Here are some tips:

1. Enrichment in broth with 5-10% defibrinated blood (sheep or horse) will increase the chances of recovering C. jejuni.

2. Consider using Campy selective supplement (e.g., from Himedia) with Hemin.

3. Don't forget about microaerophilic condition (5% O2, 10% CO2, 85% N2)

4. Be careful about the thickness of your agar medium as it determines the water content of the medium.

Good luck!

Hi,

I agree with Dr. Salaheen

according to himedia, the campylobacter colonies should be grey in color and should appear spiral on gram staining under microscope. I am getting white or transparent colonies and arenot spiral..if anyone has their own microscopic image of campylobacter please share..

Amphotericin B and vancomycin (final conc. 10 μg/ml for both) should be added in your medium to make sure the bug you isolate is Campylobacter jejuni.

Dear Vandana,

Following Dr. Salaheen's suggestions, since you are working with environmental samples which may have low numbers of campylobacters and high level of background flora, I would suggest considering Preston broth as an alternative enrichment medium instead of Bolton broth.

Detection using Preston broth is useful for samples that contain significant numbers of microflora resistant to third generation β-lactams, like cefoperazone which is used in Bolton broth as well as in the commonly used mCCD selective agar. Such microflora includes e.g. strains of E. coli which appear white on mCCD agar.

During isolation on selective media, in order to avoid confusion, you can also try using chromogenic media, like Brilliant Campy Count Agar (BCCA, Oxoid) and Campylobacter Selective Agar (CASA, AES) which are highly selective media and make Campylobacter reading easy, through the enumeration of brick red colonies.

Hope this will help you!

Hey, thankyou for your reply. I am using wesley broth instead of bolton.

Good day Ms. Kaushal

Campylobacter jejuni are considered as microaerophilic organism which growth are best observed in an environment with 5% oxygen, 10% carbon dioxide and 85% nitrogen concentrations. You can achieve this by using GasPak E2 Generating System or by a much simpler candle jar method. Isolation of C. jejuni is also prone to contamination from other bacteria such as those microbiota of our colon thus, it is recommended to incubate your media in 42 degrees Celsius to minimize this contamination.

In terms of culture medium, i suggest to use Campy-Bap. It is a selective agar mainly for isolating C. jejuni and other enteric Campylobacter spp. It contains antimicrobials such as vancomycin and polymyxin B which can also enhance your isolation for C. jejuni.

Reference:

Mahon C.R. Lehman D.C. Manuselis G. (2015) Textbook of Diagnostic Microbiology, Fifth Edition

Hello Ms. Kaushal

The use of a sterile filter placed upon a nonselective Brucella Agar Blood Plate (BAB), followed by the incubation at 37 degrees Celcius in a hydrogen-containing atmosphere (Campycheck), is a method used to recover Campylobacter spp. from complex environmental matrices. While the enumeration of these spp is performed using serial dilutions of sample homogenate onto modified Campy-Cefex media followed by incubation at 37 to 42 degrees in a micro aerobic atmosphere. Furthermore, Campy-Bap, a selective agar which contains vancomycin and polymyxin V can also be performed for the isolation of your target specie.

You may also visit this link.

Chapter Campylobacter jejuni Isolation/Enumeration from Environmental Samples