Basic operations
Sterilisation kills all microbes. Glass and media are sterilised in an autoclave (pressure cooker): 121 °C for 15 minutes. Loops and needles are heated red in a flame. Benches are wiped with 70% alcohol. Heat-sensitive liquids pass through a fine filter.
Culture media are foods for microbes: nutrient agar (bacteria), potato dextrose agar, PDA (moulds and yeasts), malt extract or sugar agar (yeasts). Agar is the jelly that makes the food solid.
Aseptic technique. Work near a flame or in a clean cabinet. Flame the loop, cool it, open a tube only a little, flame the tube mouth, and close fast. Label plates with name, date and medium. Incubate plates upside down so water drops do not fall on the colonies.
Pure culture and storage. A pure culture has one kind of microbe. Keep it on an agar slant in the fridge. Observe colonies by eye and cells under a microscope. Before throwing away, soak plates in disinfectant or autoclave them.
Isolating and culturing moulds
Take a bit of the mould from a food, using a sterile needle, and touch it in the middle of a PDA plate (point inoculation). Incubate at about 25 °C for 3 to 5 days. The colony grows outward as a circle, because hyphae spread in all directions. To get a pure culture, cut a small piece of colony edge with a sterile needle and move it to a fresh plate.
Look at colour, texture (fuzzy, powdery) and the reverse side. Under a microscope see hyphae, and the spore-bearing heads. Safety: moulds produce airborne spores, so keep plates closed and never sniff.
Isolating and culturing yeast
Make a dilution series: mix 1 g of food (for example fruit skin or idli batter) in 9 ml of sterile water, then take 1 ml into another 9 ml, and repeat. Put 0.1 ml of one dilution on sugar agar and spread it with a sterile spreader (spread plate). Incubate at 25 to 30 °C for 2 to 3 days.
Yeast colonies are round, smooth, moist and cream-white. Under a microscope see oval cells with buds. A drop of methylene blue stains dead cells blue, and live cells stay colourless. Adding acid to the agar (pH about 5) stops most bacteria from growing.
Isolating and culturing bacteria
Use the streak plate method. Touch the loop to the sample and streak the first third of a nutrient agar plate. Flame the loop, cool it, turn the plate and drag the loop from the first streak into a second area. Flame again and do a third streak. Each streak carries fewer cells, so in the last area the cells are far apart and each makes its own colony. Incubate at 37 °C for 24 hours.
Pick one colony to get a pure culture. Describe colony shape, edge, colour and size. A Gram stain shows two groups under the microscope: purple (Gram positive) and pink (Gram negative). Other methods: pour plate (mix sample into melted agar) and spread plate.
Culturing mushrooms
A mushroom is the fruit of a fungus whose body is a hidden mass of hyphae called mycelium. Oyster mushroom is easy to grow:
- Prepare the substrate. Chop paddy straw, soak it, then pasteurise it (hot water about 70 to 80 °C for 1 hour) and drain it so it is damp but not dripping.
- Spawn. Mix in or layer spawn (grain covered in mycelium) at about 3 to 5% of the straw weight in clean plastic bags with small holes.
- Spawn run. Keep bags in a dark, warm room (about 25 °C) for 2 to 3 weeks until the straw is white all through.
- Fruiting. Cut the bag, give light, fresh air and high humidity (80 to 90%) by gentle sprinkling. Small mushrooms form in a few days.
- Harvest. Pick before the caps flatten and release spores. Cook before eating.
Green or orange mould in the bag means contamination: do not use it.
Key formulas and definitions
- Autoclave: 121 °C, 15 minutes
- Dilution: 1 g in 9 ml = 10⁻¹; each further 1 ml in 9 ml adds ×10
- CFU per ml = colonies ÷ (volume plated in ml × dilution)
- Spawn rate for mushrooms ≈ 3–5% of substrate weight
- Key terms: sterilisation, aseptic, agar, slant, pure culture, colony, streak plate, spawn, mycelium, substrate
Worked examples
1. You plate 0.1 ml of a 10⁻³ dilution and count 45 colonies. Find CFU per ml of the original sample.
CFU/ml = 45 ÷ (0.1 × 10⁻³) = 45 ÷ 0.0001 = 450,000 = 4.5 × 10⁵.
2. You need to spawn 4 kg of wet straw at 5%. How much spawn?
5% of 4 kg = 0.05 × 4 = 0.2 kg = 200 g of spawn.
3. You made a streak plate but the whole plate is one solid lawn. What went wrong?
The streaks carried too many cells, or the loop was not flamed between streaks. Dilute the sample and flame the loop each time.
Common mistakes
- Not letting the loop cool, which kills the cells you want to pick.
- Incubating plates lid-up so water drops fall on the colonies.
- Sniffing or opening plates with unknown mould or colonies.
- Using straw that is dripping wet or that was not pasteurised, so contaminants take over.