22.05.13PROFILAB participated in the scientific and practical conference dedicated to the 90th anniversary of the Microbiology Department of the Military Medical Academy
The conference took place on May 22 in St. Petersburg, Russia.
There were the following relevant topics of discussion:
- Questions of Clinical Microbiology
- Actual problems of sanitary microbiology
- Laboratory diagnosis of viral infections of topical
- Microbiological quality control
PROFILAB introduced Dehydrated Culture Medias produced by Indian company Titan Biotech Ltd.
DIAGNOSTIC CAPABILITY OF MODERN CULTURE MEDIA & CONTRIBUTION OF TITAN BIOTECH LTD
Microbial culture media are used for the growth, isolation and detection of microorganisms. They generally consist of nutrients such as peptones, amino acids, meat extracts, yeast extracts, minerals and vitamins, inhibitors, solidifying agents and detection systems. Albeit traditional culture media are an important part of the microbiological testing and commonly used in microbiological examination and diagnostics for the decades, the changes in the social habits, life styles, eating habits and food production methods urge the scientific community to develop new technologies for the fast and minute level detection of microorganisms from the food, clinical, water and environmental samples.
Titan Biotech also joins to meet out the new challenges of the fast detection of microbes and extend the hands in the research and development of modern microbiological culture media. The launching of chromogenic culture media for food and water pathogens is our first step to this venture. The incorporation of traditional culture media with a suitable indicator system is the idea behind the chromogenic and fluorogenic media. The specific enzyme produced by the target microorganisms cleave chromogenic or fluorogenic substrate liberating the chromophor or fluorophor, which highlight the microorganism by fluorescence in culture media or coloration of the grown colony. We have a collection of chromogenic culture media intended for the detection and isolation of various microorganisms such as Candida, Klebsiella, Bacillus, E. coli, and Enterococcus etc. Supplementation of these chromogenic plating media with inhibitors such as antibiotics mostly or completely inhibits contaminants occurring in the samples to be tested. Utilization of the virulence factors of the target microbes in the chromogenic media can combine both the quantification and rapid detection of microbes which can make a cost effective and time saving technique.
Collection of samples from clinical specimens, veterinary animals, food, water and other environmental samples are usually performed using the sterile swabs and loops. The metal loops and normal swabs need to be sterilized each time and also not sure that sterilization will be done properly. To make the sample collections easier and convenient, Titan Biotech developed different types of disposable sterile inoculation loops, sterile screw caped vials, transposable swabs and needles. We developed special transposable swabs intended to use in each of the sample collections.
Our 12 inches blue sponge swabs are specially developed for the food and industrial sample collections. The 12 inch foam tip with a polypropylene shaft is very useful for the sampling down small pipes or curved drains. The flexible shaft allows sampling of tricky or hard to reach areas. We have another swab to use for the collection of clinical samples, especially for anaerobic organisms. This swab is made up of a flexible polypropylene shaft with viscose tip. Viscose is a semi-synthetic material made up of cellulose xanthate (obtained from wood pulp) treated with sodium hydroxide and carbon disulphide. This is an ideal transposable swab for the sampling from clinical specimens. The tip can be preserved for a longer period in sterile condition. We manufacture Amies transposable swabs also, recommended for general clinical swabbing of fastidious, non-fastidious and anaerobic microorganisms, including throat, vaginal and wound samples.
Our manufacturing includes various types of disposable inoculation loops to maintain extreme purity throughout the inoculation procedures. We have blue coloured loops with loop sizes of 10µl, 1µl etc. all are made up of γ – irradiated polystyrene materials. This is ultra pure and maintains sterility even in the prolonged storage periods. We use nontoxic materials in the manufacturing to ensure that it is not harmful to both the microorganisms and the handling persons. The droplets of sample are properly adhered in the loop because disposable loops are surface treated and also can reduce the risks of spreading microorganisms while inoculation. Titan Biotech also supplies the L – shaped spreaders. Our L – shaped disposable microbiological spreaders provide a best alternative and cost effective method to blending glass rods or pipettes for spreading the samples on agar surfaces and filters in the Petri plates. As we distribute the sterile spreaders, no need to more sterilization via flaming or autoclaving.
