Primary Isolation Media Research Articles

Molecular investigation of cytolysin genes among bacterial isolates recovered from pyospermic patients in Hilla City, Iraq

Enterococcus faecalis and Uropathogenic Escherichia coli (UPEC) are the most common causal agents of urinary tract infections (UTI) and infertility in humans. They secrete many cytolytic toxins that impact sperm functions and spermatogenesis. The current paper aimed to investigate the cytolysins among Bacteriospermia associated bacteria in pyospermic patients. 110 Seminal fluid swabs were collected from pyospermic patients (age Mean±SD, 35.5±2.12 years) from September 2020 to January 2021. All swabs were inoculated on UTI chromogenic medium for primary isolation of bacteria. Then the suspected Es. coli and En. faecalis have been confirmed by PCR using uidA and ddl genes, respectively. The results revealed that Es. coli compile 42.9% of bacteriospermia while En. faecalis 25.71%. Results of PCR for cytolysins reveal that all E. coli isolates have lta, hylA, sta, and stb (100%) genes, sheA (96.7%), stx2 (20%), and stx1(3.3%). All En. faecalis (100%) have Hyl, cylLS, cyILL genes. The current study concludes that both Es. coli and En. faecalis have a set of toxins with possible damage to sperm function, causing indirect infertility.

Bacteriological and heavy metal status of water and fish samples from Jebba Lake – Nigeria

Quantitative and qualitative analysis of bacterial isolates in water and fish samples (Oreochromis niloticus and Clarias gariepinus) from Jebba Lake were carried out using primary isolation media and microbact identification kits. The concentration of heavy metals, Lead (Pb), Copper (Cu), Cadmium (Cd), Chromium (Cr) and Arsenic (As) were determined using Atomic Absorption Spectrophotometer (AAS). Samples were collected from five (5) different stations. Station Jl.1 and Jl.2 are located at the upper course of the lake and were characterized by high human activities (washing, bathing, mining and animal husbandry operations) especially in sample station Jl.2. Sample station Jl.3 has low human activities, however it has a tributary with high mining activities. Station Jl.4 has high domestic washings but low animal husbandry operation and low mining activities. Station Jl.5 has low domestic washings, low animal husbandry operation and low mining activities. Mean faecal coliform count of sample station Jl.1 (580.83 CFU/100ml) and station Jl.2 (700.83 CFU/100ml) were above maximum permissible limit for Federal Environmental Protection Agency (FEPA). Faecal coliform count of sample stations Jl.3, Jl.4 and Jl.5 were below maximum permissible limit. Maximum faecal coliform count during the wet season was 1600.00 CFU/100ml, while the maximum count during the dry season was 920.00 CFU/100ml. Bacterial species such as Aeromonas caviae, Aeromonas hydrophila, Pseudomonas fluorescens – 25, Escherichia coli, Moraxella species, Mannheimia (pasturella) haemolytica, Pseudomonas aeruginosa, Pseudomonas fluorescens – 35, Escherichia hermannii were found in water samples and species such as Vibrio alginolyticus, Moraxella species, Escherichia hermannii, Vibrio parahaemolyticus, Aeromonas hydrophila were isolated from fish intestines and gills. Concentration of Cu, Cr and As in water was observed to be within permissible limit but the concentration of Pb and Cd were above tolerable limit. In fish organs, the concentration of heavy metals were within permissible limit except that of Pb and As in Oreochromis niloticus fish intestines (ONFI) (0.38±0.00 and 0.03±0.00 respectively) and Clarias gariepinus fish intestines (CGFI) (0.40±0.00 and 0.03±0.01 respectively) which were above maximum permissible limit by standard organizations.
 Key words: Jebba Lake, water, fish, Oreochromis niloticus, Clarias gariepinus, bacteria, heavy metals

Characterization and evaluation Typha domingensis Pers.) of a fungal growth medium composed pollens powder of cattail (

