Peptone Yeast Dextrose Broth (Cantino) – Composition, Principle, Preparation, Uses

What is Peptone Yeast Dextrose Broth (Cantino)?

• Peptone Yeast Dextrose Broth (Cantino) is a specialized medium formulated by Cantino for the cultivation of aquatic fungi, particularly Blastocladiella species. It is designed to promote the luxuriant growth of these fungi in the presence of visible light illumination and increased CO2 fixation. The medium shares a similar composition with Peptone Yeast Dextrose Agar (Cantino) and is also recommended for the cultivation of Eikenella corrodens, a bacterium that is part of the resident microflora of mucous membrane surfaces in humans.
• The main components of Peptone Yeast Dextrose Broth (Cantino) include peptic digest of animal tissue and yeast extract, which provide nitrogenous nutrients, vitamin B complex, peptides, and trace ingredients necessary for the growth of aquatic fungi and E. corrodens. Dextrose serves as the energy source for the microorganisms.
• Peptone Yeast Extract Broth Based Media, including Peptone Yeast Dextrose Broth (Cantino), are enriched nonselective media that are supplemented with hemin and vitamin K. This supplementation facilitates the recovery of more fastidious organisms such as Prevotella spp, Porphyromonas spp, and the Bacteroides fragilis group. These media can be used to determine whether anaerobic bacteria will utilize specific biochemicals or if their growth is stimulated or inhibited in the presence of these substances.
• Peptone Yeast Extract Glucose Broth, which is a nonselective broth media originally developed by the Virginia Polytechnic Institute Anaerobe Laboratory (VPI) group, is also a part of the Peptone Yeast Extract Broth Based Media. It is primarily used for the recovery of more fastidious microorganisms and for chromatographic analysis of metabolic products resulting from the fermentation of glucose. This chromatographic analysis is particularly useful in identifying clinically important anaerobic bacteria.
• To ensure the quality and integrity of the medium, Peptone Yeast Extract Broth Based Media, including Peptone Yeast Dextrose Broth (Cantino), are prepared, dispensed, and packaged under oxygen-free conditions. This precautionary measure prevents the formation of oxidized products that could potentially affect the efficacy of the medium.
• In summary, Peptone Yeast Dextrose Broth (Cantino) is a specialized medium formulated for the cultivation of aquatic fungi and Eikenella corrodens. It provides the necessary nutrients and energy sources for their growth and is commonly used in microbiological laboratories for research and diagnostic purposes. Additionally, Peptone Yeast Extract Broth Based Media, which include Peptone Yeast Dextrose Broth (Cantino), are enriched nonselective media used to recover fastidious microorganisms and for biochemical analysis in the identification of anaerobic bacteria.

Composition of Peptone Yeast Extract Glucose Broth

Ingredients	Grams/L
Pancreatic Digest of Casein	20.0gm
Yeast Extract	10.0gm
Glucose	10.0gm
L-Cysteine	0.5gm
Sodium Bicarbonate	0.4gm
Sodium Chloride	0.08gm
Monopotassium Phosphate	0.04gm
Dipotassium Phosphate	0.04gm
Calcium Chloride, Anhydrous	0.008gm
Magnesium Sulfate, Anhydrous	0.008gm
Hemin Solution, 0.1%	5.0ml
Vitamin K Solution, 1%	0.1ml

Final pH 7.3 +/- 0.5 at 25ºC.

Principle of Peptone Yeast Extract Glucose Broth

The principle of Peptone Yeast Extract Glucose Broth lies in its composition and the specific components that support the growth of anaerobic bacteria.

The broth contains a pancreatic digest of casein and yeast extract, which provide nitrogenous compounds and other essential nutrients necessary for the growth of anaerobic microorganisms. These nutrients serve as building blocks for cellular structures and metabolic processes.

Sodium bicarbonate and dipotassium phosphate act as buffers in the medium. They help maintain a stable pH, ensuring optimal conditions for bacterial growth. pH stability is crucial for the proper functioning of enzymes and other biological processes.

Glucose, a simple sugar, serves as an energy source for the anaerobic bacteria. It is metabolized through various pathways to produce ATP, the energy currency of the cell.

L-cysteine, a reducing agent present in the medium, helps maintain a low oxygen potential. This is important because anaerobic microorganisms thrive in low oxygen environments and are often inhibited by the presence of oxygen. L-cysteine helps create an environment conducive to their growth by reducing the available oxygen.

