Microbiology

There are many different procedures that microbiologists use to study the effects of various antimicrobial agents in treating an infection caused by different microorganisms. Mueller Hinton Agar is considered as best for the routine susceptibility testing since it is has batch-to-batch reproducibility, low concentration of inhibitors of sulphonamide, trimethoprim and tetracyclines and produce satisfactory results for most of the non-fastidious pathogens. Fastidious organisms which require specific growth supplements need different media to grow for studying the susceptibility patterns.

The Kirby Bauer test is a qualitative assay whereby disks of filter paper are impregnated with a single concentration of different antibiotics or any chemicals that will diffuse from the disk into the agar. The selected antibiotic disks are placed on the surface of an agar plate which has already been inoculated with test bacteria. During the incubation period, the antibiotics/chemicals diffuse outward from the disks into the agar. This will create a concentration gradient in the agar which depends on the solubility of the chemical and its molecular size. The absence of growth of the organism around the antibiotic disks indicates that, the respected organism is susceptible to that antibiotic and the presence of growth around the antibiotic disk indicates the organism is resistant to that particular antibiotic. This area of no growth around the disk is known as a zone of inhibition, which is uniformly circular with a confluent lawn of growth in the media.

The diameters of the zone of inhibition are measured (including disk) using a metric scale or a sliding caliper. The measured zone diameter can be compared with a standard chart for obtaining the susceptible and resistant values. There are zone of intermediate resistance which means that the antibiotic may not be sufficient enough to eradicate the organism from the body.

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E.g. fermentation of various sugars (carbohydrates). Morphology and cultural characters may not be able to distinguish some species of bacteria; but these same species may exhibit distinct differences in their biochemical reactions e.g. typhoid and paratyphoid bacilli (glucose and mannitol are fermented without gas production by typhoid bacilli, whereas paratyphoid bacilli produce acid and gas).

Certain serotypes of the salmonella group may resemble one another in fermentation properties.

When working with microorganisms in a microbiology lab, sterility is often a high priority to ensure accuracy of test results and avoid cross-contamination of samples. Many of the disposable or consumable lab supplies shown in this index offer guaranteed sterility and are affordable enough to be disposed of when no longer in use. (Other disposable lab supplies which do not come sterile may be sterilized using specified methods.) Choose from single-use sterile petri dishes, cell culture plates, pipette tips, swabs and more.

The Pulse BactoBank is a system intended for the long-term storage of microorganisms at low temperature using ceramic beads.

The maintenance of quality control or clinical cultures for long term storage is a routine practice by hospitals. They usually employ home-made preservation systems.  However, the availability of commercial systems for the preservation of microorganisms has revolutionized this hospital practice and has reduced workload substantially.

BactoBank contains a plastic vial with ceramic beads suspended in a cryogenic fluid. Upon inoculation into the cryogenic fluid, the microorganisms will adhere to the surface of the bead. In this manner, the microorganisms will maintain long term survival when the coated beads are stored at refrigerated temperatures.

BactoBank is available in several colour beads to allow the laboratory to identify different categories of microorganisms.  The compact vial size allows the storage of a large quantity of cultures using a minimum space².  Each individual bead can be removed from the vial without thawing the entire vial sample.  The bead system permits the microorganism to be reactivated and be transferred onto a plated media or tube media for recovery. This method has been proven to be a simple, rapid, safe and reliable procedure to be routinely used to preserve quality control cultures or cultures for other applications for long term storage.

Vials: Each vial contains ceramic beads suspended in a cryogenic fluid.

Additional Items Required: Pure Culture to be preserved. Sterile loops. Sterile pipet tips. Sterile hooks or needles.  Freezer at -20 degree C or below.  Biohazard Waste Bag.

Advantages:

• Stackable box
• Box with position indicated for better arrangement
• Plastic box good for freezing
• Multi color and single color available.
• Various kit sizes (25, 50, 100 vials)
• Long shelf life
• Color coded beads matched with color coded caps
• Leak proof cap and vial

Maintains viability for a wide variety of Microorganisms, including (not limited to): N. gonorrhoeae, H. influenzae, B. vulgatus, C. albicans, A. brasiliensis.

