BACILLUS | Detection

L.A. Shelef , in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

PEMBA medium

Polymyxin–pyruvate–egg yolk–mannitol–bromothymol blue agar (PEMBA), developed by Holbrook and Anderson in the early 1980s, also relies on the mannitol-negative, lecithinase-positive properties of the organism. The medium contains pyruvate to reduce the tendency of B. cereus to form rhizoid colonies, improve the egg yolk reaction, and enhance sporulation. After 18–24   h of incubation at 37   °C, B. cereus strains form flat, crenate to rhizoid, turquoise–peacock blue colonies, 2–5   mm in diameter, with a 'ground-glass' surface appearance. After a further 24   h of incubation at ambient temperature, all colonies turn peacock blue. B. cereus is differentiated microscopically from other mannitol-negative species by microscopic examination of the stained cells for the presence of lipid granules and spores in the cytoplasm.

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Volume 1

Cletus P. Kurtzman , ... Vincent Robert , in The Yeasts (Fifth Edition), 2011

4.4.13 Canavanine-Glycine-Bromothymol Blue (CGB) Agar for Identifying Filobasidiella neoformans (Cryptococcus neoformans) and Sister Species

Filobasidiella neoformans can be distinguished from F. bacillispora within 2–5 days by incubating cultures on CGB agar at 25°C. Isolates of F. bacillispora hydrolyze glycine and are resistant to l -canavanine. The pH of the medium rises and the color of the bromothymol blue indicator turns to blue when glycine is hydrolyzed. Most isolates of F. neoformans do not hydrolyze glycine, and the few that do are sensitive to the canavanine, so the medium remains greenish-yellow in color (Kwon-Chung et al. 1982b, Min and Kwon-Chung 1986).

A loopful of cells from a young culture is streaked onto CGB agar in either a tube or a petri plate and incubated at 25°C for up to 5 days. The color of the medium does not change if the strain is F. neoformans (Cr. neoformans), but turns blue if the strain is F. bacillispora (Cr. gattii).

Canavanine-glycine-bromothymol blue (CGB) agar. The complete medium is prepared by cooling 900   ml of bromothymol agar (below) to about 55°C and adding 100   ml of stock solution A (below).

Stock solution A. Dissolve 300   mg l-canavanine sulfate, 100   g glycine, 10   g potassium dihydrogen phosphate, 10   g of magnesium sulfate heptahydrate, and either 10 drops of Bejectal with vitamin C (Abbott Laboratories, Chicago, IL, USA) or 10   mg thiamine hydrochloride in 1 liter of demineralized water, adjust the pH to 5.6 and filter sterilize.

Bromothymol blue solution. Dissolve 0.4   g sodium bromothymol blue in 100   ml of demineralized water and filter sterilize.

Bromothymol blue agar. Dissolve 20   g agar in 880   ml of demineralized water and add 20   ml bromothymol blue solution. Sterilize by autoclaving at 121°C.

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BACILLUS | Detection by Classical Cultural Techniques

I. Jenson , in Encyclopedia of Food Microbiology (Second Edition), 2014

Holbrook and Anderson Stain

Smears may be produced from the center of a 24   h colony or the edge of a 48   h colony growing on PEMBA. Smears are air dried and fixed with minimal heating. Stain with malachite green over a boiling waterbath for 2   min. After washing the slide and blotting it dry, stain with Sudan black for 15   min. Then rinse the slide in xylol for 5   s and blot dry before staining with safranin for 20   s. Bacillus cereus will appear 4–5   μm long and 1.0–1.5   μm wide with square ends. Lipid globules, staining black, are present in vegetative cells. Spores, staining green, are ellipsoidal, central to subterminal in position, and do not swell the sporangium.

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VIBRIO | Standard Cultural Methods and Molecular Detection Techniques in Foods

Kasthuri Venkateswaran , in Encyclopedia of Food Microbiology, 1999

Fluorogenic Assay

In general, the detection of V. parahaemolyticus in foods involved a pre-enrichment step (APW or polymyxin broth) followed by streaking onto a selective agar (TCBS or BTB–teepol agars). Unfortunately, a number of other Vibrio species mimic V. parahaemolyticus, necessitating the use of additional biochemical tests for reliable identification. Hence, a more rapid and sensitive detection assay is warranted. A procedure that involves 6-h cultivation of cells in a specific medium (AG medium) followed by measuring the intracellular trypsin-like activity of V. parahaemolyticus is described (Fig. 3). Measurement of fluorescence intensity of the culture supernatant solution can be made with a fluorospectrometer. The excitation wavelength is 360   nm, and the fluorescence intensity is measured at 450   nm. Media, buffer and substrate controls should be included in the assay and fluorescence intensity is measured. One unit of trypsin-like activity is defined as the fluorescence intensity that is excited by 1   μg of trypsin (bovine pancreas origin). When the fluorescence intensity of the test sample is more than 1 trypsin unit, trypsin-like activity is recorded as positive for that sample. A typical negative control will range from 0 to 0.5; and a positive control would exceed more than 4–6 trypsin units.

