Spread Plate Technique Research Articles

English

Environmental alterations leads to fluctuations in the soil microbial population and soil enzyme activities, as different weather parameters affect microbial biota and their activities in rhizospheric soil of crop. An agroclimatic study was carried out to study the effect of environmental alterations on soil microbial population and enzyme activities in rhizospheric soil of rice and wheat crop. Rice and wheat crop were grown under field conditions and under temperature gradient tunnel which was maintained at 4-5°C higher temperature than open field temperature. Soil samples were taken from rhizospheric soil of rice and wheat. Microbial population was enumerated by serial dilution spread plating technique. Statistically significant higher microbial population of total bacteria, nitrogen fixers and P-solubilizers was found in rhizospheric soil samples taken from temperature gradient tunnel as compared to soil samples taken from rice and wheat grown under field conditions. Fungal population was found to be statistically, significantly higher in soil samples taken from field conditions in case of rice but, higher in soil samples taken from temperature gradient tunnel in case of wheat crop. Activities of alkaline phosphatase and dehydrogenase were assessed by using para nitro phenl phosphate (PNPP) and tri phenyl tetrazolium chloride (TTC) as substrates, respectively. Enzymes activities were found to be significantly higher in rhizospheric soil samples taken from temperature gradient tunnel. Microbial population and enzyme activities were found over a broad range of temperature but, maximum microbial and enzyme activity was found only at and near optimum conditions. Key words: Environmental alterations, microbial activities, rice, temperature gradient tunnel, wheat.

Microbial contamination of traditional medicinal plants sold at the Faraday muthi market, Johannesburg, South Africa

The Faraday traditional healers' trading market is the hub of the medicinal plant trade in Johannesburg, South Africa. Modes of harvesting, transporting, storage and distribution of medicinal plants render them susceptible to microbial attack, and thereby make customers, especially patients with compromised immune systems, vulnerable to infections that could increase morbidity and mortality. This study evaluated the microbial contamination on five frequently used traditional medicinal plant species sold by traders in the Faraday market. Bacterial contamination was determined using serial macro dilutions, spread plate and streak plate techniques. Fifteen bacterial contaminants were identified, the most recurrent being Pantoea sp. and five strains of Bacillus spp. (non-pathogenic). There was little variation between contamination levels of the five different traders, and the mean CFU/g per species ranged from 3.03×104 (Hypoxis sp.) to 4.22×105 (Hydnora abyssinica). While there was no overall significant levels of contamination, the CFU counts for two plant species purchased from one specific trader (viz. H. abyssinica and Acacia xanthophloea) exceeded maximum acceptable contamination limits set by the World Health Organisation (i.e. ≤105 to ≤107CFU/g). The levels of contamination varied greatly between the commercially available over the counter product and the plant samples investigated. The microbial types are predominantly opportunistic pathogens. The implementation of good processing practices therefore clearly influences the quality and safety of medicinal products, especially regarding microbial contamination. It is evident that policies and regulations need to be developed and implemented in order to address possible contamination by opportunistic pathogens.

Antimicrobial Resistance of Pathogenic Bacteria Isolated from Tube Well Water of Costal Area of Sitakunda, Chittagong, Bangladesh

Groundwater is thought to be free of microbial contamination due to the natural filtering ability of the subsurface environment. The study is undertaken to investigate the presence pathogenic bacteria and their antimicrobial resistance pattern of groundwater of Sitakunda, Chittagong. In this study bacterial contamination in tube well water has been analyzed in terms of coliform, fecal coliform and vibrio. Qualitative analysis of bacteria is performed by spread plate technique and antibiotic susceptibility test is done by disk diffusion method. In bacteriological analysis total coliform, total fecal coliform, and total vibrio spp. count are ranged from 0.65⇥102 to 2.0⇥102 cfu/ml, 0.10⇥102 to 2.20⇥102cfu/ml and 0.0 to 0.55⇥102cfu/ml, respectively. All of the coliform isolates are resistant to ampicillin, all fecal coliform isolates to penicillin and sulphamethoxazole and vibrio spp. are resistance to erythromycin and azithromycine. The coliform isolates also display multidrug resistance. The resulting contamination of tube well water with pathogen and their resistivity to antibiotic is an alarming threat to public health, so tube well water of Sitakunda, Chittagong must be treated prior to drinks.

