Microbial Testing
You'll be able to design a valid microbial testing investigation with identified variables.
The WHO's 2019 report Progress on Drinking Water, Sanitation and Hygiene found that 2.2 billion people lacked safely managed drinking water, and that inadequate drinking water services were linked to an estimated 485,000 diarrhoeal deaths per year across all faecal-oral pathogens. E. coli is not usually the killer, it is the indicator organism laboratories count to show that faecal contamination has reached the supply. Under WHO standards, any detection of E. coli in a 100 mL sample fails the safe drinking water threshold. The method used to detect it in a field laboratory is the same one used in a school science classroom.
Orient yourself
Warm up on earlier lessons, make a prediction from real colony counts, then scan the goals and the vocabulary you will need at the bench.
Three quick questions from earlier lessons. Pulling old material back to mind before you learn something new makes the new material stick better, so this is not busywork.
Practise this lesson
Four printable worksheets that build from the foundations up to exam-style questions, start at whatever level suits you.
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.
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
See why microbial testing exists
Contamination is invisible, so indicator organisms and legal thresholds do the seeing for you.
Core Content
Microbial contamination is invisible, testing is the only way to detect it
Water carrying dangerous levels of E. coli, Salmonella or Cryptosporidium looks, smells, and tastes identical to safe water, the only way to know is to test for the organisms directly.
Contaminated water and food are among the leading causes of infectious disease globally. Unlike chemical contamination, microbial contamination is invisible. 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
Microbial contamination is invisible, testing is the only way to detect it. Safe drinking water = <1 CFU of E. coli per 100 mL (Australian guidelines). E. coli is used as an indicator of faecal contamination (signalling other pathogens may be present) and because it is easy to culture and count.
Pause, copy why water must be tested and the E. coli safe-water standard into your book.
Why is E. coli used as the indicator organism for water safety?
Serial Dilution Method
Count the invisible
Serial dilution, plating, incubation and the CFU/mL calculation, including why the countable window is 30 to 300.
The viable plate count, step by step
We just saw that water must be tested directly. That raises a question: how do you actually count invisible microbes? This card answers it → the serial dilution and plate count method.
The core technique of microbial testing is the serial dilution and plate count method, diluting a sample until colonies become countable, then scaling back up.
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.
The 30 to 300 countable range has a separate reason at each end, and the HSC expects both. Below 30 colonies, random variation in how few cells happen to land on that particular plate becomes proportionally large: a swing of three or four colonies changes the calculated CFU/mL by more than ten percent, so the estimate is statistically unreliable. Above 300 colonies, neighbouring colonies merge and crowd one another out, so colonies cannot be distinguished and the count comes out too low.
Even a plate inside that range gives an underestimate of the true cell number. A CFU may be a single cell or a clump or chain of several cells that stayed together, and either way it grows into just one colony. A plate count is therefore best read as a lower bound on the viable cells present, never an exact head count.
Serial dilution & plate count: collect → serially dilute → plate → incubate (inverted) → count. Dilution is needed because concentrated samples give confluent, uncountable growth. Count plates with 30–300 colonies; CFU/mL = colonies ÷ (volume × dilution factor). Below 30, random variation makes the estimate statistically unreliable; above 300, colonies merge and the count reads low. Every plate count is a lower bound, because one clump of cells forms only one CFU.
Pause, copy the serial-dilution steps, the 30 to 300 reasoning and the CFU/mL formula into your book.
CFU/mL = colony count ÷ (volume plated × dilution _____).
The plating work you have just met is not a new invention. Isolating an organism from a sample and growing it in pure culture is step two of Koch's postulates from L03, and the agar plate is the tool that made it possible. When you streak a diluted water sample and read separate colonies, you are running Koch's isolation step, just for counting rather than for proving causation.
Koch's postulates test causation: isolate the suspected microorganism, reproduce the disease in a healthy susceptible host, then recover the same microorganism again.
Plate Counting, Colony Estimation
Design it so it is valid
Name your variables, justify both controls, and separate validity from reliability and accuracy. Then hunt errors in a flawed method.
