Recognition and Elimination of Contaminants
Stephen Pena, UTEX
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Importance:
o DNA work
o Isolation
o Competition for nutrients
Types of Contaminants
o Protists
 Rotifers – eat algae
 Some, like amoebas & paramecium, engulf algae
o Fungi
o Bacteria
o Mites – can contaminate cultures by carrying algae from flask to flask
o Artifacts
Testing for Contaminants
o Observation of culture with naked eye
 See fungus & mites
o Observation with microscope
 See bacteria
o Testing with different media (agar plates)
 LB-tryptone  peptones (provide essential amino acids) & vitamins
 Euglena Medium  beef extract, tryptone
 Trebouxia Medium  good for fungus; they like the glucose
 Sabouraud Dextrose Media  good for fungus since it contains dextrose
Removing Contaminants
o Mechanical cleaning – preferred at UTEX; won’t alter morphology
 Differential centrifugation – protocol used most at UTEX; great general protocol
to begin with
 Micropipette
o Chemical cleaning – last resort; can alter morphology or indirectly select for mutant
strains
 Antibiotics – ampicillin; triplicate antibiotics
(gentamycin/streptomycin/penicillin); also some against eukaryoties but those
are also harmful to people
 Fungicide – carbendazim in triplicate plates at 1 ug/3 ug/5 ug
Cleaning Contaminated Cultures
o Differential centrifugation
 Standard setup: 7-10 ml test tubes  6 test tubes filled approximately with 6
ml of sterile media and sterile blank tube used for inoculating plates. Can add
or reduce number of tubes as needed.
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Centrifuge at low speeds so the heavier cells, usually the ones wanted, have
barely sedimented.
 Usually at 1250 rpm/184 RCF for approximately 3 minutes.
 Range used is 340 rpm/13.5 RCF to 2010 rpm/482 RCF
Pellet is transferred to a new tube containing fresh sterile medium.
Minimum is FOUR washes
Use of sonicator, which produces shear forces that act on the surfaces of algal
cells, helping to dislodge attached microorganisms
Agar Plating
o Relatively easy way to separate contaminants from algae
 Alga MUST be able to be plated!
o Can also produce axenic cultures this way
o Mix antibiotics/fungicide into agar media
o May be able to exploit phototactic behavior of algae by having plate split into light/dark
areas
Problems That May Occur
o Some algae have a sheath
 Example of paper using Lysol Disinfecting Solution
o Contamination grows faster than algae
 Fungus/bacteria approximately 3 days for growth, while algae can be a month at
least
o Some algae do not plate on agar
 Media differences are the reason
o Some antibiotics are effective against cyanobacteria
o MORAL: Keep an axenic/clean backup stock. Sterile techniques are important.