The growth factors hemin (X factor) and vitamin K are vital for the growth of many fastidious anaerobes. Hemin supplies X factor, which is required by certain bacteria for their growth. Vitamin K is also necessary for the growth of anaerobic microorganisms and is known to promote pigment production in certain species. These growth factors are added to the medium to ensure the successful cultivation of fastidious anaerobes.

Sodium, potassium, calcium, and magnesium salts present in the broth serve as pH stabilizers. They help maintain the osmotic balance of the medium and provide critical ions required for various transport processes within the bacterial cells.

To preserve the integrity and quality of the medium, Peptone Yeast Extract Glucose Broth is prepared and processed in an atmosphere of nitrogen and hydrogen. This oxygen-free environment prevents the formation of oxidized products that could potentially affect the medium’s performance.

In summary, Peptone Yeast Extract Glucose Broth promotes the growth of anaerobic bacteria by providing nitrogenous compounds, essential nutrients, energy sources, reducing agents, growth factors, and pH stabilizers. The absence of oxygen and the specific composition of the medium create an environment suitable for the cultivation of anaerobic microorganisms.

Preparation of Peptone Yeast Extract Glucose Broth

The preparation of Peptone Yeast Extract Glucose Broth involves the following steps:

1. Suspend 5.5 grams of the medium in 1000 ml of distilled water. The medium may require some heating to aid in its complete dissolution. It is important to ensure that all the components are fully dissolved to create a homogeneous solution.
2. Once the medium is suspended and dissolved in water, it needs to be sterilized to eliminate any potential contaminants. Autoclaving is the most common method of sterilization used in laboratories. The medium should be subjected to autoclaving at 15 lbs pressure (121°C) for 15 minutes. This process effectively kills any microorganisms present in the medium.
3. After autoclaving, the sterilized Peptone Yeast Extract Glucose Broth should be mixed thoroughly to ensure an even distribution of components and to prevent settling or separation of any particles that may have formed during autoclaving.
4. The final step is to dispense the prepared broth as desired. The volume and containers for dispensing may vary depending on the specific requirements of the laboratory or experiment. The broth can be dispensed into sterile petri dishes, tubes, or other appropriate containers.

It is important to maintain aseptic techniques throughout the preparation process to prevent contamination. Proper lab practices, including the use of sterile equipment and working in a clean environment, should be followed.

Following these steps will result in a properly prepared Peptone Yeast Extract Glucose Broth ready for use in the cultivation and growth of anaerobic bacteria.

Plating Technique

The plating technique for Peptone Yeast Extract Glucose Broth involves the following steps:

1. Allow the tubes containing the broth to reach room temperature before starting the inoculation process. This helps to prevent temperature shock to the bacteria and ensures more accurate results.
2. Obtain a clean, young colony of anaerobic bacteria. The age of the colony may vary depending on the specific organism being cultured, ranging from 6 to 72 hours, depending on its growth rate. It is important to use a colony that is in the logarithmic growth phase to ensure optimal viability and growth.
3. Swirl or vortex the tube containing the Peptone Yeast Extract Glucose Broth to ensure the broth is well mixed and homogeneous. This step helps distribute the bacteria evenly throughout the broth.
4. Using a sterilized syringe, take a small portion of the broth culture. It is crucial to maintain sterile conditions throughout this process to prevent contamination.
5. To inoculate the Peptone Yeast Extract Glucose tubes, cut through the rubber section of the cap. Slowly introduce 5-10 drops of the broth culture into the tube. It is also recommended to place a couple of drops of the culture on an unselective plate of blood agar to check the viability and purity of the culture. This allows for verification of the bacterial growth and ensures the culture is free from contaminants.
6. If you are inoculating a tube that is supplemented with specific additives such as AN270A, AN270F, or AN270T, ensure that you follow the exact procedure outlined for those specific supplements. Additionally, inoculate an unsupplemented reference tube of PYG Broth at the same time using an identical inoculum.
7. Incubate the tubes at 35 degrees Celsius, which is the recommended temperature for the growth of anaerobic bacteria. Incubation conditions may vary depending on the specific requirements of the microorganism being cultured.
8. After 48 hours of incubation, the tubes should be discarded, as extended incubation may lead to overgrowth or changes in the bacterial population.
9. Note and document the results of the growth observed in the tubes. It is important to compare the growth of the tubes supplemented with additives to those that are not supplemented. This comparison helps assess the impact of the additives on bacterial growth.

By following these steps, the plating technique for Peptone Yeast Extract Glucose Broth can be successfully executed, allowing for the cultivation and evaluation of anaerobic bacteria.

