Cultural, Physiological, and Biochemical Identification of Actinobacteria

The traditional phenotypic tests are commonly used in actinobacterial identifica‐ tion. They constitute the basis for the formal description of taxa, from species and subspecies up to genus and family. The classical phenotypic characteristics of acti‐ nobacteria comprise morphological, physiological, and biochemical features. The morphology of actinobacteria includes both cellular and colonial characters. The physiological and biochemical features include data on growth at different tempera‐ tures, pH values, salt concentrations, or atmospheric conditions, and data on growth in the presence of various substances such as antimicrobial agents, the pres‐ ence or activity of various enzymes, and with respect to metabolization of com‐ pounds. The phenotype is the observable expression of the genotype. Gene expression is directly related to the environmental conditions. Actinobacterial phe‐ notype cannot be based on the simple observation of the organism. Strains of the most closely related taxa should be compared in their phenotypic analysis using identical methods. The comparisons must include the type strain of the type species of the appropriate genera. Furthermore, with the development of technology, mi‐ crobial physiological and biochemical identification technology is becoming fast, simple, and automated. range of pH as well as the optimal pH for growth. Measure growth responses from a standar‐


Introduction
The polyphasic approach [1], the comprehensive results of various methods, such as morphological, physiological, rRNA gene sequencing, chemotaxonomic markers and pathogenicity, are used to compile a description of a new species. Classification of actinobacteria is now based largely on analysis of the nucleotide sequences, especially 16S rRNA genes [2]. However, a comprehensive characterization of a new species should still be identified, because from even a complete genomic sequence, it would be difficult to predict many of the phenotypic features of a new species [3]. Physiological and biochemical characteristics are directly related to the activity of microbial enzymes and regulatory proteins. Enzymes and proteins are gene products; so the comparison on physiological and biochemical characteristics of actinobacteria is the indirect comparison of genome, and determination of physiological and biochemical characteristics is much easier than direct analysis of the genome. Therefore, the physiological and biochemical characteristics in actinobacterial systematics and identification are still meaningful [4]. In addition to a thorough phenotypic characterization of a new species, it is important to determine which phenotypic features are the ones most useful for identifying new species. Moreover, the particular methods used for characterizing an organism should always be stated, because the results of phenotypic tests can vary with methodology.
Phenotypic analysis is a very tedious task in the classification of actinobacteria. The classical phenotypic characteristics of actinobacteria comprise morphological, physiological, and biochemical features. Individually sufficient as parameters for genetic relatedness, yet as a whole, they provide descriptive information enabling us to recognize taxa [5]. The morphological traits include both cellular (cell shape and size, spore, sporangia, sporangiospore, the location of the spores or sporangia and their size, flagella, motility, intracellular structures, etc.) and colonial characters (shape and size, color, dimensions, form, etc.). The physiological and biochemical traits include data on growth at different temperatures, pH values, salt concentrations, atmospheric conditions (aerobic/anaerobic), growth in the presence of various substances such as antimicrobial agents, and data on the presence or activity of various enzymes, metabolization of compounds, and so on (Table 1). Physiological and biochemical tests should be carried out in test media and under conditions that are identically standard or at least comparable. There are three major inter-related areas in actinobacterial identification: taxonomic relevance; methodological reliability and cost effectiveness; and data portability [6]. It must be noted that novel taxa should be described based on the characteristics of more than one related strain and the type strain of the type species of the appropriate genera [7]. It should be emphasized that some phenotypes are encoded by extrachromosomal inheritance factors and the influencing factors that affect the expression of physiological and biochemical traits are complicated. To determine the genetic relationships based on physiological and biochemical characteristics,systematic classification, must be integrated with other characteristics, particularly genotype characteristics analysis.
Identification of a species is a constant basic work in any microbiology laboratory. Regardless of the type of microorganisms, the working steps are inseparable from the following three items: (i) to obtain the pure cultures of microorganism, (ii) to determinate the necessary appraisal indicators, (iii) to find authoritative identification manuals and publications, and related site information. Different organisms often have their own different identification priorities. For example, the identification of microorganisms with rich morphological characters, such as fungi, often bases on their morphological features as the main indicators; the identification of actinobacteria and yeasts, synthesizes the morphological, physiological and biochemical characteristics; the identification of bacteria lacking morphological difference, often uses more physiological, biochemical, and genetic parameters.