Microbiology stains are used to detect and identify microorganisms that would otherwise be difficult to find in clinical specimens. Simple positively or negatively charged dyes create a fast discovery. The premixed solutions artificially color to help observe movement, structure, and characteristics of live or preserved specimens. Differentiating microorganisms based on properties, color combination procedures like the Gram stain or acid-fast techniques require microbiology stains.

The following points highlight the top five types of Staining. The types are: 1. Simple Staining 2. Differential Staining 3. Gram Staining 4. Acid Fast Staining 5. Endospore Staining.

Staining Type # 1. Simple Staining:

Colouration of microorganisms by applying single dye to a fixed smear is termed simple staining. One covers the fixed smear with stain for specific period, after which this solution is washed off with water and slide blotted dry. Basic dyes like crystal violet, methylene blue and carbolfuchsin are frequently used in simple staining to determine the size, shape and arrangement of prokaryotic cells.

Staining Type # 2. Differential Staining:

These staining procedures are used to distinguish organisms based on staining properties. They are slightly more elaborate than simple staining techniques that the cells may be exposed to more than one dye or stain, for instance use of Gram staining which divides bacteria into two classes-Gram negative and Gram positive.

Staining Type # 3. Gram Staining:

It is one of the most important and widely used differential staining techniques in microbiology. This technique was introduced in 1884 by Danish Physician Christian Gram. In the first step the smear is stained with basic dye crystal violet (Primary stain) followed by treatment with iodine solution functioning as mordant. Iodine increases the interaction between cell & dye so that cell stains strongly. The smear is next decolourized by washing with ethanol or acetone. This step generates the differential aspect of Gram stains. Gram positive bacteria retain crystal violet and become colourless. Finally smear is counter-stained with a simple basic dye different in color from Crystal violet. Safranin is the most common counter stain which colours Gram negative bacteria pink to red and leaves Gram positive bacteria dark purple. The differences in staining responses to the Gram stain can be related to chemical and physical difference of cell walls. The Gram-negative bacterial cell wall is thin, complex multilayered structure and contains relatively high lipid contents in addition to protein and mucopeptide. The higher amount of lipid is readily dissolved by alcohol, resulting information of large pore in the cell wall, thus facilitate leakage of crystal- violet – iodine (CV-I) complex which results in decolorization of the bacterial cell. Which later take counter stain and appears red. In contrast the cell wall of gram+ve bacteria is thick and chemically simple, composed mainly of mucopeptides. When treated with alcohol, it causes dehydration and closure of cell wall pore, thereby does not allow the loss of (CV-I) complex and cell remain purple.

Staining Type # 4. Acid Fast Staining:

It is another important differential staining procedure. It is most commonly used to identify Mycobacterium spp. These bacteria have cell wall with high lipid content such as mycolic acid -a group of branched chain hydroxy lipids, which prevent dyes from readily binding to cells. They can be stained by Ziehl-Nulsen method, which uses heat and phenol to derive basic fuchsin into the cells. Mycrobacterium spp. were penetrated with basic fuchsin, not easily decolourized by acidified alcohol (acid alcohol) and thus are said to be acid fast. Non arid fast bacteria are decolourized by arid alcohol and thus are stained blue by methylene blue counter stain.

Staining Type # 5. Endospore Staining:

Spore formation takes place in some bacterial genera to withstand unfavourable conditions. All bacteria cannot form spores, only few bacterial genera including Bacillus, Clostridium, Desulfotomaculum produce sporulating structure inside vegetative cells called endospore. Endospore morphology and location vary with species and are valuable for identification Endospores are not stained well by most dyes, but once stained, they strongly resist decolorization. In the Schaffer-Fulton procedure, endospores are first stained by heating bacteria with malachite green, which is very strong stain that can penetrate endospores. After malachite green treatment, the rest of the cell is washed free of dye with water and is counter-stained with safranin. This technique yields a green endospore with red vegetative cell.

Product range for Microbial Staining and Fixatives:

Quantitative assay for determining the Minimum Inhibitory Concentration (M.I.C.)

MIC Test Strip is a quantitative assay for determining the Minimum Inhibitory Concentration (M.I.C.) of antimicrobial agents against microorganisms and for detecting the resistance mechanisms. MIC Test Strip are porous strips with special features (International Patent) that are impregnated with a predefined concentration gradient of antibiotic, across 15 two-fold dilutions of a conventional M.I.C. method.