Análise fenotípica e genotípica de bactérias heterotróficas e fixadoras de nitrogênio em sedimento na bacia do Rio Cuiabá-MT

Sediments are complex systems that are affected by geological, hydrodynamic, chemical and biological parameters, as determined by interactions between the sedimentary environments of each region. This study aimed to characterize the diversity of total heterotrophic bacteria and nitrogen fixers in Cuiaba River sediments using conventional techniques of microbiology and molecular biology. Sediment samples were collected bimonthly from four points: Cuiabazinho, Passagem da Conceicao, Ribeirao Cocais and Barao de Melgaco. The samples were processed in serial dilutions (10-2 to 10-7) in 0.85% saline solution. They were then cultured in Petri dishes using the Spread Plate technique in the Trypic Soy Agar (TSA) culture medium for total heterotrophic bacteria and selective medium (NFB, JMV and culture median 79) incubated at 35°C for nitrifying bacteria. The bacterial strains were then re-isolated on Nutrient Agar (NA) in order to obtain a pure culture for the Gram analysis morphotypes. This test revealed that, of the 202 bacterial isolates, 59 % were positive rods. The largest bacterial count was obtained in the TSA medium, as compared to other means of culture. The profile of the bacterial community showed that most, or 28 %, of the bacteria were of the Bacillaceae family and these were used for molecular analysis using Box-PCR, which showed a great diversity of species. These results indicate the importance of microbial diversity research in sediments in Mato Grosso State using molecular techniques.

Listeria detection in microscale solid state inoculation with minimal selective agents

The food industry needs a simple, reliable, and cost-effective primary screening protocol for routine inspection of bacterial contamination. Microscale inoculation technique is proposed as an alternative method for Listeria detection. This proposed novel technique shows a good correlation with standard spread plate technique for the enumeration of pure Listeria cultures (R2 = 0.96, P < 0.0001). The commonly used selective agents in Listeria media (PALCAM, MOX, and OCLA) were assessed for their inhibitory effect on Listeria monocytogenes, Listeria innocua, and Listeria ivanovii using the microscale inoculation technique. The concentration of selective agents was lowered to inhibit competitive bacteria with minimum impact on the growth of Listeria. At the standard concentration, all three media showed less inhibition on L. monocytogenes and L. innocua than on L. ivanovii. OCLA had less inhibitory effect on the three species than did MOX and PALCAM. The comparison between ISO 11290-1 and microscale with 25% of selective agent concentration on detection of Listeria in 36 naturally contaminated food samples revealed that the microscale technique agreed well with the ISO method (Cohen KAPPA = 0.83). Reduction of selective agent concentration to 25% of the conventional formulas resulted in substantial improvement in detectability of Listeria spp. without significantly reducing specificity. The detection sensitivity of the Listeria colonies in food samples was significantly improved by microscale inoculation with 25% of the inhibitors on the three selective media at 24 and 48 h incubation (ANOVA, P = 0.039). At 48 h incubation the improvement was from 95%, 90%, and 85% (ISO method with regular strength inhibitors) to 100%, 100%, and 90% (microscale) on OCLA, MOX, and PALCAM respectively. There was no discrepancy between the microscale and the ISO method in detection of L. monocytogenes in the food samples. The optimization of Listeria detection improves sensitivity of detection as well as reducing media volume which may reduce costs and waste generated.