Figure 3. Fluorogenic assay in the identification of V. parahaemolyticus.

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Analytical Profiles of Drug Substances and Excipients

Alison E. Bretnall , Graham S. Clarke , in Analytical Profiles of Drug Substances and Excipients, 1998

6.6.1 Spectrophotometric Analysis

A UV method for the determination of metformin in urine samples has been described [33 ]. Metformin was quantitatively extracted as an ion-pair, based upon the complexation of the compound with bromothymol blue. Once extracted, the metformin was released from its ion-pair by the addition of tetrabutyl-ammonium hydroxide, and subsequently quantitated by its UV absorbance at 232 nm. This method was adapted to determine metformin in whole blood and plasma, by precipitating out the protein present before performing the metformin ion-pair extraction and subsequent quantitation by UV [ 34].

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Profiles of Drug Substances, Excipients, and Related Methodology

Haitham Alrabiah , in Profiles of Drug Substances, Excipients and Related Methodology, 2019

5.2.1 Spectrophotometry

Srinivasu et al. [15] developed three simple, economical, and reliable spectrophotometric methods for determining levetiracetam. These methods were dependent on the formation of yellow complexes with bromocresol green, bromophenol blue, and bromothymol blue in these three assays, which are detectable at 435, 454, and 415  nm, respectively. The calibration curves for the three assays demonstrated linearity over similar ranges of levetiracetam concentration, 2.5–25, 2.5–25, and 1.5–15   μg/mL for the three assays, respectively. These methods were optimized and applied successfully for the analysis of levetiracetam in solution and in tablet dosage form.

Similarly, Hadad et al. [16] used a combination of flow injection analysis method with spectrophotometry for the analysis of levetiracetam in tablet dosage form based on reaction of the drug with ortho-phthalaldehyde and 2-mercaptoethanol at basic pH. Detection of the compound under flow conditions was carried out at 295   nm and the assay was linear over the range 5–35   μg/mL. Quarter-fraction factorial design was employed to optimize experimental parameters, including volume, pH, ionic strength, concentrations, and flow rate. Thanuja et al. [17] also developed a spectrophotometric method for levetiracetam based on the formation of drug complex with oxidized 2,4-dinitrophenylhydrazine. Detection was carried out at 455   nm and linearity was demonstrated over a wide concentration range (30–130   μg/mL). Recovery studies were used to validate the developed method.

Muralikrishna et al. [18] developed two simple spectrophotometric methods for the quantification of levetiracetam in formulations based reaction of the drug with 2-chlorophenylhydrazine or anthranilic acid. Detection was carried out at 560 and 485   nm, respectively, and the method was highly sensitive, demonstrating linearity over the concentration range 2.5–130   μg/mL. Recovery studies were used to validate the developed method, which were successfully utilized for the determination of levetiracetam in injection powder formulation. Bhaskararao et al. [19] described a robust spectrophotometric method for quantitative analysis of levetiracetam based on reaction of levetiracetam with chloranilic acid, with linearity over the range 1000–5000   μg/mL, high precision and reproducibility, which was applied to measuring levetiracetam in tablet dosage forms.

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Composition of the Body Fluid of Drosophila and the Design of Culture Media for Drosophila Cells

Guy Echalier , in Drosophila Cells in Culture (Second Edition), 2018

B pH

In spite of notable differences, insect body fluids tend to be slightly acidic. The hydrogen ion concentration in freshly drawn hemolymph from D. melanogaster third instar larvae (see Table 1.2 ) was estimated, using pH indicator dye bromothymol blue, to be between 6.6 and 6.7 pH units.

