Cat. no. K226 C Diff Banana Broth™, 16x100mm Tube, 10ml 20 tubes/box
Cat. no. FS1HD Flocked swab, nylon tip, standard-tip, standard-shaft (score at 123mm from tip), for general use, 6" overall swab length, sterile** 500 swabs/case
Cat. no. TP3F TransPRO™ Liquid Amies, multi-purpose, bacterial collection and transport swab, with advance HydraFlock™ technology, standard tip swab, easy-opening peel pouch, sterile** 50 swabs/pack
** Item sold separately


Hardy Diagnostics C Diff Banana Broth™ is recommended as a selective enrichment broth for the culture and recovery of C. difficile from environmental samples.

This product is not intended to be used for the diagnosis of human disease.


Approximately 1.7 million hospitalized patients develop nosocomial infections annually while being treated for entirely unrelated conditions; more than 98,000 (one in 17) of these patients will die due to complications from acquired infections.(5) Consequently, healthcare-associated infections (HAI) are a major cause of morbidity and mortality around the world, posing an enormous financial and physical burden on the global health care system.

In the U.S., the Centers for Disease Control and Prevention state that Clostridium difficile infection (CDI) surveillance is a core component of the CDC's Emerging Infections Program, Healthcare-Associated Infections Community Interface (HAIC).(3) C. difficile causes diarrhea linked to approximately 14,000 U.S. deaths annually, particularly in immune compromised and elderly patients who take antibiotics or are under routine medical care.(4) Deaths from C. difficile infection increased 400% from 2000 to 2007 and at least 94% of C. difficile infections are derived from contact in a medical or patient care facility. Of these, 25% of patients show symptoms in hospitals; the remaining 75% are from patients in nursing homes, treatment clinics and doctor's offices.(4) Since diarrhea is the leading symptom, C. difficile is readily spread from one care facility to another and from healthcare worker to patient due to soiled surfaces and equipment. The organism can be difficult to contain due to its ability to produce spores resistant to many commonly used disinfectants and which may lay dormant until transmission occurs. At present, it is estimated that C. difficile causes an additional $1 billion in extra healthcare costs annually.(4)

Current methods to detect pathogens like C. difficile utilize rapid techniques such as pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR), toxin-typing and mutation analysis. However, surveillance is performed after infection and involves patient testing for positive stool specimens. Thus, an efficient and easy-to-use detection method for monitoring the effectiveness of room disinfection procedures and for identifying environmental reservoirs of C. difficile spore contamination prior to the onset of disease could prevent infection, reduce HAI, and potentially reduce the number of deaths due to CDI.(2)

A study by Cadnum et al. compared the use of Clostridium difficile Brucella Broth with Thioglycolic Acid and L-cystine under aerobic conditions versus standard Clostridium difficile Brucella Broth incubated anaerobically for the recovery of C. difficile from hospital environmental samples. Their research concluded that broth containing the reducing agents Thioglycolic Acid and L-cystine was significantly more sensitive and specific at recovering C. difficile from the environment: 88% and 100%, respectively, versus 51% and 96% for Brucella Broth without these ingredients.(2) In addition, because Brucella Broth supplemented with Thioglycolic Acid and L-cystine can be incubated aerobically and samples obtained using routine environmental monitoring procedures, researchers determined that broth enrichment with these reducing agents was an effective tool at identifying environmental sources of C. difficile in healthcare facilities.(2)

Hardy Diagnostics C Diff Banana Broth™ is based on the formulation proposed by Cadnum et al. The basal medium is Brucella Broth supplemented with hemin and vitamin K to promote the growth of fastidious anaerobic microorganisms such as C. difficile. L-cystine acts as a growth factor for thiol-dependent or sulfur-containing amino-acid-requiring microorganisms and, in conjunction with the small amount of agar in the formulation, acts as an oxygen reducer to promote the growth of obligately anaerobic bacteria without the need to incubate samples anaerobically.(2,8) In addition, mannitol is used instead of glucose to prevent glucose repression of C. difficile cytotoxin synthesis and release.(7) Mannitol fermentation is also an important characteristic of C. difficile identification and can be used to differentiate this species from C. sporogenes, a similar organism.(6-8) Lysozyme, thioglycolic acid, and sodium taurocholate are included to promote C. difficile spore germination from environmental samples, and the pH of the medium is optimized to facilitate fermentation of mannitol by germinating spores of C. difficile via the alkaline up-regulated PEP-phosphotransferase (PTS) transport system.(1,2) Cycloserine and cefoxitin are selective agents to inhibit the growth of unwanted environmental organisms. Neutral red is the pH indicator and sodium bicarbonate acts as a buffering agent to maintain alkaline conditions required to promote mannitol fermentation.(10)

