Correct interpretation of actinomycete imagery using scanning electron microscopy

Keywords: Actinomycetales, Actinomycetes, Actinomycetia, Actinomycetota, growth morphology, SEM imagery, Streptomyces, taxonomy.

In Memoriam, Professor Tom Cross, University of Bradford, UK.

First discovery of a species within this group of bacteria dates to Harz in 1877¹ by the description of Actinomyces bovis. Later Buchanan named the order Actinomycetales² and in the subsequent year named the family Actinomycetecaea.³ Stackebrandt et al.⁴ proposed a new Class called Actinobacteria under the Domain Bacteria, followed by the creation of the phylum with the same name.^5,6 In this restructure order, Actinomycetales was confined to the original Hartz cluster that is comprised of non-mycelial taxa (e.g. Actinomyces).

The use of the same name both for the phylum and the class was, however, not preferable, and recently a new class named Actinomycetia was proposed by Salam et al.⁷ covering all members of the former order Actinomycetales.⁸ Here caution must be exercised as currently the order Actinomycetales covers the family Actinomyceteceae (Buchanan original description).⁹ So, families like Streptomyceteceae that formerly belonged to this order¹⁰ cannot be located under this order any longer. In addition, in 2021 the name of the phylum Actinobacteria revised again into Actinomycetota.⁹ In summary, the phylum Actinomycetota is the former phylum Actinobacteria. The proposed Class Actinomycetia is the former class Actinobacteria. Class Actinomycetia also covers the members of the former order Actinomycetales, which is different to the current order Actinomycetales. Finally, to conclude, every member under the class Actinomycetia is an actinomycete (actinomycetes in plural) in a general term that has been in use over 70 years.¹⁰

Another important point is that RNA oligonucleotide studies showed that the possession of branched hyphae (e.g. Thermoactinomyces) should not automatically place a bacterium within the class Actinomycetia, nor should the inability of an organism to form branching filaments (e.g. Arthrobacter, Cellulomonas and Rothia) necessarily exclude it from this taxon. Accordingly, genus Thermoactinomyces with its low guanine and cytosine content and endospore forming ability was removed from the actinomycete cluster and placed into the family Bacillaceae.¹¹

Whichever family they belong to, actinomycetes only display four different types of growth morphologies (Table 1). Full understanding of these structures is imperative during interpretation of the SEM images.

Table 1.  Distinctive growth morphologies of actinomycetes.^12,13

Fig. 1.  Growth morphologies of actinomycete groups captured using SEM.

It is also important that intact material is not damaged during processing for the SEM such as when gradual dehydration in alcohol is done prior to critical point drying. Any damage during processing can result in wrong interpretation of the image e.g. spore surface morphology, especially for the structures ‘rugose’ and ‘warty’.^8,14

Another important aspect is the timing of the examination for growth morphologies. Different microscope slide preparations should be prepared and examined in consecutive days. Young colonies will exhibit different morphologies as full maturation is not complete as well as too old colonies will result in collapse of spore chains. Spore surfaces also should be examined at right times (this differs for all above listed four different growth morphologies) to be able to identify spore surface structure correctly. As an example, a smooth looking spore surface might change into a ‘hairy’ or ‘spiny’ structure⁸ later in the growth cycle (Fig. 2).

Fig. 2.  Spore maturation of a Micromonospora species (USC-5041): (a) early stage, (b) mature stage where spines are formed.

As noted by the late Professor Cross (1989),¹⁵ choice of media is important as most sporoactinomycetes would require special media to allow differentiation and development of characteristic spores and pigments. His examples include, the transformation of pale, shiny, hard colonies of a Streptomyces species on nutrient agar into bright yellow colonies with a powdery white aerial mycelium and spirals of arthrospores when the organism is subcultured onto a more suitable growth medium such as oatmeal or inorganic salt starch agars.¹⁶ More recent examples of such differences in growth can be seen in recent publications of English et al.¹⁷ and Kurtböke.¹⁸

Again, the late Professor Cross (1989)¹⁵ highlighted that the actinomycete outgrowths starts from fragments of mycelium and develop into hyphae that penetrate the agar forming the substrate mycelium and hyphae that branch repeatedly and become cemented together on the surface of the agar to form a tough, leathery colony. He also added that actinomycete growth can be slow, a branching mycelium growing at the surface of transparent agar can be seen with the aid of a microscope after 24 h, and visible colonies may appear in 3–4 days, but mature aerial mycelium with spores may take 7–14-days to develop, and some very slow growing strains may require up to a month of incubation. Lengthy incubation times can result in evaporation of the medium, so thick agar plates are required. Thermophilic species incubated at high temperatures require a humid incubator.

A useful diagram of the developmental life cycle of a Streptomyces species is provided by Barka et al.¹⁹ illustrating growth from sporulation to development of substrate (vegetative) and then aerial mycelium leading to septation and formation of spores.

At all times a ‘rough, stiff loop is essential for abrading the colony and collecting sufficient mycelial fragments for an efficient transfer’. Spore suspensions can also be prepared first and used for streaking.¹⁵ They can be prepared ‘by detaching the spores from aerial hyphae with a loop or scraper and placing them in a suspending medium containing a wetting agent’. The arthrospores of streptomycetes are hydrophobic because of the enveloping sheath, and the wetting agent aids their even suspension in the diluent. Free spores may also be removed from lawns of aerial mycelium by rolling glass beads or agar cylinders over the surface.¹⁵

Best results can be achieved using inclined glass coverslip technique¹⁵ and Oatmeal agar supplemented with yeast extract.¹² Once the actinomycete is streaked plated onto this medium, round cover slips can be embedded onto the streaked lines with a 45° angle. Growth starts and developing mycelia simultaneously moves onto the coverslip. Multiple cover slips would allow replication and observations to be conducted at different growth times.

Once the growth is sufficient the cover slip can be removed, place onto SEM stubs, fixed with osmium tetroxide, gradually dehydrated in alcohol before being subjected to critical point drying and subsequently coating with gold.^20,21 In this final stage, it is imperative that experienced technical officers who have acquired knowledge on the use of SEM assist the researcher who is engaged in image interpretation.

Modified techniques were also developed, such as the one by Prakash and Nawani²² in which lyophilisation is used rather than chemical fixatives and dehydrating agents.

Again, as stated by the late Professor Cross,¹⁵ ‘one requires patience when working with actinomycetes, and the ability to plan and run several experiments concurrently to avoid wasting time’. I would like to add the importance of students understanding the long and arduous route from being a novice to becoming an expert. Hard work and perseverance is important to patiently build their knowledge and gain laboratory skills.²³ What is presented here is the outcome of 40 years of continuous learning and skill building in the field of actinomycetology.²⁴ Understanding the value of team work as well as appreciating the roles of other disciplines and experts such as technical staff members in EM units, without whom the quality micrographs cannot be produced, is also imperative for novices.

Data is embedded in the text as SEM micrographs.

The author declares no conflicts of interest.

This research did not receive any specific funding.