Enriched Chocolate Research Articles

ChocOmega-3: Innovative manufacturing of ω-3 enriched chocolate

Three-dimensional (3D) printing technology allows foods to be shaped into unique and complex. Chocolate is frequently used in food printing due to its extrusion melting capacity and attractiveness. In this study, chocolates enriched with ω-3 were produced with 3D printing technology. Using electrospraying, ω-3 was encapsulated within sodium alginate (SA) microparticles, and 3D printed chocolates were coated with those microparticles. Then, the ω-3 blend and ω-3-SA coated chocolates were compared. Before printing, the rheological properties of chocolate were analysed. In addition to their characteristics, the printed chocolates were evaluated for total phenolic content (TPC) and antioxidant capacity, in vitro gastrointestinal digestion, and ω-3 release profile. Fourier transform infrared (FTIR) spectroscopy indicated that ω-3 was successfully incorporated into the SA particles. According to the mechanical testing, the chocolate structures coated with ω-3-SA exhibited higher compressive strength than structures mixed with ω-3. The results revealed that the incorporation of alginate into pure chocolate scaffolds through the coating process resulted in an increase in compressive strength. The TPC and antioxidant capacities of ω-3-SA microparticles (NP) coated and ω-3 mixed chocolate samples were also significantly increased compared to those of pure chocolate after in vitro digestion. ω-3-SA NP-coated chocolate appeared to have a lower quality release profile. The faster release of encapsulated ω-3 at a pH value of 7 may be attributed to the fact that sodium alginate particles dissolve faster in high pH environments. This study revealed that 3D printing technology could be actively leveraged to create food-based products with the necessary ingredients to meet consumer demand.

A systematic review of randomized controlled trials on the health effects of chocolate enriched/fortified/supplemented with functional components.

The most significant contributor to global mortality are cardiovascular diseases. Dietary factors significantly impact the risk, advancement, and treatment of cardiometabolic conditions. Chocolate, known for its adaptability and capacity to stimulate pleasure centers, emerges as a promising vehicle for integrating different bioactive elements. This systematic review analyzed 10 randomized controlled trials investigating the health effects of consuming enriched, fortified, or supplemented chocolate. These trials varied in chocolate intake amounts (ranging from 5 to 101 g day-1), incorporated bioactive components (co-crystalized astaxanthin, lycopene, wood-based phytosterol-phytostanol mixture, canola sterol esters, etc.), and duration (from 2 weeks to 1 year). Some enriched chocolates were found to reduce total and LDL cholesterol and influence markers of oxidative damage, inflammation, immune function, and skin parameters. However, certain trials showed a minimal impact on health outcomes. Therefore, while enriched chocolate holds promise as a carrier for beneficial bioactive compounds, rigorous scientific inquiry and methodological rigor are crucial to fully substantiate these claims. Comprehensive evaluations covering cardiovascular health, metabolic function, immune response, and other aspects are needed to understand its potential benefits and limitations. Advancing robust research initiatives could help realize the full potential of enriched chocolate in promoting human health and well-being.

An extra virgin olive oil‐enriched chocolate spread positively modulates insulin‐resistance markers compared with a palm oil‐enriched one in healthy young adults: A double‐blind, cross‐over, randomised controlled trial

AimsTo investigate if extra virgin olive oil (EVOO) or palm oil enriched chocolate spreads consumption leads to different results in terms of plasma ceramides concentration, glucose and lipid metabolism, inflammatory markers and appetite regulation in young healthy subjects.MethodsIn a 2‐week, double‐blind, cross‐over, randomised controlled trial, 20 healthy, normal‐weight subjects with a mean age of 24.2 years (SD: 1.2), consumed chocolate spread snacks (73% of energy [%E] from fat, 20% from carbohydrates and 7% from proteins), providing 570 Kcal/day added to an isocaloric diet. The chocolate spreads were identical, except for the type of fat: EVOO oil, rich in monounsaturated fatty acids (MUFAs), or palm oil, rich in Saturated Fatty Acids (SFAs).ResultsEVOO‐enriched chocolate spread consumption led to better circulating sphingolipids and glucose profile, with reduced plasma ceramide C16:0, ceramide C16:0/ceramide C22:0‐ceramide C24:0 ratio and sphingomyelin C18:0 (P = 0.030, P= 0.032 and P = 0.042, respectively) compared to the palm oil‐enriched chocolate spread diet. HOMA‐IR and plasma insulin were lower, while the Quicki and the McAuley Index were higher after the EVOO diet compared to the palm oil diet (P = 0.046, P = 0.045, P = 0.018 and P = 0.039 respectively). Subjects maintained a stable weight throughout the study. No major significant changes in total cholesterol, triglycerides, HDL, inflammatory markers, and appetite‐regulating hormones/visual analogue scale were observed between the groups.ConclusionsPartially replacing SFAs with MUFAs in a chocolate‐based snack as part of a short‐term isocaloric diet in healthy individuals may limit SFAs detrimental effects on insulin sensitivity and decrease circulating harmful sphingolipids in young adults.

