Stability of minocycline,doxycycline, and tetracycline stored in agar plates and microdilution trays

When the agar dilution method is used for performing antimicrobic susceptibility tests, Mueller-Hinton agar plates may be prepared with varying concentrations of the drugs to be studied. With most antimicrobics, the plates can be prepared in advance and stored in the refrigerator for as long as four weeks. The present study documents the instability of minocycline when stored under refrigeration in agar plates. Doxycycline and tetracycline also lost activity but at a slower rate. Degradative changes occurred most rapidly at the lower concentrations; at 16 μg/ml or greater, the drugs were relatively stable. When diluted in Mueller-Hinton broth and stored at −60°C in microdilution trays, the three tetracyclines could be held up to six weeks with no loss of bioactivity. Concentrated stock solutions were kept at −60°C for six months with no loss of potency.     

Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

Measurement of lonized magnesium in biological fluids

A method that provides for direct in vitro measurement at 37°C of ionized magnesium in biological fluids using the Abbot bichromatic analyzer, a fully automated instrument is described. The metallochromic dye Eriochrome blue SE was used to measure ionized magnesium levels in Mueller-Hinton agar fluids. Formulation of an appropriate blank was found necessary and was made by treating agar fluids with Chelex 100 to remove divalent cations. The influence of pH in the biological range was examined and poses a significant but manageable source of error. The method allowed an appropriate assessment of the role of ionized magnesium in the susceptibility testing of Pseudomonas aeruginosa on Mueller-Hinton agar with the aminoglycoside antibiotics.     

Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest.

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.     

Survival, growth and pathogenicity of Gaeumannomyces graminis var. graminis with different methods of long-term storage

The fungal plant pathogen Gaeumannomyces graminis var. graminis was preserved with 12 different storage methods. Five strains, each with unique morphological and pathological characteristics, were used for comparison of the methods. The storage treatments included potato-dextrose agar slants, with or without mineral oil, stored at either 4 C, 28 C or ambient temperature; colonized agar plugs placed in glycerol solution at either -75 C or -20 C; colonized agar plugs placed in sterile deionized water at either 4 C or ambient temperature; and mycelial growth on intact or precut pieces of filter paper, desiccated and stored at ambient temperature. Survival was evaluated at 6, 12, 24, 36, 48 and 120 mo. The three best treatments for survival were PDA slants, with or without mineral oil, and colonized agar plugs stored in water, all at ambient temperature. All five fungal strains were recovered from all four replicates at each sampling date for agar plugs stored in water at ambient temperature. The worst treatments were agar slants and agar plugs in water stored at 4 C and agar plugs stored in glycerol at -20 C. Morphological characteristics were not affected by storage treatments. In general, there were minimal or no effects on growth and pathogenicity for all strains for all storage treatments with survival. Colonized agar plugs stored in water at ambient temperature provides an economical storage method (materials and labor) that does not need an electrical power for long-term maintenance.     

Interlaboratory variability of disc diffusion and agar dilution susceptibility tests with cefamandole and cephalothin

Reproducibility of antimicrobic susceptibility tests was estimated by examining control data accumulated during a multicenter study for evaluating cefamandole and cephalothin. The precision of agar dilution minimal inhibitory concentrations was compared with the standardized Bauer-Kirby disc method. Regression lines were established for each antimicrobic and were used to calculate the range of minimal inhibitory concentration values that corresponded to the observed ranges in zone sizes, thus permitting a comparison of the two types of procedures. The precision of the disc method was equal to or greater than that of the agar dilution method.     

Comparison of disk diffusion test and Etest for voriconazole and fluconazole susceptibility testing

The distribution for voriconazole and fluconazole susceptibility was determined by Etest and disk diffusion test in 143 clinical isolates. The majority of the strains of Aspergillus spp., Candida krusei, C. inconspicua, C norvegensis and Saccharomyces cerevisiae displayed resistance or decreased susceptibility to fluconazole in contrast to voriconazole. The absolute categorical agreement for voriconazole and fluconazole susceptibility results by the disk method and Etest was 90.5 and 74.8 % respectively. The error rate bounding analysis showed only 0.7 % of false susceptible results ( very major error) with voriconazole, but 2.8 % with fluconazole. Fluconazole can be used as a surrogate factor to predict voriconazole susceptibility but with lower reliability for susceptible-dose dependent and resistance category, especially in Candida glabrata isolates. The results of the disk method were not substantially influenced by the composition of media (Mueller-Hinton agar vs antimycotic Sensitivity Test agar), even if with the latter the results had fewer tendencies to produce false susceptibility of C.glabrata isolates to both of the triazole drugs. Disk test as well as Etest were shown to represent suitable methods for routine evaluation of susceptibility of clinical isolates of pathogenic fungi, including aspergilli, to fluconazole and voriconazole.     

