Multichannel plating unit for high-throughput plating of cell cultures

High-throughput genomic approaches to gene function or target identification have led to the development and implementation of the 96-well format for many standard molecular biology manipulations. The apparatus described here, a Multichannel Plating Unit, is designed to plate out individual cultures efficientlyfrom standard 96-well culture blocks. Following transformation, aliquots of culture are loaded onto sterile beads that are rolled along individual channels of agar media. After the beads traverse the channel, they drop into the exit alley for disposal via an exit pore. The apparatus presented has 12 individual lanes, and the spacing is compatible with a standard 12-channel pipettor Thus, the unit allows for the rapid plating of 12 individual cultures at a time. For one 96-well block of transformants, this method reduces the labeling and plating effort from 96 culture dishes that are spread individually to eight multichannel plates. The savings in time, materials, and storage space is significant     

Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares

The endometria of 77 barren mares was swabbed simultaneously using a swab guarded with a single cannula and distal, gelatin capsule (completely guarded swab - CGS) and a partially guarded swab (PGS) with an open cannula. Sheep blood (5%) agar plates were inoculated with each swab, while MacConkey's agar plates were inoculated with the swabs from 44 mares. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. Organisms present were identified, counted, and categorized as saprophytic or pathogenic flora. The endometria of all mares were biopsied immediately following swabbing. Histologic evidence of inflammation in biopsy specimens was classified as (1) none, (2) slight, discrete, focal, and (3) slight or moderate, diffuse, widespread infiltration of inflammatory cells. The number of inflammatory cells migrating through the luminal epithelium was counted and averaged. There were significantly fewer CGS than PGS cultures that yielded growth at 24 and 48 hours of incubation after being streaked on blood agar and MacConkey's agar plates. There were fewer pathogenic bacterial or fungal colonies present at 48 hours of incubation on blood agar plates after being streaked with CGS as compared to PGS. There were no differences in the number of pathogenic bacterial or fungal colonies present at 24 hours of incubation on blood agar or at 24 and 48 hours of incubation on MacConkey's agar plates. There was no correlation between CGS or PGS culture of pathogens and severity of histologic inflammation. There was a positive correlation between culture of pathogens and number of inflammatory cells migrating through the luminal epithelium.     

Simplified 48-hour IMVic test: an agar plate method.

An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h.     

Simplified 48-hour IMVic test: an agar plate method.

An agar plate method was developed for the performance of the IMVic (indole, methyl red, Voges-Proskauer, and citrate) tests in lieu of the conventional tubed liquid media. By modifying the composition of the media and adding agar, a single "X"-compartmented petri dish was prepared containing all four IMVic test media. Ease of performance and simplification of the test were achieved by inoculating all four media simultaneously from a single colony (single inoculum) on eosin-methylene blue agar. Tests with 87 cultures, representing 7 genera in the family Enterobacteriaceae, were completed with typical (correct) IMVic patterns for all cultures within 48 h. Parallel tests with conventional media showed that the agar plate method was superior, more sensitive, faster, and simpler to perform, and less time was required to identify Escherichia coli by 72 h.     

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day ^1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.     

Comparison of two techniques for obtaining endometrial bacteriologic cultures in the mare

The endometria of 39 mares were cultured simultaneously using a swab guarded with a double cannula and distal, teflon plug and an unguarded swab with a single, open cannula. Sheep blood (5%) agar, Mac-Conkey's agar, and Sabourad's agar media were innoculated with each swab. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. There were significantly more plates that failed to yield bacterial or fungal growth when streaked with swab specimens obtained with the guarded cannula than when streaked with those obtained with the unguarded cannula. It was concluded that while culturing the endometrium of mares for bacteria or fungi, the use of a guarded instrument consisting of a double cannula with a closed end will result in the recovery of fewer contaminants; therefore, it will be more likely to result in a more accurate representation of uterine bacterial and fungal flora.     

Iodometric method for detection of beta-lactamase activity in yeast cells carrying ampicillin resistance gene in chimeric plasmids

In order to determine whether an ampicillin resistance gene in a chimeric plasmid is active in transformed yeast cells, it is necessary to have a simple and quick assay procedure. We describe here a procedure for achieving this goal using an iodometric color reaction. This method is based on the fact that the ampicillin resistance gene product, beta-lactamase, can hydrolyze penicillin G and release a reducing product, which can be visualized by the discoloration of a dark blue iodine-starch complex. We have improved this method so that the assay can be carried out on agar plate and in liquid culture. It permits the detection of the beta-lactamase enzyme activity in yeast liquid culture at a concentration as low as 1 X 10(5) cells/ml within 12 h. This method is especially useful for certain yeast transformation systems, such as industrial yeast cultures, where the transformants can be selected only by drug resistance.     

Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell

Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation. Temporal analysis of image intensified light was set so that a quadratic window covered a single cell. Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase. These responses on an agar plate were similar to those from liquid cultures. The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing. Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells. The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing. In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate.     

Colonyzer: automated quantification of micro-organism growth characteristics on solid agar

Background  

High-throughput screens comparing growth rates of arrays of distinct micro-organism cultures on solid agar are useful, rapid methods of quantifying genetic interactions. Growth rate is an informative phenotype which can be estimated by measuring cell densities at one or more times after inoculation. Precise estimates can be made by inoculating cultures onto agar and capturing cell density frequently by plate-scanning or photography, especially throughout the exponential growth phase, and summarising growth with a simple dynamic model (e.g. the logistic growth model). In order to parametrize such a model, a robust image analysis tool capable of capturing a wide range of cell densities from plate photographs is required.     

Automated agar plate streaker: a linear plater on Society for Biomolecular Sciences standard plates

Several protocols for bacterial isolation and techniques for aerobic plate counting rely on the use of a spiral plater to deposit concentration gradients of microbial suspensions onto a circular agar plate to isolate colony growth. The advantage of applying a gradient of concentrations across the agar surface is that the original microbiological sample can be applied at a single concentration rather than as multiple serial dilutions. The spiral plater gradually dilutes the sample across a compact area and therefore saves time preparing dilutions and multiple agar plates. Commercial spiral platers are not automated and require manual sample loading. Dispensing of the sample volume and rate of gradients are often very limited in range. Furthermore, the spiral sample application cannot be used with rectangular microplates. Another limitation of commercial spiral platers is that they are useful only for dilute, filtered suspensions and cannot plate suspensions of coarse organic particles therefore precluding the use of many kinds of microorganism-containing substrata. An automated agar plate spreader capable of processing 99 rectangular microplates in unattended mode is described. This novel instrument is capable of dispensing discrete volumes of sample in a linear pattern. It can be programmed to dispense a sample suspense at a uniform application rate or across a decreasing concentration gradient.     

A method for estimating viability of aquatic bacteria by slide culture

To estimate the viability of freshwater bacteria, slide cultures were prepared by spreading 10 μl of water, concentrated by centrifugation, over 1 cm² of agar. After drying, a cover-slip was placed on the agar. Following incubation, microcolonies and single cells were counted under a phase contrast microscope, and the viability estimated. Incubation at 10°C on casein-peptone-starch agar for 24 or 72 h provided the highest estimates of viability ( ca 45%). Most microcolonies developed under these conditions and the total number of microcolonies and single cells did not decrease significantly during incubation. Several morphological types of bacteria were observed as both microcolonies and single cells. Most microcolonies consisted of two cells, although some larger ones were present. The method could be used as an alternative to the spread plate and other methods which assess viability from bacterial growth on agar.     

The taxonomy of the anascosporous yeast-like fungi

Conclusion The species of yeast-like anascosporogenous fungi most frequently isolated from man were found to be the following: albicans, tropicalis, pseudotropicalis, Krusei, parakrusei, stellatoidea and Guilliermondi. A restudy of old cultures maintained in culture bureaus for long periods of time has allowed investigators to add other species to this list. These old cultures, representing single isolations, were thought to be partially dissociated because both rough and smooth colonies developed on blood agar plates at 37°C. when streaked from Sabouraud's glucose broth. When the smooth colony was picked for study, the culture could be identified as one of the most frequently isolated species.The rough colonies, however, showed certain differences which allowed them to be classified as distinct species. To avoid characterizing new species on the basis of dissociative changes, freshly isolated cultures should be identified immediately by some rigidly standardized technique giving constant results.A single genus, to be used internationally, has been proposed for these fungi which were obviously too closely related to be separated generically. Since Syringospora Quinquaud 1868 was the first validly published genus, it can be replaced by a more recently published genus only by establishing the latter as a nomen conservandum with appropriate emendation and the selection of the proper type species.1. Presented at the Third International Congress for Microbiology, Sept. 2–9, 1939, New-York, New-York.Aided by a grant from the John and Mary R. Markle Foundation.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the “gold” standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.     