Table 1.2. pH, Drophila Third Larva Hemolymph

Developmental Stage Measurement Method Mean Values Refs.
Larvae Colorimetric indicators 7.1 Boche and Buck (1942)
Microelectrodes
  Glass electrode 7.1
  Quinhydrone 7.12
Mature larvae Microelectrodes Boche and Buck (1942)
  Glass electrode 7.1
  Quinhydrone 7.12
Last instar larvae Colorimetric method (bromothymol blue) 6.6–6.7 Begg and Cruickshank (1963)

Begg, M., Cruickshank, W. J. 1963. A partial analysis of Drosophila larval hæmolymph. Proc. R. Soc. Edinb., 68, 215–236.

Boche, R. D., Buck, J. B. 1942. Studies on the hydrogen-ion concentration of insect blood and their bearing on in vitro cytological technique. Physiol. Zool., 15, 293–303.

The highest figures reported by Boche and Buck (1942) might be explained by a loss of CO2 during measurement with microelectrodes, whereas in the colorimetric method, the capillary tubes used to collect hemolymph can be immediately sealed.

According to various observations from other insects, the buffering capacity of the hemolymph might be the sum of several overlapping systems of varied importance: bicarbonates, inorganic, and organic phosphates, but also and perhaps predominantly, amino acids, with their carboxylic and amino groups, and proteins. However, several authors pointed out that dissociation constants of amino acids are so different from the normal pH of the hemolymph that they are unlikely to have any appreciable buffering effect.

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Citrate

Stanley Dagley , in Methods of Enzymatic Analysis, 1965

Method: Dilute

4 ml. serum (or other biological fluid containing about 100 μg. citric acid)

to 20 ml. with distilled water, stir for 5 min. with

1 g. Amberlite CG-120

and then filter off the resin. Add to 15 ml. filtrate,

3 ml. metaphosphoric acid solution (II)

with stirring and filter after 5 min. To four graduated tubes add

3 ml. filtrate

2 small glass beads

0.1 ml. H2SO4 (solution III).

Heat the tubes in a 115–120° C oil bath and evaporate the solutions to about 1 ml. Cool to room temperature, neutralize (pH 7.2–7.6) *) each solution with ca.

0.75 ml. NaOH (solution V)

and make up to 3 ml. with distilled water. Pipette into test tubes:

2 ml. neutralized sample

0.2 ml. magnesium sulphate solution (VI)

0.55 ml. distilled water.

Mix and equilibrate at 30° C. 1.75 ml. of the mixture is analysed.

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Taxonomy of Prokaryotes

Noel R. Krieg , Penelope J. Padgett , in Methods in Microbiology, 2011

1 O/F test

•

(Method of Hugh and Leifson, 1953) Use two tubes of a semisolid medium containing the carbohydrate to be tested. The sugar is usually glucose, but some organisms may use other sugars; e.g. Novispirillum itersonii uses fructose but not glucose (Hylemon et al., 1974). The medium of Hugh and Leifson (1953) is usually used and contains the following (per litre of distilled water): peptone (pancreatic digest of casein), 2.0   g; NaCl, 5.0   g; K2HPO4, 0.3 g; bromothymol blue, 0.03  g; and agar 3.0   g; pH 7.1. Boil to dissolve the agar, dispense 4-ml portions into 13   mm×100   mm tubes and sterilize by autoclaving, Cool to 45°C, then add sufficient 10% carbohydrate solution (sterilized by filtration) to give a final concentration of 1   g/l and mix.

•

Heat the tubes in a boiling water bath for 10   min to expel excess oxygen. After the tubes have cooled, inoculate them vertically with a straight wire. Dispense sterile molten petrolatum into one tube as a seal against oxygen (this is the 'anaerobic' tube).

•

After incubation, examine the tubes daily for growth and for a colour change. If growth and an acidic change (yellowing) occur only at or near the surface of the aerobic tube, the organism can oxidize the carbohydrate (an 'O' reaction). If the anaerobic tube shows growth and is acidified, the organism can ferment the carbohydrate (an 'F' reaction). If neither tube becomes acidified but growth occurs, the organism cannot catabolize the carbohydrate. If no growth occurs in either tube, the medium may be lacking some required nutrient; try supplementing it with 0.1% yeast extract.