Microencapsulation of Phenolic Extracts from Cocoa Shells to Enrich Chocolate Bars

Cocoa bean shells were subjected to green extraction technologies, based on the absence of toxic organic solvents, to recover polyphenols; the extract was then encapsulated using a spray dryer and maltodextrin as coating agent. The best conditions observed in the spray drying tests (core-to-coating ratio 1:5; inlet temperature 150 °C; flow rate 6 ml min−1) were applied to produce the microcapsules used to enrich the same cocoa mass as the shells and processed for the preparation of the chocolate bars. Sensory analysis showed no significant differences between enriched chocolate bar and the unenriched reference one, except for the appearance. Both samples were then subjected to accelerated storage tests, at the end of which the polyphenols in the control chocolate bar (0.85 g 100 g−1) were reduced by about 50% (0.42 g 100 g−1), while in the enriched chocolate (1.17 g 100 g−1) by only 22% (0.97 g 100 g−1). The proposed process significantly enriched the chocolate bars with phenolic antioxidants recovered from cocoa waste without increasing the sensations of bitterness and astringency.

Improvement of the Flavanol Profile and the Antioxidant Capacity of Chocolate Using a Phenolic Rich Cocoa Powder.

Chocolate is made from cocoa, which is rich in (poly)phenols that have a high antioxidant capacity and are associated with the prevention of chronic diseases. In this study, a new production process was evaluated in order to obtain a dark chocolate enriched in (poly)phenols using a cocoa powder with an improved flavanol profile. The antioxidant capacity (Oxygen Radical Absorbance Capacity (ORAC) assay) and the flavanol profile (HPLC-DAD and HPLC-FL) was determined. The analysis of the enriched chocolate showed that the total flavan-3-ols (monomers) content was 4 mg/g representing a 3-fold higher than that quantified in the conventional one. Total levels of dimers (procyanidin B1 and B2) were 2.4-fold higher in the enriched chocolate than in the conventional, with a total content of 6 mg/g. The total flavanol content (flavan-3-ols and procyanidins) in the enriched chocolate was increased by 39% compared to the conventional one which led to a 56% increase in the antioxidant capacity. The new flavanol-enriched dark chocolate is expected to provide greater beneficial effect to consumers. Moreover, the amount of flavonols provided by a single dose (ca. 200 mg per 10 g) would allow the use of a health claim on cardiovascular function, a fact of interest for the cocoa industry.

Physical, sensorial and bioactive characteristics of white chocolate with encapsulated green tea extract.

Many studies have described cocoa polyphenols as being bioactive compounds with a potential positive effect on human health. Although dark chocolate is a most powerful source of antioxidants, their content is lower in milk chocolate; conversely, in white chocolate the fat-free cocoa solids are omitted. The aim of this study was to increase polyphenol content and antioxidant capacity of white chocolate by adding encapsulated green tea extract (GTE) in amounts of 60, 80 and 100 g kg-1 . GTE influenced the particle size parameters, increasing the volume weighted mean from 15.43 μm in white chocolate to 19.34 μm in chocolate with 100 g kg-1 GTE. At the same time, the viscosity of enriched chocolate also increased owing to the addition of new solid particles. The surface color of enriched chocolates changed in accordance with the amount of encapsulate, where all enriched chocolates had a slightly lighter color after 12 months of storage. Total polyphenol content (mg gallic acid equivalents (GAE)kg-1 ) increased from 0.41 in white chocolate to 2.73 in chocolate enriched with maximal GTE. This amount of GTE increased antioxidant capacity (mmol Trolox equivalents kg-1 ) from 1.22 in white chocolate to 16.12. After 12 months of storage, degradation of polyphenols was found to be a maximum of 37.27%, while antioxidant capacity decreased up to 44.14%. In addition to the impact on chocolate viscosity, GTE added value through the polyphenol content and sensorial profile of the new product with an unusual green tea flavor and a shelf life of at least 12 months. © 2019 Society of Chemical Industry.