Stability of Antibiotics and Chemotherapeutics in Agar Plates

The stability of chemotherapeutic agents incorporated into agar plates was studied by comparison of minimum inhibitory concentrations on fresh and stored plates and by direct bioassay of the chemotherapeutic agar plates. Plates were stored in sealed bags at 4 C. No loss of bioactivity was demonstrated after 30 days of storage in plates containing methicillin, erythromycin, cephalothin, tetracycline, chloramphenicol, kanamycin, streptomycin, polymyxin B, or nalidixic acid. Penicillin G, ampicillin, and nitrofurantoin showed statistically significant losses of activity after 4 weeks. None of the chemotherapeutics tested showed significant loss in activity after 1 week.     

Effect of medium composition on the susceptibility of oral streptococci to mercuric chloride

Although there is considerable interest in identifying mercury-resistant bacteria, no standardized assay exists for this purpose. In this study, the effect of the composition of the medium on the susceptibility of oral streptococci to HgCl₂ was investigated. The minimum inhibitory concentration (MIC) of HgCl₂ for 52 streptococcal strains and the reproducibility of MIC values for Hg-sensitive and Hg-resistant strains was determined with 11 different media. Addition of blood increased the MIC values, and some media (tryptone soya agar, with or without blood) could not discriminate between Hg-sensitive and Hg-resistant strains. The proportion of streptococci that appeared to be resistant to Hg was very high (>70%) on some media (mitis-salivarius, tryptone soya, Columbia), but not on others (Mueller-Hinton, Brain Heart Infusion, Isosensitest). The MICs of the control strains varied considerably on different testing occasions for tryptone soya agar (with and without blood), Isosensitest agar, and Columbia agar (with blood). Mueller-Hinton (without blood) appeared to be the most suitable medium for isolating Hg-resistant oral streptococci. Received: 21 December 2001 / Accepted: 17 January 2002     

Medium-Dependent Activity of Gentamicin Sulfate Against Enterococci

Routine disc diffusion susceptibility tests (Bauer-Kirby technique), employing 5% sheep blood-Mueller-Hinton agar and 10-mug gentamicin sulfate discs, disclosed that a significant number of clinical enterococcal isolates were sensitive to the antibiotic, as also revealed by the agar dilution technique. With few exceptions, the isolates proved resistant to this antibiotic when tested for susceptibility in Brain Heart Infusion and Trypticase soy broth or agar. The addition of 5% sheep blood to Trypticase soy and Brain Heart Infusion agars resulted in markedly enhanced activity of the antibiotic, indicating medium-dependent activity of gentamicin against enterococci. Human serum and urine failed to support optimal growth of enterococci. Thus, it was not possible to correlate the activity of gentamicin in any of the media examined with that in serum or urine.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Detection of sulfa drugs and antibiotics in milk

A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination of Dairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy.     

Carbohydrate Fermentation Patterns of Neisseria meningitidis Determined by a Microtiter Method

A carbohydrate fermentation technique has been developed and compared to the standard fermentation test with cystine-Trypticase-semisolid agar for the identification of Neisseria meningitidis. This new method utilizes Mueller-Hinton broth as a basal substrate and is performed with microtiter methods. By using Mueller-Hinton broth with and without the addition of antibiotics, the method can be adjusted to test the fermentation patterns of all of the Neisseria including N. gonorrhoeae.     

A simple method for counting Staphylococcus aureus in swimming pool water

Staphylococcus aureus counts from swimming pool water were determined by the membrane filtration technique. Water samples were passed through a membrane filter and then put on Baird-Parker media. After incubation, the filters were transferred to nutrient agar, and incubated at 37 degrees C, for 3 h. After removal of the filters, the plates were incubated at 60 degrees C for 2 h. An overlay of toluidine blue agar was added and the plates reincubated for 4 h at 37 degrees C. The formation of thermonuclease correlated with the formation of coagulase, and the results indicated that Staphylococcus aureus could be present in swimming pool water without the presence of either coliform or faecal coliform bacteria.     

