Hey, Good to see you here 😀 …… In this Article, we’re gonna discuss the Preparation of Alkaline Peptone Water….. If you have any queries, don’t forget to mention in Comments…
Before discussing the Preparation of Alkaline Peptone Water let’s take a brief intro about Enrichment media…..
Enrichment media are liquid culture media used in microbiology laboratory which contain substances that inhibit the growth of unwanted microorganisms and promotes the growth of the desired organism.
Alkaline Peptone Water (APW) is one of the commonly used enrichment liquid media which is highly recommended for the isolation of Vibrio cholerae from clinical specimens like feces as well as Non-clinical specimens like Food & Water.
It is quite helpful when there is a very minute quantity of specimen is available or the viable count is low.
The specimen is inoculated in APW medium prior to the plating on Selective medium for Vibrio cholrae, e.g. Thiosulfate-citrate-bile salts-sucrose agar (TCBS) medium.
In fact, Alkaline peptone Water is a simple modification of Peptone water which is made alkaline by adding the Sodium Hydroxide solution and pH is adjusted to 8.4 – 9.0.
This Alkaline pH of APW eases in the growth of Vibrio species and inhibits the growth of commensal intestinal bacteria which are commonly present in the Clinical Specimens.
Alkaline Peptone Water is quite helpful when it needed to isolate the organism from any of the following cases:
- Convalescent patients i.e. the patients who recover from the infection but still are a source of infection to others.
- suspected asymptomatic infections
- environmental specimens, and
- Whenever high numbers of competing organisms especially Intestinal flora are likely to be present in the Specimen.
The principle of Alkaline Peptone Water (APW)
APW is the best Enrichment medium for the isolation of Vibrio species especially the Vibrio cholerae.
Alkaline peptone water contains following substances which ease in the growth of Vibrio species and inhibits the growth of unwanted organisms.
Peptone present in APW provides carbon and Nitrogen source, Aminoacids, Vitamins, and other essential nutrients that promote the growth of microbes.
Sodium Chloride present in Alkaline Peptone water maintains the osmolarity of the medium and promotes the growth of microbe.
Sodium Hydroxide is used to make the medium Alkaline which is adjusted to 8.4 – 9.0 which promotes the growth of Vibrio species and inhibits most of the commensal Bacteria.
The composition of Alkaline Peptone Water (for 500 ml APW)
|Components||Quantity (in grams)|
|Distilled water||500 ml|
|Final pH at Room Temperature||8.6±0.2|
Procedure for the Preparation of Alkaline Peptone Water
Weigh 5 gm of Peptone & Sodium Chloride separately with the help of weighing scale.
Now take 250 ml of distilled water in a volumetric flask or in a Beaker and to this add the above-weighed Peptone and Sodium Chloride.
Swirl the flask gently to mix the contents or stir in case you are making the solution in a beaker with the help of sterile stirrer.
Gently heat the content in Flask or Beaker to completely dissolve the Peptone and Sodium Chloride.
When the content dissolves completely then add distilled water and make the final volume 500ml.
Adjust the pH of the solution to 8.6 ± 0.2 using 1M Sodium Hydroxide (NaOH) solution.
Now Dispense the Prepared Alkaline Peptone Water in 10 ml Screw-cap Glass bottles.
Now Autoclave the Bottles containing APW at 121 °C, 15 psi pressure for 15 minutes.
Note: Keep the cap of screw-cap bottles slightly loose while autoclaving it. It is because the bottles may burst due to the steam produced while autoclaving.
Now, tighten the bottle caps after the APW medium cools.
In this way, the Alkaline Peptone Water medium is prepared in laboratory.
Label the bottle clearly as Alkaline Peptone Water with the batch number, the name of the person who prepared it, date of preparation and date of expiry (Usually 2 years from the preparation), and document in the quality control log-book of your Laboratory.
Tighten the screw-cap on the bottle to prevent absorption of water vapors from the air & change in pH, and store in a cool & dark place.
Inoculation of the fecal sample in Alkaline Peptone Water (APW)
The APW medium can be inoculated with liquid/ watery stool specimen, fecal suspensions, or rectal swabs.
Inoculate the Alkaline Peptone Water bottle with the obtained specimen.
Note: The stool inoculums should not exceed 10% of the volume of the broth e.g. for 10 ml of broth medium the specimen inoculum must not exceed 1 ml.
Incubate the tube with the cap loosened at 35°C to 37°C for 6 to 8 hours.
After 6 to 8 hours’ incubation, subcultures to Thiosulfate-citrate-bile salts-sucrose agar (TCBS) should be made with 1 – 2 loopful of Alkaline Peptone Water.
Note: Always pick the inoculum from the surface or topmost area of the Cultured APW bottle because Vibrio species preferable grow on the surface. Do not shake or mix the tube before subculturing.
!Imporant! If the first cultured APW medium cannot be subcultured after 6 to 8 hours of incubation then, subculture it after 16-18 hours to a fresh tube of Alkaline Peptone Water.
This second cultured APW tube should be subcultured to a solid medium (TCBS) after 6 to 8 hours of incubation.
Observations & Identification of Vibrio cholerae
Vibrio cholerae grows rapidly in the Alkaline Peptone Water produces turbidity on and below the surface specifically within 3-6 hours.
You can follow the further steps listed below in order to confirm that whether the cultured organism is Vibrio or not:
- Hanging drop Preparation for the motility of bacteria.
- Gram staining of the culture (Vibrio cholerae appear as Gram-negative curved bacilli)
- Culture on TCBS medium.
The appearance of Medium after preparation
Alkaline peptone water appears light yellow colored clear solution without any precipitate.