Mannitol Salt Agar

 

Objectives:


  1. To introduce the concepts of selective and differential media.
  2. To demonstrate the presence of "normal" flora.
  3. To introduce the use of specific media to isolate specific organisms.

Theory:

Microbial contamination of food products could serve as a significant source of infectious disease. The food industry is aware of the organisms that are frequently responsible for outbreaks of food borne illness and has developed methods for detecting and identifying pathogens.

A "selective" medium is one which encourages the growth of desired organisms, usually by discouraging the growth of undesired organisms. A "differential" medium is one that offers visible indication of a physiological property, such as the fermentation of a carbohydrate. Mannitol salt agar(MSA) was developed by microbiologists seeking a method for isolating Staphylococcus aureus, a pathogen frequently transmitted by contaminated food. This medium contains 7.5% salt, which is inhibitory to most bacteria other than staphylococci. While most species of Staphylococcus are capable of growing in this high salt concentration, S. aureus is also capable of fermenting the carbohydrate mannitol. MSA contains the pH indicator phenol red, which has an orange color near neutrality but turns bright yellow at acidic pHs. On MSA, colonies of S. aureus are yellow and may even turn the medium around the colony yellow due to the drop in pH around the colony of a mannitol fermenter.

Numerous species of Staphylococcus, including S. aureus may be isolated from the skin, nose and throat of healthy individuals. This accounts for its presence in foods, primarily those which not heated after preparation which involved handling, such as cream puffs or potato salad. There is also a high carrier rate for S. aureus in hospital personnel, which explains the high incidence of the organism in "nosocomial" infections. Nosocomial infections are infections that develop in the course of a hospital stay and were not present in the patients upon admission to the hospital.

 

Materials per student:

1 cotton tipped swab
water
1 Mannitol Salt Agar (MSA) plate / pair
An inoculating loop and means of sterilizing it


Protocol:

  1. Moisten the swab with water just until it is damp, not dripping.
  2. Rub the swab inside a nostril or in the back of the throat. (NOT both places)
  3. Smear the swab onto about one quarter of the MSA plate, rolling it around to assure good inoculation. Discard the swab as directed.
  4. Sterilize the inoculating loop; cool; streak for isolation on the remainder of the plate.
  5. Label and incubate the inverted plate at 37*C until the next lab period.
  6. Observe the plate for typical colonies. Record observations.


Appearance of Mannitol Salt Agar plate

The Quadrant Streak Plate Technique

  1. Using a flame sterilized inoculation loop, spread (streak) the culture over a small area near the edge of the plate (A) using a continuous motion.
  2. Flame sterilize the loop and allow it to cool.
  3. Turn the plate and spread the bacteria from the end of area A across area B. (You can see the streak marks of the loop in area A.)
  4. Flame sterilize the loop and allow it to cool.
  5. Turn the plate in the same direction and spread the bacteria from the end of area B across area C.
  6. Flame sterilize the loop and allow it to cool.
  7. Turn the plate in the same direction and spread the bacteria from the edge of area C across the rest of the plate (area D).
  8. Flame sterilize the loop before setting it down.