Result and Interpretation of Peptone Yeast Extract Glucose Broth

The results and interpretation of Peptone Yeast Extract Glucose Broth, along with additional testing procedures, can be summarized as follows:

1. Positive Result (Inoculated Medium): When the medium is inoculated with bacteria, a positive result is indicated by the presence of growth. This growth is observed as slight turbidity or cloudiness in the broth. Additionally, there may be the formation of a smooth sediment, which can have a stringy, granular, or flocculent appearance.
2. Negative Result (Uninoculated Medium): In the absence of bacterial inoculation, a negative result is obtained. In this case, there is no growth observed, and the medium remains clear without any turbidity.
3. pH Measurements: Once sufficient bacterial growth is observed, pH measurements can be performed using a pH meter and a long, thin combination electrode. The pH value obtained provides insight into acid production by the bacteria.

• pH value of 5.5 to 6.0: This range indicates weak acid production by the bacteria.
• pH value of 5.5 or less: A pH value of 5.5 or lower suggests strong acid production by the bacteria.
• pH above 6.0: A pH value above 6.0 indicates no acid production.

4. Esculin Hydrolysis Detection: To detect esculin hydrolysis, add 2 to 3 drops of a one percent solution of ferric ammonium citrate to an inoculated tube of PY ESCULIN that contains sufficient bacterial growth. The blackening of the medium indicates that esculin present in the medium has been hydrolyzed by the bacteria.
5. Starch Hydrolysis Detection: To detect starch hydrolysis, add 2 to 3 drops of a dilute 1:5 Gram’s iodine solution to an inoculated tube of PY STARCH that has sufficient growth. No color change indicates the absence of starch hydrolysis by the organism, while the development of a blue/black color signifies the presence of starch hydrolysis.
6. Gelatin Hydrolysis Detection: Perform a gelatin hydrolysis test by setting up a 24-72 hour culture using an uninoculated tube at a temperature of 2-8°C. The uninoculated control tube should solidify, and if the inoculated tube fails to solidify, it indicates the hydrolysis of gelatin by the organism.
7. Growth in Bile Determination: Measure the level of turbidity in the medium containing bile and compare it against the growth in the standard Peptone Yeast Glucose medium (AS-822). This comparison helps determine if the organism is stimulated or inhibited by the presence of bile.

It’s important to note that Peptone Yeast Extract Broth Based Media may not provide all the information required for complete identification of the isolated bacteria. Additional testing procedures and different media may be necessary for a more comprehensive identification process. It is recommended to refer to reference materials and protocols for detailed instructions and further information.

Quality Control

The quality control of Peptone Yeast Extract Glucose Broth involves assessing various parameters to ensure its effectiveness and reliability. The following information provides an overview of the quality control measures for this broth:

1. Appearance: Peptone Yeast Extract Glucose Broth should appear as an off-white to yellow homogeneous free-flowing powder. Any significant deviations from this appearance may indicate potential quality issues.
2. Colour and Clarity of Prepared Medium: After preparing the medium, it should exhibit a yellow color and be clear in tubes. This visual assessment ensures that the prepared medium meets the expected appearance criteria.
3. Reaction: The pH of a 0.55% w/v aqueous solution of Peptone Yeast Extract Glucose Broth should be within the range of 6.8 ± 0.2 at 25°C. This pH range ensures the appropriate conditions for bacterial growth and metabolic activities.
4. pH: The acceptable pH range for Peptone Yeast Extract Glucose Broth is 6.60-7.00. This range confirms that the pH of the broth is within the specified limits and is conducive to bacterial cultivation.
5. Cultural Response: Cultural characteristics are observed after incubation of the broth at 25°-30°C for up to 8 days. This assessment involves observing the growth and appearance of specific organisms to ensure that they exhibit the expected cultural response in the medium.

Organism Growth and Cultural Response: Specific organisms, such as Blastocladiella emersonii (ATCC 22665), Candida albicans (ATCC 10231), Eikenella corrodens (ATCC 23834), and Saccharomyces cerevisiae (ATCC 9763), are typically used as control strains. The growth of these organisms should be “luxuriant,” indicating robust and abundant growth in the medium.

By conducting these quality control assessments, laboratories can verify that Peptone Yeast Extract Glucose Broth meets the necessary standards and ensure its reliability and performance in supporting bacterial cultivation and identification.