Molecular Characterization of Aerobic Heterohophic Bacteria Isolated from Petroleum Hydrocarbon Polluted Brackish Waters of Bodo Creeks, Rivers State Nigeria

Surface water sources in the oil producing Niger Delta region of Nigeria are highly susceptible to pollution by petroleum hydrocarbons and so it is important to understand the microbial diversity of such ecosystems. Water and sediment samples were collected between April-August, 2013 from Bodo creeks and taken to Environmental Microbiology laboratory of University of Portharcourt for analysis. A total of thirty aerobic heterotrophic bacterial strains isolated ranged from 3.0 - 7.0 × 104 cfu for surface water and 1.6 - 5.6 × 104 cfu for sediment samples of Bodo creek using serial dilution and spread plate technique. Pure cultures of bacteria were obtained on the basis of their morphological characteristics and subjected to biochemical tests and further classified on the basis of 16S rRNA gene sequence analysis. The DNA was isolated from size fractionated samples and the diversity of bacteria in each fraction was studied using PCR amplification of partial 16S rRNA. The sequences were submitted to NCBIGen bank for identification and assigning of accession numbers. The isolated aerobic heterotrophic bacteria belong to the families of Enterobacteriaceae, Bacilliceae, Alcaligenaceae, Pseudomonadaceae, Flavobactericeae and Planococcaceae.

Impact of Salt on Bioelectricity Generation in a Dual-Chambered Microbial Fuel Cell Using Sago-Processing Wastewater

Effect of NaCl on electricity generation, COD removal, reduction in carbohydrate and starch content in dual chambered, salt bridge Microbial Fuel Cells (MFCs) employing raw sago-processing wastewater with an organic load of 14,400 mg COD/l as substrate was evaluated. Four dual chambered MFCs were constructed and the study aimed to find out the impact of addition of NaCl which is carried out for effective MFC performance. Isolation and identification of microbes from initial influent and final effluent was performed using serial dilution, spread plate and selective agar techniques. Interestingly, it was found that the MFC in which NaCl was added to its cathode chamber was best in performance compared to other three MFCs, with a maximum voltage of 603mV and current of 6.03mA. It also documented that the maximum COD removal efficiency of 83% with a total reduction of carbohydrate and starch content from the wastewater was obtained. Utilizing sago wastewater for the production of bioelectricity from MFC technique is considered as a feasible and sustainable process.

Biodegradation of Total Petroleum Hydrocarbon by Aerobic Heterotrophic Bacteria Isolated from Crude Oil Contaminated Brackish Waters of Bodo Creek

Bodo creek is located in Gokana Local Government Area of Rivers state and is characterised by brackish water which is heavily contaminated with crude oil. Water samples collected from the creek were taken to Environmental Microbiology Laboratory of University of Portharcourt for isolation of aerobic heterotrophic bacteria using serial dilution spread plate technique. Isolation of morphologically distinct pure cultures was done and the isolates obtained were identified molecularly on the basis of 16S rRNA gene sequence analysis. 16S rRNA sequencing for identification of the isolates generated sequences ranging from 500 bp to 1700 bp and a 250 bp size PCR amplified fragment. The nucleotide sequence of the 16S rRNA gene was compared with published 16S rRNA sequences using BLAST search at the data base of National Centre for Biotechnology Information (NCBI). Biodegradation of petroleum hydrocarbon test on the isolates was conducted using Bony light crude oil obtained from Shell Petroleum Development Company and monitored using GC-FID (Agilent 6890 model) for 49 days. The initial quantity of TPH was 24091 and 6706 mg/l on day 0 for aerobic heterotrophic treatment and crude oil contaminated control and decreased progressively to 212.8 and 1174 mg/l respectively on day 49 indicating biodegradation in the treatment and control. Loss of TPH was statistically significant using one way analysis of variance (ANOVA) p<0.05 with time. Model for predicting TPH loss was developed and rate of biodegradation of the isolates determined using Trend line method of Microsoft Excel, 2010. The growth of bacteria cells increased progressively with decrease in TPH implying that the bacteria were responsible for biodegradation. Further application of bioremediation strategies in Bodo creek for biostimulation of the crude oil biodegrading bacteria for reclamation and restoration of an efficient ecosystem structure and function is suggested.

