You really do not need highly specialized media for cultivation of enterococci. However there is a pfizer selective enterococcus agar. I have used it. It is very good. Gives reliable growth of enterococci.

pfizer selective enterococcus agar avilable in Egypt, expensive or not?

I am not sure about Egypt. It is avaiable here in India and it is not very expensive.

Thank you

Enterococci grow very well on standard laboratory media such as blood agar - small beta-haemolytic colonies usually. Most species will also grow as pin-point lactose-fermenting colonies on MacConkey agar without crystal violet. They are aesculin positive, so media that contains aesculin is often used to isolate them as they will show up as pale colonies in a brown-black halo. kanamycin is added to media to select against the Enterobacteriaceae, which is really useful when you are culturing intestinal/faecal/sewage samples. Oxoid has a reasonably priced dehydrated medium that you can use.

Thank you very very much ,how i can keep enterococci colonies after their culture by any of the previous method you mention for long time e.g more one year .And whtat is the media suitable for that ?

Manal, it depends on what you have available. Long term storage of young cultures emulsified in brain heart infusion broth containing 10% glycerol in cryovials at -78C or lower will store them indefinitely. I usually add sterile glass beads as you then can just remove a bead from the cryovial without defrosting, which assists in preservation of the bacteria. If you don't have ultra-cool freezers, you may want to freeze-dry your young cultures. They will also keep indefinitely if stored at 4C. If you are to use them regularly then the use of agar slants i.e. blood agar kept in the fridge and sub-culture every 7 - 14 days will work. Note that this method could lead to some genetic or protein expression alterations of your colonies, so you must always test them to make sure that they retain the characteristic that you wish them to have i.e. antimicrobial susceptibility pattern, expression of certain virulence factors etc.

Thank you for interest, tell me if there is another method for long term storage of young cultures ?

There may be, but you will have to play around a bit as any other methods can lead to a decrease in survival of these bacteria i.e. standard freezer temperatures can maintain a few bacteria - I have found that even the hardy ones will decrease in number when stored like this - so you will have to test that. You will also have to test how long you can keep S. agalactiae on a slant in the fridge or at room temperature without significant loss in viability. Streptococci are relatively hardy but not as hardy as staphylococci.

Anyone about anaerobic culture i have problem with it

More details please

MacConkey Agar is most suitable medium for Enterococcus

Very right.

But it is better to prepare the medium in the lab using ingradients rather than going for a readymade medium.

One must not use crystal violet in this medium.

thanks Navaneeth , and thanks Jackie Picard :please give me all information (in steps) : for example in our lab.i culture urine sample on CLED media then check colonies by gram film (always mixed bacteria, enterococci with others) so, i subculture these colonies on bile esculin ,then check colonies again by gram film,the result enterococci with others,then i do antibiotic sensetivity for vancomycin resistant enterococci ( VRE ), if resistant.iwil keep these colonies untill collect 20 one VRE for anther steps as API, E- test, so i am afraid from keeping not pure strain of enterococci ,and failure all my work. so please tell me your experaince to obtain pure strain of enterocci.And thanks again. please explain in detals the methods of storage as you mention : skimmilked method OR Glecerol as an good methods to store the culture.

I always have contamination issues, when I don't have single colonies and when I don't purify a mixed culture from only one colony. Streak dilution or 10-fold dilution techniques, should allow you to see discrete colonies. The other problem that can arise is when using selective media, you pick-up living, but non-growing bacteria that is hiding underneath the colony.

When examining urine and wanting to look in particular for enterococci, I would do the following at the same time from the original specimen

Plate 1 CLED agar medium - for non-enterococci and enterococci (lactose-fermenting)

Plate 2 Kanamycin Bile Esculin agar medium for enterococci

Plate 3 vancomycin containing Bile esculin medium for vancomycin resistant enterococci (this is only necessary if you are really looking for them)

After 1 say incubation - sub-culture single target colonies only blood agar for purification. This media will ensure that you have pure colonies and is better for bacterial survival. Then perform any further identification and antimicrobial susceptibility tests. This gives results in 72 hours. If you want results in 48 hours, you may have to use 10-fold dilutions of the sample, to ensure that there is sufficient single colonies of a particular type to perform all the necessary tests

Thanks for valuable advice,please tell me about 10-fold dilution techniques but write to me quickly.

Enterococci are common faecal bacterium that easy to isolate and maintain in agar slants for further works.Techniques for that are available in diagnostic microbiology textbook.thank you.

I agree, I have never had a problem growing them. Therefore, I was concerned that bacterial overgrowth was the problem in this case. Hence tips on managing that. All one does is do a series of dilutions of your sample i.e. 1 ml to 9 ml PBS/normal saline (10-1 dilution), place 1 ml of this into 9ml PBS will get a 10-2 dilution). If your then remove 100ul from each tube and spread plate it using a sterile bent glass rod. you will have the following: 10-1(sample); 10-2 (1st dilution); 10-3 (2nd dilution). In mid-stream urine from infected individuals you would expect to grow quite a high number of bacteria, so you could even go one to two dilutions more. Note that the most predominant - it is usually by far- bacterium is the most likely cause of the cystitis.

I really had a problem with Enterococci identification but solved with VITEC2 system

Please suggest me, Which liquid medium is best to culture Enterococcus. I need to increase their concentration.

Macconkey agar is best suitable, since the organism is an enteric organism

Thank you@ Joseph A. Olugbojo