Enrichment of Milk Chocolate by Using EPA and DHA Originated from Various Origins: Effects on Product Quality

In this study, milk chocolate was enriched with different forms of omega-3 fatty acids, EPA and DHA after conching process followed by tempering process. Total EPA and DHA contents of the milk chocolate samples increased to 168.1 and 13.8 mg/25 g chocolate, respectively. The main quality properties (melting, rheological, textural colour, aw and sensory) of the milk chocolate samples were also determined. Viscosity and yield stress values of molten milk chocolates ranged between 7685–15,600 mPa s and 79.1–237.5 Pa, respectively. The results indicated that rheological parameters were influenced by the addition of various forms of EPA/DHA sources in different levels (P < 0.05). Also, EPA/DHA source component addition did not significantly affect the Tonset and Tend of the samples. According to the sensory analysis results, chocolate with microencapsulated form of EHA/DHA source had the highest overall acceptance among the enriched chocolate samples. Usage potential of milk chocolate as a delivery agent of EPA and/or DHA was confirmed.

Enrichment of white chocolate with blackberry juice encapsulate: Impact on physical properties, sensory characteristics and polyphenol content

White chocolate does not contain fat-free cocoa solids and thus lacks bioactive components. In the present study, white chocolate was enriched with encapsulated blackberry juice in concentrations: 60 g kg−1, 80 g kg−1, and 100 g kg−1. The results showed an increase of volume weighted mean in enriched chocolates with increasing the concentration of encapsulate from 13.06 μm in white chocolate to 18.05 μm in chocolate with 100 g kg−1 of encapsulate. Casson viscosity of chocolate mass increased at the same time (from 0.63 Pa in white chocolate to 1.22 Pa in chocolate with a maximum concentration of encapsulate) due to an increase of specific surfaces of solid particles in chocolate. Internal structure and surface gloss in chocolate with 100 g kg−1 of encapsulate are also affected where the lack of free fat phase caused mat color on the surface. However, the addition of encapsulated blackberry juice to white chocolate led to the creation of new type of enriched chocolate with attractive color and blackberry taste. Total polyphenol content (mg GAE/100 g) of white chocolate is increased from 40.75 to 75.06, 145.86, and 153.95 in chocolates with 60 g kg−1, 80 g kg−1, and 100 g kg−1 of encapsulate, respectively.

Omega-3 enriched chocolate milk: A functional drink to improve health during exhaustive exercise

Omega-3 enriched chocolate milk (O3ECM) has a suitable combination of proteins, electrolytes and carbohydrates for post-workout recovery. Herein, the effects of an O3ECM on biochemical parameters and muscle damage in sedentary rats submitted to a session of intense and acute exhaustive exercise was evaluated. Forty-eight male Wistar rats were divided into the following test groups: (a) sedentary rats fed a chocolate milk; (b) sedentary rats fed an O3ECM; (c) exercised rats fed a chocolate milk; and (d) exercised rats fed an O3ECM. After 15 days, the analysis of muscle damage and health parameters was performed. The results showed that the O3ECM consumption decreased the muscle damage (creatine kinase −20% and lactate dehydrogenase −18.7%), as well as increased the activity of endogenous enzymes (catalase +41%, superoxide dismutase +26.7% and glutathione peroxidase +35.7%) and decreased the total cholesterol (−7.8%) and triacylglycerols (−16.2%). Overall, O3ECM can minimize the deleterious effects of exhaustive exercise.

Mixed Products- How Adding Different Attributes Influences Consumer Perceptions and Product Evaluation

