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#3 Microbiological Medium Science Series

 

 

Saburo OHKAWA, Director of Ohkawa Microorganism & Medium Laboratory

Biography of Mr. Ohkawa

https://www.facebook.com/ohkawa.saburo

 

Preface

There are many types of selective medium for Staphylococcus aureus such as Mannitol Salt Agar, Mannitol Salt Agar with egg yolk, Staphylococus Agar #110, Baird-Parker Agar, KRANEP Agar, Coagulase-Mannitol Agar, Tellurite-Glycine Agar, CHROMagar SA Agar, and DOXSA.

Standard Methods of Analysis in Food Safety Regulation of Japan adopts Mannitol Salt Agar with egg yolk and Baird-Parker Agar.

Baird-Parker Agar is widely used in overseas countries, whereas Mannitol Salt Agar with egg yolk is dominant in Japan.  In this piece of column, I will deal with this Mannitol Salt Agar with egg yolk.

 

Mannitol Salt Agar with egg yolk

  

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 Mannitol Salt Agar with egg yolk

Mannitol Salt Agar with egg yolk   37°C, 48 hours


 

1. Principle

The basic principle of this medium is a discovery by Koch that only staphylococci grows in culture media containing 7.5% of sodium chloride.  And then Chapman developed a selective isolation medium for Staphylococcus aureus made by adding 7.5% of sodium chlorideaddedphenol red - mannitol salt agar (1945).  Gillespie, W. A., and V. G. Alder, et.al. reported that this Mannitol Salt agar with egg yolk added is effective for the isolation of Staphylococcus aureus (1952).


The basal medium, composed of peptone with beef extract added, is rich in nutrients.  This is designed to help and promote the growth of staphylococci stressed by high concentration of salt.  Since this medium contains 7.5% of sodium chloride as a selective agent for staphylococci, growth of most other bacteria is inhibited.  Also, mannitol, phenol red, and egg yolk are contained as differential agents for Staphylococcus aureus.  Mannitol-fermenting bacteria form yellow colonies and non-fermenting ones form red colonies.  Also, white turbidity around colonies (halo) shows lecithinase capacity, and pearlescent colonies indicate lipase activity.  The above-mentioned properties distinguish Staphylococcus aureus from other staphylococci.

 

2. Approximate formula for per Liter


Beef extract 1g
Pancreatic Digest of Casein 5g
Peptic Digest of Animal Tissue   5g
Sodium chloride 75g
D-mannitol 10g
Phenol red 25mg
Agar 15g
Egg yolk (50%) 100ml

pH 7.4±0.2

 

3. Functions of the agar components

Beef extract

Beef extract, rich in vitamin, nucleic acid, amino acid, organic acid, mineral, etc., is used as a supplemental growth-promoting substance for staphylococci rather than carbon/nitrogen source.  Beef extract is made by leaching meat in water and concentrate it (by heating).  As the heating modifies the components, high concentration of beef extract can inhibit the growth of bacteria.  Therefore, it is usually used at approximately 0.3-0.5 % of concentration.

 

Casein peptone, meat peptone

Essential nutrients for bacterial growth are nitrogen source and carbon source.  Since bacteria cannot break down protein by themselves, they cannot use protein as nutrient unless protein is digested or decomposed to be the form of polypeptide or peptide (a substance generated from digestion or decomposition of protein is called peptone). 

 

Peptones generally added in culture media are; casein peptone, soy peptone, meat peptone, myocardial peptone, and gelatin peptone.  Among various types of peptones, most suitable ones are chosen according to the composition of the medium.  Mixture of casein peptone (pancreatin digestion of the pancreas) and meat peptone (pepsin digestion of meat) are used in mannitol salt agar with egg yolk.  Casein peptone is economically superior, rich in tryptophan, and poor in sulfur amino acid.  Meat peptone is poor in tryptophan and rich in sulfur amino acid, vitamin, and other growth factors.

 

Sodium chloride

Staphylococci can grow in the presence of high concentration of salt (7.5%) whereas many other bacteria cannot.  Exploiting this property, sodium chloride is contained as a selective agent for staphylococci

 

The reason why staphylococci can grow with such a high-concentrated salt is that it can change its phospholipid composition of membrane to strengthen its barrierhood against sodium ion permeation.  (When cultured in hypersaline environment, cardiolipin increases significantly and acts as a barrier against sodium ion permeation.)  This maintains the homeostasis of monovalent cation within a bacterial body, which enables a constant osmotic pressure.

 

Mannitol

Mannitol (carbohydrate) is contained to serve as a carbon source for energy obtainment, and differentiate bacterial species by breakdown capacity of carbohydrate.  Mannitol helps to distinguish its fermenting bacteria from non-fermenting ones.