Confirmation of C. difficile should be performed on positive C Diff Banana Broth™ tubes using established methods. If confirmed, corrective measures using a sporicidal disinfectant can be taken to eliminate environmental reservoirs from facilities and equipment to prevent cross-contamination and infection.(2-4)


Ingredients per liter of deionized water:*

Brucella Broth 28.0gm
D-Mannitol 6.0gm
Sodium Bicarbonate 5.0ml
Neutral Red 5.0ml
Vitamin K1 (1mg/ml) 1.0ml
Hemin 1.0ml
Thioglycolic Acid 1.0gm
L-Cystine 1.0gm
Sodium Taurocholate 0.5gm
D-Cycloserine 500.0mg
Cefoxitin 16.0mg
Lysozyme 5.0mg
Agar 1.0gm

Final pH 7.6 +/- 0.2 at 25 degrees C.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt, store at 2-8ºC away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Sample Collection: As a general rule, potentially infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Consult laboratory procedures or listed references for information on sample collection and environmental monitoring procedures.(1-9)

Sterile swabs (Cat. no. FS1HD) pre-moistened with a sterile diluent such as Saline, 0.85% (Cat. no. R45) can be used to obtain samples from environmental surfaces. For assessing the efficacy of disinfection procedures, a neutralizing broth such as D/E (Dey-Engley) Neutralizing Broth (Cat. no. K108) should be used.

Verify the swab can be broken off into and fit securely in the C Diff Banana Broth™ tube during incubation prior to use. Flocked swabs (Cat. no. FS1HD) designed to elute the sample should be placed directly into C Diff Banana Broth™ tubes after sampling for best results. However, transport swabs such as ESwab™ or TransPRO™ Liquid Amies (Cat. no. TP3F) can also be used for surface sampling and samples taken to the lab for inoculation into C Diff Banana Broth™. Follow the manufacturer's instructions for storage and holding time prior to inoculation. See procedure step 2b for inoculation of C Diff Banana Broth™ from a transport medium.

A swab with a wooden shaft should not be used for sample collection when using molecular confirmatory methods, as this may interfere with PCR results or other molecular assays.

General Procedure for Collecting Environmental Swab Samples:

Collect the sample by aseptically rubbing a pre-moistened swab over the sample area (approximately 50cm2), reversing directions between strokes, and return the swab to the collection device or place the swab directly into C Diff Banana Broth™. When sampling bedding, curved or irregular surfaces, run the swab over the surface of the object as indicated to approximate a similar sampling area. If the sample is not immediately taken to the lab for processing, it can be refrigerated for up to 24 hours prior to analysis.

Method of Use for C Diff Banana Broth™:

1. Allow the medium to warm to room temperature prior to inoculation.

2a. Inoculate the broth by inserting the environmental sample swab directly into the bottom of the tube. Rotate the swab to emulsify the sample in the broth and carefully break the swab shaft, leaving the tip of the swab at the bottom of the tube during incubation. Replace the cap and twist down tightly to seal the tube. It is important that the caps be tightly sealed during incubation in order to create an atmosphere of reduced oxygen tension necessary for anaerobic conditions at the bottom of the tube.

2b. Alternatively, transport media systems can also be used for surface sampling, and samples taken to the lab for inoculation into C Diff Banana Broth™. Follow the manufacturer's instructions for use (IFU) for storage conditions and holding time prior to inoculation. If the transport medium contains a flocked or elution swab such as ESwab™ or TransPRO™ Liquid Amies (Cat. no. TP3F), inoculate the C Diff Banana Broth™ with 50-100µL of the transport medium and cap the tube tightly as outlined in 2a.

3. Incubate inoculated tubes with swabs aerobically with tight caps at 35-37 degrees C. for 48 to 72 hours.

4. Observe daily for development of a yellow color in the medium.

5. Confirmation of C. difficile should be performed on positive C Diff Banana Broth™ tubes using established methods, so corrective measures can be taken to eliminate environmental reservoirs from facilities and equipment to prevent cross-contamination and infection.


