PPLO Agar- Introduction, Composition, Principle, Preparation

Introduction

Table of Contents

PPLO (Pleuropneumonia-Like Organisms) Agar is a specialized growth medium used primarily for the cultivation of Mycoplasma. Mycoplasma species are unique prokaryotes that lack a cell wall and, therefore, have specific nutritional requirements that are provided by the PPLO agar.

Components of PPLO Agar:

Peptone and Yeast Extract: These are primary sources of essential amino acids, vitamins, and other nutrients required for Mycoplasma growth.
Horse Serum: Typically added as a supplement, horse serum provides additional growth factors and nutrients that benefit Mycoplasma growth.
Glucose: Serves as a primary carbon and energy source for the growing microorganisms.
Phenol Red: A pH indicator that changes color based on the acidity of the medium. This is useful for detecting acid production from glucose fermentation, a characteristic of some Mycoplasma species.
Penicillin: Sometimes added to suppress the growth of bacteria that might contaminate the culture.
Agar: Provides a solid matrix for the medium, although liquid formulations (PPLO broth) without agar are also common for Mycoplasma cultivation.

Characteristics and Uses:

Selective Growth: Since Mycoplasma spp. don’t possess a cell wall, they are not susceptible to penicillin (which targets cell wall synthesis). By adding penicillin to the medium, other contaminating bacteria are inhibited, allowing selective growth of Mycoplasma.
Differential Growth: The phenol red in the medium can help in differentiation. If a Mycoplasma species ferments glucose, it will produce acid, leading to a color change in the medium.
Clinical and Veterinary Importance: They are significant in both human and veterinary medicine. For instance, Mycoplasma pneumoniae is a known causative agent of atypical pneumonia in humans.

When cultivating Mycoplasma species or working with PPLO agar, it’s crucial to note that they are fastidious organisms. They have specific growth requirements and may take longer to grow compared to other bacteria. As always, when working in a microbiological lab, following safety and aseptic techniques is of utmost importance.

Composition

PPLO Agar is specially formulated to cater to the nutritional requirements of Mycoplasma. Here’s a general breakdown of the components commonly found in PPLO Agar:

Proteose Peptone or Peptone: It’s a source of carbon, nitrogen, vitamins, and minerals.
Sodium Chloride (NaCl): Helps maintain the osmotic balance.
Yeast Extract: Provides the B-complex vitamins required by Mycoplasma for growth.
Glucose: Acts as a carbon source and energy for the microorganisms.
Horse Serum (often added to enhance growth): Offers additional nutrients and growth factors.
Phenol Red: A pH indicator for detecting glucose fermentation. If Mycoplasma ferments glucose, they produce acids, causing the medium to change color.
Agar: The solidifying agent. It gives the medium its gel-like consistency.
Penicillin (optional): Sometimes included to inhibit the growth of gram-positive and gram-negative bacteria, making the medium more selective for Mycoplasma.
Distilled Water: The solvent for all the components.

When preparing the medium, each component is weighed out as per the desired concentration, dissolved in distilled water, and sterilized, usually by autoclaving. After sterilization, temperature-sensitive supplements like horse serum or antibiotics can be added when the medium has cooled to an appropriate temperature.

Keep in mind that there are variations in the formulation based on specific needs or strains being cultivated, so always refer to the specific recipe or protocol you are using.

Principle

The principle of PPLO (Pleuropneumonia-Like Organisms) Agar revolves around providing the specific and unique nutritional needs of Mycoplasma , facilitating their growth, while potentially suppressing unwanted contaminants.

They are distinct from other bacteria in several ways, most notably by their lack of a cell wall. This absence means they have particular growth requirements and also that they are resistant to antibiotics that target cell wall synthesis, such as penicillin.

