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In this article we will discuss about the isolation and identification of actinomycetes.
Isolation of Actinomycetes:
This group of prokaryotes are ubiquitious and are found in all ecosystems. Some genera like Actinomyces are anaerobic and others like Frankia (symbiotic) require specialised growth media and incubation condition.
To isolate them from soil, special treatments are essential.
(i) Phenol Treatment:
Principle:
This treatment is done to reduce bacterial and fungal contaminants.
Requirements:
a. 1:20 soil dilution.
b. 1:140 phenol dilutions.
c. Agar medium in Petri plates.
d. Pipettes.
e. Glass marking pencil.
f. Bunsen flame.
g. Incubator.
Procedure:
1. Add to 10 ml of a 1:140 dilution of phenol, 2 drops of 1:20 soil dilution.
2. Place one drop of the phenol soil dilution, after keeping it for 10 minutes, in a Petri plate with melted agar medium (45°C), rotate the plate gently and incubate at 37°C for 5-6 days.
(ii) Centrifugation Technique:
Principle:
This technique is used to isolate actinomycetes from soil where their population is less compared to bacteria and fungi.
Requirements:
a. Centrifuge.
b. Incubator.
c. Soil sample.
d. Mortar and pestle.
e. Blanks of 9 ml sterile water for dilutions.
f. Petri plates with agar medium (Conn’s agar).
g. Pipettes.
h. Bunsen flame.
Procedure:
1. Grind 1 g of soil in a mortar and pestle with little water in order to separate actinomycete hyphae sticking to larger soil particles.
2. Make dilution of soil.
3. Centrifuge the final dilution at 3000 rpm for 20 minutes in order that bacteria and fungal spores settle down.
4. Plate one ml of supernatant on Conn’s agar plate.
5. Incubate for 5-10 days at 37°C.
6. Observe actinomycete colonies and identify them.
(iii) Calcium Carbonate Treatment:
Soil is treated with 3% CaC03 for 8-10 days and then dilutions are made and plated on Conn’s agar/Kuster and Williams agar, etc.
Isolation of Specific Genera:
(a) Selective enrichment for Micromonospora and Streposporangium:
Heat soil at 120°C for one hour and plate dilutions on medium (in plates) with inorganic salts, arginine, glucose, and antibiotics like Penicillin and nystatin.
(b) Activation of dormant spores:
Pretreat soil suspension with 6% yeast extract and 0.5% sodium dodecyl sulphate. Use a medium with humic acid as the principal nutrient with 30 mg/lit nalidixic acid.
(iv) For Micromonospora
a. Wakisaka et al. (1982):
Plate soil samples on Bennet’s agar supplemented with low concentration of case amino acids, beef extract, yeast extract and 25-50 jig turcamycin/ml.
b. Novamura and Hayakawa (1988):
Pretreat soil with 1.5% phenol solution for 30 minutes. Plate on humic acid agar supplemented with tunicamycin (20 mg/1) and nalidixic acid (30 mg/1).
Use of Antibiotics for Selective Isolation:
Streptoverticillium:
This organism is resistant to 10-25 µg of oxytetracyclin/ml which suppresses Streptomyces spp.
Glycomyces:
A medium supplemented with 25 µg/ml and streptomycin 15 µg /mi promotes the growth of Glycomyces.
Amycolatopsis:
Vancomycin 15µ g/ml
Thermoactinomyces:
Novobiocin 25 µg/ml and cycloheximide 50 fig/ml.
Saccaromonospora:
Penicillin G (1 µg/ml), polymyxin (5 µg/ml) and cycloheximide (50 µ.g/ml) on tryptone soya agar plus lactose and casein hydrolysate.
Baiting Technique:
Actinoplanaceae: Baiting
Principle:
Baits like pollen grains, grass blades, snake skin, etc. serve as substrates for motile spores to settle and grow.
Requirements:
a. Plant material collected from shore.
b. Moist chamber.
c. Baits, pollen grains, grass leaf blades (bleached).
d. Agar medium.
e. Bunsen flame.
f. Glass marking pencil.
g. Petri plates.
Procedure:
1. Collect plant material from shores of lakes, ponds streams, etc. and incubate them in moist chamber followed by dehydration to reduce bacteria.
2. Suspend these in distilled water.
3. Plate the planospores released on suitable media.
4. Pick up zoospores using capillaries filled with phosphate buffer containing KCl and then plate them on agar medium.
Media for Actinomycetes Isolation:
(i) Glycerol Asparagene Agar:
(ii) Benedict Agar:
(iii) Starch Casein Agar (pH 7.0 to 7.2):
pH 7.0-7.2 before autoclaving.
To this cooled (45°C) medium nystatin and actidione (50 µg/ml) and sodium penicillin (1.0 µg/ml) should be added.
Observation of Actinomycetes:
(i) Direct microscopic observation:
Cultures can be mounted in lactophenol and observed directly under the microscope.
Identification of Actinomycetes:
Actinomycetes are Gram-positive, forming branching filaments which are thin (0.5-1.0 µm in diameter). Filaments produce arthrospores either singly or in chains which are straight or variously coiled or in sporangia.