BIO11/12-2 · variables, controls, validity vs reliability vs accuracy
We just saw the counting method. That raises a question: what makes a microbial test trustworthy? This card answers it → defining variables, justifying controls, and maximising validity and reliability.
A strong microbial investigation identifies its variables, justifies its controls, and is designed to maximise both validity and reliability.
The HSC requires you to be able to design and evaluate microbial testing investigations.
| 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
IV = water source; DV = colony count (CFU/mL); controlled = volume, agar, temperature, time. Negative control (sterile water) → 0 colonies; positive control (known culture) → growth. Validity = measures what it claims; reliability = consistent/repeatable; accuracy = close to true value. Use ≥3 replicates per dilution and average.
Pause, copy the IV/DV/controls and the validity vs reliability definitions into your book.
A negative control plate (sterile water only) should show:
Error Spotting, Flawed Investigation Design
Pattern B, Error Spotting
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.
- 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.
Valid Investigation Design, 7 Steps
Run it safely
BSL-1 risk assessment, PPE, aseptic handling and the disposal rules that are not negotiable.
BIO11/12-3 · BSL-1 organisms and standard precautions
We just saw how to design a valid microbial test. That raises a question: how do you run it safely? This card answers it → risk assessment and aseptic handling of cultures.
Any practical involving microorganisms requires a risk assessment, and used cultures must always be treated as potentially pathogenic.
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.
School microbial work is BSL-1, treat all cultures as potentially pathogenic. PPE: gloves, lab coat, safety glasses; no eating/drinking; no mouth pipetting. Disposal: autoclave (121°C/15 min) or 10% bleach (30 min); never reopen plates after incubation. Spills: cover with a 10% bleach-soaked towel for 20 min.
Pause, copy the PPE rules and the culture disposal methods into your book.
After incubation, used agar plates can be opened and thrown straight into the general waste bin.
Serial dilution is necessary before plating because concentrated samples would produce too many colonies to count accurately.
A colony-forming unit (CFU) on an agar plate always represents exactly one individual bacterial cell.
Across remote Aboriginal and Torres Strait Islander communities in Australia, access to safe drinking water has been a persistent public health challenge. A 2021 report by the Australian National University found that up to 40% of remote community water supplies had detectable E. coli at some point during the study period, indicating faecal contamination. In many cases, the water looked and tasted clean. The consequences are direct: gastroenteritis, particularly in children under five, is significantly more prevalent in remote communities than in urban Australia, and repeated gut infections in early childhood are linked to long-term developmental and growth impacts. The same microbial testing method used in a school laboratory, serial dilution and colony counting on selective media, is the primary tool used by environmental health officers to assess these water supplies. The difference between a safe and unsafe result is quantitative: fewer than 1 CFU of E. coli per 100 mL is safe; any detectable CFU/100 mL requires immediate action, typically a boil-water advisory issued to every household on the supply while the contamination source is traced and the water is treated or an alternative supply is arranged. You will apply this testing framework in Activity 1 and Short Answer Q3.
Consolidate before you practise
Clear the three traps examiners rely on, then copy the four summary blocks into your book.
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: below 30 random variation dominates, above 300 colonies merge and the count reads low.
- A plate count is a lower bound, one clump or chain of cells forms a single CFU.
- 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.
Assess the reliability and validity of a practical investigation testing water samples for microbial contamination.
The dot point instructs you to design and conduct a practical investigation, which cannot itself carry a written response. The examinable judgement is about the method, so it is examined with Assess — design → assess.
Plan: state the criteria, judge validity, judge reliability, judge the safety and control requirements, then reach a verdict.
The investigation will be assessed on whether the quantity measured answers the question asked (validity), on whether repeating the procedure would give consistent results (reliability), and on whether the controls and aseptic technique exclude contamination introduced by the experimenter.
Validity turns on what a colony count actually measures. Plating a water sample and counting colonies measures the number of viable culturable organisms able to grow on that medium at that temperature — not the total number of microorganisms, since many environmental species will not grow under those conditions, and not the number of pathogens, since most colonies will be harmless. The investigation is therefore valid as a measure of general microbial load and only indirectly informative about safety.