Introduction: A great progressing occurs in microbiology Ulocladium sp. after culture media have been used in culturing, isolation, identification, and reservation of microorganisms. According to their components, culture media were divided into natural, synthetic, and semisynthetic, the natural medium contain almost all the nutritional requirements but in non-defined quantities. In the current work, the validity of Typha domingensis pollens as a new substrate for fungal culture medium was tested. Several physical and chemical characteristics of medium were detected. The growth of nine fungal isolates were evaluated on semisolid and broth media. Materials and Methods: Ten gram of starchy powder free from spike tissue + 15gm agar were used for one liter of Typha pollens agar (TPA). The growth of local isolates of Candida sp., Rhodotorula sp., Aspergillus niger, , Cladosporium sp., Trichoderma sp., Penicillium sp., Chrysosporium sp., and Microsporum sp. were tested on (TPA), and (PCA). Biomass of filamentous isolates was estimated by TP broth and PC broth.ANOVA test at level 0.05 was followed to clarify the significant increasing of biomass. To qualify the nutritional value of Typha pollens, the percentage oftotal nitrogen , carbon , phosphorus , and potassium were estimated . pH of TPA were detected as well as its color, transparency , and gelatin texture were practically evaluated . Results and Discussion : The chemical composition analysis showed that pollens powder contain C=58%, N=2.16%, P= 0.19%and K=3.36%. The pH was 6.72 at room temperature . Typha pollens agar support growth of all tested fungal isolates. The semisolid TPA had a typical characteristic for culturing and diagnosis of fungi including a suitable gelatin texture and a pale transparent yellow color . All tested fungi except Penicilliumsp. showed higher growth on TPA , on the other side the fungal biomass increased significantly by TP broth in comparison with PC broth (ANOVA test at 0.05 level). Conclusions : Pollens of cattail (T. domingensis ) provide a suitable environment , and nutritive requirements for fungi .It can be usedeasily and successfully as essential substrate for preparing culture medium for primary isolation of fungi. A farther studies were needed to gain a view about using T.domingensis pollens to isolate pathogenic fungi and bacteria.

Evaluation of Adding Erythritol to Farrell Medium for Primary Isolation of the Brucella melitensis Strains

This study aims to evaluate the addition of erythritol to Farrell medium (FM) for the primary isolation of B.melitensis strains. Selective media, particularly FM, is commonly used for Brucella isolation. Isolation is the gold standard for the diagnosis of brucellosis. The isolation success of selective media depends on their ingredients. Isolation rate may decrease particularly in contaminated samples; therefore, the addition of components like erythritol that augments the growth of Brucella species on media can support isolation. In this study, organ samples associated with 14 small ruminant abortion cases caused by B.melitensis strains were utilized. Organ suspensions were inoculated to FM and Farrel medium with erythritol (F+ER) simultaneously. The media’s isolation and inhibition performance was observed during incubation. The addition of erythritol was evaluated based on whether it provides earlier isolation with the help of increasing the colony growth rate and colony size. According to the findings, F+ER is more advantageous for 12 samples among 14 through isolation as it enables faster colony growth and larger colonies. The results confirm the recommendation of adding erythritol particularly in the samples with inadequate contaminant inhibition. Therefore, high isolation performance can be achieved through erythritol addition to the selective media like FM.

CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens.

A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture medium (SuperPolymyxin). The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media. Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 101 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1. CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance.

Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens

Aim: Fungi are increasing in incidence as human pathogens and newer and rarer species are continuously being encountered. Identifying these species from growth on regular culture media may be challenging due to the absence of typical features. An indigenous and cheap medium, similar to the natural substrate of these fungi, was standardised in our laboratory as an aid to species identification in a conventional laboratory setting. Materials and Methods: Ripe banana peel pieces, sterilised in an autoclave at 121°C temperature and 15 lbs pressure for 15 min promoted good growth of hyphae and pycnidia or acervuli in coelomycetes, flabelliform and medusoid fruiting bodies of basidiomycetes and fruit bodies such as cleistothecium in ascomycetes. The growth from the primary isolation medium was taken and inoculated onto the pieces of double-autoclaved ripe banana peel pieces in a sterile glass Petri dish with some moisture (sprinkles of sterile distilled water). A few sterile coverslips were placed randomly inside the Petri dish for the growing fungus to stick on to it. The plates were kept at room temperature and left undisturbed for 15–20 days. At a time, one coverslip was taken out and placed on a slide with lactophenol cotton blue and focused under the microscope to look for fruit bodies. Results:Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum, Nattrassia mangiferae and Schizophyllum commune were identified by characteristic features from growth on banana peel culture. Conclusions: Banana peel culture is a cheap and effective medium resembling the natural substrate of fungi and is useful for promoting characteristic reproductive structures that aid identification.