(i) Nocardiform actinomycetes (Group 22):
This group forms filaments which fragment into shorter elements. Aerial growth is present in some genera which form chains of spores.
Based on wall chemotypes and presence of mycolic acids and other characters, four subgroups are present:
1. Mycolic acid containing forms.
2. Pseudonocardia and related genera.
3. Nocardioides and Terrabacter.
4. Promicromonospora and related genera.
(ii) Genera with multilocular sporangia (Group 23):
Genera included in this group have their filaments which divide by transverse and longitudinal septa. This forms coccoid-like elements which are motile in Dermatophilus and Geodermafophilus and non-motile in Frankia.
(iii) Actinoplanetes (Group 24):
Stable filaments produce sporangia having motile spores in Actinoplanes, Ampullariella Dactybsporangium and Pilimelia. Non-motile single spores are produced by Micromonospora and spores in chains by Catellatospora. Cell walls contain meso—DAP and glycine. Arabinose and xylose are present in whole cell hydrolysis.
(iv) Streptomyces and related genera (Group 25):
All members of this heterogeneous group has cell walls with L-DAP and glycine. Hyphal filaments produce extensive aerial growth with long spore chains in Streptomyces and Streptoverticillium. Genera like Intrasporangium, Kineosporia, and Sporichthya produce very little aerial hyphae and the spore types are of several forms.
(v) Maduromycetes (Group 26):
Plently of aerial hyphae which bear spores. Microbispora produce pairs of non-motile spores and that of Microtetraspora produce non-motile spores in fours. Actinomadura produce varying number of spores. Planobispora, Planomonospora and Spirillospora produce motile spores in sporangia. Streptosporangium has non-motile spores in sporangia.
There are two sub-groups:
1. Streptosporangium and related genera.
2. Actinomadura.
The cell walls contain Meso—DAP and cell hydrolysates contain madurose.
(vi) Thermomonospora and related genera (Group 27):
Aerial mycelium produce single spores in Thermomonospora and in chains in Actinosynnema and Nocardiopsis. In Streptoalloteichus spores are produced in sporangia. Cell walls contain meso-DAP and do not have any characteristic amino acids or sugars in whole cell hydrolysates.
(vii) Thermoactinomycetes (Group 28):
Thermoactinomyces has aerial mycelium that produce single endospores on both vegetative and aerial hyphae. All species are thermophilic. Cell walls have meso-DAP but no characteristic amino acids or sugars.
(viii) Other genera (Group 29):
There are three genera which cannot be assigned to other groups. They produce aerial hyphae with chains of spores.
Species of Actinomycetes can be identified based on:
(i) Morphology:
Type of mycelium, spore chain morphology formation of sclerotia, sporangia, synnemata, flagellate elements, pigment production, etc.
(ii) Cell wall types:
There are I to IV types of cell wall. Cell walls have peptidoglycans containing diaminopimelic acid (DAP) which occurs in three isomeric forms, two of which, meso and Z-forms are easily separable by paper chromatography. The third D-isomer cannot be easily separated from meso-form.
Cell wall types and whole sugar patterns of aerobic actinomycetes:
Constituents given in () are variable. Among major constituents all cell wall preparations contain major amounts of alanine, glutamic acid, glucosamine and muramic acid.
Some organisms with type IV cell walls produce α branched, β—hydroxylated fatty acids called mycolic acid. These lipids fall into three groups based on molecular weight with Mycobacterium having largest and Corynebacterium with smallest molecular weight and No- car di a being intermediate. Based on the contents of nitrogenous phospholipids, there are I to V groups.
PE-Phosphatidyl ethanol amine.
PME-Phosphatidyl methylethanol amine.
PC- Phosphatidyl choline.
GluNU-Phospholipids of unknown structure containing glucosamine.
PG-Phosphatidyl glycerol.
Generic Identification of Actinomycetes:
The genera included under Group 22-29 in Bergey’s Manual contains filamentous, aerobic Gram-positive, branched filaments or hyphae which remain as stable mycelium or break up into rod-shaped or coccoid elements. The genera in some produce motile flagellated spores. The genera are divided into eight groups (22-29) based on morphology.
The morphological characters include:
Mycelium:
Most of the members of the groups 22-29 have stable mycelium. Oerskovia spp-Group 22 produce flagellated spores. Most of the genera produce substrate and aerial mycelium but many of them only substrate and some of them produce only aerial mycelium (rare), e.g. Sporichthya of Group-25. Intercalary vesicles without spores are produced by Intra- sporangium, of Group 25 or with spores by Frankia of Group 23.
Conidia:
Conidia or asexual spores are produced in several ways:
(a) Single conidia in several genera. Thermoactinomyces of Group 28 produce endospores which are thermostable.
Non-thermostable conidia are found in:
(b) Pairs of conidia:
(c) Short chains of conidia upto 20 spores:
(d) Long chains of conidia:
(e) Synnemata with motile spores:
(f) Sporangia with spores:
(g) Other structures:
Multilocular sporangia formed as a result of division in several planes; organisms of Group 23.
Spherical structures having drops of condensed water that enclose a curled chain of spores or hyphae embedded in an amorphous mass.