A double layer plaque assay using spread plate technique for enumeration of bacteriophage MS2

Bacteriophage MS2 is used widely as a model organism to estimate pathogenic virus survival in various environments, and is usually quantified by plaque assay. Although current plaque assays work well in enumeration of MS2 in environmental samples, quantification of MS2 calls for better visibility and higher consistency. In an attempt to improve the visibility and consistency of the current plaque assay, spread plate technique was introduced, instead of the pour plate technique used commonly in existing methods. Other parameters that influence the outcome of the plaque assay were also compared. Using spread plate technique resulted in an increase of plaque size by approximately 50% and contributed to a better visibility. Addition of supplements (glucose, CaCl2 and thiamine); reduction of agar thickness and hardness, also contributed to enhanced plaque visibility and increased plaque count. Among all the conditions tested, a supplemented thin bottom agar (10ml 1% agar) and a supplemented thin top agar (10ml 0.45% agar) with spread plate technique gave the maximum countable plaques with a minimum standard deviation. When compared to other methods, it produced significantly higher plaque count and lower variation. The optimized plaque assay significantly improved visibility and consistency of the existing plaque assay methods and could be used in quantification of MS2.

Gari agar as culture media for mycological studies

Gari agar was prepared by weighing 28 g of Gari, 14 g of agar powder and 8 g of Hibiscus rabdariffa powder to 1 L of sterile water. A conventional media, Sabouraud Dextrose Agar (SDA) was prepared as control according to manufacturer’s procedure. Aliquot of appropriate dilutions of 1 g of agricultural soil was inoculated onto SDA and Gari agar by pour plate technique and spread plate technique. After 2 days and 4 days of inoculation, the growth of moulds was examined. Fungal colonies on Gari agar were very healthy and compared favourably with fungal growth on Sabouraud dextrose agar which is a conventional medium. Gari agar produced healthy moulds as determined by diameter of growth. Furthermore, conventional media (SDA) supported the growth of bacteria while Gari agar inhibited the growth of bacteria and promoted that of fungi. Cassava can be successfully grown in poor soils with low labour requirement, low capital and ease of cultivation. Gari extract agar can now be used successfully for quantitative count of moulds. The result of this study will go a long way in solving the problem of high cost of conventional media used as culture media for mycological studies.Keywords: Gari agar, Hibiscus rabdariffa, Sabouraud Dextrose agar, quantitative count, moulds, cassava.

Influence of pre- and post-usage flushing frequencies on bacterial water quality of non-touch water fittings

BackgroundNon-touch fittings have been reported to be susceptible for Pseudomonas aeruginosa accumulation. A number of factors may contribute to this, including the frequency of usage, duration of water stagnation, or presence of plastic materials. Programmable non-touch fittings are appearing which allow regular automated post-flushing with cold water to prevent water stagnation. However, the ideal duration of post-flushing is unknown as well as the effect of pre-rinsing with cold water before use.MethodsEight non-touch fittings with brass valve blocks were mounted on a mobile test sink and connected to the same central water pipe source, differing only in presence or absence of water connection pipes, length of connection pipe, frequency of usage, and time intervals for pre- and post-usage water flush. The total bacteria colony-forming unit (cfu) counts were obtained by the spread plate technique.ResultsLow frequency of water use in combination with a long stagnating water column resulted in high bacterial cfu counts. Post-usage flushing for 2 seconds did not differ from no flushing. Flushing for 10 seconds with cold water after use or 30 seconds flush before use were both the most effective measures to prevent non-touch fittings from biofilm formation over a period of 20 weeks.ConclusionFurther improvements in water fitting technology could possibly solve the problem of bacterial water contamination in health care settings.

Evaluation of soil biological properties of 9- and 15-year-old stands in the oil palm plantation in Perak, Malaysia