On one side of the strip is indicated a M.I.C. scale in μg/mL and a code that identify the antimicrobial agent. For ESBL, MBL, GRD, AmpC and KPC detection, the double-sided gradient carries the appropriate diagnostic reagents. MIC Test Strip are available in a large variety of configurations. Each configuration is available in packages of 10, 30 and 100 tests.

Method Principle
When the MIC Test Strip is applied onto an inoculated agar surface, the preformed exponential gradient of antimicrobial agent is immediately transferred to the agar matrix. After 18 hours incubation or longer, a symmetrical inhibition ellipse centered along the strip is formed. The MIC is read directly from the scale in terms of μg/mL at the point where the edge of the inhibition ellipse intersects the strip MIC Test Strip. Other growth/inhibition patterns may also be seen for resistance detection methods.

BIBLIOGRAPHY
• CLSI M100-S26 , 2016. Performance Standards for Antimicrobial Susceptibility Testing.
• CLSI M7-A10, 2015. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.
• CLSI M11-A8 Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria.
• CLSI M11-S1 Performance Standards for Antimicrobial Susceptibility Testing of Anaerobic Bacteria.
• CLSI M27-A3. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard – Third Edition.
• CLSI M27-S4. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Fourth Informational supplement.
• EUCAST. Breakpoint tables for interpretation of MICs and zone diameters Version 6.0 , January 2016.

The Liofilchem Quanti-CultiControl™ are lyophilized, quantitative microorganism preparations to be used in industrial laboratories for Quality Control purposes. Processed as directed, these preparations provide a challenge of <100 CFU per 0.1 mL, which is a concentration usually suitable for several pharmaceutical applications, including growth promotion testing of culture media to be employed in sterility testing. These microorganism preparations are derived from the American Type Culture Collection (ATCC®) or other authentic reference culture collection. The Quanti-CultiControl™ are suitable for the Growth Promotion Test recommended by the international Pharmacopoeiae, without need of intermediate dilution steps, since each rehydrated suspension generates less than 100 UFC for 0.1 mL inoculum. Each suspension (1 mL) allows 10 inocula.

A dip slide (or dipslide) is a test for the presence of microorganisms in liquids. The use of dip slides is the method most frequently used to measure and observe microbial activity in liquid-based systems. It is often used in testing cooling systems. Dip slides are often used to determine the presence of slime forming bacteria in cooling & industrial water systems. The Health and Safety Executive’s (HSE) recommends the use of dipslides to monitor the general activity of aerobic bacteria. The dip slide test consists of a sterile culture medium, on a plastic carrier that is dipped into the liquid to be sampled. The culture is then incubated, allowing for microbial growth. Most Dip slides consist of 1 – 2 agars attached to a flexible plastic paddle, this allows full contact of the agar onto the desired area for testing. Most Dipslides come in a circular clear shatterproof tube that can be inserted into a dip-slide incubator.

Most Dip slides are incubated at 30^oC for 48 hours after being dipped into the sample to ensure that the results are accurate. Some dipslides require different temperatures and incubation times depending on the type of agar used, and which organism is being tested for. After being dipped into the sample the dip slide is returned and secured in its original container for the incubation process. Multiple tests at varying periods are recommended to increase accuracy. It is important that biocides are not applied to the water prior to testing as this would significantly alter the results. The clarity of the sample is not necessarily reflective of the presence of bacteria as seemingly clear water may still have bacteria present. For some water systems a weekly dip slide test is recommended. For multiple tests the incubation period and temperature should be the same each time a new sample is assessed. Bacteria present in the sample liquid will grow and form colonies. A bacterial reference chart is used to determine the number of bacteria in the sample. Appropriate treatment is applied to the water source once abnormal levels of bacterial activity are noticed. Once water treatment is effective the bacterial count produced by the dip slide test should be low, approximately <10⁴. Dip slides are normally used when microbiological activity is relatively high (1,000 – 100,000 CFU per milliliter of water).

The dip slide results should be used only as a guide as the accuracy of the dip slide is limited as a result of the small sample size that is analyzed and the method used to obtain results. Nevertheless, dip slides may be very useful as they are very convenient, simple to use and cost effective.

