Culturing microorganisms
To study microbes we grow them. A culture medium is food for microbes. Agar is a jelly that sets in the Petri dish; we add nutrients to it. Everything is sterile (free from living germs) and we work carefully so that no other microbes enter: this is aseptic technique. After spreading the sample, the plate goes into an incubator, a warm box (about 37 °C for body germs).
A mixed sample (for example soil or stool) has many kinds of microbes. A selective medium contains something that lets only the wanted microbe grow (for example salt, a dye or an antibiotic that the others cannot stand). A differential medium lets all grow but shows differences by colour. Colonies from the selective plate are streaked again until we get a pure culture (one kind only).
Growth curve and controlling growth
Bacteria multiply by splitting in two. In a tube of broth the number follows a growth curve with four phases:
- Lag: cells get used to the food; little increase.
- Log (exponential): they double again and again. After n doublings, N = N₀ × 2ⁿ.
- Stationary: food runs out and waste builds up; births equal deaths.
- Death: more cells die than are born.
To control growth we use antimicrobial agents: disinfectants (on objects), antiseptics (on skin) and antibiotics (inside the body). The antibiogram tests a microbe against several antibiotics. We spread the microbe over the plate, put discs soaked in drugs, and measure the clear inhibition zone after incubation. A big zone means sensitive (S); no or a tiny zone means resistant (R); in between is intermediate (I). The doctor picks a drug marked S.
Identifying microorganisms
Identification is detective work with clues, from simple to specific.
- Morphology (look): colony colour and shape; under the microscope the cell shape (cocci = balls, bacilli/rods = sticks, spirilla = spirals) and how cells group (pairs, chains, clusters).
- Gram stain: cells are stained violet, then washed. Thick-walled Gram-positive cells keep the violet. Thin-walled Gram-negative cells lose it and take the pink counter-stain.
- Metabolic tests (what the microbe does): catalase test (bubbles with hydrogen peroxide), oxidase test, can it ferment a sugar (colour change), can it use citrate. Test strips with many tiny wells do many tests at once.
Strategy: start with cheap, fast tests (Gram stain and shape), then use a few metabolic tests that split the remaining possibilities, and compare the pattern with a key or table. Always confirm with a pure culture.
Counting microorganisms
Direct count under the microscope. A special slide (counting chamber) has a grid; the volume above one big square is known (for example 1 mm × 1 mm × 0.1 mm = 10⁻⁴ mL). Count cells in several squares and take the mean. Cells per mL = mean per square ÷ volume. Quick, but it counts dead cells too.
Count after solid culture (plate count). Dilute the sample step by step (1 mL into 9 mL gives 1 : 10, so 10⁻¹, then 10⁻², …). Spread a known volume (say 0.1 mL) on plates. After incubation, count colonies on a plate that has 30 to 300 colonies: fewer is not reliable, more crowd together.
CFU/mL = colonies × (1 ÷ dilution) ÷ volume plated. It counts only living cells that can form colonies.
Try it
In the 3D: go to step 5 and slide the dilution. Before you read the answer, predict which plate is "countable". Then check that the green ring plate has between 30 and 300 colonies. Next, in step 2 tick and untick the selective agar and count the colonies each time.
At home (safe): put one drop of food colouring in a glass of water. Take 1 spoon of it into 9 spoons of clean water, mix, and see how the colour gets lighter. Do it three times. This is what a serial dilution does to the number of cells. Do not grow germs at home; use only a classroom lab with a teacher.
Key formulas and definitions
- N = N₀ × 2ⁿ (n = number of doublings = time ÷ doubling time)
- CFU/mL = colonies × dilution factor ÷ volume plated (mL)
- Dilution factor = 10ᵉ for a 10⁻ᵉ plate
- Direct count: cells/mL = mean cells per square ÷ volume of the square (mL)
- Use plates with 30 to 300 colonies
Worked examples
1. One bacterium doubles every 30 minutes. How many cells after 3 hours (no lag)?
3 h = 180 min = 6 doublings. N = 1 × 2⁶ = 64 cells.
2. A 10⁻³ plate, spread with 0.1 mL, shows 142 colonies. Find CFU/mL.
CFU/mL = 142 × 10³ ÷ 0.1 = 1.42 × 10⁶.
3. A 10⁻⁴ plate spread with 1 mL shows 45 colonies. Find CFU/mL.
45 × 10⁴ ÷ 1 = 4.5 × 10⁵ CFU/mL.
4. Plates of the same sample: 10⁻² has >300, 10⁻³ has 142, 10⁻⁴ has 15. Which plate do you count?
Only the 10⁻³ plate is in the 30 to 300 range. 10⁻² is crowded, 10⁻⁴ has too few.
5. Inhibition zones: A = 24 mm, B = 16 mm, C = 8 mm, D = 0 mm. Which drugs would you consider?
A and B have big zones: the microbe is sensitive. C is weak. D shows no zone, so the microbe is resistant to D. Choose A first.
6. A counting chamber square has volume 10⁻⁴ mL. The mean count is 25 cells per square. Find cells/mL.
25 ÷ 10⁻⁴ = 2.5 × 10⁵ cells/mL.
7. A sample is diluted three times, each time 1 mL into 9 mL. What is the dilution?
Each step is 1 : 10, so after three steps 10⁻³, i.e. 1 : 1000.
8. A pink, rod-shaped, catalase-positive microbe is found. What do the clues say?
Pink means Gram-negative, rod means bacillus; catalase-positive removes some groups. Next you would run sugar and citrate tests and compare with a key.
Common mistakes
- Counting a plate with 500 colonies and trusting it. Only 30 to 300 is reliable.
- Forgetting the dilution factor or the volume plated in the CFU/mL formula.
- Thinking a Gram stain gives the species name. It only splits microbes into two groups.
- Opening a plate of unknown microbes in class. Keep it closed and use aseptic technique.