BLOGS > IN VITRO EVALUATION OF OLUS® AC34 AGAINST SALMONELLA AND E. COLI

Study methods

• Laboratory: The study was conducted by the Institute of Research and Community Outreach (IRCOH), Sargodha, Pakistan, under the supervision of Prof. Dr. Zafar Hayat.
• Report date:12 May 2026.
• Test organisms: The antimicrobial activity of Olus® AC34 was evaluated against E. coli (Avian Pathogenic E. coli, APEC) and Salmonella (Salmonella gallinarum).
• Assays: Agar well diffusion, qualitative bactericidal recovery assessment, and time-kill assessment.
• Controls: A positive growth control containing the test organism without Olus® AC34, an uninoculated negative control, and inoculum verification to confirm the starting bacterial density. Test samples containing Olus® AC34 were evaluated separately at the specified concentrations, allowing bacterial growth in treated samples to be compared with normal growth in the positive control.

Concentration-dependent inhibition

In the agar well diffusion assay, inhibition increased as the concentration of Olus® AC34 increased. At 100 µL/mL, the mean inhibition zone measured 27.67 mm for Salmonella and 21.67 mm for E. coli. At 50 µL/mL, the corresponding zones were 21.00 and 15.67 mm, and at 25 µL/mL they were 12.67 and 12.00 mm. No inhibition was observed at 12.5 µL/mL for either organism.

The four tested laboratory concentrations were:

• 100 µL/mL
• 50 µL/mL
• 25 µL/mL
• 12.5 µL/mL

No inhibition was observed at 12.5 µL/mL for either organism.

E. coli (left) and Salmonella (right) agar well diffusion plates across tested concentrations.

The concentration-dependent trend shows that product concentration influenced bacterial inhibition under the conditions of the agar diffusion assay. Zone diameter in this type of assay is also influenced by how a substance diffuses through agar, so comparisons are only meaningful when method, organisms, media, concentrations, and incubation conditions are equivalent.

The qualitative bactericidal recovery assessment provided an additional perspective. Weak or partial bactericidal activity was observed in one of three replicates for each organism, while the other replicates showed no bactericidal effect.

Change in bacterial counts over time

The time-kill curves tracked viable bacterial counts over 24 hours at concentrations described as 1X, 2X, and 4X the apparent MIC, with measurements at 0, 2, 4, 6, 8, and 24 hours.

The laboratory preparations consisted of 1 mL of distilled water combined with 1, 2, or 4 mL of Olus® AC34 for the 1X, 2X, and 4X preparations, respectively. In the technical follow-up supplied by Dr. Kashif Waqas, these were described as corresponding to 1, 2, and 4 L of Olus® AC34 per 1,000 L of water, equivalent to 1, 2, and 4 mL/L.

An initial response was already measurable at the first 2-hour measurement, with viable bacterial counts declining across all tested preparations.

For comparison, the commercial recommended dose is 0.5 to 1.0 mL/L, equivalent to 500 to 1,000 mL per 1,000 L. The 1X time-kill concentration therefore corresponds to the upper end of the commercial range, while the 2X and 4X preparations are higher. The time-kill assay used one initial exposure, whereas commercial application supplies the product continuously.

For Salmonella, counts declined at all three concentrations through 8 hours. The greatest reduction was observed at 8 hours, reaching 0.42 log10 CFU/mL in the 4X preparation. At 24 hours, counts remained below their respective starting levels, with reductions of approximately 0.26 to 0.28 log10 CFU/mL across the three preparations.

For E. coli, counts declined through approximately 6 hours. The largest reduction was 0.32 log10 CFU/mL at 6 hours. Counts then increased again and, by 24 hours, were approximately 0.19 to 0.20 log10 CFU/mL above their respective starting values.

One possible explanation proposed in the technical follow-up is that the effectively available product concentration may decrease during incubation as active molecules interact with bacterial membranes or other components in the broth. This mechanism was not measured in the study.

What this means in practice

Across the four assessments, Olus® AC34 showed concentration-dependent inhibitory activity against both organisms, with a stronger inhibitory response observed for Salmonella under the tested conditions.

• The time-kill data showed measurable changes from the first 2-hour measurement.
• The time-kill dose comparison provides a practical reference for interpreting the laboratory data alongside commercial use: 1X corresponds to the upper end of the commercial recommendation, while 2X and 4X are higher. The application pattern also differs, with one initial exposure in the time-kill assay versus continuous product supply in commercial use.
• Under field conditions, the recommended application rate for Olus® AC34 is 0.5 to 1.0 mL per liter of drinking water (500 to 1,000 mL per 1,000 liters), administered continuously over a 12- to 16-hour period.

At a glance

• Concentration-dependent response: inhibition increased as the tested Olus® AC34 concentration increased.
• Organism-specific response: Salmonella showed larger inhibition zones than E. coli at the concentrations where inhibition was observed.
• Early response: viable bacterial counts were already declining at the first 2-hour measurement.
• Different time-kill patterns: Salmonella counts remained below their starting levels at 24 hours, while E. coli showed regrowth after the initial decline.

Together, these results provide a useful technical evidence layer for Olus® AC34 and a basis for further application-specific research.

Interpretation note

These findings are based on in vitro testing under controlled laboratory conditions and should be interpreted as formulation-level evidence rather than proof of efficacy in poultry or under commercial conditions. The report does not fully specify whether the reported replicates were biological or technical, what the reported variability values represent, or which statistical analysis was applied. Application-specific and in vivo studies are needed to confirm practical relevance.

Evidence note

Source: In vitro antimicrobial activity report for Olus® AC34, Institute of Research and Community Outreach (IRCOH), Sargodha, Pakistan (May 12, 2026).

Scientific context: Appleton et al. (2024), Use of Monoglycerides and Diglycerides to Mitigate Poultry Production Losses.

About the author

Dr Kashif Waqas – Regional Manager | Southeast Asia

Dr. Kashif is a qualified veterinarian with specialized expertise in monoglycerides, emulsifiers, phytobiotics and their application in animal nutrition and health. With a strong scientific approach and practical industry experience, he focuses on delivering effective, innovative solutions that enhance gut health, performance, and sustainability in livestock and aquaculture. His work bridges research and field application, supporting the advancement of modern animal production systems. 

Reach out to Dr. Kashif for guidance on integrated poultry gut health and nutritional management strategies.