Contamination is one of the most common and disruptive problems in a microbiology lab. This free guide helps you identify the type of contamination you are dealing with — bacterial, fungal, phage, cross-contamination, or sterilisation failure — using a rapid symptom-based checker, and provides evidence-based causes, identification criteria, and remediation steps used by research labs, clinical settings, and industrial microbiology facilities.
⚠️ Diagnosis
How to Use the Lab Contamination Guide
Use the Contamination Type Checker above to quickly diagnose contamination based on where it appeared, what it looks like, and when it was noticed. The checker maps your symptom combination to the most likely contamination type and provides immediate recommended actions. The full visual reference guide below covers all major contamination types in detail.
Using the Contamination Checker
Select the location where contamination appeared (agar plate, broth, both, or uninoculated media), then describe the visual appearance of the contamination, and finally indicate the timing of onset. The three-parameter combination is cross-referenced against a database of contamination patterns to return the most probable diagnosis. If your combination is not matched, a set of general troubleshooting recommendations is provided instead.
Understanding the Five Major Contamination Types
Fungal / Mould Contamination is the most common contaminant in standard microbiology labs. Airborne fungal spores settle on plates and media during pouring and streaking and germinate into visible fluffy or powdery growth over 2–5 days. The presence of coloured growth (green, black, orange) is strongly indicative of mould. Primary prevention is working inside a biosafety cabinet (BSC) at all times when handling open plates or media.
Bacterial Contamination introduces foreign bacteria into a pure culture or sterile medium. It manifests as unexpected colony morphologies on plates (different size, edge, or pigmentation from the expected strain) or turbidity in broth controls. Poor aseptic technique, contaminated pipette tips, improperly flamed loops, or leaving tubes open too long are the most common root causes.
Bacteriophage Contamination is the most difficult contamination to eliminate because phage particles are extraordinarily small, are not inactivated by standard ethanol wiping, and can persist on surfaces. A phage event is characterised by cultures that grow initially and then clear suddenly. Immediate aggressive decontamination with SDS or bleach is essential, and all liquid waste must be autoclaved before disposal.
Autoclave / Media Sterilisation Failure is detected by growth in uninoculated media controls. This indicates the problem is upstream of your technique — the media itself was never sterile. The entire batch must be discarded and the autoclave must be validated with indicator strips before any further media preparation.
Cross-Contamination Between Strains is identified when two distinct colony morphotypes appear on the same plate, or when PCR or sequencing results show an unexpected profile. Working with two strains open at the same time, using unlabelled equipment, or failing to re-streak from glycerol stocks are common causes.
When to Use This Guide
Use this guide any time you observe unexpected growth, culture behaviour, or experimental anomalies that suggest the presence of a contaminant. It is particularly valuable when troubleshooting recurring contamination events, training new lab members in contamination recognition, auditing aseptic technique after a failed experiment, or preparing standard operating procedures (SOPs) for contamination prevention and response.
Common Mistakes That Lead to Contamination
1. Pouring plates on an open bench. Even briefly, an open plate in still lab air collects fungal spores. Always pour inside a laminar flow BSC or directly next to a flame.
2. Skipping blank media controls. Without an uninoculated control run alongside your culture, autoclave and media failures go undetected until experimental data is already compromised.
3. Not maintaining glycerol stocks. When contamination occurs, recovery depends on having a verified clean frozen stock to re-streak from. Labs that do not maintain −80°C stocks risk losing strains permanently after a contamination event.
General Contamination Prevention Checklist
- Aseptic technique: Always work near a flame or inside a biosafety cabinet (BSC).
- Personal hygiene: Wash hands, wear gloves and lab coat. Change gloves between strains.
- Surface decontamination: Wipe bench with 70% ethanol before and after every experiment.
- Controls: Always include uninoculated media controls to detect media/autoclave failures.
- Labelling: Label every tube, plate, and flask with strain name, date, and antibiotic resistance.
- Incubator hygiene: Clean incubators monthly with 70% ethanol. Do not store spilled cultures.
- Waste disposal: Autoclave all liquid waste and contaminated solid waste before disposal.
- Glycerol stocks: Always maintain backup glycerol stocks at −80°C for all important strains.
Frequently Asked Questions
How do I tell the difference between fungal and bacterial contamination on an agar plate?
Fungal contamination appears as fluffy, woolly, or powdery growth that may be white, grey, green, black, or orange, and spreads rapidly over 2–5 days with visible aerial mycelium rising above the agar. Bacterial contamination produces discrete smooth, slimy, or mucoid colonies that are opaque, more uniform in shape, and typically appear within 12–24 hours at standard incubation temperatures. Under a stereo microscope, fungal colonies show thread-like hyphal structures while bacterial colonies appear dense and granular.
What causes a bacterial broth culture to suddenly clear after growing well?
A culture that reaches visible turbidity and then rapidly clears is the classic hallmark of bacteriophage lysis. Lytic phages infect bacterial cells, replicate inside them, and lyse the cells to release new phage particles, clearing the broth within hours. This is distinct from bacterial contamination, which causes increased turbidity rather than clearing. If OD600 values rise and then drop sharply, or if this pattern recurs across multiple cultures in the same lab area, a phage contamination event is highly likely. Decontaminate immediately with 1% SDS or 10% bleach and autoclave all liquid waste before disposal.
What should I do if my uninoculated (blank) media control shows growth?
Growth in uninoculated media controls indicates a sterilisation failure, not a technique problem. The most common cause is an incomplete autoclave cycle — incorrect temperature (should be 121°C), insufficient pressure (15 psi), too short a run time (20–30 min), or an overloaded chamber that prevented steam penetration. Discard the entire media batch and run a biological indicator (Geobacillus stearothermophilus spore strip) to validate the autoclave before preparing any new media.
How can I prevent fungal contamination when pouring agar plates?
Fungal spores are ubiquitous in lab air and settle readily on open plates during pouring. The most effective prevention is to pour plates inside a laminar flow biosafety cabinet. If a BSC is unavailable, work quickly near a Bunsen burner flame to create an upward convection current. Allow molten agar to cool to approximately 50°C before pouring to minimise condensation on lids, and seal long-term incubation plates with parafilm. Consider adding cycloheximide (50–100 µg/mL) to media when culturing bacteria that are not sensitive to it.
How do I confirm that my pure culture has become cross-contaminated with a second strain?
Cross-contamination is confirmed by observing two distinct colony morphologies on the same agar plate — differences in size, shape, colour, opacity, or edge texture. Streak single colonies of each morphotype separately onto fresh selective plates, then verify strain identity by colony PCR using strain-specific primers or 16S rRNA sequencing. Cross-contamination can also manifest as unexpected growth on antibiotic plates the target strain should be sensitive to, or anomalous phenotypic results in a functional assay. Always maintain glycerol stocks at −80°C so you can re-streak a verified pure culture if cross-contamination is confirmed.