Maintaining and preparing cultures of bacteria and yeasts
Notes on maintaining stock cultures, and preparing actively growing cultures for use by students.
Instead of re-purchasing whenever needed, it may be convenient to maintain a stock of a pure culture. Most of those recommended are relatively easy to maintain on the appropriate growth medium, but maintenance of stock cultures needs to be well-organised with attention to detail.
Date stamp new cultures on arrival from the supplier.
Cultures on streak plates are not suitable as stock cultures. Cultures of bacteria and fungi are normally kept in Universal/ McCartney bottles or (for a short time) in test tubes in which the agar has been allowed to set at a slope. Dispense the agar in the normal way (5 ml is sufficient in a Universal bottle) and, after autoclaving, prop the bottle (or tube) at an angle, for example, around the edges of a pile of bench mats, until the agar has set. Make sure that the sloping agar does not quite reach the neck of the bottle (or tube). Caps should remain loose until the agar slope has solidified. If a lot of slopes have to be prepared, it might be worthwhile making a d-i-y construction to hold the containers at the correct angle.
Slope cultures in bottles with screw-caps (rather than cotton wool plugs or plastic lids) are preferred because the cap reduces evaporation and dehydration and the caps cannot be accidentally knocked off. Slope cultures are also preferred to broth (liquid medium) cultures because the first sign of contamination is more readily noticed on an agar surface.
Prepare two stock cultures; a ‘permanent’ stock which is opened once only (to prepare the next two stock cultures) and a ‘working’ stock for taking sub-cultures for class. If a particular culture is used a lot in a short period, it is much more convenient to prepare several working sub-cultures at once.
Incubate at an appropriate temperature until there is good growth. For growing strict aerobes it may be necessary to slightly loosen the cap for incubation (but close securely before storage) if there is insufficient air in the headspace. If you are not sure whether the strain is a strict aerobe or not, leave the cap loose until the culture is established.
As soon as there is adequate growth, and the caps are screwed tight, the cultures may be stored in a refrigerator (one in which human foodstuffs are never kept), but they will remain viable in either a cupboard or a drawer at room temperature. A dark place at a temperature of 10-15 °C is ideal.
Be prepared to transfer most cultures four times a year to maintain viability. Label each culture clearly including the date of transfer. Do not destroy old cultures until the new subcultures have become established. Some bacteria need more frequent sub-culturing to maintain viability (for example Lactobacillus sp).
Checking purity of cultures
It is sensible to check purity on suspicion of contamination of the working stock, and of the permanent stock when preparing new stock cultures. Evidence of purity is given by the uniformity of colony form on a dilution streak plate, cell form on stained microscope slides, and consistency with the appearance of the original culture.
If a culture becomes contaminated, go back to the working stock or permanent stock cultures, or buy in fresh supplies.
Preparing cultures to use in investigations
Microbial cultures cannot be taken from a shelf and instantly be ready for use. It is necessary to begin to prepare cultures well in advance otherwise the outcome might not be as expected. The key is to transfer cultures several times in advance to ensure that they are growing well and are presented as young, fully-active cultures on the day of the practical class.
To transfer cultures, scrape a small amount of bacteria or yeast cells off the agar slope of a culture using a sterile loop. Wipe the loop over a fresh agar slope to seed a new culture directly or, preferably, shake the loop in a small volume of sterile nutrient broth. After allowing the transferred cells time to multiply until the broth becomes cloudy (1-2 days, or longer with slower-growing organisms or large volumes of broth), the microbes are ready to be used for investigations or to prepare new agar-slope cultures.
Progress of growth can be followed by observation with the naked eye, looking for growth on an agar surface or turbidity in a broth culture. It is usual to grow moulds on the surface of an agar medium, allowing an incubation period of several days to a week.
Use a sterile loop, syringe or Pasteur pipette to transfer bacteria or yeast cells from the broth.
The main points to observe are:
- use of an adequate amount of inoculum
- an appropriate culture medium
- an appropriate incubation temperature
- adequate aeration for a strictly-aerobic organism in a single large volume (more than 20 cm3) of liquid culture.
Suggestions and tips
1 It will save time preparing large numbers of cultures of bacteria and yeast for a class if the inoculum is taken by Pasteur pipette from a well-growing, turbid broth culture. A line of growth on a slope culture inoculated by a wire loop is easy for students to observe – but you can achieve almost the same effect with a pipette. With a broth (rather than a slope) the risk of spills is greater, so slopes are preferred with younger or inexperienced students.
2 Regular practice of aseptic technique is necessary to ensure that the manipulations involved in culture transfer and handling sterile solutions become second nature.
3 The choice of loop or pipette for transfers between test tubes and screw cap bottles depends on whether they contain agar slopes, liquid media or sterile solutions.
4 To inoculate a tube or bottle from a separate colony on a plate, a wire loop is usually satisfactory. A straight wire is sometimes needed for small colonies (for example in pure cultures of Streptococcus and Lactobacillus) or on plates used to isolate cultures from natural samples.
Health & Safety and Technical notes
Carry out a full risk assessment before planning any work in microbiology (see note 1 for more details).
Society for General Microbiology – source of Basic Practical Microbiology, an excellent manual of laboratory techniques and Practical Microbiology for Secondary Schools, a selection of tried and tested practicals using microorganisms.
MiSAC (Microbiology in Schools Advisory Committee) is supported by the Society for General Microbiology (see above) and their websites include more safety information and a link to ask for advice by email.
(Websites accessed October 2011)