If bromothymol blue is inhibitory or not sensitive enough to detect small pH changes, substitute with phenol red (0.01   g/l). For marine organisms, use the modified medium of Leifson (1963), which contains the following (per litre): pancreatic digest of casein, 1.0   g; yeast extract, 0.1   g; (NH4)2SO4, 0.5   g; Tris buffer, 0.5   g; phenol red, 0.01   g; agar, 3.0   g; and distilled water, 500   ml. Adjust the pH to 7.5, sterilize by autoclaving and cool. Separately autoclave 500   ml of artificial seawater, cool and combine with the first solution. An artificial salt mixture closely resembling the composition of the dissolved salts of ocean water can be obtained commercially (Sigma-Aldrich, Cat. no. S9883).

•

Sometimes acid production can be masked by formation of alkaline substances from the peptone; in these instances, the peptone-deficient medium of Board and Holding (1960) may be satisfactory: (NH4)H2PO4, 0.5   g; K2HPO4, 0.5   g; yeast extract, 0.5   g; bromothymol blue, 0.03   g; mineral salts solution, 20   ml; agar, 5.0   g; and distilled water, 880   ml. Dissolve the phosphates and yeast extract in the water, add the bromothymol blue and a mineral salts solution. After adjusting to pH 7.2, add the agar and boil to dissolve it. Dispense 9-ml portions into tubes, autoclave for 1–2   min at 22   lb/in2 and cool to 45°C. Add 1.0   ml of a 5% filter-sterilized solution of glucose (or other desired sugar) solution, mix and allow to cool.

Prepare the mineral salts solution from two solutions, A and B. To prepare Solution A, dissolve 10.0   g of nitrilotriacetic acid in 950   ml of distilled water; then add MgSO4 (anhydrous), 14.45   g; CaCl2·2H2O, 3.335; (NH4)6Mo7O24·4H2O, 0.00925   g; and FeSO4·7H2O, 0.099   g. Add 50   ml of Solution B and adjust the pH to 6.8 with ca. 7.3   g of KOH. Solution B contains the following (per litre of distilled water): EDTA, 2.5   g; ZnSO4·7H2O, 10.95   g; FeSO4·7H2O, 5.0   g; MnSO4·H2O, 1.54   g; CuSO4·5H2O, 0.392   g; Co(NO3)2·6H2O, 0.248   g; Na2B4O7·10H2O, 0.177   g. Add a few drops of sulfuric acid to Solution B to decrease precipitation.

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Opportunistic Mycoses

Jennifer L. Horan-Saullo MD, PharmD , Barbara D. Alexander MD, MHS , in Murray and Nadel's Textbook of Respiratory Medicine (Sixth Edition), 2016

Diagnosis

The diagnosis of pulmonary cryptococcosis is based on symptoms, chest radiography, culture, and/or histopathologic findings (Fig. 38-1) and cryptococcal antigen testing. Cryptococcus can be cultured from respiratory specimens including sputum and bronchoalveolar lavage (BAL); blood cultures are only positive in disseminated infections. Cryptococcus is easily identified under the microscope as 5 to 10 µm spherical to oval yeast cells with a surrounding capsule. Biochemical testing is used to confirm the identification; canavanine-glycine-bromothymol blue agar can be used for differentiating C. gattii from C. neoformans. 37 New methods are under development for rapid identification and speciation of cryptococci, including polymerase chain reaction (PCR) 38 and matrix-assisted laser desorption/ionization time of flight spectrometry. 39

In tissue samples, specialized stains such as Mayer's mucicarmine, which stains fungal melanin, are also helpful in establishing a diagnosis. 37 While the serum cryptococcal antigen assay has a high sensitivity and specificity in disseminated infection and cryptococcal meningoencephalitis, it can be negative in patients with isolated pulmonary infection. In a study of patients with SOT, serum cryptococcal antigen was detectable in 73% of patients (22 of 30) with isolated pulmonary involvement and was more likely to be negative with solitary pulmonary nodules than with multiple nodules and more extensive pulmonary disease. Titers of serum cryptococcal antigen were higher in those patients with concurrent extrapulmonary infection. 40

Determining the presence of disseminated infection and extent of organ involvement is crucial to the selection of appropriate therapy. Thus, cerebrospinal fluid evaluation (e.g., cell count, culture, and cryptococcal antigen) should be performed in all immunosuppressed patients with pulmonary cryptococcosis. Whether cerebrospinal fluid analysis is essential in immunocompetent patients with pulmonary cryptococcosis is less clear. Factors associated with higher likelihood of disseminated disease and the need for cerebrospinal fluid analysis include neurologic findings, signs of systemic infection, such as fever and weight loss, and serum cryptococcal Ag titer of at least 64. 41

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