Organism Tested	Results	Time
Bacteroides vulgatus	Growth	24 hrs
Bacteroides fragilis	Growth	24 hrs
Prevotella melaninogenica	Growth*	24 – 48 hrs
Fusobacterium necrophorum	Growth*	24 – 48 hrs
Fusobacterium nucleatum	Growth*	24 – 48 hrs
Clostridium perfringens	Growth	24 hrs
Peptostreptococcus anaerobius	Growth*	24 hrs
Clostridium novyi, Type A          (PY and PYG only)	Growth	48 – 72 hrs
Propionibacterium acnes           (PY, PY ADONITOL, PYG, and PY GLYCEROL only)	Growth	24 – 48 hrs
Staphylococcus aureus               (PY and PYG only)	Growth	24 hrs
Peptoniphilus asaccharolyticus (PY FRUCTOSE, PYG GELATIN, PYG BILE, and PYG only)	Growth*	24 – 48 hrs

*No growth in PYG BILE

Storage and Shelf Life

PYG Broths must be kept at room temperature, in an upright location and kept away from light. If stored in this manner, the medium will last a shelf-life of 26 weeks starting from the date of manufacturing.

Uses of Peptone Yeast Extract Glucose Broth

Peptone Yeast Extract Glucose Broth has various uses in microbiology, particularly in the cultivation and identification of obligately anaerobic microorganisms. Here are some of its key applications:

1. Cultivation of Obligately Anaerobic Microorganisms: Peptone Yeast Extract Glucose Broth provides a suitable environment for the growth of obligately anaerobic bacteria. These microorganisms require an oxygen-free environment for their survival and growth. The broth supports their nutritional needs and facilitates their propagation in the laboratory.
2. Biochemical Identification of Anaerobic Bacteria: The broth is utilized for the biochemical identification of anaerobic bacteria. It enables the identification of short-chain acid end products produced during glucose fermentation by anaerobic bacteria. This information is crucial for distinguishing different bacterial species and for their accurate identification from clinical specimens.
3. Glucose Fermentation Determination: Peptone Yeast Extract Glucose Broth is used to determine the glucose fermentation capability of anaerobic bacteria. By observing their growth and metabolic activities in the broth, the ability of bacteria to ferment glucose can be assessed. This information aids in the identification and classification of anaerobic bacteria.
4. Gas-Liquid Chromatography (GLC) Analysis: The broth is suitable for the growth of anaerobic bacteria that are to be identified through gas-liquid chromatography (GLC) analysis. GLC analysis is a technique used to identify and quantify the volatile metabolic products produced by bacteria during fermentation. Peptone Yeast Extract Glucose Broth supports the growth of these bacteria, allowing for the collection and analysis of their metabolic products.

In summary, Peptone Yeast Extract Glucose Broth finds applications in the cultivation and biochemical identification of obligately anaerobic microorganisms. It is used to determine glucose fermentation, identify short-chain acid end products, and provide a suitable growth medium for anaerobic bacteria that will undergo gas-liquid chromatography analysis. These uses contribute to the understanding and characterization of anaerobic bacteria in clinical and research settings.

Limitations of Peptone Yeast Extract Glucose Broth

Peptone Yeast Extract Glucose Broth has certain limitations that should be taken into consideration. Here are the key limitations:

• Additional Testing for Final Identification: While Peptone Yeast Extract Glucose Broth provides a suitable environment for the cultivation and preliminary identification of anaerobic microorganisms, it is important to note that additional testing is required for final identification. This includes morphological, biochemical, and/or serological tests. These additional tests are necessary to confirm the findings obtained from the broth and provide a comprehensive identification of bacterial isolates.
• Incomplete Information for Identification: Peptone Yeast Extract Glucose Broth alone does not provide complete information for the identification of bacterial isolates. While it supports the growth and biochemical assessment of anaerobic bacteria, it may not provide all the necessary data to definitively identify the specific bacterial species. To achieve accurate identification, a combination of different tests, media, and techniques are typically employed.
• Reliance on Supplementary Tests: To overcome the limitations of Peptone Yeast Extract Glucose Broth, supplementary tests such as morphological, biochemical, and serological tests become essential. These additional tests provide a more comprehensive analysis of the bacterial isolates, including their phenotypic characteristics, metabolic activities, and antigenic properties. These tests contribute to a more accurate and reliable identification of the microorganisms.

FAQ

References

• https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU62148.pdf
• https://exodocientifica.com.br/_technical-data/M671.pdf
• https://anaerobesystems.com/products/broth-media/peptone-yeast/
• https://www.sigmaaldrich.com/IN/en/search/peptone-yeast-extract-broth?focus=products&page=1&perpage=30&sort=relevance&term=peptone%20yeast%20extract%20broth&type=product_name