SOP: Lactic Acid Bacteria Count
Lactic acid bacteria are the good guys in yogurt, cheese, and fermented sausages, but the same organisms spoil beer and vacuum-packed meats. Counting them tells the fermenter whether the culture is thriving and the brewer whether contamination crept in.
Microbiological testing protects consumers from pathogens and tracks hygiene across the plant. Because living organisms are the analyte, aseptic technique and precise incubation conditions are not optional extras; they are the measurement itself. A single lapse in sterility or temperature control invalidates the result.
Purpose
To enumerate lactic acid bacteria by selective plating for culture monitoring and spoilage assessment.
Scope
Applies to dairy cultures, fermented meats, beverages, and vacuum-packed products.
Equipment and Materials
- de Man Rogosa Sharpe (MRS) agar
- Anaerobic jars or overlay technique materials
- Incubator at 30 C (or 37 C for thermophilic cultures)
- Stomacher, diluent blanks, pipettes
- Colony counter
Procedure
- Prepare the 1:10 homogenate and decimal dilutions.
- Pour 1 mL of dilutions into duplicate plates with MRS agar, or spread 0.1 mL.
- For the pour plate method, overlay with a thin layer of MRS agar after solidification to create microaerophilic conditions.
- Incubate at 30 C for 48 to 72 hours (use 37 C for yogurt thermophilic cultures).
- Count white to cream colonies, typically 1 to 2 mm.
- Confirm representative colonies as Gram-positive rods or cocci, catalase negative, if required.
Recording Results
Report CFU per g or per mL to two significant figures, noting the incubation temperature. For starter cultures, compare against the supplier specification.
Quality Control
Verify each new lot of culture media with reference strains before use: the target organism must show typical growth and morphology, and a non-target strain must be inhibited or show atypical growth as expected. Include a positive control (reference strain) and a negative control (sterile diluent) with every test batch. Incubate an uninoculated plate from each poured batch as a sterility check. Monitor incubator temperatures twice daily with a calibrated thermometer; do not use incubators found outside tolerance until corrected and re-verified. Track contamination rates in blanks; a rising blank contamination rate signals a systemic problem requiring investigation. Participate in proficiency testing at least annually and investigate any unsatisfactory scores with documented corrective action.
Troubleshooting
- Low counts on active cultures: MRS pH or incubation temperature wrong for the strain. Check the culture supplier conditions.
- Spreading colonies: plates too wet. Dry plates before use or use the overlay method.
- No growth: the agar was poured too hot or the culture needs anaerobic conditions. Verify tempering and try anaerobic jars.
- Incubator temperature alarm overnight: assess all plates incubated during the excursion; document the impact decision and repeat affected tests.
- Reference strain atypical: the culture may have mutated or been mislabeled. Obtain a fresh certified strain and re-verify media.
Interferences and Limitations
Injured or stressed cells may fail to grow on selective media, causing false negatives; the resuscitation steps in this procedure address this but cannot recover all injured populations. Viable-but-nonculturable organisms are not detected by culture methods. High background flora can mask target colonies on plating media, which is why confirmation of suspect colonies is mandatory rather than optional. Antimicrobial residues in the sample (sanitizers, preservatives) suppress growth and must be neutralized during homogenization. Results apply to the analytical unit tested; heterogeneous contamination requires larger or composite sampling plans.
Safety Notes
- Generally low hazard (Biosafety Level 1), but treat unknown isolates with standard precautions.
- Autoclave plates before disposal.