Validity is improved by testing for an indicator organism rather than for pathogens directly. Escherichia coli is used because it is abundant in faeces, easily cultured, and rarely persists long in clean water, so its presence indicates recent faecal contamination and therefore the possible presence of pathogens that are individually too rare and too hazardous to test for. This is an indirect measure, so it can mislead in both directions — a pathogen from a non-faecal source would be missed, and E. coli from a non-hazardous source would raise a false alarm.
Reliability requires replication at every level. A single plate per sample cannot distinguish a real difference from ordinary plating variation, so at least three plates per sample and a mean count are needed, along with serial dilution so that plates carry a countable number of colonies rather than a confluent lawn. Consistency between replicates supports reliability; wide scatter shows the result is not repeatable regardless of the apparent difference between samples.
Two controls are essential and both are frequently omitted. An uninoculated plate incubated alongside the others shows whether the medium or the technique introduced organisms, and a sterile-water plate shows the same for the sampling equipment. Aseptic technique throughout — flaming the loop, minimising plate exposure, sealing plates — is what makes the count attributable to the sample. Plates should also be incubated at a temperature that does not select for human pathogens and never opened after incubation, which is a safety requirement as much as a methodological one.
A replicated, controlled version of this investigation is reliable and valid as a comparison of microbial load between water sources, and weak evidence about whether a particular sample is safe to drink. Its usefulness therefore depends on wording the conclusion to match what was measured — that one source carried more culturable organisms than another, rather than that one is safe.
The investigation will be assessed on whether the quantity measured answers the question asked, on whether repeating it would give consistent results, and on whether controls and aseptic technique exclude contamination introduced by the experimenter. Validity turns on what a colony count measures: plating a water sample and counting colonies measures viable culturable organisms able to grow on that medium at that temperature — not the total number of microorganisms, since many environmental species will not grow under those conditions, and not the number of pathogens, since most colonies are harmless — so it is valid as a measure of general microbial load and only indirectly informative about safety. Validity improves by testing an indicator organism rather than pathogens directly: Escherichia coli is used because it is abundant in faeces, easily cultured and rarely persists in clean water, so its presence indicates recent faecal contamination and therefore possible pathogens too rare and too hazardous to test for individually — an indirect measure that can mislead both ways, missing a pathogen from a non-faecal source and raising a false alarm on harmless E. coli. Reliability requires replication at every level, since a single plate cannot separate a real difference from plating variation: at least three plates per sample with a mean count, and serial dilution so plates carry countable colonies rather than a confluent lawn, with consistency between replicates supporting reliability and wide scatter showing the result is not repeatable. Two controls are essential and frequently omitted — an uninoculated plate incubated alongside shows whether medium or technique introduced organisms, and a sterile-water plate shows the same for the equipment — while aseptic technique throughout makes the count attributable to the sample, and plates should be incubated at a temperature that does not select for human pathogens and never opened afterwards. A replicated, controlled version is therefore reliable and valid as a comparison of microbial load between sources and weak evidence about whether a sample is safe to drink, so its usefulness depends on wording the conclusion to match what was measured.
Explain why water quality testing uses indicator organisms rather than testing for pathogens directly.
Explain asks why the indirect method is preferred over the direct one, which is counter-intuitive — so the answer must show that the indirect method is better, not merely easier.
Plan: name the principle, take each reason as a linked pair, then close on what the approach cannot do.
The purpose of water testing is to decide whether water is safe, which means detecting the possibility of faecal contamination rather than confirming the presence of any particular organism. An indicator organism is chosen because it reliably accompanies that contamination, so its presence signals the hazard even when the specific pathogen is absent or undetected.
Pathogens are present in far smaller numbers than the harmless faecal organisms accompanying them, and they are distributed unevenly, so a sample of a litre may contain none even when the supply is contaminated. A negative pathogen test on such a sample would be reassuring and wrong. Escherichia coli, by contrast, is present in enormous numbers wherever faecal contamination has occurred, so it is detectable in a sample small enough to be practical.