Development of turbidity and a yellow color change in the medium after incubation is indicative of growth and a positive mannitol fermentation reaction and suggests the presence of C. difficile. Perform confirmatory tests on positive tubes as outlined in the references or as established by laboratory procedure.(4,10)

Lack of yellow color development is indicative of a negative mannitol fermentation reaction and suggests the absence of C. difficile.


Established confirmatory methods such as C. difficile latex tests (e.g. Cat. no. M41), enzyme immunoassays (EIA) for toxins A and B or glutamate dehydrogenase, spectrophotometric analysis, and PCR tests for toxin B genes are required for confirmatory testing following culture in C Diff Banana Broth™.(2)

This product should be incubated with the caps tightly sealed to create an atmosphere of reduced oxygen tension required to support the growth of C. difficile. Failure to incubate with tight caps may produce delayed reactions or erroneous results.

Sample testing should take into account the confirmatory assay being performed and the type of swab recommended for use with the assay as indicated by the manufacturer's technical literature or instructions for use (IFU).

Failure to place flocked swabs directly into C Diff Banana Broth™ upon sampling or to incubate environmental sample swabs in C Diff Banana Broth™ may result in erroneous results.

When using a transport medium for sampling with delayed inoculation into C Diff Banana Broth™, follow the manufacturer's instructions for use (IFU) for storage conditions and holding time prior to use. For flocked or elution swab transport systems such as ESwab™ or TransPRO™ Liquid Amies (Cat. no. TP3F) with delayed inoculation into C Diff Banana Broth™, inoculate tubes with 50-100µL of the transport medium.

The design and implementation of a microbiological environmental monitoring program should be established prior to initiating sample collection to ensure the program has established a sampling and site plan, detects change, captures trends, and implements appropriate countermeasures.(9)


Standard microbiological supplies and equipment such as loops, swabs (Cat. no. FS1HD), applicator sticks, diluents such as Saline, 0.85% (Cat. no. R45) or D/E Neutralizing Broth (Cat. no. K108), transport swabs (Cat. no. TP3F), other culture media, confirmatory tests such as C. difficile latex test (Cat. no. M41), incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Clostridium difficile
ATCC® 9689
B 48-72hr 35°C Aerobic** Growth; color change from red to yellow
Clostridium perfringens
ATCC® 13124
B 48-72hr 35°C Aerobic** Growth; slight turbidity, no color change
Enterococcus faecalis
ATCC® 29212
B 48-72hr 35°C Aerobic Inhibited

** Tubes incubated with tight caps to create an atmosphere of reduced oxygen tension.


Physical Appearance

C Diff Banana Broth™ should appear hazy, and cherry red in color.

C Diff Banana Broth

An uninoculated tube of C Diff Banana Broth™ compared to a positive tube of Clostridium difficile (ATCC® 9689) grown for 48 hours at 35 degrees C.


1. Buggy, B.P., C.C. Hawkins, and R.Fekety. 1985. Effect of Adding Sodium Taurocholate to Selective media on the Recovery of Clostridium difficile from Environmental Surfaces. J. Clin. Microbiol. 21(4):636-637.

2. Cadnum, J.L., K.N. Hurless, A. Deshpande, M.M. Nerandzic, S. Kundrapu, and C.J. Donskey. 2014. Sensitive and Selective Culture Medium for Detection of Environmental Clostridium difficile Isolates without Requirement for Anaerobic Culture. J. Clin. Microbiol. 52(9):3259.

3. Centers for Disease Control and Prevention. Emerging Infections Program - Health care-associated Infections Projects. Web. Accessed 9 January 2015.

4. Centers for Disease Control and Prevention. Stopping C. difficile Infections. Web. Accessed 20 January 2015.

5. Cimiotti, J.P., L.H. Aiken, D.M. Sloane, E.S. Wu. 2012. Nurse staffing, burnout, and health care-associated infection. Am. J. Infect. Control. 40:486-490.

6. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

7. Kazamias, M.T. and J.F. Sperry. 1995. Enhanced Fermentation of Mannitol and Release of Cytotoxin by Clostridium difficile in Alkaline Culture Media. Appl. Environ. Microbiol. 61(6):2425-2427.

8. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

9. United States Pharmacopoeia and National Formulary <1116> (USP-NF). Rockville, MD: United States Pharmacopeial Convention.

10. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual , 6th ed. Star Publishing Company, New York, N.Y.

ATCC is a registered trademark of the American Type Culture Collection.