Selenite F Broth – Composition, Principle, Preparation, Results, Uses

What is Selenite F Broth?

• Selenite F Broth, also known as Selenite Broth or Selenite-Feces Broth, is a selective enrichment medium used for the isolation of Salmonella species from various specimens, including feces, urine, water, foods, and other materials of sanitary importance. It was originally developed by Leifson as an effective medium to inhibit coliforms and certain other microbial species, such as fecal streptococci, while promoting the growth of Salmonella.
• The composition of Selenite F Broth includes Lactose Peptone Broth as a buffering agent, along with the addition of Sodium Biselenite as the selective agent. Tryptone provides nitrogenous substances for bacterial growth, and lactose helps maintain a stable pH within the medium. The presence of selenite in the broth serves as a selective agent, inhibiting the growth of coliforms and other competing bacteria. As Salmonella grows in the medium, it reduces selenite and produces alkali, which increases the pH and reduces the inhibitory effect of selenite. The acid produced by lactose fermentation helps maintain a neutral pH and further supports the growth of Salmonella.
• The purpose of using Selenite F Broth as an enrichment medium is to enhance the detection of Salmonella, especially during the non-acute stages of illness when the organism may be present in low numbers in the feces. It is also useful for epidemiological studies and for detecting low numbers of Salmonella in asymptomatic or convalescent patients. After incubating the specimen in Selenite F Broth for 12-18 hours at 35-37°C, subculture is done on selective agar media, such as Bismuth Sulphite Agar, Brilliant Green Agar, or XLD Agar, for further isolation and identification of Salmonella.
• While Selenite F Broth has been widely used for the enrichment and isolation of Salmonella, the Rappaport-Vassiliadis (RV) medium is now being recommended as an alternative for the selective enrichment of Salmonella from food and sewage-polluted water. RV medium offers improved selectivity and sensitivity compared to Selenite Broth or Tetrathionate broth.
• In summary, Selenite F Broth is a selective enrichment medium that aids in the isolation of Salmonella species from various specimens. It inhibits the growth of coliforms and other competing bacteria while promoting the growth of Salmonella, allowing for their subsequent isolation on selective agar media.

Principle of Selenite F Broth

The principle of Selenite F Broth is based on the selective inhibitory effects of selenite and the ability of Salmonella to overcome this inhibition. The components of the medium work together to create a suitable environment for the growth of Salmonella while inhibiting the growth of other bacterial species.

1. Nitrogenous Substances and Carbon Compounds: Casein enzymic hydrolysate present in the medium provides nitrogenous substances and carbon compounds that serve as essential nutrients for bacterial growth.
2. Carbohydrate Source: Lactose is included as a carbohydrate source in Selenite F Broth. It plays a role in maintaining the pH of the medium and provides a substrate for lactose fermentation by certain bacteria.
3. Reduction of Selenite: Bacterial growth in the medium leads to the reduction of selenite present in the form of sodium hydrogen selenite (Sodium biselenite). This reduction results in the production of alkali, which increases the pH of the medium.
4. Inhibition of Other Bacteria: Selenite is known for its inhibitory properties against many species of gram-positive and gram-negative bacteria, including enterococci and coliforms. The toxicity of selenite is reduced as the pH of the medium increases, allowing for the overgrowth of other bacteria.
5. pH Regulation: The acid produced by bacteria through the fermentation of lactose helps maintain a neutral pH in the medium. Sodium phosphate is added to further stabilize the pH and lessen the toxicity of selenite.

It is important not to incubate the broth longer than 24 hours, as the inhibitory effect of selenite decreases after 6-12 hours of incubation. Prolonged incubation may result in the overgrowth of non-target bacteria and can affect the selectivity of the medium.

By providing a selective environment that favors the growth of Salmonella, Selenite F Broth enables the enrichment and subsequent isolation of this pathogen from various specimens.