CHROMagar as a primary isolation medium for rapid identification of Candida and its role in mixed Candida infection in sputum samples

Introduction: Candida species occurs as commensal organisms but in immunocompromised patients and those on long term antibiotics, these unrecognized opportunistic fungi may become fatal. Although C. albicans is the most frequently isolated pathogen, the incidence of infections due to non-albicans Candida are on the increase especially with fluconazole resistant strains. Identification of Candida to species level therefore becomes important for selecting the appropriate antifungal agents. The need for rapid identification and the difficulty in detecting mixed cultures on the traditional Sabouraud’s dextrose agar (SDA) have led to the development of chromogenic media. Aim: This study was done to evaluate the usefulness of CHROMagar Candida for rapid identification of Candida species and to identify mixed Candida infection from sputum samples. Materials and methods: A total of 126 sputum samples were inoculated on CHROMagar Candida. The plates were incubated at 37?C for 48 hrs and the growth identified based on the colour of the colony. Results: Single yeast infection was observed in 113 samples (89.7%) and 11 samples showed mixed infection (8.7%). In our study, C.albicans (61.5%) was the predominant species and among the non-albicans Candida, C. tropicalis (18.5%) was the commonest species. Conclusion: Certainly CHROMagar Candida will be useful as a primary culture medium for identifying the yeast infections directly from the clinical specimens and also for the detection of mixed yeast infections which cannot be done on conventional SDA. Hence it helps us in selecting the appropriate antifungal agents for therapy and prophylaxis. Keywords: Candida albicans, Non-albicans Candida, CHROMagar, Mixed yeast infections, Antifungal agents

Isolation of Dermatophytes (and Other Fungi) from Human Nail and Skin Dust Produced by Podiatric Medical Treatments in Australia.

Background: Podiatric physicians routinely use electric drills for the treatment of nail and skin conditions. The grinding process produces human nail and skin dust that is generally vacuumed into bags in the grinding unit. Many of the nails are thought to be mycotic, particularly because they are obtained from patients with symptoms of dermatophyte infections. Currently, there is limited information available on the detection of fungi from nail dust samples. Herein, we attempt to address this situation and outline some of the difficulties that pathology laboratories face in isolating and identifying dermatophytes from nail samples. Methods: Fifty nail dust bags from podiatric medical clinics across all of the states and territories of Australia were collected and analyzed. Samples from the bags were inoculated onto primary isolation media. Fungal colonies that grew were then inoculated onto potato dextrose agar for identification using standard morphological (macroscopic and microscopic) features. Results: One hundred fifty-one colonies of dermatophytes were identified from 43 of the 50 samples. In addition 471 nondermatophyte molds were isolated, along with some yeasts and bacteria. Conclusions: The most common dermatophytes isolated were from the Trichophyton mentagrophytes/interdigitale complexes. Trichophyton rubrum, Trichophyton tonsurans, Trichophyton soudanense, and Epidermophyton floccosum were also isolated. An unidentified group of dermatophytes was also present. The three most common genera of nondermatophyte molds were Aspergillus, Penicillium, and Scopulariopsis, all of which have been implicated in onychomycosis and more general disease. The presence of viable fungal pathogens in the dust could potentially pose a health problem to podiatric physicians.

Isolation of dermatophytes (and other fungi) from human nail and skin dust produced by podiatric medical treatments in Australia.

Podiatric physicians routinely use electric drills for the treatment of nail and skin conditions. The grinding process produces human nail and skin dust that is generally vacuumed into bags in the grinding unit. Many of the nails are thought to be mycotic, particularly because they are obtained from patients with symptoms of dermatophyte infections. Currently, there is limited information available on the detection of fungi from nail dust samples. Herein, we attempt to address this situation and outline some of the difficulties that pathology laboratories face in isolating and identifying dermatophytes from nail samples. Fifty nail dust bags from podiatric medical clinics across all of the states and territories of Australia were collected and analyzed. Samples from the bags were inoculated onto primary isolation media. Fungal colonies that grew were then inoculated onto potato dextrose agar for identification using standard morphological (macroscopic and microscopic) features. One hundred fifty-one colonies of dermatophytes were identified from 43 of the 50 samples. In addition 471 nondermatophyte molds were isolated, along with some yeasts and bacteria. The most common dermatophytes isolated were from the Trichophyton mentagrophytes/interdigitale complexes. Trichophyton rubrum, Trichophyton tonsurans, Trichophyton soudanense, and Epidermophyton floccosum were also isolated. An unidentified group of dermatophytes was also present. The three most common genera of nondermatophyte molds were Aspergillus, Penicillium, and Scopulariopsis, all of which have been implicated in onychomycosis and more general disease. The presence of viable fungal pathogens in the dust could potentially pose a health problem to podiatric physicians.

