Plate culture is one of the most fundamental skills in microbiology. Whether you are isolating bacteria from a clinical sample, counting colonies from food, or maintaining pure cultures in the lab — this technique is your starting point. This guide covers everything you need to know, step by step.
What is Plate Culture?
- Definition: Plate culture is the technique of growing microorganisms on the surface of a solid nutrient medium contained in a Petri dish (plate)
- Purpose: To isolate, identify, count, or maintain bacterial or fungal colonies
- Invented by: Robert Koch and Julius Richard Petri in the 1880s — a breakthrough that transformed microbiology
- Key principle: A single cell, when placed on solid agar, multiplies to form a visible colony — and all cells in that colony are genetically identical (a pure culture)
- Applications: Clinical diagnostics, food microbiology, environmental monitoring, pharmaceutical quality control, research
Bacterial colonies growing on a nutrient agar plate — each colony originates from a single cell. Image: Wikimedia Commons
Types of Culture Media
- Nutrient Agar (NA): General purpose medium — supports growth of most non-fastidious bacteria. Contains beef extract, peptone, agar
- Blood Agar (BA): Contains 5–10% sheep/horse blood. Used to culture fastidious organisms and detect haemolysis patterns (alpha, beta, gamma)
- MacConkey Agar: Selective and differential. Selects gram-negative bacteria; differentiates lactose fermenters (pink) from non-fermenters (colourless)
- Mannitol Salt Agar (MSA): Selective for Staphylococcus. High salt (7.5% NaCl) inhibits most organisms; mannitol fermentation differentiates S. aureus (yellow halo)
- Sabouraud Dextrose Agar (SDA): Low pH (5.6) selects for fungi and yeasts — inhibits most bacteria
- Chocolate Agar: Heated blood agar — releases NAD and haem for fastidious organisms like Haemophilus and Neisseria
Key Concept
Agar is a polysaccharide extracted from red algae. It melts at ~85°C and solidifies at ~42°C — critical properties that make it ideal for culture media. Bacteria cannot digest agar, so it remains solid throughout incubation.
Three Main Plating Methods
Method 1: Streak Plate Method (Isolation)
Purpose: To obtain isolated, well-separated colonies from a mixed sample — the gold standard for obtaining pure cultures.
- 1Label the bottom of the agar plate with sample name, date, and your name
- 2Sterilise the inoculating loop by flaming it until it glows red hot. Allow it to cool for 10 seconds
- 3Pick up a loopful of sample (broth culture, colony, or specimen)
- 4Zone 1: Streak back and forth 4–5 times across one quarter of the plate
- 5Reflame and cool the loop. Rotate the plate 90°
- 6Zone 2: Pass through the end of Zone 1 two times, then streak into fresh agar 4–5 times
- 7Reflame, cool, rotate 90° again
- 8Zone 3: Pass through end of Zone 2, streak into fresh agar. Continue similarly for Zone 4
- 9Invert the plate (agar side up) and incubate at 37°C for 18–24 hours
💡 Student Tip: Inverting the plate prevents condensation water from dripping onto colonies, which would cause spreading and contamination. Always incubate inverted.
Method 2: Spread Plate Method (Quantitative)
Purpose: To count the number of viable bacteria in a sample — used extensively in food microbiology and water testing.
- 1Prepare serial dilutions of your sample (10⁻¹, 10⁻², 10⁻³... up to 10⁻⁶ or further)
- 2Pipette exactly 0.1 mL of the appropriate dilution onto the centre of a pre-dried agar plate
- 3Sterilise a glass spreader (Drigalski spatula) by dipping in alcohol and flaming — allow to cool
- 4Spread the inoculum evenly across the entire agar surface using gentle, circular/back-and-forth motions
- 5Allow to absorb for 5 minutes, then invert and incubate
- 6Count colonies after incubation. Countable range: 30–300 colonies per plate
- 7Calculate: CFU/mL = colony count ÷ (volume plated × dilution factor)
Method 3: Pour Plate Method (Quantitative)
Purpose: Alternative quantitative method — can detect anaerobes and organisms that grow within the agar.
- 1Prepare serial dilutions of sample
- 2Pipette 1 mL of sample into a sterile, empty Petri dish
- 3Pour approximately 15–20 mL of cooled (but still liquid) agar — held at 45–50°C — into the dish
- 4Gently swirl the plate in a figure-8 motion to mix sample and agar evenly
- 5Allow to solidify completely on a flat surface (5–10 min)
- 6Invert and incubate. Colonies grow both on surface and within agar
⚠️ Warning: If agar is too hot (>55°C) when poured, it will kill the bacteria. If too cool (<42°C), it solidifies before mixing. Temperature control is critical in the pour plate method.
Reading and Describing Colonies
After incubation, describe each colony type using these standard characteristics:
- Size: Measure in mm (pinpoint <1mm, small 1–2mm, medium 2–5mm, large >5mm)
- Shape: Circular, irregular, filamentous, rhizoid, spindle
- Margin/Edge: Entire (smooth), undulate (wavy), lobate, erose (irregular), filiform
- Elevation: Flat, raised, convex, umbonate (central raised knob), crateriform
- Surface: Smooth, rough, wrinkled, glistening, dull
- Colour/Pigmentation: White, cream, yellow, orange, pink — note any diffusible pigment into agar
- Transparency: Opaque, translucent, transparent
- Consistency: Butyrous (buttery), viscid, membranous, brittle — test with inoculating loop
- Odour: Some species have characteristic smells (Pseudomonas — grape-like; Proteus — fishy)
A correctly streaked plate showing isolated single colonies in zones 3 and 4. Image: Wikimedia Commons
Common Mistakes & How to Avoid Them
- Too much inoculum: Colonies grow into each other (confluent growth) — no isolation possible. Always streak away from the previous zone.
- Not cooling the loop: Hot loop kills bacteria. Always wait 10 seconds after flaming before picking colonies.
- Plate too wet (condensation): Colonies spread and merge. Pre-dry plates by inverting with lid ajar in incubator for 30 min before use.
- Not streaking through the previous zone: Defeats the purpose of dilution by streaking. Always pass 2–3 times through end of the previous zone.
- Contamination: Never leave plates open unnecessarily. Work near a flame or in a laminar flow hood.
- Agar too hot in pour plate: Kills bacteria and gives falsely low counts.
- Incubating right-side up: Condensation drops ruin colonies. Always incubate inverted.
🎓 Exam Practice Questions
- Why is the plate inverted during incubation?
- What is the countable range of colonies for quantitative methods and why?
- What is the difference between selective and differential media? Give an example of each.
- Why is agar used instead of gelatin as the solidifying agent in culture media?
- A spread plate of a 10⁻⁵ dilution gives 150 colonies. What is the original count in CFU/mL?
- What is the principle behind the streak plate method? Why does the colony number decrease from zone 1 to zone 4?