Microbial Testing
In remote Australian communities, tap water that looks clear can carry E. coli. Regular microbial testing of water and food is the only reliable way to detect contamination before people get sick — and the method used in a school lab is the same one used by public health authorities.
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Think First
A student testing water samples from three sources — tap water, a local creek, and a rainwater tank — gets the following results after 48 hours of incubation on nutrient agar plates:
- Tap water: 3 colonies visible
- Creek water: 214 colonies visible
- Rainwater tank: 0 colonies visible
Before reading: what conclusions can and cannot be drawn from these results alone? Write down two things the data tells you and two things it does not tell you.
Come back to this at the end of the lesson.
Know
- The purpose of microbial testing of water and food
- The standard method: serial dilution, plating, colony counting
- Key variables: independent, dependent, controlled
- Validity, reliability, and accuracy in microbial investigations
Understand
- Why serial dilution is necessary before plating
- Why colony counts are estimates, not exact counts
- How to identify and control sources of error in microbial testing
Can Do
- Design a valid microbial testing investigation with identified variables
- Calculate colony counts per mL from serial dilution data
- Evaluate the validity and reliability of a microbial investigation
📚 Know
- Key facts and definitions for Microbial Testing
- Relevant terminology and conventions
🔗 Understand
- The concepts and principles underlying Microbial Testing
- How to explain the reasoning behind key ideas
✅ Can Do
- Apply concepts from Microbial Testing to exam-style questions
- Justify answers using appropriate biological reasoning
Core Content
Misconceptions to Fix
Wrong: Common misconception for this lesson.
Right: Correct understanding with explanation.
Why Microbial Testing of Water and Food Is Essential
Contaminated water and food are among the leading causes of infectious disease globally. Unlike chemical contamination, microbial contamination is invisible — water containing dangerous levels of E. coli, Salmonella, or Cryptosporidium looks, smells, and tastes identical to safe water. The only way to detect it is through microbial testing.
Microbial testing determines whether a sample contains harmful microorganisms — and at what concentration. In public health, water is considered safe for drinking if it contains fewer than 1 colony forming unit (CFU) of E. coli per 100 mL. Food safety standards set similar thresholds for Salmonella, Listeria, and other pathogens.
Drinking water
Recreational water
Food safety
School investigations
The Standard Method — Serial Dilution and Plate Counting
Microbial testing of water or food samples follows a standard procedure. The core technique is the serial dilution and plate count method — also called the viable plate count or colony count method.
Collect and prepare the sample
Collect a measured volume of water (e.g. 1 mL or 10 mL) or a weighed food sample (e.g. 1 g dissolved in 9 mL sterile water) using aseptic technique. Aseptic technique — sterile equipment, closed containers, no talking over open plates — is essential to prevent contamination of the sample.
Serial dilution
Transfer 1 mL of sample into 9 mL sterile water — this is a 1:10 (10⁻¹) dilution. Repeat to create 10⁻², 10⁻³, 10⁻⁴ dilutions. Serial dilution is necessary because heavily contaminated samples produce uncountable plates (confluent growth). Countable plates have 30–300 colonies.
Inoculate agar plates
Using a sterile loop or pipette, spread 0.1 mL or 1 mL from each dilution onto a labelled nutrient agar plate. Include a negative control (sterile water only) to check for contamination of the medium. Include a positive control (known bacterial suspension) to confirm the medium supports growth.
Incubate
Invert plates (to prevent condensation dripping onto colonies) and incubate at 25°C–37°C for 24–48 hours. Higher temperatures encourage faster growth but may select for different species. Temperature is a key controlled variable.
Count colonies and calculate CFU/mL
Count colonies on the plate with 30–300 colonies (most accurate range). Calculate: CFU/mL = colony count ÷ (volume plated × dilution factor). Multiple replicates should be averaged for reliability.
Koch's postulates provide the logical framework for proving that a specific microbe causes a specific disease. Each step eliminates alternative explanations.