Characteristics
The difference between groups Adaptability of temperature The optimal, the lowest, and highest growth temperature and die

Cultural characteristics of actinobacteria
Cultural characteristics of actinobacteria refer to the growth characteristics and morphology in various kinds of culture media. Pure culture should be taken before morphological observation. The pure culture of actinobacteria can be obtained through the use of spread plates, streak plates, or pour plates and are required for the careful study of an individual microbial species [ Figure 1]. Cultural characteristics on 4 to 6 media are usually determined after incubation 14 to 28 days at 28°C strictly according to the methods used in the International Streptomyces Project (ISP) [8]. Sometimes, other media can be chosen, such as nutrient agar and czapek's agar. The colors of substrate and aerial mycelia and any soluble pigments produced were determined by comparison with chips from the ISCC-NBS color charts [9].

Physiological and biochemical characteristics for identification of actinobacteria
Some phenotypic characteristics of actinobacteria are of such primary importance to a genus or species description. Several problems must be considered when planning the physiological and biochemical tests of actinobacteria. One of them, according to the phylogenic information based on 16S rRNA analyses, strains of the most closely related taxa and the type strain of the type species of the appropriate genera should be chosen for comparison in their phenotypic traits. Other problems are concerned with methodology. In classifying actinobacteria, it is desirable to use an established approach based on common sense, and to use tests that are pertinent. If novel methods are used, the researcher must provide evidence that the new methods produce comparable results to established methods. Furthermore, phenotypic characteristics of actinobacteria are influenced by cultural conditions and other factors, so tests should be performed in duplicate or triplicate. More importantly, design reasonable positive and negative controls in the experiments.

Temperature range and optima for growth
Incubate cultures at a range of temperatures; using constant temperature incubators or water baths, measure the growth response of the actinobacteria. The tested temperature range is usually from 0°C to 75°C. In general, temperature experiments employ solid medium instead of broth in order to better observe. The basal medium is Bennett's medium or YIM38 medium or nutrient medium. Note: If the temperature is above 100°C, use screw-cap culture tubes or screw-cap glass bottles and seal the screw caps to prevent the evaporation of the medium. Incubate the cultures in commercial water baths filled with dimethyl silicone oil. If the temperature is below 0°C, use an ethylene glycol water bath. For marine actinobacteria that require seawater, open ocean seawater has NaCl concentration of about 35 g/L or 3.5% (wt/vol).

Optimum pH and pH range for growth
An essential part of the description of any actinobacteria is the range of pH values at which it can grow, as well as the optimal pH for growth. Measure growth responses from a standar-dized inoculum using basic medium (Bennett's medium or YIM38 medium or nutrient medium) at various pH values. Liquid medium is to be used for pH tests, to measure the growth responses turbidimetrically. The selection of buffer is critical. A buffer should be used in most media to maintain a stable pH for growth of the test strain. Buffers are most effective at their pK a values and should be chosen with this in mind. Some useful biological buffers are listed in Table 2. Some buffers such as citrate, succinate, or glycine may be metabolized by the test organism. Others may be toxic. Sometimes, a combination of buffers may be helpful. Certain buffers (Good buffer) are non-metabolizable, non-toxic, have low reactivity with metal ions, and have other desirable features [10]. Phosphate salts are most commonly used because they are effective in the growth range of most bacteria, are usually non-toxic, and provide a source of phosphorus for the organism.    [11]. Negative controls for each buffer were used and the final pH was determined by using an indicator of acidity.

NaCl ranges and optima for growth
Salt tolerance experiments mainly test the tolerance ability of the organism to NaCl and other salts, and determine the optimum concentration for growth. Inoculate liquid media containing a range of NaCl (usually 0-30%, W/V, or relative molar concentrations) concentrations and measure the growth response turbidimetrically. Bennett's medium or YIM38 medium or nutrient medium can be used as basal medium.
For some marine actinobacteria, NaCl alone may not substitute for filtered seawater, which should be sterilized by filtration and added aseptically to the sterilized medium. Even if seawater is used, it may need to be aged for a few weeks in a glass vessel in the dark to be effective. Seawater contains 3% NaCl, and testing marine organisms for growth at levels below this can be done by using various proportions of distilled water to seawater in the medium, or by using artificial seawater in which the level of NaCl can be varied.

