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!