Mice were euthanized at the indicated time points. Lungs were removed aseptically and homogenized in 1 ml of mycoplasma broth with glass homogenizers (Sankyo Co., Ltd., Tokyo, Japan) . Ten-fold serial dilutions were prepared and an aliquot of 10 µl of each dilution was plated onto PPLO agar medium, which was made by dissolving 35 g of PPLO agar (Becton Dickinson and Company, Sparks, MD, USA) with 750 ml of distilled water, autoclaved for 15 min, allowed to cool at 52-54 ˚C, and then 150 ml of heat-inactivated horse serum (GIBCO), 100 ml of 25% fresh yeast extracts (Oriental Yeast), and 1,000,000 units of ampicillin sodium salt (Sigma) were added. The total number of colony-forming unit (CFU) per lung from each animal was determined under a stereomicroscope after incubation for 10 days at 37˚C in an incubator with 5% CO2.
Pplo agar
Manufactured by BD
Sourced in United States
PPLO agar is a microbiological culture medium used for the isolation and cultivation of Mycoplasma species. It provides essential nutrients and growth factors required for the growth of these fastidious bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Glucose, by Avantor (1 mentions)
Horse serum, by Merck Group (1 mentions)
β nad, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Lab M (1 mentions)
Ampicillin sodium salt, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Heat inactivated horse serum, by Thermo Fisher Scientific (1 mentions)
3 protocols using pplo agar
1
Quantifying Mycoplasma Lung Infection in Mice
2
Characterization of Moderate Virulent Hps
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Strain of Hps, previously isolated in National Veterinary Research Institute was selected for the experimental infections (isolate PIWetHps192/2015). Strain originating from lung of pig from Polish herd and the analysis of the 16S rRNA gene sequences [33 (link)] revealed 99% similarity to Hps isolate CN9–2 described by Olvera et al. [34 ] (classified as moderate virulent serovar 15).
To prepare the inoculum, the strain was streaked onto a pleuropneumonia-like organism (PPLO) agar (Becton Dickinson, USA), supplemented with 10 μg/ml of β-NAD (Sigma-Aldrich, Germany), 1 mg/ml glucose (Avantor Performance Materials, Poland) and 5% horse serum (Sigma-Aldrich, Germany), which was incubated for 24 h at 37 °C in an atmosphere of 8% CO2. Colonies were harvested and suspended in phosphate-buffered saline (PBS) (Life Technologies, USA) to 0.5 McFarland turbidity (which corresponds with approximately 1.5 × 108 colony forming units (CFU)/ml). A plate count was also performed to quantify the accurate number of viable bacteria (final result 1.2 × 108 CFU/ml of Hps strain).
To prepare the inoculum, the strain was streaked onto a pleuropneumonia-like organism (PPLO) agar (Becton Dickinson, USA), supplemented with 10 μg/ml of β-NAD (Sigma-Aldrich, Germany), 1 mg/ml glucose (Avantor Performance Materials, Poland) and 5% horse serum (Sigma-Aldrich, Germany), which was incubated for 24 h at 37 °C in an atmosphere of 8% CO2. Colonies were harvested and suspended in phosphate-buffered saline (PBS) (Life Technologies, USA) to 0.5 McFarland turbidity (which corresponds with approximately 1.5 × 108 colony forming units (CFU)/ml). A plate count was also performed to quantify the accurate number of viable bacteria (final result 1.2 × 108 CFU/ml of Hps strain).
3
Sterilization of Montanide ISA 70 Adjuvant
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According to Bekele and Assefa (2018) , sterilization of the oil adjuvant (Montanide ISA 70) used in the immunization was achieved using a dry autoclave at 160°C for 1 h. These experiments were conducted at 37°C over a period of 7 d using thioglycolate (Lab M Limited), tryptic soy broth (Lab M Limited), tryptose agar (Lab M Limited), Sabouraud agar (Lab M Limited) and PPLO agar (Becton Dickinson). After the validation of the growth inactivation procedure, Montanide oil adjuvant was used to sufficiently emulsify the bacterial biomass. As a final step, the inactivated culture was sterilized and rendered safe.
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