Utilization of carbon source
Utilization of carbon source tests usually uses turbidimetric method. Use a chemically defined basal medium that lacks a carbon source, but otherwise is suitable for growth of the actinobacteria being tested. The basal medium is Pridham and Gottlieb carbon utilization medium [8]. Add carbon sources to a concentration (sugar alcohols 0.5-1%, others 0.1-0.2%). After growth has occurred, measure the growth response turbidimetrically with a spectrophotometer. Note: 1. Use liquid medium to avoid the influence of agar.

2.
Thermolabile carbon sources should be sterilized by filtration (filter sterilize 10% solution through bacteriological filter) or ether sterilization (weigh an appropriate amount of the dry carbon source and spread in a pre-sterilized Erlenmeyer flask fitted with a loose cotton plug. Add sufficient acetone-free ethyl ether to cover the carbohydrate. Allow ether to evaporate at room temperature under a ventilated fume hood overnight or longer. When all ether has evaporated, add sterile distilled water aseptically to make a 10% w/v solution of the carbon source).

3.
Controls required for the test: No carbon source (negative control); D-glucose (positive control).

4.
For marine actinobacteria, instead of distilled water, use a synthetic seawater and sterilize the media by filtration.

Utilization of nitrogen source
Use the turbidimetric method to test the utilization of nitrogen source, especially sole nitrogen sources. Basal medium that omit nitrogen source but include a suitable carbon source are used. Add nitrogen source to a concentration (usually 0.5%). Note:

Basic medium for nitrogen source utilization (G/Liter)
1. Use liquid medium to avoid the influence of agar.

2.
Thermolabile nitrogen sources should be sterilized by filtration or ether sterilization.

3.
Controls required for the test: negative control (no carbon source).

Enzymological characteristics
Some enzymological characteristics are of such primary importance to a genus or species description that they must appear in every published description.

Oxidase test
This method tests for an enzyme that transfers electrons from a donor molecule to O 2 , thereby forming H 2 O. Oxidase-positive organisms are usually aerobes or microaerophiles that can use O 2 as their final electron acceptor. The test reagent, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), acts as an artificial electron acceptor for the oxidase and the reduced form is the colored compound indophenol blue.
Prepare a 1% (wt/vol) solution of TMPD in certified-grade dimethylsulfoxide (DMSO). The solution is stable for at least a month under refrigeration. Test methods: a. Method described by Kovacs [12]: Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry. Use a loop and pick a well-isolated colony from a fresh (18-to 24-h culture) bacterial plate and rub onto treated filter paper (please note on recommended media and loops). Observe for color changes. Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 sec. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 sec. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 min.
b. Method of Tarrand and Gröschel [13]: Place a piece of Whatman no. 40 ashless filter paper, quantitative grade, in a petri dish and wet it with 0.5 ml of the TMPD-DMSO solution.
Use a cotton-tipped swab to pick up one large isolated colony and allow the inoculum on the swab to dry for 5 sec. Tamp the swab lightly 10 times on the wet filter paper. Development of a blue-purple color in 15 sec is a positive test. Prepare the following medium (per liter of distilled water): peptone 1 g; NaCl 5 g; glucose 1 g; KH 2 PO 4 2 g; phenol red 0.012 g; agar 15 g; pH 6.8-6.9. Autoclave and cool to 55°C. Add 30% (W/V) filter-sterilized (or ether-sterilized) solution of urea to make final concentration of 2%. Mix and dispense appropriate portions into tubes or plates. Prepare a control medium lacking urea. Inoculate the surface of the slant or plate for 4 days. Look for development of a red-violet color compared to an uninoculated control. The medium can also be prepared as a liquid medium by omitting the agar [14].
For other organisms that grow poorly or not at all on Christensen's medium, use a test medium of the composition (per liter of distilled water): BES buffer 1.065 g; urea 20.0 g; phenol red 0.01 g; pH 7.0. Also prepare a control medium lacking urea. Sterilize both media by filtration and dispense 2.0-ml portions into sterile tubes. Culture the test organism in a suitable liquid medium. Centrifuge the cells and suspend them in sterile distilled water to a dense concentration. Add 0.5 ml of the suspension to the test medium and the control medium. Incubate the tubes for 24 h at the optimal temperature for the organism. Look for the development of a red-violet color in the test medium, but not in the control medium [15].