Preponderance of bacterial isolates in urine of HIV-positive malaria-infected pregnant women with urinary tract infection.

This study examined HIV and malaria co-infection as a risk factor for urinary tract infections (UTIs) in pregnancy. The study group included 74 pregnant women, 20 to 42 years of age, who attended the antenatal clinic at the Specialist Hospital at Akure, Ondo State, Nigeria. Forty-four of the pregnant women were either HIV seropositive with malaria infection (HIV+Mal+) or HIV seropositive without malaria (HIV+Mal-). The remaining thirty pregnant women served as controls and included women HIV seronegative but with malaria (HIV-Mal+) and women HIV seronegative without malaria. UTI was indicated by a bacterial colony count of greater than 10⁵/mL of urine, using cysteine lactose electrolyte deficient medium (CLED) as the primary isolation medium. Bacterial isolates were characterized using convectional bacteriological methods, and antibiotics sensitivity tests were carried out using the disk diffusion method. A total of 246 bacterial isolates were recovered from the cultures, with a mean of 3.53 isolates per subject. Women who were HIV+Mal+ had the most diverse group of bacterial isolates and the highest frequency of UTIs. The bacterial isolates from the HIV+Mal+ women also showed the highest degree of antibiotic resistance. While pregnancy and HIV infection may each represent a risk factor for UTI, HIV and malaria co-infection may increase its frequency in pregnancy. The higher frequency of multiple antibiotic resistance observed among the isolates, particularly isolates from HIV+Mal+ subjects, poses a serious public health concern as these strains may aggravate the prognosis of both UTI and HIV infection.

Direct Drug Susceptibility Testing of Mycobacterium tuberculosis for Rapid Detection of Multidrug Resistance Using the Bactec MGIT 960 System: a Multicenter Study

Conventional indirect drug susceptibility testing of Mycobacterium tuberculosis with liquid medium is well established and offers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibility testing (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substantial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in Bactec MGIT 960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed specimens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT 960 instrument using the 21-day protocol (Bactec 960 pyrazinamide [PZA] protocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time savings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three different countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% concordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the Bactec MGIT 960 system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.

Development of a selective culture medium for primary isolation of the main Brucella species.

Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis.

Evaluation of CHROM-Pal medium for the isolation and direct identification of Candida dubliniensis in primary cultures from the oral cavity

Candida albicans is the species most frequently isolated from oral specimens, but the recovery of other Candida species such as Candida dubliniensis is increasing. Differentiation of C. dubliniensis from C. albicans requires special tests and both species are misidentified in some studies. CHROM-Pal (CH-P) is a novel chromogenic medium used in our laboratory for differentiation between C. albicans and C. dubliniensis on the basis of colony colour and morphology, and chlamydospore production. The performance of CH-P and CHROMagar Candida (CAC) was compared for primary isolation and presumptive identification of yeasts from oral specimens from human immunodeficiency virus (HIV)-infected and uninfected individuals. The identification of Candida species on both media was compared with two reference identification methods (API ID 32 C and multiplex PCR). A total of 137/205 oral swabs (66.8 %) plated onto CH-P and CAC media were positive by culture and resulted in the growth of 171 isolates of Candida species on CH-P, whilst only 159 isolates grew on CAC. C. albicans was the most frequently isolated species in both groups of patients, followed by Candida parapsilosis in the HIV-negative group, and by C. dubliniensis in the HIV-infected group. The other Candida species isolated were Candida guilliermondii, Candida glabrata, Candida krusei, Candida tropicalis, Candida famata, Candida rugosa, Candida kefyr, Candida pelliculosa and Candida pulcherrima. The sensitivity and specificity for identifying C. albicans, C. krusei, C. tropicalis and C. dubliniensis on CH-P were over 98.5 %, always equal to or higher than those obtained when CAC was used. CH-P is a simple reliable medium for primary isolation and presumptive identification of yeast isolates from oral samples. The ability of CH-P to discriminate between C. dubliniensis and C. albicans was significantly higher (P <0.05) than that of CAC.

