bacterial culture fundamentals how to address common
TRANSCRIPT
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Credible Leads to Incredible™
Bacterial Culture Fundamentals: How to Address Common Propagation ChallengesNancy Krueger, MSSenior Biologist, ATCC
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About ATCC
Founded in 1925, ATCC is a non-profit organization with HQ in Manassas, VA, and an R&D and Services center in Gaithersburg, MD
World’s largest, most diverse biological materials and information resource for bacterial culture – the “gold standard”
Innovative R&D company featuring gene editing, microbiome, NGS, advanced models
cGMP biorepository
Partner with government, industry, and academia
Leading global supplier of authenticated cell lines, viral and microbial standards
Sales and distribution in 150 countries, 19 international distributors
Talented team of 450+ employees, over one-third with advanced degrees
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Agenda
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1. Propagation Methods2. Nutritional and Atmospheric
Considerations3. New bacterial isolates4. Troubleshooting5. Quality Control Testing
Actinocrinis puniceicyclus (ATCC® BAA-2771™)
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Propagation Methods
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Starting a culture from a preserved state
Freeze-dried−Batch vials−Serum vials
Frozen−Cryovials−Mini’s
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Propagation Methods
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Basic Method Use a single tube of broth: 5 to 6 mL Rehydrate the entire pellet Aseptically transfer this aliquot back into
the broth tube and mix well Inoculate a secondary broth Inoculate an agar slant and/or plate Incubate all tubes and plate
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Propagation Methods
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Open a batch vial
Product Information Sheet−Includes specific detail for the item−Follows the procedure tested at ATCC−Contact Customer Care if there isn’t one available
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Propagation Methods
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Open a batch vial
Considerations for recovery−Typical cell count−Thaw – refreeze−Over dilution
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Agenda
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1. Propagation Methods2. Nutritional and Atmospheric
Considerations3. New bacterial isolates4. Troubleshooting5. Quality Control Testing
Actinocrinis puniceicyclus (ATCC® BAA-2771™)
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Nutritional Requirements
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Common growth media−Nutrient−Tryptic Soy−LB
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Nutrient rich media−Chocolate (GC)−Bordet-Gengou−Buffered Charcoal Yeast Extract (CYE)−Brucella
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Facultative
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This group includes:EscherichiaSerratiaKlebsiellaEnterobacter
Gram-negative Generate ATP by aerobic respiration in
the presence of oxygen. Switch to fermentation in its absence. Ferment carbohydrates Many are pathogenic
Klebsiella pneumoniae (ATCC® BAA-1898™)MacConkey agar, positive for lactose fermentation
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Non-Enteric
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This group includes: PseudomonasAcinetobacterBurkholderia
Gram-negative Large diverse group Found in a wide variety of habitats Can be opportunistic pathogens Do not ferment carbohydrates
Pseudomonas chlororaphis subsp. chlororaphis(ATCC® 9446™)Pseudomonas F agar, positive for fluorescence
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Fastidious
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This group includesBordetellaMoraxellaFrancisella
Gram-negative Slow-growing Have complex or specific nutritional
requirements May require additional CO2
Bordetella pertussis (ATCC® 9306™)Bordet-Gengou agar
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Nutritional Requirements
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Supplements
Bases for blood agar media: −Brucella−Columbia based blood agar−Tryptic soy based blood agar−Brain heart infusion w/ 0.5% yeast extract
Supplements to enhance growth: −5% sheep, horse, or rabbit blood−Vitamin K1 (1 µg/mL)−Hemin (5 µg/mL)−Fetal Bovine Serum (ATCC® 30-2020™)−Horse serum
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Atmospheric Conditions
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Aerobic/Ambient Microaerophilic or Anaerobic−Automatic jar system−Jars and gas generating sachets−5% CO2 Incubators
Anaerobic−Anaerobic Chamber−Needle and gas exchange
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Extremophiles
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What are extremophiles?−Thermophile – 45°C and up−Psychrophile – 15°C and below−Halophile – high salt concentration−Acidophile – pH 3.0 or below−Alkaliphile – pH 9.0 or above
These are just a few examples
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Extremophiles
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Methanosarcina barkeri (ATCC® BAA-2329™)−Grows in a gas mixture of 80% H2 - 20% CO2 (5 PSI)−Requires the use of Hungateor Balch tubes−Growth inhibited by both nitrogen and oxygen
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Microaerophilic
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This group includes:HelicobacterNeisseriaCampylobacter
Require a reduced oxygen concentration to grow. Require nutrient rich media. May grow best in a biphasic
environment
Campylobacter hyointestinalis (ATCC® 35217™)Biphasic growth
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Microaerophilic
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Magnetospirillum magnetotacticum−Unusual microaerophile−Produces magnetite−Highly motile
Magnetospirillum magnetotacticumImage courtesy of https://en.wikipedia.org/wiki/Magnetospirillum
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Agenda
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1. Propagation Methods2. Nutritional and Atmospheric
Considerations3. New bacterial isolates4. Troubleshooting5. Quality Control Testing
Actinocrinis puniceicyclus (ATCC® BAA-2771™)
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New Bacterial Isolates
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Unique characteristics May require specific medium May have other requirements−Light/dark−Shaking−Atmosphere−Additives
When depositing at ATCC−Provide details−Specific formulations
Cytophaga hutchinsonii Winogradsky(ATCC® 33406™)
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Agenda
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1. Propagation Methods2. Nutritional and Atmospheric
Considerations3. New bacterial isolates4. Troubleshooting5. Quality Control Testing
Actinocrinis puniceicyclus (ATCC® BAA-2771™)
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Troubleshooting
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Chromobacterium violaceum Bergonzini(ATCC® 31532™)
Antibiotic susceptibility test strips
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More Troubleshooting
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Medium−Components−Commercially-made medium
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Mollicutes
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Mycoplasma and Ureaplasma−Smallest bacteria−Depend on their hosts for nutrition−Lack a cell wall−Do not grow well on common media−Sensitive to overgrowth
Why they are so challenging
Mycoplasma hyopneumoniae (ATCC® 25095™)
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Mollicutes
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Grow initial cultures in a serial dilution Transfer every 24 hours Most do not grow well on agar
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Agenda
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1. Propagation Methods2. Nutritional and Atmospheric
Considerations3. New bacterial isolates4. Troubleshooting5. Quality Control Testing
Actinocrinis puniceicyclus (ATCC® BAA-2771™)
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Quality Control
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Viability Purity Sequencing Phenotypic testing Additional tests
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Conclusion
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• Follow a standard propagation method
• Use appropriate medium, growth conditions and length of incubation
• Consider specific techniques necessary for specialized strains.
Gloeothece sp. (ATCC® 27152™)
Porphyromonas catoniae(ATCC® 51270™)
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From Our Lab To Yours
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Safety Tips for Returning to the LabTo help you get back to work, we have put together a free kit containing best practices to help keep your lab infection-free: Poster Infographic DecalsOrder the kit here:
www.atcc.org/backtothelab
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