科技革命与实验动物科学的发展

近代的3次科技革命促进了实验动物学由原始的、简单的动物实验向现代实验动物学的质的转变,最终使实验动物学形成一门独立的系统科学。科技革命可以看作是实验动物科学发展的源动力。中国实验动物科学必须适应科技发展的需要,不断地创新。这不仅是对科技发展的被动反应,更对科技发展具有促进作用。     

Method for the passaging of microcarrier cultures to a production scale for producing high titre Disabled Infectious Single Cycle-Herpes Simplex Virus Type-2

A complementary cell line CR2 is currently used to propagate the Disabled Infectious Single Cycle Herpes Simplex Virus Type 2 (DISC HSV-2) on a small laboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up our production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production times were factors which were taken into consideration whilst developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the small scale study for an envisaged manfacturing process.     

实验动物皮肤病原真菌DNA快速少量提取法

PCR技术应用于实验动物皮肤病原真菌检测,方法简单、省时。但是,真菌的DNA提取较为困难。本文推荐一种既简单又经济快速的提取皮肤真菌DNA的方法,并能成功用于实验动物皮肤病原真菌质量检测研究。     

Treatment of water for aquatic bacterial growth studies

The composition of a water is inextricably linked to its nutrient status and hence influences the behaviour of bacteria in artificial water systems. It has not yet been possible to devise a defined liquid medium representative of the complex composition of a treated water. Instead sterilized natural or distilled waters are used to study the growth or survival of aquatic bacteria in the laboratory. This has led to conflicting data and opposing opinions when the same water has proved toxic in some laboratory studies and growth-supporting in others. These differences may be explained by the variations in chemical compositions which occur when water is collected, transported and treated or stored in the laboratory. This study describes a simple, membrane filtration method of preparing a fresh sample of water collected from the environment or a building water system such that it is both sterile and chemically unaltered. The availability of such water may enhance understanding of the behaviour of bacteria in the aquatic environment.     

Treatment of water for aquatic bacterial growth studies

The composition of a water is inextricably linked to its nutrient status and hence influences the behaviour of bacteria in artificial water systems. It has not yet been possible to devise a defined liquid medium representative of the complex composition of a treated water. Instead sterilized natural or distilled waters are used to study the growth or survival of aquatic bacteria in the laboratory. This has led to conflicting data and opposing opinions when the same water has proved toxic in some laboratory studies and growth-supporting in others. These differences may be explained by the variations in chemical composition which occur when water is collected, transported and treated or stored in the laboratory. This study describes a simple, membrane filtration method of preparing a fresh sample of water collected from the environment or a building water system such that it is both sterile and chemically unaltered. The availability of such water may enhance understanding of the behaviour of bacteria in the aquatic environment.     

Laboratory-dependent bacterial ecology: a cautionary tale

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.     

Simple preparation method of PCR fragments for automated DNA sequencing

In an effort to find a simple and inexpensive purification method of polymerase chain reaction (PCR) reaction before cycle sequencing reaction, we compared a commercial system with a precipitation protocol performed in our laboratory. We found that, particularly with small PCR products, our method works with greater success than the method compared. Our precipitation method may be used on a larger PCR fragment before cycle sequencing reaction as well. Furthermore, it has the advantage of being simple as the well-known dilution method; in contrast to the dilution method, the precipitation method removes excess primers as well as possible primer dimers.     

An exercise on the determination of the primary structure of proteins

An exercise is described which illustrates how a simple laboratory investigation may be complemented with suitable related logical and numerical problems to increase its educational scope. The exercise extends the students' analysis of an unknown dipeptide to the larger problem of the primary structure of a polypeptide. As far as possible it reiterates techniques already used in the laboratory and involves photographs and facsimilies of actual data mounted on large display cards. Thus it is an integral part of the laboratory work, rather than being a mere appendix to it.     

A Cheap,Effective Gastric Cooling Apparatus for the Control of Gastric Hemorrhage

Gastric cooling (as opposed to freezing) is a useful adjunct in the management of upper gastrointestinal hemorrhage. Commercial machines for this purpose are expensive. A simple system is described in which cooled tap water is circulated through an extragastric reservoir, using an applicator modified slightly from the original design described by Wangensteen. The entire system can be made up in any hospital workshop at a cost of less than ten dollars. Tested in the laboratory and clinically, it has been found to be easy to set up, readily monitored, and safe. It has effectively controlled bleeding in seven of eight patients treated by this device.     

Laboratory-Dependent Bacterial Ecology: a Cautionary Tale

Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.     

An automated point-of-care system for immunodetection of staphylococcal enterotoxin B

An automated point-of-care (POC) immunodetection system for immunological detection of staphylococcal enterotoxin B (SEB) was designed, fabricated, and tested. The system combines several elements: (i) enzyme-linked immunosorbent assay–lab-on-a-chip (ELISA–LOC) with fluidics, (ii) a charge-coupled device (CCD) camera detector, (iii) pumps and valves for fluid delivery to the ELISA–LOC, (iv) a computer interface board, and (v) a computer for controlling the fluidics, logging, and data analysis of the CCD data. The ELISA–LOC integrates a simple microfluidic system into a miniature 96-well sample plate, allowing the user to carry out immunological assays without a laboratory. The analyte is measured in a sandwich ELISA assay format combined with a sensitive electrochemiluminescence (ECL) detection method. Using the POC system, SEB, a major foodborne toxin, was detected at concentrations as low as 0.1 ng/ml. This is similar to the reported sensitivity of conventional ELISA. The open platform with simple modular fluid delivery automation design described here is interchangeable between detection systems, and because of its versatility it can also be used to automate many other LOC systems, simplifying LOC development. This new POC system is useful for carrying out various immunological and other complex medical assays without a laboratory and can easily be adapted for high-throughput biological screening in remote and resource-poor areas.     