A Biological Indicator (BI) is a well-characterized preparation of a specific microorganism with known resistance to aspesific sterilization process (USP Biological Indicators – Resistance Performance Tests).

BIs are regulated by: ISO 111-1:2017 Sterilization of health care product — Biological Indicators — Part 1: General requirements. USP pharmacopeia  Biological Indicator for sterilization.

A BI is ready for use microbiological test system, consisting of spores that are added to a carrier (suspension, paper strip, glass, plastic or other) packed to maintain the integrity and the viability of inoculated carrier.

Microorganisms widely recognized as suitable for biological indicator are spore-forming bactreia, more resistant than microflora:

– Geobacillus stearothermophillus (steam, hydrogen peroxide sterilization)

– Bacillus athropeus (dry heat, ethylene oxide sterilization)

– Bacillus pumilus (gamma irradiation sterilization).

If the spores grow after a sterilization cycle the process is considered to have failed. On the contrary the sterilization process turns out to be successful if spores are completely killed after the sterilization cycle.

Since 1983 Liofilchem® has committed its own Research & Development department in the production of high quality and reliable microbiology products. Our dedication to Customer satisfaction and exceptional manufacturing versatility have granted Liofilchem continuous growth, as a result of the increasing quality of our products and the worldwide distribution of the Liofilchem ® brand name. Liofilchem® currently has over 100 employees, including direct scientific and commercial representatives. The complete manufacturing processes and quality control are carried out in house by our staff at our two facilities in Roseto degli Abruzzi of total over 7000 m² area. The newest of them was opened in August 2010 and represents the state-of-the-art for the diagnostic manufacturing.

Liofilchem® produces a variety of chromogenic media of its own formulation, called Chromatic™, in continuous expansion. Available as both dehydrated format and ready to use in petri dish or bottle.

Contact plates 55 mm
Available in double or triple envelop, gamma or beta irradiated, packed either singularly intransparent peelable blister or in pile. Manufactured under the maximum hygiene control: A grade Clean Room.

Latex Agglutination Kits, these tests depend on the agglutination of antibody coated latex particles with antigens from corresponding microorganisms from various sources such as biological fluids from patients, liquid broth media etc. These Latex Agglutination tests are extremely rapid (typically less than 15 minutes) and simple to perform, can be read by eye and do not require expensive equipment. Moreover the reagents are stable in the refrigerator (2-8 C) for 13 months. These tests also have very high sensitivity and specificity, making them an excellent choice for a variety of broad applications such as rapid screening of multiple samples for infection, microbial contamination or microbial identification.

Chemical Indicator strips and challenge packs capture sterilization or disinfection failures through immediate results, while external indicator tapes and labels provide confirmation that a pack has been exposed to the sterilization process and helps distinguish between processed and unprocessed packs.

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A growth medium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.

The two major types of growth media are those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or fungi. The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (lysogeny broth). Liquid media are often mixed with agar and poured via a sterile media dispenser into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar (the exception being some species in the genera: Cytophaga, Flavobacterium, Bacillus, Pseudomonas, and Alcaligenes). Bacteria grown in liquid cultures often form colloidal suspensions.

The difference between growth media used for cell culture and those used for microbiological culture is that cells derived from whole organisms and grown in culture often cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo. In the case of animal cells, this difficulty is often addressed by the addition of blood serum or a synthetic serum replacement to the medium. In the case of microorganisms, no such limitations exist, as they are often unicellular organisms. One other major difference is that animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. In contrast, bacteria such as Escherichia coli may be grown on solid or in liquid media.

An important distinction between growth media types is that of defined versus undefined media. A defined medium will have known quantities of all ingredients. For microorganisms, they consist of providing trace elements and vitamins required by the microbe and especially defined carbon and nitrogen sources. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media.