Lipase test
Lipase activity can be shown by using Tweens, for example, Tween 80 (polyethylene sorbitan monooleate, an oleic acid ester), Tween 40 (a palmitic acid ester), and Tween 20 (a stearic acid ester). Lipolytic organisms split off the fatty acid, and the calcium salts of the fatty acids produce opaque zones around the colonies.
Prepare a basal medium containing the following (per liter of distilled water): peptone 10.0 g; NaCl 5.0 g; CaCl 2 2H 2 O 0.1 g; agar 9.0 g; pH 7.4. Sterilize by autoclaving (121°C, 20 min). Autoclave the desired Tween separately (121°C, 20 min). Cool the basal medium to 45-50°C, add the Tween to give a final concentration of 1.0%, shake until the Tween is completely dissolved and pour into plates. Inoculate the cultures as lines on the surface of the agar. Incubate for up to 7 to 14 days, inspecting daily. Look for an opaque halo around the growth [16].

Gelatin liquefaction
The gelatin hydrolysis tests for an organism's ability to break down the protein gelatin, which is derived from collagen. Gelatin causes the media to thicken, especially at cooler (below 28°C) temperatures. If the organism can release gelatinase enzymes, the gelatin is broken down or liquefied. The media is checked over a period of about a week after inoculation and incubation at room temperature, for gelatinase activity. The tube is placed on ice for a few minutes; and if the media fails to solidify, it is considered a positive test. The gelatinase reaction may be slow or incomplete.
The conventional methods require long periods of growth, long periods for development, or are difficult to interpret. Now, commonly use the trichloroacetic acid (TCA) enhancement to be more rapid and sensitive [17].

Coagulation and peptonization of milk
Milk coagulation and peptonization test the ability of actinobacteria to produce protease. Coagulation is that mild protein is preliminarily degraded into big pieces by organism. Further degradation is peptonization.
Prepare milk coagulation and peptonization medium (per liter of distilled water): skim milk powder 200 g; CaCO 3 0.2 g. Dispense 3-5 ml portions into narrow tubes and sterilize by autoclaving (115°C, 15 min) or fractional sterilization for 2-3 times. Inoculate the tubes and observe in 5, 10, 20, 30 days, respectively. Milk solidification occurrence is the phenomenon of coagulation. Clots further hydrolyzed into liquid, is the phenomenon of peptonization. Peptonized exudates is translucent, typically begins after coagulation.

Starch hydrolysis
Starch hydrolysis tests the ability of an organism to produce certain exoenzymes, including aamylase and oligo-1, 6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. Make a single spot of the test organisms on a plate of the agar (within 5 mm in diameter) and incubate.
Prepare a basal medium containing the following (per liter of distilled water): soluble starch 10 g; K 2 PO 4 0.3 g; MgCO 3 1 g; NaCl 0.5 g; KNO 3 1 g; agar 15 g; pH 7.2-7.4. Prepare Gram's iodine solution by grinding 1.0 g of iodine crystals together with 2.0 g of KI in a mortar (slowly add 300 ml of distilled water while grinding until the iodine is dissolved). After growth occurs, flood the plate with the iodine solution. Starch stains blue with iodine, so look for colorless areas around the microbial growth.

Cellulose hydrolysis
Cellulose hydrolysis tests the ability of an organism to produce cellulase.
Other method [19]: Prepare mineral agar culture media in which cellulose is to be provided as a sole carbon source (per liter of distilled water): KNO 3 0.5 g; K 2 HPO 4 1.0 g; KCl 0.5 g; MgSO 4 7H 2 O 0.5 g; and agar, 15.0 g. Add 0.5 ml of a suitable trace metals solution. Autoclave, cool, and dispense into plates. Prepare a series of dilutions of the organism to be tested and spread 25-50 μl portions over the surface of the plates. Place a sterile disc of lens paper on the seeded surface of the plates and incubate for 3-7 days. Look for colonies that form visible holes in the paper. To increase visibility of the holes, stain the paper on the plates or after the paper is removed with 0.2% irgalan black in 2% acetic acid.