A simple flow meter for bioreactors applicable to oscillating feed systems

Summary A new simple electronic device for liquid flowrate measurement in laboratory or semipilot scale fermenters is presented. The flow meter is also applicable to pulsing or oscillating flow rates. This system allows either a direct measurement of the flow or implementation on a data acquisition system, if available.     

Development of a Soviet diagnostic kit for determining class M immunoglobulins to the hepatitis A virus by immunoenzyme analysis (a test system for anti-HAV IgM)

A new test system Diagn-A-Hep for the laboratory diagnosis of hepatitis A (HA) by means of the enzyme immunoassay has been developed at the Institute of Poliomyelitis and Viral Encephalitides (Moscow). The sensitivity and specificity of the newly developed test system have proved to be similar to those of the well-known commercial diagnostic system HAVAB manufactured by Abbott Laboratories (USA). Diagn-A-Hep permits the diagnosis of HA with 96-100% effectiveness both in patients with the acute form of the disease and in patients with its anicteric or inapparent forms. This system is simple and convenient, it may be employed in inadequately equipped laboratories or even under field conditions. The rules for the selection of immunobiological preparations to be included in the test system have been worked out.     

An endocrinology laboratory exercise demonstrating the effect of confinement stress on the immune system of mice

This article describes a simple laboratory exercise for examining the effect of stress on the immune system in mice. Mice are subjected to confinement stress for 1 h, after which a sample of blood is collected via the caudal vein. Blood samples are smeared onto microscope slides, air dried, and stained with Wright's Giemsa stain. When differential white blood cell counts are performed, there are noticeable differences between the neutrophil and lymphocyte counts of stressed versus control mice. The protocol is simple enough for students to perform, and the entire experiment can be completed within 3 h. Examples of ways in which the basic protocol can be modified to accommodate a shorter laboratory class are provided. This hands-on laboratory experiment provides students with experience using the scientific method to investigate the interaction between the endocrine and immune systems in response to stress.     

Not only students can express alcohol dehydrogenase: goldfish can too!

This article describes a novel approach to study the metabolic regulation of the respiratory system in vertebrates that suits physiology lessons for undergraduate students. It consists of an experimental demonstration of the goldfish's (Carassius auratus) adaptations to anoxia. The goldfish is one of the few vertebrates showing strong enzymatic plasticity for the expression of alcohol dehydrogenase (ADH), which allows it to survive long periods of severe anoxia. Therefore, we propose two simple laboratory exercises in which students are first asked to characterize the distribution of ADH isozymes in the goldfish by performing cellulose acetate electrophoresis. The second part of this laboratory lesson is the determination of liver glycogen. To further student comprehension, an interspecies comparative component is integrated, in which the same subjects are studied in an anoxia-sensitive species, the brook charr (Salvelinus fontinalis). ADH in goldfish is restricted to skeletal muscles, where it catalyzes alcoholic fermentation, permitting ethanol excretion through the gills and therefore preventing lactate acidosis caused by sustained glycolysis during anoxia. Electrophoresis also reveals the occurrence of a liver isozyme in the brook charr, which ADH catalyzes in the opposite pathway, allowing the usual ethanol degradation. As for the liver glycogen assay, it shows largely superior content in the goldfish liver compared with the brook charr, providing goldfish with a sustained energy supply during anoxia. The results of this laboratory exercise clearly demonstrate several physiological strategies developed by goldfish to cope with such a crucial environmental challenge as oxygen depletion.     

Handheld device for real-time, quantitative, LAMP-based detection of Salmonella enterica using assimilating probes

A simple handheld instrument was designed to enable real-time detection of the LAMP reaction in a standard PCR tube using newly described assimilating probes as sequence-specific reporter molecules. The system was validated using DNA isolated from Salmonella enterica, demonstrating accurate temperature control with little power and little overshoot of setpoint temperatures, with rapid and accurate detection often in less than 30 min and within 20 min for reactions with high (>10⁵) genome copy numbers. The system could be used for quantitative determination of pathogen DNA, with a limit of detection of about 15 genome copies in purified DNA or 25 cells in DNA extracts from chicken rinsate – comparable to values obtained when running the same reaction on a commercial benchtop real-time PCR instrument. Positive classification of standards nominally containing a single genome equivalent was demonstrated, and no false positives were reported. Detection of S. enterica in rinsate from a contaminated chicken sample required 48 h enrichment prior to the LAMP reaction or plating on semi-selective media. The new system demonstrates a major compelling advantage of the LAMP reaction, in that it may be enabled in simple, low-power, handheld devices without sophisticated custom miniaturized disposables. This new diagnostic system is especially promising for on-site diagnostics in the food and agricultural industries where laboratory space is often primitive if it is available at all.     

Safety Considerations for In Vitro Toxicology Testing

A range of hazards may be encountered in in vitro toxicology laboratory work. Such hazards are primarily chemical reagents for which the level of risk, particularly with long-term exposure, may be difficult to ascertain. Cells and tissues may also present biosafety concerns and it is important that risk assessments are prepared for each procedure before commencing project work. Ideally control measures should be simple and combine the use of appropriate equipment with documented procedures supported by risk assessments. In any laboratory staff and procedures change significantly over time, thus monitoring of procedures (including waste disposal) and a comprehensive training programme for all staff are essential components to assure both the quality and safety of laboratory work.     

Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term "half-seed" to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5-7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T1 generation by phenotypic assay using the herbicide Liberty (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method.