Opening land for oil palm cultivation provokes many debates around the world regarding on the fate of biodiversity. A study was conducted to evaluate and compare soil biological properties of 9-year-old (P1) and 15-year-old (P2) stands of an oil palm plantation in Bikam, Perak, Malaysia. Composite samples were collected at depths of 0-15 cm (topsoil) and 15-30 cm (subsoil) located within six subplots (20 m × 20 m). The microbial population count was estimated using a spread-plate technique, and the Fluorescein diacetate (FDA) hydrolysis assay was used to measure microbial enzymatic activity. A rapid ethanol-free chloroform fumigation extraction technique was used for microbial biomass extraction, and the extracts were respectively analyzed by wet dichromate oxidation and Kjeldahl digestion for biomass carbon (C) and nitrogen (N). At the 0-15 cm depth, the microbial biomass C and N contents in the soils from both plots were significantly different (P<0.05). At the 15-30 cm depth, only microbial enzymatic activity was significantly different between plots. Although the addition of fertilizers to the soil is believed to be a predisposing factor, no significant differences in P1 and P2 plots for the biomass C and N in soils at the 15-30 cm depth were observed. Variations in the MBC/MBN ratio in soils of the P1 and P2 plots indicate that changes occurring in the soil microbial composition are due to the availability of soil organic substrates and N. Thus establishment of an oil palm plantation does contribute to changes in soil biological properties. Key words: Oil palm, different ages, microbial population, enzymatic activity, biomass C and N, rapid chloroform-fumigation extraction technique.

Determination of the Soil Microflora of a Soil Near Microbiology Laboratory at the University of Ilorin Main Campus

The microflora of a soil near microbiology laboratory at the University of Ilorin, Ilorin, Nigeria was analyzed in this study. Total bacterial count and total fungal count of the soil samples were determined using standard spread plate technique. Six soil samples were collected with interval of two weeks between two samples. Identification of the isolates was done using their colonial, morphological, and biochemical characteristics and established procedures were followed. The total bacterial counts ranged from 1.4 x 10 8 to 1.7 x 10 8 cfu/g and the total fungal counts ranged from 1.4 x 10 5 to 1.5 x 10 5 cfu/g. The bacteria isolated were Pseudomonas aeruginosa, Staphylococcus epidermidis, Acetobacter species, Lactobacillus species, Erwinia species, Klebsiella species, Bacillus subtilis, Clostridium species, and Bacillus cereus. Isolated fungi were Rhizopus oryzae, Aspergillus niger, Aspergillus flavus, Mucor hiemalis, Penicillium chrysogenum, Fusarium oxysporium, and Trichophyton rubrum.

STATUS OF SOIL MICROBIAL POPULATION, ENZYMATIC ACTIVITY AND BIOMASS OF SELECTED NATURAL, SECONDARY AND REHABILITATED FORESTS

Substantial clearance of forests and conversion of forest into various land use types contribute to deterioration of soil fertility and associated nutrients loss. Soils from natural and rehabilitated forest in Chikus Forest Reserve and also enrichment planting forest and secondary forest of Tapah Hill Forest Reserve, Perak, Malaysia were selected in order to assess the influence of land use change on biological properties. This study was carried out to provide fundamental information on soil biological properties and also to compare the differences between natural forest, mono-rehabilitated forest, mixed planting forest and natural regenerated forest (secondary forest). Six subplots (20×20 m) were established at each study plot and soil samples were collected at the depths of 0-15 cm (topsoil) and 15-30 cm (subsoil). Soil microbial population was determined using spread-plate technique. Fluorescein Diacetate (FDA) hydrolysis was used to assess the amount of microbial enzymatic activity for each forest plot. Soil Microbial Biomass C (MBC) and N (MBN) were extracted using chloroform fumigation extraction technique and the amount of MBC was determined by dichromate digestion, while MBN via Kjeldahl digestion technique. Soil acidity was determined by pH meter and moisture content was elucidated using gravimetric method. The levels of microbial population of bacterial and fungal at natural significantly exceeded the corresponding values of rehabilitated and secondary forest. However, microbial population is much higher in rehabilitated forest of Tapah Hill compared to that of secondary forest and also Chikus Forest Reserve planted forest which proves that rehabilitation activities do help increase the level of microbial community in the soils. Longer period of time after planting as in enrichment planting compared to mono planting of S. leprosula plantation showed that restoring and recovery of the planted forest needed time. Deforestation activities decrease soil biological activities; however, proper forest management and rehabilitation activities are able to restore the condition of degraded forest land to its original state.