A good example of a growth medium is the wort used to make beer. The wort contains all the nutrients required for yeast growth, and under anaerobic conditions, alcohol is produced. When the fermentation process is complete, the combination of medium and dormant microbes, now beer, is ready for consumption. The main types are

• Cultural media
• Minimal media
• Selective media
• Differential media
• Transport media
• Indicator media

Liofilchem is based in Roseto degli Abruzzi, Italy, where its products are manufactured. Since 1983 Liofilchem s.r.l. has committed its Research and Development department to the production of reliable and high quality

microbiology products. Our dedication to Customer Satisfaction and exceptional manufacturing Versatility have granted Liofilchem continuous growth, as a result of the increasing Quality of our products and the Worldwide Distribution of the Liofilchem brand name. The complete manufacturing processes and quality control are carried out in house by Liofilchem staff at our 7000 m²  factory.

The term was coined by Russian biologist Ilya Ilyich Mechnikov, who advanced studies on immunology and received the Nobel Prize for his work in 1908. He pinned small thorns into starfish larvae and noticed unusual cells surrounding the thorns. This was the active response of the body trying to maintain its integrity. It was Mechnikov who first observed the phenomenon of phagocytosis in which the body defends itself against a foreign body.

Prior to the designation of immunity from the etymological root immunis, which is Latin for “exempt”, early physicians characterized organs that would later be proven as essential components of the immune system. The important lymphoid organs of the immune system are the thymus,^[13] bone
marrow, and chief lymphatic tissues such as spleen, tonsils, lymph vessels, lymph nodes, adenoids, and liver. When health conditions worsen to emergency status, portions of immune system organs, including the thymus, spleen, bone marrow, lymph nodes, and other lymphatic tissues, can be surgically excised for examination while patients are still alive.

Many components of the immune system are typically cellular in nature and not associated with any specific organ, but rather are embedded or circulating in various tissues located throughout the body.

Endotoxins are small bacterially-derived hydrophobic lipopolysaccharides (LPS) molecules locating in the outer cell membrane of gram-negative bacteria. Endotoxins consist of a core polysaccharide chain, O-specific polysaccharide side chains (O-antigen) and a lipid compenent, Lipid A, which is responsible for the toxic effects. Bacteria shed endotoxin in large amounts upon cell death and when they are actively growing and dividing. A single Escherichia coli contains about 2 million LPS molecules per cell.

Endotoxin can easily contaminate labwares, and its presence can significantly impart both in vitro and in vivo experiments. And for parenteral products, parenteral products contaminated with endotoxins including LPS can lead to the development of fever, induction of inflammatory response, shock, organ failure and death in human. For dialysis products, LPS can be transferred through membrane with large pore size by back-filtration from the dialysis fluid to the blood, inflammatory problems may be caused accordingly.

Endotoxin is detected by the Lyophilized Amebocyte Lysate (LAL). Bioendo has been dedicated to researching, developing and producing LAL reagent for more than four decades. Our products cover all techniques employed to detect endotoxin, which are gel-clot technique, turbidimetric technique, and chromogenic technique.

Xiamen Bioendo Technology Co., Ltd., founded in 1978, is the expert in the field of endotoxins and Fungi (1,3)-β-D-glucan testing. Bioendo works on promoting application and development of lyophilized amebocyte lysate, and dedicates to researching, developing, producing, and marketing amebocyte lysate for four decades. Our products are registered at CFDA (China Food and Drug Administration) since 1988. We participate in preparing national standard amebocyte lysate and Reference Standard Endotoxin and standardizing Control Standard Endotoxin for the Government of China.

Cooperating with School of Life Science, Xiamen University, Xiamen Medical College, Xiamen Ocean Vocational College, and other universities, Bioendo is excellent at R&D. And supported by strong R&D strength and advanced equipment, Bioendo produces high-quality Lyophilized Amebocyte Lysate and endotoxins assay kits. Bioendo’s products mainly contain amebocyte lysate and endotoxins assay kits in gel clot technique, kinetic chromogenic, kinetic turbidimetric technique and end-point chromogenic technique, endotoxins assay kits for water and dialysate, endotoxin removal kits, clinical diagnostic assay kit for detection of fungi (1,3) β-D-glucan or invasive fungal infection, and supported items for endotoxins detection like control standard endotoxin, water for bacterial endotoxins test, endotoxin-free buffers, endotoxin challenge vials.

Bioendo also provides instruments such as Kinetic Incubating Microplate Reader, dry heat incubator, laboratory water purify system, centrifuge and other accessories.

Product range from Bioendo