Campylobacter Agar Base

Intended Use

Campylobacter Agar Base, when supplemented with blood or other additives and antimicrobial agents, is used for the primary isolation and cultivation of Campylobacter jejuni subsp.
jejuni from human fecal specimens. Several prepared selective media formulations are provided for the same purpose.Summary and ExplanationIn 1972, Dekeyser et al. reported that C. jejuni was isolated from the feces of patients with diarrhea and acute gastroenteritis using a filtration technique and a blood-containing selective medium with antimicrobics to suppress the normal enteric flora.1 Subsequently, Skirrow and other investigators reported similar blood-based selective media that differed in the numbers and types of antimicrobics.2-6 Bolton et al. reported that charcoal can effectively replace the blood in selective media for campylobacters.7
In 1978, Blaser et al. reported success in isolating C. jejuni with a medium containing four antimicrobics incorporated into Brucella Agar supplemented with 10% defibrinated sheep
blood.3,4 Subsequently, cephalothin was incorporated to increase its ability to inhibit the normal bacterial flora associated with fecal specimens.5
In 1983, Reller et al. introduced an improved selective medium containing cefoperazone, vancomycin and ampho tericin B (CVA) for isolation of C. jejuni.6 They reported that this combination of antimicrobial agents provided better inhibition of normal fecal flora for easier detection of C. jejuni than the selective blood agar plate developed previously. Karmali et al., in 1986, evaluated a blood-free, charcoal-based selective medium (designated CSM) in parallel with a Skirrowtype selective medium containing lysed horse blood. They reported that the quality of Campylobacter growth on CSM (luxuriant growth with smooth and effuse colonies) was similar to that seen on blood-based media and was significantly more selective than Skirrow medium.8

Principles of the Procedure

These media support the growth of Campylobacter species due to their content of peptones, yeast extract and other digests, extracts and components specific for the individual formulations provided. Campylobacter isolation relies, in addition, on a medium’s selectivity, which depends on the antimicrobial agents in the medium, a microaerophilic environment and the incubation temperature of 42°C, which suppresses the growth of most normal bacteria.9
The antimicrobial agents required to make Skirrow’s and Blaser’s formulations are provided as Campylobacter Antimicrobic Supplement Skirrow and Campylobacter Antimicrobic Supplement Blaser, respectively.
Campylobacter Agar with 5 Antimicrobics and 10% Sheep Blood supports the growth of Campylobacter species due to its content of peptones, dextrose, yeast extract and blood. The peptones supply nitrogenous compounds, carbon, sulfur and trace ingredients. Yeast extract is a source of the B-complex vitamins. Dextrose is utilized as an energy source. Sheep blood supplies additional nutrients. The incorporation of the antimicrobial agents (amphotericin B, cephalothin, polymyxin B, trimethoprim and vancomycin) suppresses the growth of the normal microbial flora in fecal specimens, thereby facilitating isolation of C. jejuni.
Skirrows Medium contains, in addition to the usual nutritional components, laked horse blood, which supplies the X factor (heme) and other growth requirements. Vancomycin inhibits
gram-positive bacteria, polymyxin B inhibits most gramnegative bacilli except Proteus and trimethoprim is inhibitory for Proteus spp.
Campy CSM Agar consists of Columbia Agar Base supplemented with activated charcoal, hematin, sodium pyruvate and three antimicrobial agents (cefoperazone, cycloheximide and vancomycin). The charcoal, hematin and sodium pyruvate improve the aerotolerance of Campylobacter species; it has been suggested that these supplements act as quenching agents of photochemically-produced toxic oxygen derivatives.8 Cefoperazone is a cephalosporin antibiotic that suppresses the growth of gram-negative enteric bacilli and some grampositive
species. Vancomycin is a glycopeptide antibiotic that inhibits many species of gram-positive bacteria. Cycloheximide is an antifungal agent.
Campy CVA Agar consists of Brucella Agar, a general-purpose medium that supports the growth of Campylobacter species. Defibrinated sheep blood provides additional nutrients. Antimicrobial agents are incorporated to suppress the growth of normal fecal flora that could mask the presence of C. jejuni. Cefoperazone is a cephalosporin antibiotic that suppresses the growth of gram-negative enteric bacilli and some gram-positive species. Vancomycin is a glycopeptide antibiotic that inhibits many species of gram-positive bacteria. Amphotericin B is an antifungal agent.