 

Phenol red

Phenol red, a type of pH indicator, is a pigment whose color changes depending on pH.  It turns yellow with pH6.8 or below and red with pH8.4 or above.  Mannitol-fermenting bacteria form yellow colonies and non-fermenting ones form red colonies.

 

If mannitol is fermented, acid is produced and it acidifies and yellow-discolors the culture medium.  If mannitol is not fermented, no acid is produced.  In this case, only ammonia is produced by degradation of peptone, and the ammonia alkalifies and red-discolors the medium.

 

Agar

Agar is used to solidify media.  Raw material of agar is seaweed such as Gelidiaceae and Gracilaria vermiculophylla.  The latter one is generally used to make agar for a culture medium (for its reasonable price).  

 

A principal component of agar is agarose with linear sugar, which makes bacterial degradation difficult.  Agar can contain relatively high weight of water molecules and has a spongy structure.  It can maintain water and store nutrition in it.

 

For that reason, agar is suitable for a culture medium of bacteria.  The temperature at which agar medium begins to melt by heating is called “melting point”, and that melted agar medium begins to solidify is called “congealing point”.  The melting point of agar is 85-93 °C and the congealing point is 33-45 °C.  These figures vary according to the ingredients mixed into agar.  Quality of agar determines quality of the medium.  Fine agar has superior transparency, jelly strength, viscosity, and water retentivity. 

 

Egg yolk

Egg yolk contains lecithin (phospholipid) and triglyceride (lipid).  Although Staphylococcus aureus can break down these two components, many other staphylococci cannot.  Harnessing this property, egg yolk is added as a selective agent for Staphylococcus aureus.  If lecithin is degraded, circumference of colonies will become white-turbid by lecithin’s crystallization (halo phenomenon).  Also, breakdown of triglyceride will make surface of colonies pearlescent, which is called lipovitellin lipase reaction.

 

 

4. How to use (quantitative culture)

# Conducted if positive in a qualitative method.

 Dilute 10% food emulsion tenfold in phases.

 Deliver 0.1ml of emulsion of each diluting phase by drops onto Mannitol Salt agar with egg yolk and spread it with a bacteria spreader.

Culture them for 48hours at 37°C.

Count colonies having positive reaction for both mannitol fermentation and egg yolk, and figure out the bacterial count per gram.

  

5. Limitation of this culture medium

1. Bacteria other than Staphylococcus aureus have a positive reaction to egg yolk.

Bacteria other than Staphylococcus aureus, such as Bacillus cereus, Micrococcus luteus, S. saprophyticus, and S. schleiferi, can grow and generate turbidity around colonies (halo) similar to that of Staphylococcus aureus. Even when typical colonies are formed in this culture medium, a textbook-based test for identification of Staphylococcus aureus is needed.

 

2. Grown colonies cannot be used for coagulase tests.

Coagulase tests using colonies grown in this medium will produce false-negative results.  (Colonies grow in non-selective media should be used for coagulase tests.)

 

3. Some Staphylococcus aureus show a negative reaction to egg yolk.

Around 10% of animal-derived Staphylococcus aureus strain show negative reaction to both egg yolk and mannitol fermentation.  This medium is not suitable for the testing of food materials for dairy products such as cheese.

 

4. This medium is not suitable for detecting Staphylococcus aureus from heat-treated materials (the recovery rate is low).

Damaged Staphylococcus aureus in food grow insufficiently.  If cell membrane or cell wall of a bacterium in food is damaged by heating, drying, freezing or other production processes, growth of the damaged bacterium is limited or stopped.  Especially damaged bacteria are apt to be influenced by high-concentrated sodium chloride and their growth will be insufficient.  This can lead to an inaccurate measurement of Staphylococcus aureus count. 

  

 

References:

 Koch.1942.Zentralbl.Nakteriol.Parasitenkd.Abt.1Orig.149:122

 Chapman.1945.J.Bacteriol.50:201

 Gillespie, W. A., and V. G. Alder. 1952. J. Pathol. Bacteriol. 64:187–200.

 Baird-Parker A. C. and Davenport E. 1965. J. Appl.Bact. 28. 390-402.

 BarakE. Ricca and S. M. Cutting: 2005Mol. Microbiol.55 33 0-338

 Tomochika, K., Journal of Antibacterial and Antifungal Agents, 30:85-90, 2002.

 Sakazaki, R., New Medium Science, Kindai Shuppan, 1988.

 Ikeda, T., REPORT of the Hokkaido Institute of Public Health, 59, 61-62, 2009.

 

 

The basic principle of this medium is a discovery by Koch that only staphylococci grows in culture media containing 7.5% of sodium chloride.  And then Chapman developed a selective isolation medium for Staphylococcus aureus made by adding 7.5% of sodium chlorideaddedphenol red - mannitol salt agar (1945).  Gillespie, W. A., and V. G. Alder, et.al. reported that this Mannitol Salt agar with egg yolk added is effective for the isolation of Staphylococcus aureus (1952).

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