Nitrate reduction
Many organisms can respire anaerobically by using NO 3 as a terminal electron acceptor for an electron transport system (nitrate respiration or dissimulator nitrate reduction). Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO 3 ) to nitrite (NO 2 ) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. The Griess reaction has more recently been employed to detect nitrite and nitrate as products of nitric oxide synthase in bacterial identification [20]. Zinc dust must be nitrate-and nitrite-free. Protocol: For either substrate, NO 3 -or NO 2 -, inoculate the medium with a heavy inoculum from wellisolated colonies of the test organism. Incubate at 28°C for 18-24 h, some actinobacteria need 7-14 days. When sufficient growth is observed in the tube, test the broth for reduction of the substrate.
For NO 3 -substrate: Observe for gas production in the Durham tube. Mix two drops each of reagents A and B in a small test tube. Add approximately 1 ml of the broth culture to the test tube and mix well. If the test organism has reduced the NO 3 -to NO 2 -, a red color will usually appear within 2 min, indicating the presence of NO 2 -in the tube. If no color change is seen within 2 min, there are several possible reasons. Either the organism (i) was unable to reduce NO 3 -at all, (ii) was capable of reducing NO 2 -, or (iii) reduced NO 3 -directly to molecular nitrogen. Zinc is a powerful reducing agent. If there is any NO 3 -remaining in the tube (option (i) above), a small amount of zinc dust will rapidly reduce it to NO 2 -. Therefore, the appearance of a red color after the addition of zinc dust to a colorless reaction tube indicates a negative reaction, i.e., the organism has failed to reduce NO 3 -. Zinc is added to the tube by dipping a wooden applicator stick in nitrate-and nitrite-free zinc powder, just enough to get the stick dirty, and then dropping it into the tube containing the culture broth and the reagents. If too much zinc is added, the color reaction may fade rapidly. If the broth remains colorless after the addition of zinc, the organism has also reduced the NO 2 -, intermediate product to N 2 gas or some other nitrogenous product. N 2 gas is usually visible in the Durham tube. In the absence of gas, the product is assumed to be other than N 2 gas.
Occasionally, a lighter pink color will appear after the addition of zinc dust because of partial reduction, i.e., some of the primary NO 3 -substrate remains in the tube. The original tube may be reincubated and retested the following day.
For NO 2-substrate: Observe for gas production on the surface and in the Durham tube. Mix two drops each of reagents A and B in a small test tube. Add approximately 1 ml of the broth culture to the test tube and mix well. If the test organism has reduced the NO 2 -, there will be no color change, indicating that all of the original NO 2 -is gone, i.e., reduced. Reduction is often confirmed by the presence of N 2 gas in the Durham tube or on the surface of the broth, but other nitrogenous products may be produced. Therefore, the absence of gas does not rule out reduction of NO 2 -. If a red color appears, it indicates the presence of NO 2 -, and therefore a negative reaction. Occasionally, a lighter pink color will appear because of partial reduction, i.e., some of the primary NO 2 -substrate remains in the tube. The original tube may be reincubated and retested the following day. There is no need to add zinc dust to this reaction.

Note:
1. Be sure to run a negative control, uninoculated broth, to illustrate that the remaining NO 2 will be reduced by zinc dust, producing a red color.

2.
Because reduction of NO 3 -is assumed to be anaerobic, many published procedures warn that the medium needs to be anaerobic or deep enough to support an anaerobic process. However, later experiments have shown that the metabolism on the surface of the broth for most organisms that grow well in the broth will reduce enough dissolving oxygen for the reaction to take place.