The prospectus of Rapid Biochemical Identification Systems is also to be utilized for the identification of clinical specimens. The use of Ready to Use Kits developed by Titan Biotech helps the accurate identification of a comprehensive range of microorganisms in 18 – 24 hours with a one step inoculation procedure. This technology allows confirming the identification of clinically significant bacteria, yeasts and fungi even in small laboratories. Titan Biotech already developed biochemical identification Ready to Use Kits for different microorganisms such as Salmonella, Mycobacterium, E. coli, and Enterococcus etc. The kits for the detection of Gram negative glucose non-fermenting bacteria, oxidase negative Enterobacteriaceae, anaerobic bacteria, Listeria species, Corynebacterium clinically significant Staphylococci and yeasts etc. are our next concerns in this area. We developed special kits for the rapid identification of tuberculosis and for malaria parasite identification also as both of these are the most prevalent pandemics in most of the countries. These laboratory kits have benefits in high disease burden countries like India and other countries. We offer a cheap and high quality standardized products for the easier procurement processes and to reduce the incidence of stock outs and service disruptions.
The delayed laboratory testing of clinical samples may cause the false and unnecessary prescription of antibiotics by the physician which may somehow lead to further complications to the patients due to increasing drug resistance. The on – site diagnosis of infectious diseases is an important and unmet need of the modern world. The development of an Aura image technology using the nanosensors will be a big leap to the fast detection of the infectious diseases. The nanosensors coupled with highly amplified signals generated by an electrocatalytic reporter system allow the direct analysis of clinical samples and the physician can offer the rapid treatments even inside the clinic. The BAX systems for PCR assays are also a new technology for a fast, accurate and safe diagnosis of microbial pathogens and other microbes directly from the food and environmental samples. The system breaks down samples at the genetic level, using the power of the PCR to detect bacteria and other microbes with certainty.
Exact counting and enumeration of bacteria and accurate distinction of live and dead bacteria in a sample is an important factor for many of the experiments in molecular biology. Titan Biotech took this challenge for the researchers and put forward a Bacteria Counting Kit for flow cytometry. This kit includes a high affinity nucleic acid stain which can easily penetrate into the bacteria (both into Gram positive and into Gram negative bacteria) and produce an extremely brilliant green fluorescent signal. The Kit also contains a calibrated suspension of polystyrene microspheres. Signals from both the stained bacteria and the beads are easily detected in the green fluorescence channel and can be distinguished on a plot of forward scatter versus fluorescence. The density of the bacteria in the sample can be determined from the ratio of bacterial signals to microsphere signals in the cytogram. The calibrated suspension of beads in the microsphere standard has size characteristics and relatively low fluorescence appropriate for use in combination with any type of bacteria that may be present in the sample. The Bacteria Counting Kit is particularly valuable for monitoring antibiotic sensitivity, as it provides a convenient and accurate means for assessing changes in a bacterial population over time. Bacteria and microsphere particles can be easily distinguished in a graph plotting forward scatter against fluorescence. The density of bacteria in the sample is determined from the ratio of bacterial signals to microsphere signals in the cytogram. To obtain the best result, it is recommended to use low phosphate medium or buffers that do not contain phosphate such as Tryptone Soya Broth or other low phosphate media. The medium should be filtered using 0.2 µm pore size filters to remove the debris and other solid particles and dilute the bacterial culture with 0.15M NaCl.