During recent decades, the number of mixed attribute products (henceforth mixed products), which have both utilitarian and hedonic benefits, has increased dramatically. Despite these products` growing popularity, academic research has paid little attention to them, and there remains a gap between theory and the real world. Hence, our study was undertaken to understand consumers` perceptions about and behaviors toward mixed products, as well as factors affecting the evaluation and choice of these products. We divided mixed attribute products into two categories: mixed utilitarian products (utilitarian products adding hedonic attributes) and mixed hedonic products (hedonic products adding utilitarian attributes). We then showed how adding different attributes affects consumers` perception, willingness to pay (WTP), and the choice of mixed attribute products compared to pure utilitarian or pure hedonic products. We conducted an experiment using a within-subject design. A total of 160 office workers and college students participated in the study. The pure utilitarian product used in the study was orange juice, and the mixed utilitarian product was carbonated orange juice. The pure hedonic product was chocolate, and the mixed hedonic product was polyphenol enriched chocolate. Results showed that consumers perceived a mixed utilitarian product to be less utilitarian, less pleasurable and more guilty than a pure utilitarian product. On the other hand, a mixed hedonic product was perceived to be more utilitarian, less pleasurable and less guilty than a pure hedonic product. Also, WTP for a mixed hedonic product was higher than WTP for a pure hedonic product, but WTP was lower for a mixed utilitarian product than for a pure utilitarian product. Furthermore, mixed hedonic products were likely to be evaluated more favorably when they were presented together with pure hedonic products, more so than when they were presented alone. Finally, when compared to low self-control participants, high self-control participants chose mixed hedonic products more frequently. The present study contributes to the existing literature on utilitarian and hedonic consumption by adding to the sparse literature on the consumption of products that have both utilitarian and hedonic purposes. Also, our research findings provide several useful implications for practitioners in related fields. First, the current study provides marketers with a useful guide for understanding consumers` perceptions of these types of products, and helps to predict how adding different attributes influences these products. Second, this study has examined the conditions that may moderate the evaluation and choice of hedonic base products and this finding will serve as a good reference for marketers of mixed hedonic products in marketing communication strategy, in-store marketing and targeting. Specifically, comparative advertising with a pure hedonic product will be beneficial for a mixed hedonic product. Also, displaying mixed hedonic products near pure hedonic products may enhance the effectiveness of in-store marketing of mixed hedonic products.

BiotecVisions 2013, February

BiotecVisions 2013, February

Ulceroglandular Tularemia

A 14-year-old boy presented with fevers and nonspecific flu-like symptoms, as well as an enlarging ulcerated plaque involving the upper back, lymphadenopathy, and bilateral pulmonary nodules. Bacterial cultures of ulcer tissue grew Francisella tularensis on enriched chocolate agar plates. Making the diagnosis requires a high index of suspicion, and communication with the laboratory to successfully and safely culture these highly pathogenic bacteria is imperative.

Etiology and Incidence of positive Microbiological tests in the Department of Pneumology

The support of microbiological diagnosis for the management of the lower respiratory tract infections is of great utility. It is known, however, that for a correct therapeutic approach is essential to obtain clinical data and images diagnostic. Sometimes, in fact, to important clinical symptoms can coincide with microbiological results little worth, or the isolation from a sputum of certain bacteria, can be an expression of colonization or trivial contamination.The objective of this work was to evaluate the frequency of bacterial species responsible for respiratory tract infections, through direct (culture) and indirect (serology) methods. Between October 2007 - October 2008, 192 sputum cultures were examined, from the Department of Pneumology and out-patients.The isolation of bacterial strains was performed by seeding samples on the following media (Dasit): Columbia blood Agar (A.) CNA, mannitol salt A., Wurtz lactose A., Sabouraud A., enriched chocolate A. + bacitracin. For serological tests, immunoassays in microplates (Vircell) were used. The data show that of 192 samples deriving from Department of Pneumology, 41 (22%) were positive, while among the out-patient samples, the percentage of positive cultures was 41% (20 positives on the total of 49 samples).The bacteria more frequently isolated in the department of Pneumology, was Pseudomonas aeruginosa (n.8) while in out-patients was Escherichia coli (n.8). Serological tests resulted positive for anti-Mycoplasma pneumoniae antibodies in 3 cases, and for anti-Chlamydia pneumoniae in one case, compared to 53 negative samples. The findings that over 75% of cultures have demonstrated negative results, is suggestive of an origin non-bacterial infections of the lower respiratory tract.Among the bacterial isolates are predominant “opportunistic bacteria” (46%) presumably responsible of nosocomial infections, while in the out-patients are Enterobacteriaceae the prevalent microrganisms isolated.

Optimum use of selective plated media in primary processing of respiratory tract specimens from patients with cystic fibrosis.

