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Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments

Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments. Julie Huang Hopkins Microbiology Course July 15, 2011. Lactic Acid Bacteria. General Features: Gram +, acid-tolerant, halotolerant, aerotolerant, rod or cocci Metabolism: Glucose  2 pyruvate  lactate Homofermentative

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Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments

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  1. Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments Julie Huang Hopkins Microbiology Course July 15, 2011

  2. Lactic Acid Bacteria General Features: • Gram +, acid-tolerant, halotolerant, aerotolerant, rod or cocci Metabolism: Glucose  2 pyruvate  lactate • Homofermentative Glucose → 2 lactate • Heterofermentative Glucose → lactate + ethanol + carbon dioxide

  3. Conditions for Enrichment Enrichment from sauerkraut • High salinity- added 3% (w/w) NaCl to cabbage • Anaerobic- added H20 and compressed cabbage layers • Incubate jars at 30°C Isolation on agar plate • Glucose and CaCO3 plates • Dissolution of CaCO3

  4. After 4 Days of Fermentation Streaked for isolation On glucose- CaCO3 plate starting pH: 6.5-7 ending pH: 3.5-5.5 Smells like sauerkraut!

  5. Lactic Acid Bacteria After Fermentation LAB from final isolation (100x) ID: Leuconostoc? (ovoid cocci and often form chains)

  6. Propionic Acid Bacteria General Features: Gram +, non-motile, non-sporeforming, anaerobic, rod Metabolism: Lactate  propionate + acetate + carbon dioxide Brown colonies- requires B12 as part of their metabolism White colonies- use the acrylate pathway (no B12)

  7. Conditions for Enrichment Enrichment from Swiss Cheese • Fill screw capped tube with swiss cheese • Add lactate yeast extract to cheese • Bubble CO2 to bottom of tube for 30 seconds • Incubate at 30°C in the dark

  8. Conditions for Enrichment Enrichment from Microbial Mat Why? • Found evidence of lactate dehydrogenase in transcriptome study • Found propionate, acetate, and formate, but not lactate in the mat • Cyanobacteria ferment at night when competition for oxygen is high Inoculum • Area underneath the cyanobacteria layer

  9. After 4 Days of Fermentation (cheese inoculum) Brown and white colonies isolated from cheese Lactate- CaCO3 plate

  10. Propionic Acid Bacteria after Fermentation (cheese) Mostly rods (100x)

  11. Enrichment Using Mat Inoculum Streak for isolation

  12. Propionic Acid Bacteria after Fermentation (mat) White colonies- ovoid cocci (100x) Brown colonies-mostly rods (100x)

  13. Catalase Test for LAB (-) and PAB (+) 2 H2O2 → 2 H2O + O2 Before peroxide After peroxide

  14. Thank you!

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