lactic acid bacteria and propionic acid bacteria enrichments julie huang hopkins microbiology course...
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Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments
Julie HuangHopkins Microbiology Course
July 15, 2011
Lactic Acid Bacteria
General Features:– Gram +, acid-tolerant, halotolerant,
aerotolerant, rod or cocci
Metabolism:Glucose 2 pyruvate lactate
– HomofermentativeGlucose → 2 lactate
– HeterofermentativeGlucose → lactate + ethanol + carbon dioxide
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
After 4 Days of Fermentation
Streaked for isolation
starting pH: 6.5-7 ending pH: 3.5-5.5Smells like sauerkraut!
On glucose- CaCO3 plate
Lactic Acid Bacteria After Fermentation
LAB from final isolation (100x) ID: Leuconostoc? (ovoid cocci and often form chains)
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 metabolismWhite colonies- use the acrylate pathway (no B12)
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
Conditions for EnrichmentEnrichment 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
After 4 Days of Fermentation (cheese inoculum)
Brown and white colonies isolated from cheese
Lactate- CaCO3 plate
Propionic Acid Bacteria after Fermentation (cheese)
Mostly rods (100x)
Enrichment Using Mat Inoculum
Streak for
isolation
Propionic Acid Bacteria after Fermentation (mat)
White colonies- ovoid cocci (100x) Brown colonies-mostly rods (100x)
Catalase Test for LAB (-) and PAB (+)2 H2O2 → 2 H2O + O2
Before peroxide After peroxide
Thank you!