Faecally contaminated water may carry bacteria, viruses and protozoan cysts, and each would require its own detection method, medium and expense. Testing for all of them is impractical, and testing for a few would miss the rest — whereas one indicator test covers the whole category, because whatever the pathogen, it arrived by the same route.
Culturing pathogens deliberately is hazardous and requires containment facilities, which routine and school testing do not have, and results are needed quickly enough to act on. E. coli grows overnight on inexpensive selective media at a temperature that does not favour dangerous organisms, so the test is safe to perform and fast enough to close a water supply before people drink from it.
The method's limitation follows from its logic. Because it detects faecal contamination rather than pathogens, it will miss a hazard that arrives by another route — a chemical contaminant, or a pathogen from a non-faecal source — and it will report contamination where E. coli is present without any pathogen. It is therefore a screening test whose negative result means faecal contamination is unlikely, not that the water is safe in every respect.
The purpose of water testing is to decide whether water is safe, which means detecting the possibility of faecal contamination rather than confirming any particular organism, and an indicator organism is chosen because it reliably accompanies that contamination, so its presence signals the hazard even when a specific pathogen is absent or undetected. Pathogens are present in far smaller numbers than the harmless faecal organisms accompanying them and are unevenly distributed, so a one-litre sample may contain none even when the supply is contaminated and a negative pathogen test would be reassuring and wrong — whereas Escherichia coli is present in enormous numbers wherever faecal contamination has occurred and is detectable in a practical sample. Faecally contaminated water may also carry bacteria, viruses and protozoan cysts, each needing its own method, medium and expense, so testing for all is impractical and testing for a few would miss the rest, while one indicator test covers the whole category because whatever the pathogen, it arrived by the same route. Culturing pathogens deliberately is additionally hazardous and requires containment that routine and school testing lack, and results are needed quickly enough to act on — E. coli grows overnight on inexpensive selective media at a temperature that does not favour dangerous organisms, so the test is safe and fast enough to close a supply before people drink from it. The limitation follows from the logic: because it detects faecal contamination rather than pathogens, it misses hazards arriving by another route, whether a chemical contaminant or a pathogen from a non-faecal source, and reports contamination where E. coli is present without any pathogen — so it is a screening test whose negative result means faecal contamination is unlikely, not that the water is safe in every respect.
Practise independently
Attempt at least one response in your own words. Model answers support checking; they do not replace the attempt.
A fresh set drawn from this lesson's question bank, feedback shown immediately. +5 XP per correct · +25 XP all correct
Pick your answer, then rate your confidence, that tells the system what to drill next.
UnderstandBand 3(3 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.
1 mark: serial dilution procedure correctly described · 1 mark: why dilution is necessary · 1 mark: why 30–300 range
EvaluateBand 5(3 marks) 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.
1 mark: first limitation linked to validity/reliability · 1 mark: second limitation linked to validity/reliability · 1 mark: overall evaluative statement
ApplyBand 5(4 marks) 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.
1 mark: result indicates water is unsafe · 1 mark: E. coli as faecal indicator · 1 mark: E. coli is practical to culture and count · 1 mark: appropriate immediate public health action
Show all answers
Multiple choice
MC answers and full explanations are shown inline as you complete each question. Use the retry button to attempt a fresh set from the lesson bank.
Short Answer Model Answers
Q1 (3 marks): 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, 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.
Q2 (3 marks): 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.
Q3 (4 marks): 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 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, 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.
Retrieve and reflect
Use the Review session first, take on the boss battle, then compare how your thinking changed.
A full module quiz covering every lesson in this module, not just this one. Set aside a decent block of time and treat it like a real assessment.
Start the module quiz →Face the boss using your knowledge of microbial testing methods and Koch's postulates. Pool: lessons 1–5.
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. The WHO's 2019 water safety report used the same threshold concept: any detection of E. coli in a 100 mL sample fails the safe drinking standard. Inadequate drinking water services are linked to an estimated 485,000 diarrhoeal deaths per year globally, caused by the full range of faecal-oral pathogens that an E. coli count is designed to flag. That number makes real what a colony count on an agar plate actually means.
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.