Peptone Yeast Extract Broth Based Media are enriched nonselective media which include hemin and vitamin K to aid in the recovery of aggressive organisms like Prevotella Spp., Porphyromonas species, as well as the Bacteroides fragileis group.
In addition to a specific biochemical, Peptone Yeast Extract Broth-based Media can be used to test whether anaerobic organisms will use the biochemical, or if the growth is inhibited or stimulated. The PeptoneYeast Extract Glucose Broth is an unselective broth medium that was originally developed in scientists from the Virginia Polytechnic Institute Anaerobe Laboratory (VPI) group to aid in the extraction of more discerning microorganisms, and to aid in the chromatographic analysis of metabolic products resulting from the fermentation of glucose.
This type of chromatographic analysis is helpful in identifying vital clinically relevant anaerobic bacteria. It is then prepared and dispersed and then packaged in conditions that are oxygen-free to stop the growth of oxidized substances prior to their use.
Composition of Peptone Yeast Extract Glucose Broth
|Pancreatic Digest of Casein||20.0gm|
|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
Peptone Yeast Extract Glucose Broth is a digest of the pancreas of yeast extract and casein that provides nitrogenous compounds as well as other nutrients needed for the development of anaerobic bacteria. Dipotassium phosphate and sodium bicarbonate are buffers. Glucose functions as an energy source, while L-cysteine acts as a reducing agent, and has a low oxygen potential, which helps in stimulating the growth of microorganisms in anaerobic conditions.
Hemin, one of the growth factors that provides X factor as well as vitamin K are needed to grow by a variety of fastidious anaerobes. They are also recognized to boost pigment production in some anaerobic species. Calcium, sodium, potassium magnesium and magnesium salts stabilize pH and aid in maintaining osmotic balance and supply the critical ions needed for transport. The media is prepared and refined by a nitrogen atmosphere and hydrogen.
Preparation of Peptone Yeast Extract Glucose Broth
- Add the components to tap water and reduce volume to 1.0 L.
- Mix thoroughly.
- Begin gently heating and then bring to a boil.
- Distribute into flasks, tubes or tubes.
- Autoclave for 15 minutes at 15 psi pressure 121°C.
- Pour the mixture into sterilized Petri dishes or place in tubes.
- Let tubes warm up to the temperature of room before the inoculation.
- Get a clean, young colony of anaerobic bacteria (6 up to 72 hours, depending on the rate of growth for the organism)
- Swirl or vortex the tube to ensure that the broth is homogeneous.
- With a sterilized syringe take a small portion of broth.
- To inoculate the PeptoneYeast glucose tubes, cut through the rubber section of the cap. Slowly introduce 5-10 drops broth culture. A couple of drops should be placed on an un-selective plate of blood agar to check the viability and purity for the culture. If you are inoculating a tube supplemented with (AN270A, AN270F or AN270T) ensure that the exact procedure is followed, except that an unsupplemented reference tube PYG Broth is inoculated at a similar time using an identical inoculum.
- Incubate the tubes for 35 degrees Celsius.
- Eliminate tubes after 48 hours.
- Note and document the results. Compare the growth of tubes supplemented with those that are not supplemented.
Result and Interpretation of Peptone Yeast Extract Glucose Broth
Positive test results of PYG Bloth shows evident an increase in turbidity (growth) within the tube. This means that the organism is able to grow without inhibition within 20% bile. The test that is negative indicates there is no growth after the incubation.
The PYG Broth that is supplemented with formate-fumarate or arginine. Tween results are determined through looking at the growth of the supplemented and non-supplemented tube. Note any increased growth of the tube supplemented because this could help to identify the anaerobic strain.
Other tests for serology or biochemical analysis are required on the colonies of pure culture to confirm the identity of the colony.
- Don’t use if the medium appears reddish or purplish-brown because this could indicate that oxygen has been introduced into the tube.
- PYG Broth is an ideal medium for the examination of GLC of anaerobic strains as described by the CDC
- The anaerobe that is tested must be pure and functional. If the anaerobe does not grow on the blood plate , but expands in the broth, you can subculture your broth in order to establish if it is an isolate or from a contaminant.
When sufficient growth is seen, the pH may be determined directly from the tube using a pH tester and a long , thin combination electrode.
Interpretation of pH
- A pH between 5.5 up to 6.0 is considered to be moderate acid producer.
- A pH in the range of 5.5 or less indicates strong acid production.
- A pH higher than 6.0 indicates that there is there is no acid production.
Esculin Hydrolysis Detection (AS-833 PY ESCULIN)
Include 2 to 3 drops one percent solution of ferric ammonium citrate to an inoculated tub of PY ESCULIN that has sufficient for bacterial growth. The blackening of the medium is a sign that the esculin present is Hydrolyzed by bacteria.
Starch Hydrolysis Detection (AS-829 PY STARCH)
Include up to 2-3 drops dilute 1:5 of Gram’s Iodine to an inoculated tub of PY STARCH that has sufficient growth. There is no change in color that indicates the starch’s hydrolysis in the organism. The development of a blue/black color signifies that there is starch in the body.
Gelatin Hydrolysis Detection (AS-860 PYG GELATIN)
Set up a 24-72 hour culture using an uninoculated tube the 2-8 degC temperature. The control tube that is not inoculated will become solid, and if the inoculated tube fails to form a solid, then the gelatin has hydrolyzed. The solidification of the tube is not accompanied by hydrolysis of gelatin.
Growth in Bile Determination (AS-857 PYG BILE)
The level of turbidity in the medium is measured against the PYG (AS-822) to determine whether the organism is stimulated or inhibited by the bile.
Peptone Yeast Extract Broth Based Media cannot provide all the information required to determine bacteria isolated. Additional testing procedures and media are needed for full identification. Refer to reference materials for more details.
Following organisms are commonly utilized in Anaerobe Systems for quality assurance test of the performance of Peptone Yeast Extract Broth Based Media.
|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
- It is utilized for the growing and the biochemical identification of anaerobic microorganisms.
- It’s used to detect the short chain acid’s end products as well as to determine the extent of glucose fermentation in anaerobic bacteria to identify them from clinical samples.
- It also allows for the development of anaerobic bacteria that can be detected by gas-liquid chromatography (GLC) examination.
Limitations of Peptone Yeast Extract Glucose Broth
- Additional tests like biochemical, morphological and/or serological tests are recommended to verify the findings.
- Peptone Yeast Extract Glucose Broth is not able to provide all the information needed to determine the identity of the bacterial isolates.