User Quality Control

Identity Specifications
Campylobacter Agar Base
Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:                       3.95% solution, soluble in purified water upon
                                    boiling. Solution is medium to dark amber, clear
                                    to slightly opalescent.
Prepared Appearance:     Plain – Medium to dark amber, very slightly to
                                    slightly opalescent.
                                    With 10% sheep blood – Cherry red, opaque.
Reaction of 3.95%
Solution at 25°C:           pH 7.4 ± 0.2
Cultural Response
Campylobacter Agar Base
Prepare the medium per label directions; e.g., with 10% sterile defibrinated sheep blood and antimicrobic supplements (Skirrow or Blaser). Inoculate and incubate at 42°C under microaerophilic conditions for 40-48 hours.

Formula

Campylobacter Agar Base
Approximate Formula* Per Liter
Proteose Peptone No. 3............................................. 15.0 g
Liver Digest................................................................ 2.5 g
Yeast Extract.............................................................. 5.0 g
Sodium Chloride......................................................... 5.0 g
Agar........................................................................ 12.0 g
Campylobacter Antimicrobic Supplement Skirrow
Formula Per 5 mL Vial
Vancomycin.............................................................. 5.0 mg
Polymyxin B.........................................................1250.0 units
Trimethoprim............................................................ 2.5 mg
Campylobacter Antimicrobic Supplement Blaser
Formula Per 5 mL Vial
Vancomycin................................................................ 5.0 mg
Polymyxin B...........................................................1250.0 units
Trimethoprim.............................................................. 2.5 mg
Cephalothin................................................................ 7.5 mg
Amphotericin B............................................................ 1.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 39.5 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes. Cool the medium to 45-50°C.
4. Aseptically add 5-7% sterile lysed horse blood or 10% sterile defibrinated sheep blood. Mix thoroughly.
5. To prepare Skirrow’s medium: aseptically rehydrate one vial of Campylobacter Antimicrobic Supplement Skirrow with 5 mL of sterile purified water. Rotate in an end-over-end motion
to dissolve the contents completely. Store the rehydrated vials at 2-8°C. Use within 24 hours after rehydration.
To prepare Blaser’s medium: aseptically rehydrate one vial of Campylobacter Antimicrobic Supplement Blaser with 5 mL of sterile purified water. Rotate in an end-over-end motion to
dissolve the contents completely. Store the rehydrated vials at 2-8°C. Use within 24 hours after rehydration.
Aseptically add 1% of the desired antimicrobic supplement (10 mL of supplement to 1 L or 5 mL of supplement to 500 mL of medium base). Mix thoroughly, avoiding the formation of air bubbles and dispense into sterile Petri dishes. 
6. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Use standard procedures to obtain isolated colonies from specimens. If immediate inoculation of a Campylobacter agar cannot be performed, the use of a suitable holding medium (e.g., Campylobacter Thioglycollate Medium with 5 Antimicrobics) is recommended. Incubate inoculated plates at 42°C in an atmosphere conducive to the primary isolation and cultivation of microaerophilic organisms. This atmosphere can be achieved by using one BBL™ CampyPak™ Plus disposable gas generator envelope in a GasPak™ 100 jar, three envelopes in a GasPak 150 jar or using the Bio-Bag™ Type Cfj or GasPak EZ Campy systems. Alternatively, the atmosphere can be achieved using evacuation of GasPak vented jars and replacement with cylinder gases, or by using the Fortner principle.10
Examine plates at 24 and 48 hours.
NOTE: If plates are to be examined after 24 hours of incubation, treat plates as if they were anaerobic cultures; i.e., examine plates quickly and place them back into a reduced oxygen atmosphere immediately after examination.

Expected Results

Campylobacter jejuni produces two types of colonies. One is small, raised, grayish-brown, smooth and glistening with an entire translucent edge. The other colony type is flat, mucoid,
translucent, grayish and has an irregular edge. A small percentage of strains may appear tan or slightly pinkish.11 Colonies tend to spread, especially when initially isolated from fresh clinical specimens.

Limitations of the Procedure

1. Due to the presence of 15 mg/L of cephalothin, growth of C. fetus subsp. fetus will be inhibited on Campylobacter Agar with 5 Antimicrobics and 10% Sheep Blood; therefore, this medium is not recommended for the isolation or culture of this subspecies.
2. Since C. jejuni is thermophilic, it is important to incubate the plates at 42°C; otherwise, growth will be delayed. Also, the higher temperature improves selectivity by inhibiting the
normal flora.
*Store at 2-8°C.

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