The core principles behind PPLO Agar are:

Nutritional Support: The ingredients in PPLO Agar are carefully chosen to meet the specific nutritional needs of Mycoplasma species. Components such as peptone, yeast extract, and glucose offer the essential amino acids, vitamins, minerals, and energy sources required for their growth.
Selective Growth: The potential addition of penicillin to the medium exploits the unique characteristic of Mycoplasma lacking a cell wall. Penicillin targets cell wall synthesis. Therefore, it inhibits many other bacteria but doesn’t affect Mycoplasma, giving them a selective advantage on this medium.
Differential Aspect: The presence of phenol red, a pH indicator, in the medium can help in distinguishing Mycoplasma species based on their ability to ferment glucose. Those that can ferment glucose produce acid, leading to a change in the color of the medium.
Protection from Osmotic Lysis: Due to their lack of a cell wall, They are more susceptible to osmotic changes. The sodium chloride (NaCl) in the medium helps in maintaining osmotic balance, protecting the Mycoplasma from lysis.
Solidifying Agent: Agar serves as a solidifying agent, allowing Mycoplasma to grow on a solid substrate. This is especially useful for isolation techniques.

Preparation Requirements

The preparation of PPLO Agar involves careful measurement and combination of its ingredients, followed by sterilization and, if necessary, the addition of temperature-sensitive components post-sterilization. Below are the requirements and steps for preparing PPLO Agar:

Requirements:

Ingredients:
- Proteose peptone or peptone
- Sodium Chloride (NaCl)
- Yeast Extract
- Glucose
- Phenol Red
- Agar
- Penicillin (optional)
- Distilled water
Supplements (if necessary):
- Horse Serum: Often added post-sterilization to enhance Mycoplasma growth.
Equipment and Utensils:
- Weighing balance and weighing papers or boats
- Measuring cylinders or pipettes
- pH meter (for adjusting and checking the pH if necessary)
- Autoclave (for sterilizing the prepared medium)
- Magnetic stirrer or a manual stirring rod
- Sterile containers or flasks for holding the medium
- Thermometer
- Sterile filter (if filtration is used as a sterilization method for some components)

Preparation

Here’s a standard protocol for preparing PPLO (Pleuropneumonia-Like Organisms) Agar:

Ingredients for 1 liter of PPLO Agar:

Proteose Peptone: 10 g
Sodium Chloride (NaCl): 5 g
Yeast Extract: 2 g
Glucose: 1 g
Phenol Red: 0.3 g
Agar: 15 g
Distilled water: up to 1 liter

Optional components (based on need):

Penicillin
Horse Serum

Procedure:

Weighing and Dissolution:
- Measure out each of the dry ingredients using a balance.
- In a suitable container, dissolve the dry components in about 800 ml of distilled water, using a magnetic stirrer or stirring rod.
Adjustment of pH:
- Check the pH of the solution. For PPLO Agar, the pH should typically be adjusted to 7.8 – 8.0.
- If necessary, adjust the pH using 1N NaOH or 1N HCl.
Volume Adjustment:
- Once the pH is adjusted, make up the volume to 1 liter with distilled water.
Sterilization:
- Transfer the medium mixture into an autoclavable flask, leaving enough space for expansion.
- Sterilize the medium by autoclaving at 121°C under 15 psi pressure for 15 minutes.
Cooling and Supplement Addition:
- After autoclaving, cool the medium to approximately 45-50°C.
- If you’re using additives like penicillin or horse serum, this is the time to add them. Make sure they’re sterile and added under aseptic conditions.
Pouring Plates:
- Under sterile conditions (preferably in a laminar flow hood), pour the medium into sterile Petri dishes.
- Allow the agar to solidify at room temperature.
Storage:
- After solidification, invert the plates (agar side up) and store them in a refrigerator at 4°C. Inverting prevents condensation droplets from dropping onto the agar surface.
Quality Control (Optional but recommended):
- Inoculate a plate with a known Mycoplasma strain to ensure the medium’s appropriateness for growth.