Composition of Selenite F Broth

Ingredients	Grams/liter
Casein enzymic hydrolysate	5.0
Lactose	4.0
Sodium phosphate	10.0
Sodium hydrogen selenite	4.0

pH at 25°C  7.1 ± 0.2

Preparation of Selenite F Broth

The preparation of Selenite F Broth involves the following steps:

1. Add 4.0 grams of sodium selenite powder to distilled or deionized water.
2. Add the remaining 19.0 grams of powder to the solution from the previous step.
3. Bring the volume to 1.0 liter, ensuring thorough mixing of the ingredients.
4. Gently heat the solution and bring it to a boil.
5. Once boiled, dispense the broth into sterile test tubes. The test tubes should have a depth of at least 5 cm to ensure an adequate volume of the medium.
6. Sterilize the test tubes containing the broth. This can be done by either placing them in a boiling water bath or subjecting them to free-flowing steam for 10 minutes.
7. It is important to note that Selenite F Broth should not be autoclaved, as it can cause changes in the selective properties of the medium.
8. Allow the broth to cool to room temperature before use.

Following these steps ensures the proper preparation of Selenite F Broth, which can then be used as an enrichment medium for the isolation of Salmonella and other relevant microorganisms from various specimens.

Storage conditions and Shelf life

Storage conditions and shelf life are important considerations for maintaining the quality and effectiveness of dehydrated and prepared Selenite F Broth. Here is the information based on the provided content:

Dehydrated Medium:

• Store the dehydrated medium at a temperature between 10-30°C.
• It is crucial to use the medium before the expiry date mentioned on the label.
• Proper storage conditions help preserve the integrity of the dehydrated medium and ensure reliable results when it is reconstituted and used.

Prepared Medium:

• Store the prepared Selenite F Broth at a temperature of 2-8°C.
• Keep the medium away from light to prevent any potential light-induced changes.
• The prepared medium can be used up to eighteen months from the date of preparation, provided there are no visible signs of contamination or deterioration.
• It is essential to regularly inspect the medium for any changes in appearance, such as discoloration, presence of particles, or other abnormalities. If such changes occur, the medium should not be used.

By following appropriate storage conditions, both for the dehydrated and prepared forms of Selenite F Broth, laboratories can ensure the longevity and quality of the medium. Adhering to the specified storage temperatures and monitoring for any signs of contamination or deterioration is crucial to obtain accurate and reliable results when using Selenite F Broth for the isolation and detection of Salmonella and other relevant microorganisms.

Inoculation of Selenite Broth

The inoculation procedure for Selenite Broth involves the following steps, as described in the provided content:

1. Inoculation:

• Take approximately 1-2 grams of the stool sample or other relevant samples (approximately 10-15% of the volume of the broth).
• Inoculate the Selenite Broth tube with the sample.
• Emulsify the broth and the sample together to ensure proper mixing.

2. Incubation:

• Incubate the inoculated Selenite Broth tube in ambient air at a temperature of 35-37°C.
• The incubation period should be up to 24 hours, as longer incubation may allow coliforms to overgrow the pathogens.

3. Subculture:

• After 12-18 hours of incubation, transfer a portion of the Selenite Broth to selective and differential enteric culture media, such as XLD (Xylose Lysine Deoxycholate) or HE Agar (Hektoen Enteric Agar).
• Streak the subculture onto the media to isolate individual colonies.

4. Incubation of Culture Plates:

• Incubate the culture plates in ambient air at a temperature of 35-37°C.
• The plates should be incubated for 18-24 hours.

5. Colony Morphology:

• After the incubation period, examine the culture plates for the presence of typical colony morphology.
• Note that the specific colony morphology may vary depending on the type of media used.

Important Note:

• Selenite Broth should not be used as the sole isolation medium for the isolation of Salmonella.
• It is recommended to use Selenite Broth in conjunction with selective and nonselective plating media to increase the likelihood of isolating pathogens, especially when they may be present in small numbers.

By following this inoculation procedure, laboratories can enhance the chances of detecting and isolating Salmonella and other relevant microorganisms from different types of samples. The combination of Selenite Broth with selective and differential media improves the probability of isolating pathogens while reducing the overgrowth of coliforms.

