What is a microbial culture?
A culture is a large number of microbes grown on purpose from a small number. Microbes are too small to study one by one. When millions grow together, we can see them, count them and test them.
Microbes need: food (a carbon source like sugar, a nitrogen source like protein, mineral salts), water, the right temperature and pH, and for many, oxygen. Some microbes die in oxygen (anaerobes). Their food mix is called the culture medium.
- Liquid medium (broth): in a flask or tube. Good for growing a lot of cells.
- Solid medium: broth plus agar, a jelly from seaweed. Microbes do not eat agar; it only makes the medium solid so colonies stay in place.
- Selective medium: lets only some microbes grow (for example, high salt).
- Differential medium: colonies of different microbes show different colours.
Sterilisation: killing every germ first
Sterilisation means killing all living things, even tough spores. If we skip it, unwanted microbes (contaminants) grow and spoil the result.
- Autoclave: steam under pressure at 121 °C for 15–20 minutes. Used for media and most tools. Like a big pressure cooker.
- Hot-air oven: dry heat, about 160 °C for 2 hours. Used for glass and metal.
- Flame: the wire loop is held in a flame until it glows red.
- Filtration: a filter with tiny holes (0.2 µm) holds back bacteria. Used for liquids that heat would spoil, like vitamins.
- Radiation and chemicals: UV light or gamma rays for plastics and benches; 70% alcohol for wiping (this disinfects but may not kill all spores).
Pasteurisation (for example 72 °C for 15 seconds for milk) is NOT sterilisation. It kills most harmful germs but not all.
Aseptic technique and the streak plate
Aseptic technique means working so no new germs get in: wash hands, wipe the bench, work near a flame (hot air rises and carries dust away), open lids only a little and for a short time, and flame the loop before and after use.
Inoculation means putting microbes into the medium. In the streak plate method, the loop spreads a sample in zig-zag lines in zone 1, is flamed, then drags a few cells from zone 1 into zone 2, and so on. Each zone has fewer cells. In the last zone, single cells sit far apart.
After incubation (keeping the dish warm, often 25–37 °C, upside down so drops do not fall), each single cell becomes a colony. All cells in one colony come from one cell, so they are the same. Picking one colony gives a pure culture. Other methods: the spread plate (a diluted drop spread with a glass spreader) and the pour plate (sample mixed into warm agar).
Counting: diluting a sample 10, 100, 1000 times, then counting colonies, tells how many live cells were in the start (colony-forming units, CFU).
The bacterial growth curve
Bacteria divide in two (binary fission). The time to double is the generation time; for some gut bacteria at 37 °C it is about 20 minutes. Growth in a closed flask has four phases:
- Lag: cells get used to the new food and make enzymes; numbers barely change.
- Log (exponential): cells double again and again; numbers rise very fast.
- Stationary: food runs low and waste builds up; new cells = dying cells.
- Death: more cells die than are born.
We measure growth by counting colonies, by cloudiness (optical density in a colorimeter), or by weighing cells. Formula: N = N₀ × 2ⁿ, where n = number of generations = time ÷ generation time.
Uses and safety
Cultures are used to: find which germ causes a disease and which antibiotic kills it; make products in big tanks called fermenters (bioreactors) – curd, cheese, bread, antibiotics, enzymes, insulin, vaccines; test water and food for safety; and pick better strains (for example a yeast that makes more alcohol, or a strain improved by mutation or genetic engineering). After fermenting, the product is separated and purified: this is downstream processing.
Safety at school: grow only at 25 °C or lower, tape plates shut, never open them after incubation, and have them sterilised before throwing away. Some microbes from the air or skin can make people ill.
Try it at home
Curd test: take three small bowls of warm milk. Add a spoon of curd to two. Keep one of those in the fridge and one in a warm place. Leave the third without curd. Check after 8 hours. Which set? This shows inoculum and temperature. In the 3D, step 6 lets you test the same idea with the temperature list.
Key formulas and definitions
- Number of cells: N = N₀ × 2ⁿ
- Generations: n = time ÷ generation time
- Autoclave: 121 °C, 15–20 min (steam under pressure)
- Hot-air oven: 160 °C, about 2 h
- Cells in sample = colonies counted × dilution factor ÷ volume plated
Worked examples
1. A bacterium doubles every 20 minutes. Starting from 1 cell, how many cells after 2 hours in the log phase?
2 hours = 120 min. n = 120 ÷ 20 = 6 generations. N = 1 × 2⁶ = 64 cells.
2. A flask has 1000 cells. The generation time is 30 minutes. How many cells after 3 hours?
n = 180 ÷ 30 = 6. N = 1000 × 2⁶ = 1000 × 64 = 64 000 cells.
3. 0.1 mL of a sample diluted 1000 times gives 50 colonies. How many live cells per mL were in the original?
Cells = 50 × 1000 ÷ 0.1 = 500 000 = 5 × 10⁵ CFU per mL.
4. Why is vitamin solution filter-sterilised and not autoclaved?
Vitamins break down in heat. A 0.2 µm filter removes bacteria without heating, so the vitamins stay active.
Common mistakes
- Thinking agar is food for microbes. It is only a jelly that makes the medium solid; the food is the sugar, protein and salts.
- Calling pasteurisation sterilisation. Pasteurisation kills most harmful germs, but sterilisation kills everything, including spores.
- Not flaming the loop between streak zones. Then too many cells are carried over and you get no single colonies.
- Mixing up the phases: numbers grow fastest in the log phase, not the lag phase.