Test Procedure

The use of PPLO Agar is typically for the isolation and cultivation of Mycoplasma. When you refer to the “test procedure,” I assume you’re referring to the method used to inoculate and incubate the agar with samples to detect the presence of Mycoplasma. Here’s a general procedure for this:

Test Procedure of PPLO Agar:

Sample Collection and Preparation:
- Depending on the context, this could be a clinical sample like sputum, throat swab, or a sample from a suspected contaminated cell culture.
- Process the sample as required. This might involve diluting, centrifuging, or filtering the sample to concentrate the Mycoplasma or remove other organisms or debris.
Inoculation:
- Using a sterile loop or swab, streak the prepared sample onto the surface of the PPLO Agar plate. If working with liquid samples, you can also pour or spread a measured volume onto the agar surface.
- Employ a streaking technique that facilitates isolation of individual colonies, especially if you suspect a mixed culture.
Incubation:
- Seal the inoculated plates with parafilm or place them in a suitable container to prevent dehydration.
- Incubate the plates at 37°C. The incubation period can vary, but it’s typically between 1 to 2 weeks since Mycoplasma spp. are slow growers. Some species might require a microaerophilic environment, so be sure to provide the necessary conditions based on the species you’re working with.
- Check the plates periodically for growth.
Examination:
- Look for colonies on the agar surface. Mycoplasma colonies may appear as fried-egg-shaped or have a film-like appearance.
- Note any change in the color of the medium (due to the phenol red indicator), which could indicate acid production from glucose fermentation.
Confirmation:
- Once growth is observed, further tests might be required to confirm that the isolates are indeed Mycoplasma This can include biochemical tests, PCR, serological assays, or electron microscopy.
Subculturing (if necessary):
- If you need to maintain or amplify the culture, you can subculture the Mycoplasma colonies onto fresh PPLO Agar plates or broth.
Safety and Disposal:
- Always follow biosafety guidelines when working with potential pathogens. Dispose of all cultures and contaminated materials properly, typically by autoclaving before disposal.

Result -Interpretation

Interpreting results on PPLO (Pleuropneumonia-Like Organisms) Agar revolves around observing colony morphology and potential changes in the medium’s color due to pH changes. Here’s a guide on interpreting results:

1. Colony Morphology:

Mycoplasma Colonies: Typically, colonies of Mycoplasma on PPLO Agar can appear in a few characteristic ways:
- Fried-egg appearance: This is the most classic representation. The center of the colony is raised and dense, while the periphery is flat and translucent, resembling a fried egg.
- Film-like growth: Some Mycoplasma species might spread along the agar surface, producing a thin film.
- Granular or speckled colonies: These can also be seen for some strains.
Other Organisms: While PPLO Agar can support the growth of Mycoplasma, other organisms, if present, especially if the medium isn’t supplemented with selective agents like penicillin, might also grow. These colonies can differ in appearance, size, and color. It’s essential to differentiate these from Mycoplasma colonies.

2. pH Indicator Change:

Yellowing of the Medium: If the medium turns yellow, it indicates a drop in pH, suggesting that acids have been produced from the fermentation of glucose. Some Mycoplasma spp. can ferment glucose, leading to this color change.
No Color Change (Remains Red or Pinkish): This suggests either no growth or growth of Mycoplasma that do not ferment glucose.

Interpreting the Results:

Presence of fried-egg colonies (or other typical Mycoplasma morphologies) with or without a yellow color change in the medium indicates a positive result for Mycoplasma.
Absence of typical Mycoplasma colonies or presence of colonies that are clearly of different bacteria indicates a negative result for Mycoplasma.
Inconclusive or mixed cultures: Sometimes, the growth may be mixed, or the results might be hard to interpret. In these cases, further purification (re-streaking) or confirmatory tests might be required.

