I agree absolutely with the previous answer. The colony inspection on specific agar media are very discriminative in the case of S. aureus. Obviously the VITEK system adds the value of automated discrimination. The molecular methods are also very reliable, but most costly and time consuming.

Hi Caroline,

There are various chromogenic media that you can use that will reduce the number of isolates you need to analyse. If you could get access to a MALDI-TOF, that would be the quickest and cheapest way to confirm identity. Otherwise, you could use a real-time PCR assay. Good luck.

J.

using mannitol salt agar media , the charge color in grow of S. aureus

but no charge in grow of S. epidermis

There are two selective plates that may help isolate S. aureus and S. epidermidis from complex samples; One use Blood Agar containing Colistin Nalidixic acid, that selects for Gram positives.

Second, test all colony types on Mannitol Salt Agar plates: . High salt helps select/ identify Staphs. You could do Coagulase (Rabbit plasma) to differentiate S. aureus (Positive, forms a clot within 3 to 12 hours) from S. epidermidis (Negative does not clot plasma)

Mannitol salts agar is quick and easy presumptive test

I normally use mannitol salt agar medium (medium changes color with Staphylococcus aureus due to fermentation mannitol) and confirm the result with biochemical tests (API staph and catalase test)

Hi Caroline,

You can use following steps for identification of S. aureus:

1- Blood agar:Hemolysis property of Staphylococci can be seen on blood agar.

2- Mannitol Salt Agar Phenol Red: Mannitol positive S. aureus converts the colour of the medium from red to yellow. S. epidermis is mannitol negative.

3- Coagulase test

4- Baird Parker Agar: S. aureus forms black colonies on baird parker agar. You can see lysis around the colonies. Lysis confirms that colonies are coagulase positive staphylococcus aureus

A very accurate method for the differentiation of these strains is using a technique PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM using dnaJ gene and two restriction endonucleases. Fingerprint patterns obtained give the 100% possibility of differentiation of these two species.

More efficiently you can isolate using blood agar. More selective - using chromogenic media for species isolation. Chromogenic Staph 24 Agar (Thermo Fisher) for S. aureus isolation could be useful however, I don't know chromogenic medium for S. epidermidis on the market today.

In any case identification of genera and species needs to be performed for reliable results: for genera: gram-positive cocci (not too big) in clusters, catalase positive, furazolidone susceptible. For species: biochemical tests (i.e. rapID Staph Plus, Thermo Fisher), or molecular technique (PCR or sequencing). Any other tests (plasma, latex agglutination, etc., pigment) useful as additional tests but did not detects species itself.

Ive got one easier method for you. Chromagar. BD makes selective agar plates for S. aureus and S. epidermidis - these are known as Chromagar plates. You can buy these here: http://www.bd.com/ds/productCenter/214982.asp.

Once you have done that I would do whole colony PCR using MLST primers to determine the clonal type.

I think, at the beginning, you should use agar Baird Parker. Staphylococci grows very well, like black colonies, and the aureus species produce an halo of hydrolysis of lecitina around the colony. Then use any commercial system applied in clinical identification.

I would recommend Blood agar and Mannitol salt agar for isolation of the said organisms.

Simply using Mannitol Salt Agar (MSA) will selectively isolate and differentiate the two species within 24 to 48 hours. On MSA, Staphylococcus aureus will appear yellow while the species epidermidis will grow as pink colonies.

There are a different species of Staphylococcus that break mannitol as well as much species that do not break it. Thus detection of mannitol fermentation is not useful for species identification. Macrococcus spp. and related bacteria from some environment grow on Mannitol Salt Agar as well (and on Baird Parker Agar too).

For author: do you need only to obtain some isolates of those two species for further investigations or do you need to detect them (just to know fact: present or absent in sample). From that depends the selection of methods, media, etc.

Thanks for all your answers which give me work lines. I really appreciate it. Modestas, i need to describe the population structure, so I need to isolate them for further characterization.

In the staphylococcus genus, one meets a score of species of which three are frequently found in human pathology. It is about S. aureus, S. saprophyticus and S. epidermidis. S. aureus is a positive coagulase and the two last species are at negative coagulase. However of another criterion can allow their identifications (table below).

S. aureus S. saprophyticus S. epidermidis

Mannitol + + -

DNase + - -

coagulase + - -

protein + - -

apart from s aureus, what other type of colonies can grow on baird parker agar ?

Some other Staphylococcus species, Micrococcus spp., Proteus mirabilis. It also depends on the composition (company produced this medium). However, this medium quite clearly select S. aureus that has black (not grey) collonies with clear halo. 

Sir, what could be the reason for not getting halo formation even after 48 hours, although the biochemical tests shows 2+ for coagulase and positive for thermonuclease.

Different S. aureus strains may have different expression of lecithinase, thus not all strains may form collonies  with clear zone. If specific thermonuclease gene of S. aureus was detected by PCR the species (S. aureus) identification is  proved.