Evaluation of extracts of Jatropha curcas and Moringa oleifera in culture media for selective inhibition of saprophytic fungal contaminants

Most fungi occur in nature and utilize simple sources of carbohydrates and nitrogen for growth. Sabouraud's dextrose agar has been an ideal medium for primary isolation of fungi from clinical specimens, but for specimens from nonsterile sites or heavily contaminated ones, it has been necessary to include inhibitory substances such as antibiotics like chloramphenicol (antibacterial) and cycloheximide (antifungal). The problems we have in the our laboratory owing to frequent contamination of cultures and the delays in the procurement of cycloheximide have stimulated a search for alternatives in our local environment to enhance effective laboratory diagnoses of fungal infections. Purified extracts of the leaves and bark of Jatropha curcas and Moringa oleifera (common plants in our locality) were tested against clinical isolates of fungi at various concentrations to determine the minimum inhibitory concentration at which common fungal contaminants are inhibited, without affecting the growth of the pathogenic fungi sought for. At a concentration of 0.75 mg ml(-1) contaminants were totally inhibited by the leaf extracts. The bark extracts did not inhibit any fungus even at higher concentrations. From the results it was evident that the leaf extracts of both plants have potentials for use as inhibitory substances in culture media against contaminant fungi including Aspergillus spp., Penicillium spp., etc. J. curcas and M. oleifera are very common plants in our locality. They can be obtained at almost no cost and at any time needed. The benefits of these findings to mycology laboratories in a developing country are enormous.

DETECTION THE OCCURRENCE OF HELICOBACTER PYLORI AMONG DYSPEPTIC PATIENT AND THEIR SUSCEPTIBILITY TO ANTIBIOTIC AGENTS

Fifty dyspeptic patient include 31 female and 19 male were subjected to esophageal gastroduodenoscopy and gastric biopsy and gastric biopsy specimens were taken from antrum and body were applied for microbiological analysis which include : urease test bacterial culture using selective and non selective media and histological examination for detecting H. pylori, and studying their susceptibility to various to antimicrobial agents. Results showed that occurrence of H.pyloria was (100%) among gastric ulcer patient,71% among duodenal ulcer and these with normal endoscopic appearance, as well as, (70%) of gastric patient were found to be infected with H.pylori. Biopsy urease test were proved to be most sensitive method for detecting H.pylori infection when 76% gave positive result for presence of H.pyloria using it, followed by histological examination when 70% of gastric tissues showed the existence of H.pylori organisms. Results also showed that using selective media was batter than not selective media for primary isolation of bacteria, with a recovery rate 61% using brain heart infusion agar and 39% was recovery rate for non selective blood agar. When isolates of H.pylori were subjected to the sensitivity test against (8) antibiotic groups, result, showed that ciprofloxacin was the most effective antibiotic against the isolates when the sensitivity percentage 90%pencilin G,tetracycline, amoxicillin and metronidazal on other hand, were the last Effective antibiotic antibiotics when bacterial sensitivity range between 30% and 90%.

Molecular Detection of Streptomyces griseus Isolated from Isfahan Soil

The aim of the present study was to detect Streptomyces griseus from Isfahan soil using biochemical, morphological and molecular techniques. Soil samples were collected from different regions (compost, 50 year old garden, river bed, clove, wheat farms and domestic yards) of Isfahan in two different seasons. One gram of each sample was serially diluted and cultivated in a primary isolation medium. Morphological shapes of colonies and vegetative hyphae were initially used to separate the grown bacteria of the samples. Various biochemical tests based on Bergey's manual of systemic bacteriology were performed to separate the Streptomyces from other related Genera. In order to further separate Streptomyces grisesus, Polymerase Chain Reaction (PCR) was used to detect the presence of aphE and strA genes in isolated colonies. Biochemical and morphological results, showed the presence of several species of Streptomyces in the collected samples. The expected band of 924 bp belonging to strA gene was observed in the positive control bacterial DNA (PTCC 1125). Several other bands were also observed in the positive control sample. From the total of 10 colonies that undergone molecular detection for the presents of strA gene, 6 colonies (W1, W3, W5, F4, F5, F26) showed only one band at 750 bp and four (W1, W3, F5, F26) showed an extra band of approximately 924 bp band as well. The expected band of 671 bp belonging to aphE gene was detected in positive control bacterial DNA (PTCC 1125). None of the soil samples, however, showed the presence of the aphE gene (data not shown).