Designing a Valid and Reliable Investigation (BIO11/12-2)
The HSC requires you to be able to design and evaluate microbial testing investigations. This means identifying variables, justifying controls, and explaining how to maximise validity and reliability.
| Design Element | What It Means | Example in Microbial Testing |
|---|---|---|
| Research question | A clear, testable question linking independent and dependent variables | "Does water from a local creek contain higher bacterial concentrations than tap water?" |
| Hypothesis | A testable prediction based on prior knowledge | "Creek water will contain significantly higher CFU/mL than tap water due to runoff contamination" |
| Independent variable | What is deliberately changed | The water source (tap, creek, rainwater tank) |
| Dependent variable | What is measured in response | Colony count (CFU/mL) on nutrient agar after 48 hours |
| Controlled variables | What is kept constant | Volume plated, agar type, incubation temperature and time, dilution method, colony counting method |
| Negative control | Rules out contamination from equipment/media | Plate inoculated with sterile distilled water only — should show 0 colonies |
| Positive control | Confirms the medium and conditions support growth | Plate inoculated with known E. coli suspension — should show predictable growth |
| Replication | Repeating measurements to improve reliability | At least 3 plates per dilution per sample; average the counts |
Validity vs Reliability vs Accuracy
Risk Assessment and Safe Working (BIO11/12-3)
Any practical investigation involving microorganisms requires a risk assessment. In a school laboratory, water and food microbial testing involves BSL-1 (Biosafety Level 1) organisms — generally considered low risk for healthy individuals but requiring standard precautions.
Common Misconceptions
Misconception: Each colony on an agar plate represents one bacterium from the original sample.
Each colony grows from a colony forming unit (CFU) — which may be one bacterium or a cluster of bacteria that were not fully separated during dilution or spreading. Colony counts are therefore estimates of the minimum number of viable bacteria in the original sample, not exact counts. This is why the method is called a "viable count" — it counts only living bacteria capable of forming colonies, not dead cells or cells that cannot grow under the specific conditions.
Misconception: A negative result (zero colonies) means the water is completely free of all pathogens.
A zero colony count on nutrient agar only means no bacteria grew under those specific conditions (medium type, incubation temperature and time). Some pathogens require special media (e.g. Campylobacter requires microaerophilic conditions), some are viruses (which cannot be cultured on agar), and some organisms (e.g. Cryptosporidium) are detected by different methods entirely. A clean colony count is reassuring but not a guarantee of complete safety.
Misconception: The negative control and positive control serve the same purpose — they are both just "comparison plates."
They serve opposite purposes. The negative control (sterile water plated) tests for contamination of the agar medium or equipment — it should show zero colonies. If it shows growth, the experiment is invalid because the medium or equipment was contaminated. The positive control (known bacterial suspension plated) tests whether the medium and conditions support growth — it should show predictable colony growth. If it shows no growth, the medium may be defective. Each detects a different type of experimental error.
Serial Dilution and Plate Count
- Purpose: estimate bacterial concentration (CFU/mL) in water or food.
- Serial dilution: 1 mL sample into 9 mL sterile water = 10⁻¹; repeat to 10⁻⁴.
- Count plates with 30–300 colonies for accuracy.
- CFU/mL = colonies ÷ (volume plated × dilution factor).
Controls and Their Purpose
- Negative control (sterile water): rules out medium/equipment contamination — should give 0 colonies.
- Positive control (known culture): confirms medium supports growth — should give expected colonies.
- Both are required for a valid investigation.
Validity, Reliability, Accuracy
- Validity: measuring what you claim — use correct medium, proper controls, aseptic technique.
- Reliability: consistent results — multiple replicates, standardised volumes and conditions.
- Accuracy: close to true value — calibrated pipettes, 30–300 colony range.
Safety in Microbial Testing
- Aseptic technique: flame loops, no talking over plates, closed containers.
- Gloves and lab coat; no eating or drinking in the lab.
- All used plates: autoclave or 10% bleach for 30 min before disposal.
- Spills: 10% bleach, leave 20 min before cleaning.
Valid Investigation Design — 7 Steps
Activities
Analysing Microbial Testing Data — Remote Water Supplies
An environmental health officer collected water samples from four sources in a remote community and performed serial dilution plate counts. The results are shown below.
| Water Source | Dilution Plated | Volume Plated (mL) | Colony Count (avg of 3 plates) | E. coli Detected? |
|---|---|---|---|---|
| Bore water (household A) | 10⁻² | 0.1 | 47 | Yes |
| Rainwater tank (household B) | 10⁻¹ | 0.1 | 8 | No |
| Community tap (treated) | Undiluted | 1.0 | 0 | No |
| Creek (upstream) | 10⁻⁴ | 0.1 | 183 | Yes |
| Negative control | — | 0.1 sterile water | 0 | No |
| Positive control | — | 0.1 known E. coli | 112 | Yes |
- Calculate the CFU/mL for the bore water (household A) and the creek water. Show all working.