Metabolic products
Thousands of characterization tests have been described in the microbiological identification. Those that follow are designed for detection of metabolic products and they are useful for physiological characterization beyond the more general features of an actinobacterial genus or species.

a. MR test (Methyl red test)
A type of fermentation called the mixed acid fermentation results in the formation of formic acid, acetic acid, lactic acid, succinic acid, ethanol, CO 2 , and H 2 in a buffered medium. The combination of acids in the mixed acid fermentation usually lowers the pH of the culture below 4.2. The test is used mainly in the differentiation of enteric bacteria. The organism being tested must be capable of catabolizing glucose [21].
Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K 2 HPO 4 5.0 g; glucose, 5.0 g; pH 7.5. Dispense 2-3 ml portions into narrow tubes and sterilize by autoclaving. Inoculate the tubes lightly and incubate for 4 days at the optimum temperature for the organism. Add one drop of methyl red reagent (0.25 g methyl red dissolved in 100 ml of ethanol). Look for a red colour (MR positive). A weakly positive test is red orange and a yellow or orange color indicates a negative test.

b. V-P test (Voges-Proskauer test)
Some fermentative organism catabolizes glucose by the butanediol pathway, in which acetoin (acetylmethylcarbinol) occurs as an intermediate in the formation of 2, 3-butanediol. In the presence of KOH and O 2 , the acetoin is oxidized to diacetyl, which in turn reacts with the guanidine group associated with arginine and other molecules contributed by peptone in the medium to form a pink-to red-colored product. The α-naphthol intensifies this color [21].
Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K 2 HPO 4 5.0 g; glucose, 5.0 g; pH 7.5. Make reagent A by dissolving 5.0 g of α-naphthol in 100 ml of absolute (100%) ethanol; the reagent must not be darker than straw color.
Prepare reagent B by dissolving 40.0 g of KOH in 100 ml of distilled water. Inoculate the tubes lightly and incubate for 2 days (routine test) and for 4 days (standard test) at the optimum temperature for the organism being tested. Add 0.6 ml of reagent A and agitate to aerate the medium. Add 0.2 ml of reagent B and again agitate the medium. Slant the tube to increase the aeration. Allow to stand for 15-60 min. Look for development of a strong cherry red color at the surface of the medium. A negative reaction shows no color or a faint pink to copper color.

c. Tryptophan decomposition (indole production)
Organisms that possess tryptophanase can carry out the following reaction: L-tryptophan → indole + pyruvic acid + NH 3 . The indole can be detected by its ability to react with p-dimethylaminobenzaldehyde to form a quinoidal red-violet condensation compound [21].
Xylene extraction test is more sensitive than the conventional test. Grow the test organism in a suitable culture medium supplemented with 0.1-1.0% tryptophan. Avoid using media containing carbohydrates, nitrate or nitrite, as these may interfere with the test. Distribute in 2-3 ml portions and sterilize by autoclaving. When cool, inoculate with the organism to be tested and incubate for up to 3 days.
Prepare Ehrlich's reagent as follows: 1.0 g of p-dimethylaminobenzaldehyde, 95 ml of 95% ethanol and 20 ml of HCl.
Add 1 ml of xylene to the broth culture, shake vigorously, and allow the mixture to stand for about 2 min. Then add 0.5 ml Ehrlich's reagent slowly down the side of the tube so as to form a layer between the medium and the xylene. Do not shake the tube after addition of the Ehrlich's reagent. Look for development of a pink or red ring below the xylene layer.