Screening method for detecting staphylococci with reduced susceptibility to teicoplanin

The incidence of infections caused by staphylococci with decreased susceptibility to teicoplanin (MIC>/=8 microg/ml) is increasing, but the disk diffusion test has difficulty detecting this low level of resistance. In addition, detection is complicated because of the heterogeneous phenotypes for teicoplanin. In this study, we evaluated an agar screening method to detect staphylococci with decreased susceptibility to teicoplanin or heterogeneous resistance. First, to investigate the inoculum density and teicoplanin concentration of screening agar, we used 10(5) and 10(6) CFU/ml and Mueller-Hinton agars supplemented with 6 and 8 microg of teicoplanin/ml to test 39 genetically distinct staphylococcal strains (15 strains with teicoplanin MICs>/=8 microg/ml and 24 strains with teicoplanin MICs/=8 microg/ml or showing heteroresistance could be detected. These findings indicate that the method can be used as a reliable screening method for detecting staphylococci with reduced susceptibility to teicoplanin.     

Effect of incubation temperature, ageing, and bisulfite content of unsupplemented Brucella agar on aerotolerance of Campylobacter jejuni

A mutant strain of Campylobacter jejuni ATCC 29428 was isolated that grows on unsupplemented Brucella agar at O2 levels as high as 21% at 37 degrees C. While measuring the degree of aerotolerance of this mutant on unsupplemented Brucella medium and comparing it with that of the wild type, we found considerable variation among our estimates. As measured by colony counts on unsupplemented Brucella agar incubated at various oxygen levels, the degree of aerotolerance was affected by incubation temperature and the age of the medium. Aerotolerance was consistently higher on plates incubated at 42 degrees C than at 37 degrees C. When the commercial dehydrated Brucella medium that was used to prepare the Brucella agar was stored in a beaker for 2.5 months, the degree of aerotolerance of C. jejuni was decreased. Addition of 0.01% sodium bisulfite reversed this inhibition. Storage of bottles of hydrated Brucella agar for 1.5 months also resulted in a decreased aerotolerance; again, in addition of 0.01% bisulfite reversed the effect. Aerotolerance was greatly decreased when Brucella agar was prepared from all its individual components except 0.01% bisulfite. The results indicate that the bisulfite component of Brucella agar deteriorates during storage of the dehydrated and hydrated media, and that this deterioration can affect measurements of aerotolerance.     

Expression of glycocalyx by coagulase-negative Staphylococcus species isolated from bovine milk

Two hundred and six strains of coagulase-negative Staphylococcus species were assessed for expression of glycocalyx on serum soft agar, india ink and adherence techniques. The organisms were maintained on trypticase soy agar plates at 4 degrees C for 30 d (120 strains) or stored at -80 degrees C in skim milk for 90 d (60 strains). Additionally, 26 milk samples from cows known to have excreted coagulase-negative staphylococci were used to inoculate serum soft agar directly. Nine of 26 direct culture samples and 43 of 180 strains maintained for an extended period had diffuse-type growth on serum soft agar. The proportion that exhibited an unstained halo by india ink was similar regardless of storage time. Slime production determined by in vitro adherence revealed a higher proportion of positive strains than had been predicted by serum soft agar or india ink techniques. More strains of Staphylococcus chromogenes, Staph. epidermidis, Staph. hominis, Staph. simulans and Staph. warneri expressed glycocalyx than other coagulase-negative Staphylococcus species. These results suggest that most coagulase-negative staphylococci produce slime rather than a capsule. However, evidence for classical encapsulation was demonstrated in several strains by india ink. The finding that Staphylococcus species other than Staph. aureus isolated from bovine milk are capable of glycocalyx production may be of importance in investigations on the relationship between staphylococci and host defence mechanisms.     

Suppression of initiation-negative strains of Escherichia coli by integration of the sex factor F.

Data are presented suggesting that the most critical factor determining whether an Hfr dnaAts strain can synthesize deoxyribonucleic acid and form colonies at temperatures that are nonpermissive for corresponding F- strains is neither the site of insertion of F nor the presence of additional mutations in the F particle or the chromosome; it is whether the particle is capable of autonomous replication at the temperature used. Consequently, suppression of the DnaA phenotype in Hfr strains occurs at 40 C but not, in most of them, at 42 C without the occurrence of additional mutations. The site of insertion of F may also be important since it is shown that in one Hfr dnaA strain partial suppression does occur at 42 C. In addition, it is shown that strains exhibiting suppression by integration of F at 40 C on minimal agar plates do not do so at this temperature on nutrient agar plates.