Uses

PPLO (Pleuropneumonia-Like Organisms) Agar, designed specifically for the cultivation of Mycoplasma, has several important uses in microbiological research, clinical diagnostics, and other areas. Here are some of the primary uses:

Clinical Diagnostics:
- Respiratory Infections: Mycoplasma pneumoniae is a common cause of atypical pneumonia in humans. Clinical samples, such as throat swabs or sputum, can be cultured on PPLO Agar to diagnose this infection.
- Genital Infections: Mycoplasma hominis and Ureaplasma urealyticum can be involved in genital infections. PPLO Agar can be used to isolate these organisms from urogenital samples.
- Joint Infections: Certain Mycoplasma species have been implicated in joint infections, and PPLO Agar can be utilized for their detection from synovial fluid.
Veterinary Diagnostics:
- Mycoplasma cause various diseases in animals, such as bovine mastitis by Mycoplasma bovis. PPLO Agar can be used to detect Mycoplasma from animal samples.
Cell Culture Contamination Detection:
- Mycoplasma species are notorious contaminants of mammalian cell cultures in research labs. They can profoundly influence research outcomes. PPLO Agar is frequently used to check cell culture supernatants for Mycoplasma contamination.
Research Purposes:
- Scientists studying the biology, genetics, or pathogenic mechanisms of Mycoplasma might grow these organisms on PPLO Agar for various experiments.
- It aids in the biochemical characterization of Mycoplasma species.
Vaccine Production:
- Mycoplasma-free cell cultures are essential for producing viral vaccines. PPLO Agar can help in ensuring that cultures used for vaccine production are not contaminated with Mycoplasma.
Antibiotic Sensitivity Testing:
- For patients infected with Mycoplasma, appropriate antibiotic treatment is crucial. PPLO Agar can be modified to assess the sensitivity of isolated Mycoplasma strains to various antibiotics.
Environmental Surveys:
- PPLO Agar can be used in studies aiming to detect the presence of Mycoplasma in different environments or ecosystems.

Keynotes

Here are some keynotes on PPLO (Pleuropneumonia-Like Organisms) Agar:

Purpose:
- PPLO Agar is specifically formulated for the isolation and cultivation of Mycoplasma species.
Composition:
- The medium typically contains proteose peptone, sodium chloride, yeast extract, glucose, phenol red (a pH indicator), and agar. Supplements like horse serum and antibiotics like penicillin can be added based on requirements.
Indicator Role:
- Phenol red is included as a pH indicator. Acidic by-products from glucose fermentation by certain Mycoplasma species can lead to a color change in the medium, turning it yellow.
Colony Morphology:
- Mycoplasma species often produce characteristic “fried-egg” colonies on this medium, though other morphologies, like film-like or granular growth, can also be observed.
Selective Nature:
- While PPLO Agar primarily supports the growth of Mycoplasma, it isn’t inherently selective. The addition of certain antibiotics, like penicillin, can make it more selective by inhibiting other bacterial contaminants.
Diagnostic Utility:
- PPLO Agar is employed clinically to detect Mycoplasma infections, including respiratory infections caused by Mycoplasma pneumoniae and genital infections from organisms like Mycoplasma hominis.
Research Applications:
- PPLO Agar is essential in research labs, especially for detecting Mycoplasma contamination in cell cultures. Such contamination can significantly impact cellular research findings.
Veterinary Relevance:
- The agar is also used in veterinary diagnostics to identify Mycoplasmas causing diseases in animals, such as bovine mastitis.
Storage:
- Prepared and uninoculated PPLO Agar plates should be stored in a refrigerator, usually at 4°C, and should be used before their expiration.
Interpretation:

Positive growth is indicated by the presence of typical Mycoplasma colonies (e.g., fried-egg appearance) and possibly a change in medium color due to pH alteration.

Safety:

As with all culture media, maintaining aseptic conditions during preparation and inoculation is essential to prevent contamination.

PPLO Agar- Introduction, Composition, Principle, Preparation Requirements, Test Procedure, Result -Interpretation, Uses, and Keynotes