The Examination of Foods for Enterobacteriaceae using a Test of the Type Generally Adopted for the Detection of Salmonellae

S ummary : In countries that have recently increased their bacteriological inspection of foods, many products have shown considerable improvement in microbiological quality; simultaneously, however, discrepancies between salmonella and coli-aerogenes tests, especially on dehydrated and frozen foods, have tended to become more frequent. These discrepancies have been eliminated by applying the following measures: incorporating glucose in culture media so as to reveal all Enterobacteriaceae; placing reliance on growth rather than gas formation so as to avoid missing anaerogenic organisms, and, especially, examining quantities of foods commensurate with those used in salmonella tests.
For this purpose a procedure is recommended in which 10 g of well homogenized food are enriched in 100 ml of buffered brilliant green-bile-glucose broth, with no attention being paid to gas formation; the enrichment cultures are then streaked on to MacConkey's glucose agar. Single colonies so obtained are tested for fermentative attack on glucose and may be further examined for other characteristics. The same enrichment fluid can be used for the so-called 'nonselective pre-enrichment' of samples of food containing salmonellae impaired by periods spent in conditions of low water activity, low pH, etc.     

Membrane-assisted culture of fungal mycelium on agar plates for RNA extraction and pharmacological analyses

Fungal mycelium grown in liquid culture is easy to harvest for RNA extraction and gene expression analyses, but liquid cultures often develop rather heterogeneously. In contrast, growth of fungal mycelium on agar plates is highly reproducible. However, this biological material cannot be harvested easily for downstream analyses. This article describes a PVDF (polyvinylidene difluoride) membrane-assisted agar plate culture method that enables the harvest of mycelium grown on agar plates. This culture method leads to a strongly reduced variation in gene expression between biological replicates and requires less growth space as compared with liquid cultures.     

Effect of incubation temperature on isolation of Campylobacter jejuni genotypes from foodstuffs enriched in Preston broth

Preston broth and agar incubated at either 37 or 42 degrees C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42 degrees C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37 degrees C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37 degrees C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42 degrees C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42 degrees C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Optical sensors for measuring dynamic changes of cytosolic metabolite levels in yeast

Optical sensors allow dynamic quantification of metabolite levels with subcellular resolution. Here we describe protocols for analyzing cytosolic glucose levels in yeast using genetically encoded F?rster resonance energy transfer (FRET) sensors. FRET glucose sensors with different glucose affinities (K(d)) covering the low nano- to mid- millimolar range can be targeted genetically to the cytosol or to subcellular compartments. The sensors detect the glucose-induced conformational change in the bacterial periplasmic glucose/galactose binding protein MglB using FRET between two fluorescent protein variants. Measurements can be performed with a single sensor or multiple sensors in parallel. In one approach, cytosolic glucose accumulation is measured in yeast cultures in a 96-well plate using a fluorimeter. Upon excitation of the cyan fluorescent protein (CFP), emission intensities of CFP and YFP (yellow fluorescent protein) are captured before and after glucose addition. FRET sensors provide temporally resolved quantitative data of glucose for the compartment of interest. In a second approach, reversible changes of cytosolic free glucose are measured in individual yeast cells trapped in a microfluidic platform, allowing perfusion of different solutions while FRET changes are monitored in a microscope setup. By using the microplate fluorimeter protocol, 96 cultures can be measured in less than 1 h; analysis of single cells of a single genotype can be completed in <2 h. FRET-based analysis has been performed with glucose, maltose, ATP and zinc sensors, and it can easily be adapted for high-throughput screening using a wide spectrum of sensors.     

Simple screening method for molds producing intracellular mycotoxins in pure cultures.

A simple screening method for molds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine C, sterigmatocystin, verruculogen, viomellein, and xanthomegnin was developed. After removing an agar plug from the mold culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within seconds and transferred directly to a thin-layer chromatography plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known thin-layer chromatographic procedures are carried out. The substrate (Czapek yeast autolysate agar) and growth conditions (25 degrees C for 7 days) used by Penicillium taxonomists proved suitable for the production of the mycotoxins investigated when 60 known toxigenic isolates and 865 cultures isolated from foods and feedstuffs were tested with this screening method.     

光周期对飞虱虫疠霉离体培养的生物量及其产孢节律的影响

飞虱虫疠霉（Ｐａｎｄｏｒａｄｅｌｐｈａｃｉｓ）是侵染飞虱和叶蝉等重要作物害虫的昆虫病原真菌。通过在液体及平板培养中研究光周期对菌丝及菌落生长量、产孢节律和产孢量的影响,作者发现光照对液培菌丝的营养生长无显著影响,但长光照液培菌丝的产孢延迟并且产孢量减少,而全黑暗液培菌丝产孢早且产孢量大。但是,光照明显促进全黑暗下液培菌丝的产孢。在平板培养中,长光照能促进菌落生长和产孢;半光照半黑暗虽有利菌落生长,但产孢量很低;光照９ｈ和１８ｈ产孢量较大,但菌落生长较小。无论液体还是平板培养,飞虱虫疠霉均生长良好,但建议在全黑暗条件下进行液体培养,在长光照下进行平板培养。