Result Interpretation on Selenite F Broth

The interpretation of results on Selenite F Broth involves observing the growth of organisms in the medium and their subsequent subculture on MacConkey Agar. Here is the interpretation based on the provided information:

1. Positive Result:

• Growth of organisms in Selenite F Broth is indicated by turbidity in the medium.
• When subcultured on MacConkey Agar after incubation at 35-37°C for 18-24 hours, positive results are characterized by colorless colonies and good growth.
• Examples of organisms that can yield positive results include Salmonella Typhimurium, Shigella sonnei, Salmonella enteritidis, and others.

2. Negative Result:

• Negative results are characterized by pink colonies with bile precipitate or no growth on MacConkey Agar.
• Inhibition or no growth on MacConkey Agar indicates the absence of the target organisms.
• Examples of organisms that can yield negative results include Escherichia coli, Enterococcus faecalis, and others.

The interpretation of results on Selenite F Broth helps in determining the presence or absence of Salmonella and other relevant microorganisms in the tested specimen. Positive results indicate the growth of target organisms, while negative results suggest the absence of these organisms. Further confirmatory tests and identification techniques may be required for accurate identification and differentiation of specific bacterial species.

Uses of Selenite F Broth

Selenite F Broth has several uses in microbiology, as mentioned in the provided content:

1. Selective Enrichment for Salmonella spp.:

• Selenite F Broth is primarily used as a selective enrichment medium for the cultivation of Salmonella species.
• The broth contains sodium selenite, which inhibits the growth of certain bacteria while promoting the growth of Salmonella.
• It provides a favorable environment for the recovery and enrichment of Salmonella from various specimens, such as feces, urine, water, and food samples.

2. Enrichment Medium for Shigella:

• Selenite F Broth can also be used as an enrichment medium for the isolation of certain species of Shigella.
• Similar to its use for Salmonella, the broth helps in the recovery and enrichment of Shigella from clinical specimens and other sources.

3. Transport Medium for Vibrio cholerae:

• Selenite F Broth is recommended for the transport of strains of Vibrio cholerae.
• Vibrio cholerae organisms can survive for 2 to 5 days in Sodium Selenite Broth, allowing for their transportation to the laboratory for further analysis and identification.

Overall, Selenite F Broth is an essential tool in the microbiology laboratory for the selective enrichment and isolation of Salmonella and Shigella species. It also serves as a transport medium for Vibrio cholerae, ensuring the viability of the organisms during transit.

Limitations of Selenite F Broth

Selenite F Broth has certain limitations that should be considered, as mentioned in the provided content:

1. Toxicity of Sodium Hydrogen Selenite:

• Sodium hydrogen selenite, the selective agent in Selenite F Broth, is a very toxic and corrosive agent.
• It is important to handle it with great care and follow proper safety precautions when working with the broth.
• In case of contact with the skin, immediate and thorough washing with a large amount of water is necessary.

2. Enrichment Broth as a Sole Isolation Medium:

• Selenite F Broth should not be used as the sole isolation medium for the detection and isolation of pathogens.
• It is designed as an enrichment medium and should be used in conjunction with selective and non-selective plating media.
• The use of additional media increases the probability of isolating pathogens, especially when they may be present in small numbers.

3. Nutritional Variation:

• Due to the variation in nutritional requirements among different strains of bacteria, some strains may grow poorly or fail to grow on Selenite F Broth.
• It is important to consider this limitation and use alternative enrichment or isolation media if poor growth or lack of growth is observed for specific strains.

These limitations should be taken into account when working with Selenite F Broth to ensure accurate and reliable results in the detection and isolation of Salmonella, Shigella, and other relevant microorganisms. Adhering to safety precautions, using additional media, and considering nutritional variations can help overcome these limitations and enhance the effectiveness of the broth.

FAQ

References

• http://exodocientifica.com.br/_technical-data/M052.pdf
• https://microbeonline.com/selenite-broth-composition-uses/
• https://legacy.bd.com/europe/regulatory/Assets/IFU/US/L007497(09)(0207).pdf