Our Bacterial Viability and Bacteria Counting Kit can be used to distinguish and quantify live and dead bacteria with the aid of flow cytometry. The Kit can also be used to distinguish the bacteria from a mixture of population containing a various range of bacterial species. This kit contains a combination of two nucleic acid stains, a green fluorescent dye and a red fluorescent propidium iodide to determine the viability of the cells along with a calibrated suspension of microspheres to measure the accurate volume of the samples. For the trial, we tested the suspensions of live and dead S. aureus and E. coli. The dead cells are alcohol treated and the live cells remain untreated and the mixed culture is stained with the mixture of the nucleic acid dyes mentioned earlier and then analyzed using the flow cytometry. The green or red fluorescence versus side scatter cytogram was used to gate the bacterial document and the bead population (left and right boxes respectively). Events in the bacteria region of each cytogram are also displayed in red fluorescence versus green fluorescence cytograms. Live and dead bacteria/mL can be calculated from either the fluorescence versus side scatter cytogram or the green fluorescence versus red fluorescence cytogram, depending on which one shows the best separation of the live and dead populations. The position of the live and dead populations in these cytograms may be dependent on cell type and gram character. Some samples may exhibit events that fall outside the defined regions and should be evaluated appropriately.
Our products also include the Yeast Viability Kit which is used to measure the yeast metabolic activities also, and the kits for the studies of viruses, fungi and other parasites. The same technologies of bacterial viability count kit are utilized for the Yeast viability kit and other kits also. The Yeast Viability Kit combines a cell permeable esterase substrate with a membrane integrity indicator to evaluate the vitality of yeast cells by flow cytometry or microscopy. The kit contains the cell permeant esterase substrate 5-carboxyfluorescein diacetate (CFDA) and the cell impermeant nucleic acid stain, propidium iodide. With an appropriate mixture of the CFDA, AM and propidium iodide stains, esterase-active yeast with intact cell membranes stain fluorescent green, whereas yeast with damaged membranes stain fluorescent red.
We are well aware about that the microbes are also responsible for the destruction of cultural heritages, along with various environmental impacts, ageing and the chemical structure of the substrate. There are much more studies by the researchers and review articles about the role of microbes in various artistic materials such as mural painting, stone, wood, paper, masonry, leather, parchment, glass, and metals. The biofilms are also commonly formed in architectural features, structural building blocks and in the interior part of water and industrial pipelines. The efforts to restore the objects not always yield the expected results and sometimes make the aesthetic works worse. Growth of microorganisms on objects of art can result in visible aesthetic and structural damage. Using scanning electron microscopy (SEM) microorganisms present on a small piece of an object can be directly visualized and assessed which groups of microorganisms are present. Further this SEM pictures can show the interaction of a biofilm with the substrate they grow on. The microbial sample collection from the mural arts can be done using the combination of various sampling techniques. The microbial biofilm can be removed with a sterile scalpel, pencil or with a cotton swab. The samples thus collected are allowed to undergo rehydration (for the dry biofilms) by diluting them with physiological water containing polythene glycol with peptone and polysorbate 80, for about two hours in 40C.Then inoculate the sample in any of the solid or liquid media depending on the microbes which needs to be investigated. Sometimes the microorganisms may be directly transferred to the solid agar media by gently pressing the agar plate on the object or by using the adhesive tape strips. Considering the risks and time lapse of these procedures we developed a Biofilm Tracer, using a modified protocol in our bacterial viability kit, to accelerate biofilm research by making it easier to examine bacteria within biofilm communities. It is very simple to use with its technology and protocols by just adding the stain, incubate and then observe to monitor the various aspects of biofilm biology. Our Biofilm tracer and viability kit differentially stains live and dead cells in the biofilms based on its membrane integrity. The live bacteria exhibit green fluorescence and dead bacteria exhibit red fluorescence, even when the population contains a mixture of bacterial species.
Taking high concern of the vegan society Titan Biotech develops Veg. dehydrated media for the cultivation of microbes utilizing in food industry. An animal product is any material derived from animals for human use such as meat, poultry, seafood, eggs, dairy products, honey, fur, leather, wool, and silk. Most complex nutrient media used in a general microbiology lab contains animal-derived ingredients, such as casein, tryptone, or meat extracts. We prepared media which are free from all type of animal products and also support the growth of most of the microorganisms comparable to the commercially available animal based media as we wanted to develop a cost effective veg. based substitutes in the animal based media. Soybean is a best alternative for vegetable proteins as it is known as the vegetable meat. We tested several combinations of soy flour and other additives on different species of bacteria and compared quantitatively the amount of growth on these plates with the standard of animal based nutrient agar plates. From all of the trails we tested, we found that a mixture of toasted soy flour and yeast extract powder provided a best yield and maximum growth of microbes in the medium. Quantitative and qualitative analyses were performed to compare growth of bacterial cultures in our soy based media to growth in the standard animal based media. Finally, we tested the soy based medium side-by-side against animal-based media typically recommended by experimental protocol during standard microbiology labs.