Microbial assessment of bottled drinking water of Kathmandu valley

INTRODUCTION: Due to increased demand and consumption of bottled water in Kathmandu valley, there has been a growing concern about the microbiological quality of this product. The objective of this study was to assess the microbial contamination of heterotrophs and total coliform. MATERIALS AND METHODS: This cross sectional study was done in randomly selected 30 different commercial brand of bottled water available in commercial market of Kathmandu valley, Nepal. Total coliform and heterotrophic bacteria were enumerated by the membrane filtration and spread plate technique, respectively. RESULTS: Nineteen samples (63.3%) showed the heterotrophic bacterial count above the acceptable range (i.e. &lt;50 cfu/ml) and 56.6% (n=17) samples showed the total coliforms count crossed the WHO guidelines (i.e. 0 cfu/ml). 83.3% (n=25) brands of bottled water were not safe for drinking as they exceed the heterotrophic and/or total coliform count above the WHO guidelines. CONCLUSIONS: The presence of heterotrophs and the coliform in the bottled water is a serious concern for public health. Concerned authority should prioritize this issue and continuously monitor the quality of mushrooming bottled water manufacturers in Kathmandu valley to provide safe drinking water to the population. DOI: http://dx.doi.org/10.3126/ijim.v1i2.7399 Int J Infect Microbiol 2012;1(1):84-86

Microbial Contamination in Well Water of Temporary Arranged Camps: A Health Risk in Northern Pakistan

High number of refugees, internally displaced people (IDP) and nomadic camps were established in the aftermath of intensive military operations and militant activities from 2000 onwards in different regions, especially areas near Afghanistan border of Khyber Pakhton Khwa (KPK), Pakistan. In this study we analyzed the quality of well drinking water in three (A, B, C) different camps situated in Kohat Division in April 2011. Well water samples were serially diluted and inoculated in respective media by spread plate technique standard procedure, and incubated at 37±1 °C for 24–36 hours. The viable count of tested microorganism was mentioned in form of CFU. Daily uptake of microbial load/person/day was calculated and each source contribution was measured by using ANOVA. Results showed that total viable count, fungal count and their daily uptake were significant in all stations, while Fecal Coliform (E. coli), Salmonella, Shigella and S. cocci loads were enormous in station B followed by station C. In station A microbial load was comparatively lesser than in station B and C. Moreover, stations A, B, and C have higher enteric pathogenic microbial load than are WHO recommended standards for potable water.

Isolation and identification of cadmium- and lead-resistant lactic acid bacteria for application as metal removing probiotic

The purpose of the present study was to isolate and identify the metal-resistant lactic acid bacteria from sediments of coastal aquaculture habitats for removal of cadmium and lead from ambience. Collected sediment samples were used to isolate the cadmium- and lead-resistant bacterial colonies by spread plate technique using agar media (De Man, Rogosa and Sharpe) supplemented with cadmium or lead at 50 mg/l. Isolates were identified by bacterial colony polymerase chain reaction and sequencing of 16S ribosomal deoxyribonucleic acid. Metal removing probiotic was determined by characterizing the lactic acid yield in culture media, viability in fish intestine, metal-resistant and metal-removal efficiencies. 16S ribosomal deoxyribonucleic acid sequencing data of five (Cd10, Cd11, Pb9, Pb12 and Pb18) and other all isolates clearly showed 99 % similarities to Enterococcus faecium and Bacillus cereus, respectively. The Pb12 exhibited higher lactic acid yield (180 mmol) than that of the remaining E. faecium strains and excellent viability without pathogenicity; therefore, further study was carried out using Pb12 strain. The selected Pb12 strain showed elevated metal resistant (minimum inhibitory concentrations 120 and 800 mg/l for cadmium and lead, respectively) and removal efficiencies [Cadmium 0.0377 mg/h/g and lead 0.0460 mg/h/g of cells (wet weight)]. From the viability and metal removal points of view, it can be concluded that isolated metal-resistant E. faecium Pb12 strains might be used as potential probiotic strains for removing heavy metals from fish intestinal milieu to control the progressive bioaccumulation of heavy metals in the fish.