- The Australian Drinking Water Guidelines specify that drinking water must contain fewer than 1 CFU of E. coli per 100 mL. Based on your calculations and the E. coli detection results, which water sources are unsafe for drinking? Justify your answer.
- What is the purpose of the negative control in this investigation? What would it indicate if the negative control showed 15 colonies?
- The rainwater tank shows 8 colonies but no E. coli. Explain what this result indicates about the safety of the rainwater for drinking.
- The creek was tested at a 10⁻⁴ dilution while bore water was only tested at 10⁻². What does this difference in dilution choice suggest about the relative bacterial concentration expected in each source?
Write your responses here or in your book.
Error Spotting — Flawed Investigation Design
A student designed the following investigation to compare the bacterial contamination of tap water and creek water. The method contains four significant errors in experimental design or procedure. Identify each error, explain why it is a problem, and describe how to correct it.
Student's method (contains 4 errors)
- Collected 10 mL of tap water and 10 mL of creek water in the same unwashed glass bottle, then divided into two samples in the lab.
- Added 1 mL of each sample directly to a nutrient agar plate without serial dilution and spread with a sterile loop.
- Incubated all plates right-side up at 37°C for 24 hours, then counted all visible colonies.
- Used only one plate per sample and one dilution level. Concluded that creek water had "more bacteria" because it had more colonies.
- Identify all four errors in the method above.
- For each error, write one sentence explaining specifically why it affects the validity or reliability of the investigation.
- Rewrite the method as a corrected, improved procedure using appropriate scientific technique.
Write your responses here or in your book.
Revisit Your Thinking
You were shown three colony counts — tap water (3), creek water (214), rainwater tank (0) — and asked what the data does and does not tell you.
What the data tells you: creek water has a substantially higher total bacterial count than tap water; the rainwater tank showed no growth under these specific conditions; tap water has low but detectable bacteria.
What the data does not tell you — and this is the critical part: it does not tell you whether any of these bacteria are pathogens. Total colony count on nutrient agar counts all bacteria that can grow under those conditions — including harmless environmental organisms. It does not tell you whether E. coli (the faecal indicator) is present, whether viruses or protozoa are present (these don't grow on nutrient agar), or whether the rainwater is truly safe (zero colonies could mean no bacteria grew under these conditions, not that the water is free of all pathogens). A clean total count is reassuring, but full safety assessment requires selective media and indicator organism testing.
If you identified that the data doesn't confirm the absence of pathogens or viral contamination — excellent reasoning. The limitations of any testing method are as important as the results.
Assessment
Multiple Choice
5 random questions from a replayable lesson bank — feedback shown immediately
Short Answer — 10 marks
1. Describe the serial dilution and plate count method for estimating the bacterial concentration of a water sample. In your answer, explain why serial dilution is necessary and why only plates with 30–300 colonies are used for counting. (3 marks)
1 mark: serial dilution procedure correctly described | 1 mark: why dilution is necessary (confluent growth at high concentrations) | 1 mark: why 30–300 range (accuracy — too many or too few introduces counting error)
2. A student investigating microbial contamination of food samples uses only one plate per sample with no replicates and no controls. Evaluate this investigation design, identifying two specific limitations and explaining how each affects the validity or reliability of the results. (3 marks)
1 mark: first limitation correctly identified and linked to validity or reliability | 1 mark: second limitation correctly identified and linked to validity or reliability | 1 mark: overall evaluative statement about the adequacy of the design
3. An environmental health officer tests drinking water from a remote community bore and finds 4 CFU of E. coli per 100 mL. The Australian Drinking Water Guidelines allow fewer than 1 CFU of E. coli per 100 mL. Explain what this result indicates about the water's safety, why E. coli is used as the test organism rather than testing directly for all possible pathogens, and describe one immediate public health action that should be taken. (4 marks)
1 mark: result indicates water is unsafe for drinking — exceeds guideline | 1 mark: E. coli as indicator of faecal contamination and correlation with other pathogens | 1 mark: E. coli is practical to culture and count | 1 mark: appropriate immediate public health action (e.g. boil-water advisory, investigate and treat source)
Answers
- Q1 — C: CFU/mL = colonies ÷ (volume plated × dilution factor) = 85 ÷ (0.1 × 10⁻³) = 85 ÷ 0.0001 = 850,000 = 8.5 × 10⁵ CFU/mL. A common error is forgetting to divide by both the volume AND the dilution factor — both reduce the number of bacteria plated, so both must be in the denominator.
