for making safety in the laboratory a part of his research planning.