Chromogenic staphylococcus agar is a new and improved version of media for the accurate isolation and detection of Staphylococcus aureus within 24 – 48 hours. We also made a modified version exclusively for a cost effective and more selective isolation of the MRSA (Methicillin Resistant Staphylococcus aureus) just within 4 hours. The success of a screening program depends on the speed, reliability and cost effectiveness of the method used. Chromogenic MeReSA Agar is a selective chromogenic medium that can permit primary growth, selectivity and differentiation of MRSA colonies on a single plate. It is both fast and reliable and, unlike other rapid methods such as PCR, it requires no capital outlay and is accessible to every hospital laboratory. The use of a new surrogate marker for methicillin resistance in Chromogenic MeReSA Agar makes the higher sensitivity, specificity and positive predictive values.
A chromogenic and modified version of media is developed for the quick and easy detection and enrichment and confirmation of Salmonella from the food, animal feed and environmental samples. Chromogenic Salmonella Agar provides a highly selective and easy-to-read medium for the presumptive identification of Salmonella from food and environmental samples. The inhibitory compounds added in this medium specifically targets Escherichia coli, which can sometimes be present in high numbers in samples. Additional compounds are added to suppress the growth of other non-target organisms. An inhibitory compound is comprised of two components, combined together by a bond that can only cleave by a specific enzyme. When bound together, the inhibitor compound is not toxic and therefore can exist in a medium without harming microorganisms. Once inside the cell, the bond will be cleaved if the target enzyme is present. When the bond is cleaved, the inhibitor molecule is released and disrupts cell wall synthesis, causing death of the organism. As cells die and lyse, free inhibitor is released but cannot be taken up by other cells, resulting in targeted inhibition. The inhibitors in Chromogenic Salmonella Agar targets Escherichia coli. Novobiocin and Cefsulodin, presented as a freeze-dried supplement, are added to the medium to inhibit the growth of other competing flora such as Proteus spp. and Pseudomonas spp. Differentiation of Salmonella from other organisms that grow on Chromogenic Salmonella Agar is achieved through the inclusion of two chromogens that target specific enzymes: caprylate esterase and ß-glucosidase. The action of the enzymes on the chromogens results in a build-up of colour within the colony. The colour produced depends on which enzymes the organism possesses. The Salmonella rapid detection method combines the benefits of one Broth – Chromogenic Salmonella Agar and the Salmonella Latex Test to reduce time to result over conventional culture methods. The action of caprylate esterase, present in all salmonellae, results in a purple colony. Some other Enterobacteriaceae species also produce caprylate esterase, but these are either inhibited or differentiated from Salmonella by the ß-glucosidase substrate. This results in blue colonies, which are easy to distinguish from the purple Salmonella colonies.
Mycobacterium tuberculosis is very clinically important microbe and most likely to infect the laboratory workers through the air because the minimum number of organisms required for infection of M. tuberculosis is too low, only less than 10 bacilli. The organism is included in the Risk Group III of WHO classification of risk in 1983. Titan Biotech believes that protecting the laboratory workers against infection should be the first consideration in mycobacteriology. The recent increase in multidrug resistant tuberculosis made more critical to laboratory operation and urges the need of more safety measures. Most laboratory manipulation of clinical specimens and cultures of microorganisms create aerosols. Although it is impossible to prevent aerosol production and the resultant droplet nuclei, aerosol formation can be minimized and controlled. Protective clothing, autoclave sterilization, ultraviolet light, and disinfectant all contribute to protecting laboratory workers and others from infection in the laboratory. Culture isolation and enrichment of this bacterium is more sensitive than smear examination and need more safety measures. Traditionally mycobacteria are cultured both in Lowenstein Jensen (LJ) medium and in Middlebrook medium. We recognize that both of these media have advantages and disadvantages and complement each other. The chromogenically modified LJ medium is developed to tackle the disadvantages and for fast diagnosis. The chromogenic LJ medium is also used for drug susceptibility testing. To make the diagnosis rapid and instant we develop the HPLC analysis system which is highly specific in mycobacterial identification and also expects to replace most of the biochemical tests as a standard for speciation.