- Q2 — B: When plates are incubated right-side up, water vapour from the warm agar condenses on the lid and drips back onto colonies, causing them to spread, merge, or run together — making counting impossible and potentially causing multiple colonies to appear as one. Inverting plates keeps condensation on the lid, away from the colony surface.
- Q3 — A: A negative control should show zero colonies — it contains only sterile water, which by definition should not produce growth. Any colonies indicate contamination of the medium, equipment, or environment — invalidating the entire experiment, because there is no way to distinguish experimental colonies from contaminant colonies. There is no acceptable threshold for negative control growth.
- Q4 — D: E. coli is used as an indicator because its presence signals faecal contamination, which implies other pathogens may be present. It is not the most dangerous pathogen (many pathogens are more dangerous), it can be cultured on selective media (not on all standard nutrient agar alone), and colony size does not aid identification. The practical advantages — easy to grow, easy to count, reliable indicator of faecal input — make it the standard.
- Q5 — B: The 10⁻³ plate with 247 colonies falls within the 30–300 countable range. The 10⁻² plate is confluent (uncountable — colonies overlap and cannot be individually distinguished). The 10⁻⁴ plate has only 28 colonies — below 30, which introduces statistical error (a small count means random variation has a proportionally larger effect on the estimate). Averaging all three would include an uncountable plate, which is not valid.
SA1: The serial dilution and plate count method begins by transferring 1 mL of the water sample into 9 mL of sterile distilled water, creating a 10⁻¹ dilution. This step is repeated using 1 mL of each new dilution to produce a series of dilutions (10⁻², 10⁻³, 10⁻⁴ etc.). A measured volume (typically 0.1 mL or 1.0 mL) from each dilution is spread onto a labelled nutrient agar plate using aseptic technique. Plates are inverted and incubated at a set temperature for 24–48 hours, then colonies are counted. Serial dilution is necessary because heavily contaminated samples produce confluent (lawn) growth when plated directly — individual colonies cannot be counted when bacteria are so numerous that their colonies overlap and merge. Only plates with 30–300 colonies are used because below 30 colonies, random statistical variation becomes proportionally significant (a few misidentified colonies greatly affect the estimate), while above 300, colonies are too crowded to distinguish individually, and multiple bacteria from the original sample may produce a single colony, leading to underestimation.
SA2: Limitation 1: Using only one plate per sample means the results cannot be assessed for reliability — if a single plate gives an unusual result due to uneven spreading, a stray contaminant, or counting error, there is no way to identify this as an outlier. Reliability requires at least three replicates per condition so results can be averaged and variability assessed. Limitation 2: Having no negative control means there is no way to determine whether any colonies that appear on sample plates came from the sample itself or from contamination of the agar medium or equipment. If the medium was contaminated, all colony counts would be artificially inflated and the results would be invalid — but without the negative control, this cannot be detected. Overall, this investigation design is inadequate for drawing reliable or valid conclusions: it cannot distinguish genuine results from experimental error, and the absence of replicates means the results cannot be confirmed or reproduced.
SA3: A result of 4 CFU of E. coli per 100 mL indicates the water is unsafe for drinking. The Australian Drinking Water Guidelines specify that fewer than 1 CFU per 100 mL is required — this bore water contains four times the maximum acceptable concentration of E. coli. E. coli is used as the indicator organism rather than testing directly for all possible pathogens for two reasons: first, E. coli is a reliable indicator of faecal contamination — its presence in water shows that sewage, animal waste, or contaminated runoff has entered the supply, and wherever E. coli is present, other faecal pathogens (Salmonella, Campylobacter, Cryptosporidium) may also be present. Second, testing for every possible pathogen individually would be impractical, time-consuming, and expensive — E. coli is easy to culture on selective media, straightforward to identify and count, and provides a reliable proxy for the entire spectrum of faecal pathogens in a single test. An appropriate immediate public health action is to issue a boil-water advisory to all households using the bore, requiring water to be boiled before drinking, while the contamination source is investigated and the bore water treated or an alternative supply identified.
Boss Battle — Microbial Testing!
Face the boss using your knowledge of microbial testing methods and Koch's postulates. Pool: lessons 1–5.