d. Hydrogen sulphide production
Some anaerobic and facultatively anaerobic actinobacteria can produce abundant H 2 S by the anaerobic reduction of S 2 O 3 2-. The H 2 S can be detected by its reaction with iron salts contained in the medium, which form a black precipitate of FeS. A different type of H 2 S production is based on the ability of some organisms to form low levels of H 2 S from sulfur-containing amino acids (cysteine, cystine, and/or methionine) by means of amino acid desulfurases. The gaseous H 2 S so produced is detected by its reaction with lead acetate strips suspended above the surface of the medium.
Thiosulfate iron H 2 S test [22]: Prepare peptone-iron agar medium (Tresner medium) as follows (per liter of distilled water): peptone 10.0 g; ferric ammonium citrate 0.5 g; agar 15 g. To achieve more satisfactory results, inoculums from actively growing cultures were used to streak the surface of the agar slants. After a short incubation period (15 to 20 h) at 28°C, the slants were observed. A pronounced bluish-black discoloration of the medium surrounding the colonies effected no change and indicated the production of hydrogen sulfide, whereas those organisms not producing H 2 S in the medium emitted only faint tints of other colors.
Paper strip method [21]: Inoculate a suitable semisolid (0.2% agar) growth medium that contains a peptone or other source of sulfur amino acids. Suspend a strip of sterile, lead acetateimpregnated paper about a centimeter above the surface of the culture, fold the upper end over the lip of the tube, and hold it in place with the screw cap or cotton plug. During growth of the organisms, H 2 S gas reacts with the lead acetate to form black PbS, beginning at the lower part of the strip. Lead acetate strips can be prepared by soaking 5-cm strips of filter paper in a 5% aqueous solution of lead acetate, sterilizing them separately in tubes by autoclaving and drying them in an oven.

Relation to oxygen
Aerobes use O 2 as a terminal electron acceptor for an electron transport system, can tolerate a level of O 2 equivalent to or higher than that present in an air atmosphere (21% O 2 ), and have a strictly respiratory type of metabolism. Anaerobes are incapable of O 2 -dependent growth and cannot grow in the presence of 21% O 2 . Facultative anaerobes can grow both in the absence of O 2 and in the presence of 21% O 2 . Microaerophiles respire with O 2 but cannot grow, or grow very poorly, under 21% O 2 . They grow best at low O 2 levels; some require levels as low as 1%. Some microaerophiles can also respire anaerobically with electron acceptors other than O 2 .
Semisolid agar method [23]: Autoclave a narrow culture tube that has been filled to 60% of its capacity with an appropriate culture medium containing 0.2% agar. After the medium has cooled to 45°C, add the inoculum, mix to distribute the organisms uniformly and then allow the agar to solidify. Alternatively, inoculate the medium by stabbing with an inoculating needle after the agar has gelled; this avoids the mixing that otherwise might add dissolved O 2 to the medium. Growth occurring only at the surface of the medium suggests that the organism is aerobic. However, a fermentable substrate should be present in the medium, because the organism might be a facultative anaerobe that not only respires with O 2 but also grows anaerobically by fermentation. Growth occurring only in the bottom region of the tube suggests that the organism is anaerobic. However, some extremely oxygen-intolerant anaerobes may not be able to grow even in the lowest region of the medium, because of the presence of small amounts of O 2 dissolved in the medium during the addition of the inoculum. Growth occurring throughout the tube suggests that the organism is a facultative anaerobe. It is important that no potential terminal electron acceptors other than O 2 should be present, as some aerobes can respire anaerobically. Growth occurring only in a disc several millimeters below the surface of the medium suggests that the organism is a microaerophile. Motile microaerophiles usually exhibit negative or positive aerotaxis, which results in their migration to a zone where the rate at which O 2 is diffusing to them matches the rate it is used by the organisms.

Susceptibility to antibiotics
Antibiotic sensitivity is the susceptibility of actinobacteria to antibiotics. Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic [ Table 3] will be most successful in treating a bacterial infection in organism.
Testing for antibiotic sensitivity is often done by the Kirby-Bauer method [24]: wafers containing antibiotics are placed on an appropriate agar plate where actinobacteria have been placed, and the plate is left to incubate. If an antibiotic stops the actinobacteria from growing or kills the actinobacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium. A stronger antibiotic will create a larger zone, because a lower concentration of the antibiotic is enough to stop growth.  Table 3. Common antibiotics and the suitable concentration

Antibacterial activity detection
Actinobacteria are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of the discovered bioactive secondary metabolites, notably antibiotics, antitumor agents, immunosuppressive agents, and enzymes. Because of the excellent track record of actinobacteria in this regard, it is necessary to preliminarily screen antibacterial activity of isolated actinomycetes. Cross streak method [25]: The actinomycetes isolates were inoculated at the centre of the sterile agar plates and the plates were incubated at 28°C for 5-7 days. After incubation, the nutrient broth (peptone 5 g; beef extract 3 g; NaCl 5 g; distilled water 1000 ml) cultures of test bacteria were streaked perpendicular to the growth of the actinomycete isolates. The plates were incubated at 37°C for 24-48 h and the extent of growth inhibition of the test bacteria was observed. The absence of growth or a less dense growth of test bacteria near the growth of actinomycete isolate was considered positive for the production and secretion of antibacterial metabolite.