Food safety is a matter of serious concern in the modern world. Food borne illness is caused by harmful pathogens from the contaminated food products and can potentially affect the health of the millions of people worldwide. To ensure the food safety Titan Biotech develops a powerful pathogen detection method, a combination of streamlined sample preparation, sensitive and specific assays, specific instrumentation with unique installed software. These molecular tools for food safety testing aid in the rapid detection of harmful food pathogens and provide an accurate result within a short period of time. The newly developed Rapid Analyzer is a validated screening system for shiga toxigenic E. coli (STEC) and 6 other non-STEC E. coli strains from the meat products such as beef, pork, poultry and other processed foods within just 10 hours and with the lowest probability for the false results obtained. This is the most advanced and fastest method compare to the current methods supported by USDA Microbiology Laboratory Guidebook (MLG) for isolating single pathogens in food samples; which is also accurate and sensitive but the time to get the final result will take several days and also requires a lot of open tubes and wet reagents. But use of our technology enables accurate same-day detection of all pathogenic STECs and clearance of negative 375 g beef samples in as little as 10 hours. Our technology emphasis the simultaneous detection of E. coli (O157:H7) and the non-O157 E. coli (The big 6) in 375 g ground beef samples. The two-stage assay design allows clear negative samples in a single reaction and confirm presence of E. coli O157:H7 and big 6 in a second assay for screened positives. Both screening and confirmation assays can be done using the same single DNA sample preparation and only one volume transfer is required to set up the real-time PCR. The assays can also be done with multiplex PCR to provide high levels of accuracy.
Listeria is another major threat to the food industry. Listeria thrives all along the food processing chain. Contamination can occur in food-processing equipment, cooling units, freezer compartments, work surfaces, wet floors, damp walls, standing water, floor drains, mats, conveyor belts, and loading docks. Employee personal hygiene is also a factor, particularly in meat- and poultry processing environments. The most important fact is that Listeria resists heat, salt, nitrite, acidity, and temperatures as low as 1°C. Freezing temperatures (0°C) can slow Listeria growth, but may not stop it; allowing it to multiply in refrigerated foods during storage, shipping, and retail display. Listeria monocytogenes is one of six species of the genus Listeria and the only identified species to cause listeriosis, a disease with a high fatality rate in susceptible populations, including newborns, immunocompromised individuals, and the elderly, causing the highest mortality rate among food borne illnesses. Listeriosis is particularly dangerous for pregnant women, potentially causing miscarriage or stillbirth. An effective way to prevent Listeria monocytogenes contamination is to monitor Listeria at all stages of food production because the presence of Listeria sp. can be an early indicator of Listeria monocytogenes contamination. We developed a special Listeria Detection Kit which offers a fast and effective procedure for detecting Listeria sp. in foods and food-processing facilities. Our Listeria Detection Kit detects all species of Listeria in just over 24 hours, with high specificity and sensitivity in a variety of foods and food processing and storage surfaces, including meat products, seafood products, milk, stainless steel, plastic, ceramic, rubber, sealed concrete etc. The kit is based on real-time PCR. Real-time PCR is a proven method for pathogen detection and has been applied successfully to a wide range of food borne pathogens, including Salmonella, Listeria monocytogenes, and E. coli O157:H7. We have the kits for the detection of Salmonella and E. coli O157: H7 using the same technology.