Agar plate

About: Agar plate is a research topic. Over the lifetime, 4419 publications have been published within this topic receiving 95488 citations.

A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine

Abstract: Screening for cellulase-producing microorganisms is routinely done on carboxymethylcellulose (CMC) plates. The culture plates are flooded either with 1% hexadecyltrimethyl ammonium bromide or with 0.1% Congo red followed by 1 M NaCl. In both cases, it takes a minimum of 30 to 40 minutes to obtain the zone of hydrolysis after flooding, and the hydrolyzed area is not sharply discernible. An improved method is reported herein for the detection of extracellular cellulase production by microorganisms by way of plate assay. In this method, CMC plates were flooded with Gram’s iodine instead of the reagents just mentioned. Gram’s iodine formed a bluish-black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes. The new method is rapid and efficient; therefore, it can be easily performed for screening large numbers of microbial cultures of both bacteria and fungi. This is the first report on the use of Gram’s iodine for the detection of cellulase production by microorganisms using plate assay.

Selective and differential medium for isolation of Clostridium difficile.

Abstract: Clostridium difficile is a recognized cause of pseudomembranous (antimicrobial agent-associated) colitis and may be one of the causes of antimicrobial agent-induced diarrhea. A selective and differential agar medium that contains cycloserine, cefoxitin, fructose, and egg yolk (CCFA) was developed to facilitate the isolation of C. difficile from fecal specimens. Quantitative cultures of 16 stock strains of C. difficile on this medium (and on a medium containing cycloserine, fructose, and egg yolk) yielded counts equivalent to those obtained on blood agar; other media selective for clostridia, including Clostrisel agar, reinforced clostridial agar plus 0.2% para-cresol, and egg yolk-neomycin agar (the latter was inoculated with cultures subjected to prior heat shocking), were also tested and found to be inhibitory to the growth of C. difficile. Of 28 fecal or colostomy effluent specimens cultured on the above media, 14 yielded C. difficile. CCFA was found to be the most sensitive and selective of these media for the recovery of C. difficile. Colonies of C. difficile growing on CCFA had distinctive morphological and fluorescent properties which were sufficient for presumptive identification. CCFA should provide a rapid method for the screening of fecal specimens from patients with antimicrobial agent-associated diarrhea or colitis for C. difficile.

Simplified agar plate method for quantifying viable bacteria

Abstract: We have developed a simple and reproducible technique that we have termed “track dilution” for enumerating viable bacteria, which reduces time and materials required compared to standard agar plate colony counting methods. Many microbiological applications require an accurate determination of bacterial numbers prior to inoculation into, or subsequent to their isolation from, experimental models and systems. For example, our experimental infection models require injection of predetermined numbers of viable colony-forming units (cfu) (1,4). The model further requires careful monitoring of bacterial growth rates at specific time points during infection. In our models of rodent gastrointestinal ecology (3), bacterial concentration must be determined from extraordinarily large numbers of fecal samples. The most commonly used technique for enumerating bacterial cfu involves serial dilution of samples followed by spreading 50–100 μL of each dilution onto agar media. Subsequent to incubation, colonies are counted and bacterial concentrations in the original sample estimated. This technique, though relatively accurate, has some drawbacks, including time required and a considerable expenditure of disposable plastics and media. To meet criteria for statistical accuracy of bacterial numbers in a given specimen samples are usually plated in triplicate, and cfu counted only from plates yielding between 20 and 200 visible colonies (2). Furthermore, since the actual number of bacteria present in a specimen at the completion of an experiment is unknown, samples must frequently be plated from dilutions ranging from 10-1 to 10-7. The result of such exhaustive measures is that most plated samples will yield either too many colonies to count or no colonies at all. For example, an overnight bacterial broth culture containing 109 cfu/mL, serially diluted 10fold over a range of 10-1 to 10-7 and plated (100 μL per plate) in triplicate, will yield only three agar plates with countable and statistically valid numbers of cfu; the remaining 18 plates must be discarded. To simplify bacterial enumeration and reduce time and material expenditures, we have developed a track-dilution technique whereby six 10-fold serial dilutions of a sample containing an unknown quantity of bacteria are plated onto a single agar plate. We compared track dilution with the standard spreadplate method by: (i) recording time required to process samples, (ii) recording materials expended and (iii) statistically analyzing the data for accuracy and reproducibility. Enterococcus faecalis and Staphylococcus aureus were propagated in brain-heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) overnight at 37°C without aeration. Bacillus subtilis and Escherichia coli DH5α (Life Technologies, Gaithersburg, MD, USA) were propagated in LB overnight at 37°C with aeration. Cultures were serially diluted 10-fold by sequential transfer of 100 μL into 900 μL phosphate-buffered saline (PBS). Experiments were performed in triplicate to allow statistical comparisons between the two techniques. For conventional spread-plate counting, cultures were diluted by a factor of 10-7, from which 100-μL samples were spread onto the surface of prewarmed BHI 1.5% agar plates (100× 15-mm round plates; Fisher Scientific, Pittsburgh, PA, USA) using a sterile bent-glass rod and an inoculating turntable (Fisher Scientific). Plates were incubated upright for ap-

Plating of isolated tobacco mesophyll protoplasts on agar medium

Abstract: A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.