Commercial multitest system for identification of actinobacteria
In recent 40 years, with the development of microelectronics, computer, molecular biology, physics, chemistry, and subject crossing universality, a significant contribution of scientific and technological development to the clinical microbiology was the development of miniaturized identification systems based on classical method. Several systems are commercially available [ Table 4], and new systems are being developed continually. These systems were mostly based on modifications of classical methods and were improved by the incorporation of highly sophisticated, computer-generated identification databases tailored for each system [26,27]. Each manufacturer provides charts, tables, coding systems, and characterization profiles for use with the particular multi-test system being offered. These systems offer the advantages of miniaturization and are usually used in conjunction with a computerized system for identification of the organisms. As mentioned earlier, the use of these systems can increase standardization among various laboratories because of the high degree of quality control exercised over the media and reagents. Now actinobacteria is defined as a phylum of Grampositive bacteria with high G+C content in their DNA. Although classical actinobacteria have the largest and most complex bacterial cells, some groups of actinobacteria possess the small and simple cell. For these simple (rod, cocci-shaped, without hyphae differentiated) actinobacteria, physiological and biochemical experiments are more important. Their physiological and biochemical tests can be carried by the automatic identification systems like the common bacteria. Trek Diagnostic Systems, Ltd., East Grinstead, West Sussex, UK (http://www.trekds.com). Table 4. Some commercial multitest systems for prokaryote identification

API Numerical identification system
API (analytical profile index) is a classification of bacteria based on experiments, allowing fast identification. This system is developed for quick identification of clinically relevant bacteria. Because of this, only known bacteria can be identified. It was invented in the 1970s in the United States by Pierre Janin of Analytab Products, Inc. Presently, the API test system is manufactured by bioMérieux [28]. The API range introduced a standardized, miniaturized version of existing techniques, which up until then were complicated to perform and difficult to read.
API systems can determinate simultaneously more than 20 items of biochemical indicators. Choose appropriate API strip according to different bacterial groups. The API strip consists of more than 20 microtubes containing dehydrated substrates. The conventional tests are inoculated with a saline bacterial suspension which reconstitutes the media. During incubation, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The assimilation tests are inoculated with a minimal medium and the bacteria grow if they are capable of utilizing the corresponding substrate. The reactions are read according to the Reading Table and the identification is obtained by referring to the Analytical Profile Index or using the identification software. Such as API 20NE is an identification system for non-fastidious, non-enteric Gram-negative rods [ Figure 2].

Biolog automatic bacterial identification system
The Biolog Microbial ID System (Biolog, Inc., Hayward, Calif.) can rapidly identify over 2,500 species of aerobic and anaerobic bacteria, yeasts, and fungi. These easy-to-use systems provide reference laboratory quality identifications. Biolog Systems do this without the labor-intensive requirements of conventional strips or panels [29,30]. Biolog's latest generation redox chemistry enables testing and microbial identification of aerobic Gram-negative and Grampositive bacteria in the same test panel. Gram stain and other pre-tests are no longer needed. A simple 1-min setup protocol and microbial samples are ready to be analyzed. Expanded GEN III database is designed to meet the needs of Biolog's broad customer base covering diverse disciplines of microbiology. All Biolog Microbial Identification Systems (manual, semiautomated, or fully automated) use the powerful new GENIII MicroPlate, allowing users to determine the most appropriate system to fit their current budget and level of throughput [ Figure 3]. It is important to realize that most such systems are designed for the identification of particular taxa and not for determining the physiological features of other taxa or new taxa. Indeed, a particular system may not even be applicable to other taxa. With these precautions in mind, multitest systems can provide useful information about the physiological characteristics of other organisms. For describing new taxa, the characterization systems that are used, as well as the inoculum age and size and the incubation temperature, must always be stated because reactions may not always agree with the results from classical characterization tests or with the results with other multitest systems.