Prospective evaluation of the new chromogenic medium CandiSelect 4 for differentiation and presumptive identification of the major pathogenic Candida species

The rapid identification of pathogenic yeasts is a crucial step in ensuring that effective antifungal treatment is started as early as possible. CandiSelect 4 (CS4; Bio-Rad) is a new chromogenic medium for the isolation of fungi, the direct identification of Candida albicans and the presumptive identification of the major pathogenic Candida species. The performance of CS4 was compared with that of another chromogenic medium, CHROMagar Candida (CA; Becton Dickinson). For primary cultures, 502 of the 1549 (32 %) samples were culture-positive. A total of 542 yeasts were isolated including 465 monomicrobial and 37 mixed cultures: 392 C. albicans, 60 Candida glabrata, 25 Candida tropicalis, 12 Candida krusei and 53 other Candida species. The percentage of C. albicans isolates that could be identified directly after 24, 48 and 72 h culture was 31.6, 82.9 and 92.1 %, respectively, for CS4, and 32.9, 82.9 and 91.1 % for CA. The presumptive identification of C. glabrata, C. tropicalis and C. krusei was evaluated after 48 h incubation. The percentage of strains with morphologically typical colonies was 80, 68 and 84.6 %, respectively, for CS4 compared with 75, 76 and 76.9 % for CA. For pure subcultures, from 24 h, all isolates of C. albicans (n=21) were directly identifiable on the two chromogenic media CA and CS4. At 48 h, the proportion of typical strains observed on the two chromogenic media was identical for C. glabrata (85 %) and C. krusei (100 %). A slight difference in favour of CS4 was observed for C. tropicalis (100 vs 95 %). CS4 also allowed the growth of several other fungi. CS4 can be recommended as a primary isolation medium for the identification of C. albicans, and for the rapid and effective differentiation of the major pathogenic Candida species.

Isolation ofCryptococcus gattiiandCryptococcus neoformansvar.grubiifrom the flowers and bark ofEucalyptustrees in India

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii was isolated from two samples of flowers of Eucalyptus terreticornis. C. neoformans var. grubii was recovered twice from the bark of Eucalyptus camaldulensis, initially from one of three samples, and again 2 months later, from one of four samples collected beneath the canopy of the tree. The primary isolation medium was Nigerseed agar, and brown colonies were presumptively identified as C. gattii or C. neoformans. The species identification was confirmed by morphological and biochemical characteristics. Using the Crypto-Check kit (Iatron, Tokyo, Japan), the first two isolates were identified as serotype B (C. gattii) and the other two were serotype A (C. neoformans var. grubii). PCR analysis of the isolates of C. neoformans var. grubii revealed that they possessed the MATalpha mating type allele. Molecular typing by amplified fragment length polymorphism markers indicated that both isolates of C. neoformans var. grubii possessed the same genotype. This study demonstrates that C. neoformans var. grubii, as well as C. gattii, may be associated with Eucalyptus trees.

UTILITY OF URICHROM II – A CHROMOGENIC MEDIUM FOR UROPATHOGENS

Purpose: Chromogenic media are increasingly being used as versatile tools in early differentiation and identification of bacterial isolates from clinical specimens. We have evaluated the Urichrome II, a newly introduced chromogenic medium, for its utility as a primary isolation and identification medium for urinary microbial isolates. Materials: This study included 5497 consecutively collected midstream and/or catheter catch urine samples obtained from patients attending several clinical services. They were inoculated on blood, MacConkey, cystine lactose electrolyte deficient, and Urichrom II agar plates for isolation of uropathogens. Results: The Urichrom II medium allowed the growth, and primary identification, of the predominant uropathogens like E.coli, Klebsiella spp. and Enterobacter spp. (KES group). The different coloured colonies produced by the breakdown of the chromogenic substrates by the specific enzymes of the bacteria were very useful in the presumptive identification of these organisms even from mixed cultures by the colour differences of the colonies. The medium also supported growth and differentiation of gram positive organisms like Staphylococcus aureus and enterococci. Conclusions: Urichrom II can be used as a primary culture medium for predominant uropathogens like E.coli, KES group and enterococci. It is an easy to use primary screening medium that considerably reduces the daily workload and thus minimizes or limits the use of identification tests.

Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon

Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 @ 10 3 bacterial cells ml −1 in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31×10 3 to 65×10 3 ml −1 on the first day to 4–16×10 3 ml −1 on the 45th day. Similarly, the phenol oxidase activity declined from 12–32 units on the first day to 11–14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46–67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.