Non-Toxicity of Water Soluble Multi-Walled Carbon Nanotube on Escherichia-coli Colonies

Carboxylic acid functionalized water soluble carbon nanotube (wsCNT) shows no toxic effect against the growth of Escherichia-coli (E. coli), a gram-negative bacteria. Treatment up to 8 microg mL(-1) of wsCNT did not show any toxic effect on E. coli growth that was followed by using bacterial growth kinetics and Spread Plate Technique (SPT). The number of E-coli colonies counts with and without wsCNT showed nearly no change and the bacterial growth kinetics in both the cases showing no toxic effect of wsCNT on the growth of E. coli.

Practical coliforms and Escherichia coli detection and enumeration for industrial food samples using low-cost digital microscopy

This research explored several alternative coliforms and E. coli detection strategies proposed for industrial application especially low-resource settings and less advanced food manufacturers. The colony count using 2 industrial favourites (i.e., PetrifilmTM by 3 M and regular pour plate techniques) were contrasted to 4 alternative lowcost strategies. Two modified conventional protocol (i.e., pour and spread plate techniques) in standard mini Petri dishes and two drop plate techniques in microtiter plate formats (i.e., 24 - and 96-well plate) were applied to count industrial frozen food samples. The colony detection in all treatments was visually facilitated by low-cost digital microscopy technique comparing colony count, the detection time and the colony area in pixels. All experiments except for the PetrifilmTM E. coli/Coliform (EC) Plate utilized Chromocult® Coliform Agar (CCA). The inoculum sizes were varied depending on the cell count technique used; 10 μl and 5 μl for the 24- and 96-well microtiter plates, 50 μl for the mini-plate (both pour and spread plate techniques), and 1000 μl for the full-size Petri dishes and PetrifilmTME. coli/Coliform (EC) Plate. The incubation temperature was fixed at 35±2°C. The number of colonies from the conventional pour plate technique was plotted against those of the other techniques. In all treatments, the relationship plots showed highly linearity from the lower detection limit (100 CFU/ml depending on technique used) to the upper detection limit (10,000 CFU/ml). The slopes of all regression lines were close to unity showing very high correlation of the values of colony counts from different techniques. All techniques were applied to evaluate actual swap samples from the production line and returned highly consistent colony count numbers and good separation of E. coli from coliforms. The colony counts from the ready-to-eat product samples showed similar and comparable results to the two routine factory techniques. The results indicated that all six colony count methods were simply interchangeable to perform colony enumeration in the low-resource industrial setting.

Antimicrobial Activity of Antibiotic Producing Streptomyces macrosporus

The aim of the present study was to isolate and to identify the actinomycetes having antimicrobial activity. Actinomycetes strain isolated from soil samples collected at the Coimbatore region, Tamilnadu, India. Actinomycetes were isolated by spread plate technique and screening was done by parallel streak & agar well diffusion method. Upon primary screening by parallel streak method out of 180 actinomycetes isolates around 25 isolates showed antimicrobial activity against test organisms. On secondary screening out of 25 isolates, 9 isolates showed activity. According to antimicrobial activity and spectrum broadness strain 7 was selected for further study. Based on the morphological, physiological, biochemical and phylogenetic characterization the strain 7 was identified as Streptomyces macrosporus. In conclusion the isolated strain has broad spectrum of antimicrobial activity against the test organisms. I. Introduction Search for new antibiotics effective against multidrug resistant pathogenic bacteria is presently an important area of antibiotic research. Natural products having novel structures have been observed to possess useful biological activities, soil is a natural reservoir for microorganisms and their antimicrobial products (1). Of all known drugs 70% have been isolated from actinomycetes of which 60% are used in medicine & agriculture respectively. Streptomyces spp produce about 75% of commercially and medically useful antibiotics that are natural in origin such as erythromycin, Neomycin, tetracycline and Cefoxitin. Microorganisms constitute an in exhaustible reservoir of compounds with pharmacological, physiological, medical (or) agricultural applications.