When fungal spores or bacteria-laden microscopic particles make contact with your plates, broths, and tubes colonies happily reproduce and your precious media eventually resemble something out of an abandoned full refrigerator. One can’t recognize individual colonies when the plates are covered with fuzz! No untreated surface in the lab is sterile, and nearly all dust and other particles have spores or active cells on their surfaces. Obviously, then, all labware and all media must be sterilized before use. Sterilization can be accomplished in several ways including the use of moist heat, filtration, ethylene oxide, radiation, or ultraviolet light. Moist heat will be employed for most sterilization procedures in the laboratory. Other methods are necessary for materials that won’t stand up to heat, such as untempered glass, some plastics, or labile chemicals such as most antibiotics.
A steam autoclave is an instrument that is designed to deliver steam into a chamber, generating high heat and pressure at the same time. Heating media to above 120 degrees C for 4 to 20 min. destroys nearly all living cells and spores. High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 degrees, which can’t be accomplished with steam at one atmosphere. We use an autoclave that starts timing when the temperature reaches 121 degrees, and exhausts the steam slowly after the prescribed time above 121 degrees (to prevent exploding bottles!). The autoclave is effectively a giant pressure cooker.
To properly use an autoclave
Know the instrument – some are fully automatic, some are fully manual
Prepare supplies properly – the more layers or greater the volume, the longer it will take for the interior to heat up
Check the steam pressure and ensure that the instrument is set for slow exhaust if liquids are to be sterilized
Ensure that the door is closed properly and securely
Check that the time and/or automatic cycle are set properly
Ensure that the temperature is well below 100 degrees before attempting to open the door
Crack the door to allow steam to vent, keeping face and hands well away from the opening
Handle freshly autoclaved bottles and tube with care – use protective clothing and gloves. Materials may be superheated and explode or boil over on you.
Agar plates
Tryptic soy agar consists of a pancreatic digest of casein (milk sugar) and a papaic digest of soybean meal, with sodium chloride and agar. It is a general purpose medium for the culture of fastidious and nonfastidious microorganisms. Whatever we give you will grow on tryptic soy agar provided that you inoculate the plate with living material and culture it at an appropriate temperature.
Media are purchased as dehydrated granules or powder, and are rehydrated by mixing a measured amount of medium per measured volume of deionized water. Instructions for rehydration are usually printed on the container (40 gms/liter for tryptic soy agar). Since dry media tend to form clumps when dumped in water, it is best to layer the material on the surface and let it soak in. Containers used for media must have vented tops and should be capable of holding more than the intended volume of medium, to allow for expansion during sterilization. Agar does not distribute uniformly when melted. A safe way to ensure a uniform distribution for pouring plates or tubes is to drop a magnetic stir bar in the flask or bottle, then gently stir the medium after sterilization, while it cools. Stirring distributes the agar evenly. If screw cap bottles are used, the cap must be loosened prior to sterilization.
***CAUTION*** Exposing tightly stoppered bottles to variable pressures invites explosion and injury. When heating any liquids using any method, take care disturbing the flask or bottle. Material near the bottom may be superheated and boil over when moved. Stoppers, caps, covers, must be vented – never make them fit tightly.
Agar media for plates will be sterilized for 15 minutes in an autoclave in slow exhaust mode, stirred and cooled to about 50 degrees C, and plates prepared in the laminar flow hood. We use 1 liter bottles for plate media, with 500 ml medium per bottle. Sterile 100 mm plastic dishes come in sleeves of 20. To pour plates several stacks of new plates are placed in the hood and a minimum of 20 ml poured into each plate using aseptic technique. The hood prevents fungal contamination of the rich medium, which nearly always happens when we pour plates out on the lab benches.
Dishes can be left in the hood in a single layer with lids ajar or stacked with only the top lid ajar. Either way, we often get condensation which can lead to contamination of plates. To reduce condensation we may choose to leave fresh dishes for a period of time in the hood. One can expect to get 20 to 25 plates per bottle.
Broth tubes
The only difference between broth and agar media is that broths are liquid and agars provide a solid support for growth. Use broth for faster growth, to get a uniform inoculum for dilution streaking, or for assays. In broth a species may display a characteristic pattern of association among individual cells, such as chains or clusters, that is not as obvious in agar cultures. Your unknown cultures will be prepared in broth, then mixed to give proportional amounts of each species prior to passing them out. Dry medium is layered onto the surface of a measured volume of water as with agar media, mixed, and distributed into individual capped tubes in racks. Racks are autoclaved and then allowed to cool, and caps tightened to prevent evaporation.
Agar slants
An agar slant consists of agar based medium in a culture tube. It is called a slant because the tube is placed at an angle during cooling to give a large slanted surface for inoculation. The tube can be tightly capped for relatively long term storage of an isolate with low risk of contamination or drying out of the culture. To prepare an agar slant, agar media are rehydrated just as they are for agar plates, however the agar should then be melted in a microwave oven, stirred to distribute it evenly, and distributed into capped tubes. The tubes should be filled just sufficiently to allow the agar to flow to just below the neck when the neck is laid over a horizontal 10 ml glass pipet. The tubes are autoclaved with caps loose as with all media, then laid on their sides using a pipet to keep them tilted up just enough to create a long slanted surface. After cooling, the caps are tightened and the tubes are ready for use.
Aseptic technique
The media on which you culture desirable microorganisms will also grow undesirable contaminants, especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact.
Never leave a culture dish open, even for a short time when viewing colonies of organisms, unless you intend to destroy it.
When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface.
For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum.
Flame a loop or needle to red-hot just prior to use, burning off any organic material
Cool the instrument by touching the sterile agar or liquid surface prior to touching a culture (or else you will kill it)
Re-sterilize the instrument after performing the procedure, putting down safely without burning the bench, you, or another student.
Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking off the cap. Hold the cap with opening down, and the tube horizontal or nearly so. Convection from the heated neck will prevent dust from falling into the opening. Flame again before putting the cap back [see ‘preparing a bacterial smear’ in the staining section]
Use sterile disposable pipets to remove samples from a broth culture that must be kept uncontaminated.
Always be aware of where your hands are, where your face is, and whether or not your culture is in a position to be contaminated. If you have long hair, make sure it does not hang into your plate. Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms.
If you have an open flame, long hair that is not tied back or loose clothing can be hazardous to your health.
A contaminated culture can often be rescued, however there is always the risk that you will re-isolate the wrong microorganism. Besides, you don’t have that kind of time to waste. Exercise extreme care to keep your cultures pure. You will have ample opportunity to practice media preparation, assay techniques, staining and observation, and especially proper culture and handling of microorganisms