A total of 258 respiratory tract specimens from patients with cystic fibrosis were inoculated onto nine different plated media, and the rates of recovery of potential pathogens were compared. Media included sheep blood agar, enriched chocolate agar, MacConkey agar for gram-negative bacilli, chocolate agar containing bacitracin for Haemophilus spp., bromcresol green agar for yeasts, cetrimide agar for Pseudomonas spp., sheep blood agar containing colistin and nalidixic acid for gram-positive cocci, mannitol salt agar for Staphylococcus aureus, and oxidation-fermentation agar containing 300 U of polymyxin B per ml and 2 U of bacitracin per ml (OF-PBL medium) for Pseudomonas cepacia. With two exceptions, all of these media proved useful in recovering potential pathogens from respiratory tract specimens from patients with cystic fibrosis. The two exceptions were cetrimide agar and colistin-nalidixic acid-supplemented sheep blood agar, which were found to be superfluous. In addition, the results of this study further delineated the prevalence of selected bacteria and fungi in respiratory tract secretions from patients with cystic fibrosis. In rank order of frequency of isolation, we recovered isolates of Pseudomonas aeruginosa, Haemophilus parainfluenzae, Candida albicans, S. aureus, Haemophilus influenzae, molds, members of the family Enterobacteriaceae, yeasts other than Candida albicans, miscellaneous gram-negative bacilli, beta-hemolytic streptococci, P. cepacia, and Streptococcus pneumoniae.

Varied Presentations and Responses to Treatment of Infections Caused by Mycobacterium haemophilum in Patients with AIDS

We describe three patients with AIDS who developed clinically significant infection with Mycobacterium haemophilum. One patient had skin and bone involvement and suspected laryngeal involvement; the second had extensive abdominal adenopathy with partial bowel obstruction; and the third presented with limited skin involvement. Each patient responded transiently to antimycobacterial therapy, but disease recurred and progressed in all three cases. Recovery of M. haemophilum requires a high level of clinical suspicion and special handling of mycobacterial cultures by the microbiology laboratory, including cultivation on enriched chocolate agar or heme-supplemented media and incubation at 30 degrees C for up to 8 weeks. Characteristic patterns of drug susceptibility for this organism have been only partially defined. Reported responses to antimycobacterial therapy in AIDS patients with M. haemophilum infection have been poor, and the optimal therapeutic regimen is not yet known. The prognosis for recovery appears to depend heavily on host-related factors, particularly the degree of immunosuppression.

Haemophilus ducreyi and chancroid: Practical aspects for the clinical microbiology laboratory

Most clinical microbiology laboratories can isolate H. ducreyi from clinical specimens. The essential factors include collecting the specimen properly in clinically appropriate situations; delivering the specimen to the laboratory quickly; rapidly inoculating it onto appropriate media such as enriched chocolate agar; incubation in a very high humidity with added CO 2; and recognizing the distinctive colony morphology after 48 h or more of incubation. Subsequently identification can be made readily by SPS disk susceptibility, the RapID-ANA system, and other routine tests.

Problems with current recommendations for susceptibility testing of Haemophilus influenzae

We compared results of MIC and disk susceptibility tests on Haemophilus test medium (HTM) and those on comparative media. Ampicillin MICs were determined with seven ampicillin-resistant, non-beta-lactamase-producing (AmprNBLP) isolates by using HTM and supplemented brain heart infusion (sBHI) agar. Ampicillin and amoxicillin-clavulanate disk tests with 16 AmprNBLP strains, 18 ampicillin-susceptible (Amps) isolates, and 17 ampicillin-resistant, beta-lactamase-producing (AmprBLP) strains were performed by using five media: laboratory-prepared HTM (PHTM), commercial HTM (CHTM), sBHI, enriched chocolate agar, and Mueller-Hinton chocolate agar. We observed that five of seven and three of seven AmprNBLP strains were misclassified as susceptible with PHTM (MIC, less than 2 micrograms/ml) with inocula of 10(3) and 10(5) CFU, respectively, but were resistant with sBHI (MIC, greater than or equal to 2 micrograms/ml). Whereas Mueller-Hinton chocolate agar and enriched chocolate agar plates supported the growth of all 51 strains by the disk tests, 37% (19 of 51) and 8% (4 of 51) of strains did not grow on PHTM and CHTM, respectively. Lack of growth on PHTM was observed for all three phenotypes; 7 of 18 Amps, 4 of 17 AmprBLP, and 8 of 16 AmprNBLP strains did not grow. The four strains that did not grow on CHTM were all AmprNBLP isolates. Zone sizes were significantly larger on PHTM than on the other media. Of the strains that were evaluable by the new National Committee for Clinical Laboratory Standards guidelines with either PHTM or CHTM, all Amps strains were classified as susceptible. Among the AmprBLP strains, CHTM correctly identified all as resistant, whereas PHTM detected two isolates to be intermediate. Among the AmprNBLP strains, CHTM and PHTM misclassified four (33%) and five (62%) isolates, respectively, as susceptible; an additional isolate was identified as intermediate on both media. We conclude that there is strain-dependent growth on HTM, that adoption of this medium for routine Haemophilus susceptibility testing is problematic due to this growth variability, and that detection of AmprNBLP isolates would be unreliable.

Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria.

Twelve Capnocytophaga and seven DF-2 strains were tested for their susceptibility to 14 antimicrobial agents using an agar dilution and an agar diffusion method. Twenty-three other antibiotics were evaluated using the diffusion test only. All strains were fully susceptible to penicillin, ampicillin, cefuroxime, cefotaxime, erythromycin, clindamycin, chloramphenicol, doxycycline, rifamycin and ofloxacin using both methods. Clindamycin, rifamycin and cefotaxime were most active. Using agar dilution some strains were susceptible to gentamicin, but agar diffusion showed total resistance. One Capnocytophaga strain was susceptible and another moderately susceptible to metronidazole, other strains were resistant. The agar diffusion test showed that both Capnocytophaga and DF-2 were resistant to most other aminoglycosides, to fosfomycin, polymyxin and trimethoprim. All strains of both taxa were fully susceptible to piperacillin, cefoxitin, imipenem and fusidic acid and showed different susceptibilities to the other agents. Susceptibility testing by means of agar diffusion using an enriched chocolate agar and 5% CO2 atmosphere could be used to test Capnocytophaga and DF-2 strains and gives sufficient accuracy for routine use, when revised inhibition zone breakpoints are employed.

Ampicillin disk diffusion susceptibility testing of Haemophilus influenzae

A total of 114 strains of Haemophilus influenza were characterized with respect to beta-lactamase production and ampicillin MIC. Of this total, 41 strains produced a TEM-type beta-lactamase, and ampicillin MICs for these strains were greater than or equal to 2.0 microgram/ml. It was found that 54 strains lacked TEM-type beta-lactamase activity, and ampicillin MICs for them were less than or equal to 0.5 microgram/ml. The remaining 19 strains were beta-lactamase negative, but ampicillin MICs were greater than or equal to 2.0 micrograms/ml. Disk diffusion susceptibility tests were performed with two media, i.e., Mueller-Hinton agar containing 1.0% hemoglobin and 1.0% IsoVitaleX supplement (CHOC-MHA) and enriched chocolate agar (CHOC), by using disks containing 10 and 2 micrograms of ampicillin. If strains of H. influenzae for which ampicillin MICs were greater than or equal to 2.0 micrograms/ml were considered resistant, while strains for which MICs were less than or equal to 0.5 microgram/ml were considered susceptible, the following zone diameter interpretive criteria were identified as indicating ampicillin susceptibility: CHOC-MHA (10-micrograms disks), greater than or equal to 20 mm; CHOC-MHA (2-micrograms disks), greater than or equal to 17 mm; CHOC (10-micrograms disks), greater than or equal to 25 mm; and CHOC (2-micrograms disks), greater than or equal to 20 mm. In all cases, zones of inhibition less than those listed above would be interpreted as indicating resistance. Each of these four combinations was found to be essentially equivalent in identifying susceptible and resistant strains of H. influenzae, irrespective of beta-lactamase production.

In vitro chloramphenicol susceptibility testing of Haemophilus influenzae: disk diffusion procedures and assays for chloramphenicol acetyltransferase.

The activity of chloramphenicol against 100 different strains of Haemophilus influenzae was assessed by a macrotube broth dilution technique and by a standardized disk diffusion method using both enriched chocolate agar (CHOC) and Mueller-Hinton agar containing 1.0% hemoglobin and 1.0% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) supplement (CHOC-MHA). Filter disks containing 30 micrograms of chloramphenicol were used with the disk diffusion procedure. The following zone diameter interpretive criteria were defined: CHOC-MHA, less than or equal to 25 mm = resistant [corrected] and greater than or equal to 26 mm = susceptible [corrected]; CHOC, less than or equal to 28 mm = resistant [corrected] and greater than or equal to 29 mm = susceptible [corrected]. all of the H. influenzae strains examined were also characterized by using two rapid assays for chloramphenicol acetyltransferase (CAT) activity: a 1-h tube method (t-CAT) and a 30-min procedure which used commercially available reagent-impregnated disks (d-CAT). The t-CAT procedure was found to be significantly more accurate than the d-CAT procedure as a means for demonstrating production of CAT.