Identification of bacteria is not always an art. The process of recording, collating and interpreting their common diagnostic characters can be mechanized and used in a visual sorter (Olds, 1966, 1970) based on the Peek-a-boo system (Wildhack & Stern, 1958; Yourassowsky et al., 1965). Hand-sorted punched cards have been used for recording quite varied taxonomic information (Wood, 1957) and, in the first edition of this Manual, Cowan & Steel suggested that the ‘tables could form the basis of a set of diagnostic punched cards to be used with similar cards on which the characteristics of the unknown [isolates] are punched’. Soon after that was written, Schneierson & Amsterdam (1964) described a punched card used for identifying bacteria at the Mount Sinai Hospital, New York, but unfortunately they did not give details of the ‘authoritative reference’ sources from which they obtained the characters for their master cards – information which is of course essential for assembling and using tables of characters.

A disadvantage of diagnostic tables is that as more and more detail is included they become less easy to use. For the second edition of this Manual, the early drafts of what became Tables 6.1 and 7.1 stretched to more than twenty columns with up to ten lines of characters; to check them Cowan made a set of punched cards with one (or more) cards for each genus.

It is assumed that the reader of this Manual has some knowledge and experience of bacteriology and of elementary chemistry and that the basic principles including those of laboratory safety are understood. Thus, though many other essential details are given in the Appendices, how to determine the pH value of a medium, or how to make a normal or molar solution, is not described; nor are details given about how to use anaerobic jars or microscopes. Serology is not discussed but methods commonly used in the preparation of extracts for grouping streptococci are described as the Lancefield serological groups are referred to in Table 6.3b. Details of sterilization temperatures and times are also given as these so-called standard procedures still vary from one laboratory to another.

This Manual is intended to help those who have isolated a bacterium and want to identify it. The methods used by clinical bacteriologists to isolate organisms from specimens sent to the laboratory are not described as to do so would be to enter everchanging fields, and our recommendations might well be out of date. We stress, however, that before identification of any organism is attempted, it must be obtained in pure culture. Some advice on how to recognize that a culture is impure, and on the steps to be taken to purify it, is therefore given.

Taxonomy is not every man's meat, but neither is it everyone's poison. It can be likened to a cocktail: a skilful blend in which it is not easy to discern the individual ingredients. In taxonomy the ingredients are (i) classification, or the orderly arrangement of units, (ii) nomenclature, the naming or labelling of the units, and (iii) identification of the unknown with a unit defined and named by (i) and (ii). The subdivisions should be taken in the order indicated, for without adequate classification it is impossible to name rationally, and without a system of labelled units it is impossible to identify others with them or to communicate the results.

Classification

Before discussing the identification of bacteria, the principles of classification and nomenclature must first be dealt with briefly. Since this book is essentially a practical manual, theoretical speculations about the validity of bacterial species (Lwoff, 1958; Cowan, 1962a; Lapage et al., 1975) are not considered.

For this Manual, the concept of bacterial species is therefore accepted as a convenient unit. However, as it so obviously has different values in different groups of bacteria, no attempt is made to define it, or to analyse the qualities that distinguish one species from another. Nor is any attempt made to determine whether a taxonomic group (taxon) is a species, a variety or a subspecies; or to estimate the value or importance of different kinds of bacterial characters (Cowan, 1968, 1970b).

Following the 1976 revision of the Bacteriological Code (see Appendix G), the Approved Lists of Bacterial Names was published in the International Journal of Systematic Bacteriology (IJSB) on behalf of the Ad Hoc Committee of the Judicial Commission of the International Committee on Systematic Bacteriology (ICSB) of the International Association [now Union] of Microbiological Societies (Skerman et al., 1980). It took effect on 1st January 1980 and was subsequently reprinted in book form in 1980 by the American Society for Microbiology. For information, and with permission, the explanatory Introduction to the Approved Lists is reproduced here. All subsequent valid alterations and additions as well as proposals for change are published in the IJSB.

Approved Lists of Bacterial Names

edited by

V.B.D. SKERMAN, VICKI McGOWAN, AND P.H.A. SNEATH

Department of Microbiology, University of Queensland, St Lucia, Queensland 4067, Australia and MRC Microbial Systematics Unit, University of Leicester, Leicester LE1 7RH, England

on behalf of

The Ad Hoc Committee of the Judicial Commission of the ICSB (International Journal of Systematic Bacteriology, vol. 30, pp. 225–420, 1980)

Isolation methods

Isolation begins with the collection of the specimen. Normally the clinician takes the specimen and sends it to the laboratory, but there are occasions when the bacteriologist should go to the patient or vice versa, so that fresh material can be examined while ‘hot’.

Direct microscopy. Since amoebic and bacillary dysentery cannot be distinguished clinically, it is essential when amoebic dysentery is endemic or is suspected, to examine a freshly passed stool on a warmed microscope stage to see the characteristic movements of vegetative Entamoeba histolytica; even better specimens may be obtained at sigmoidoscopy. Only by seeing the movement of E. histolytica can it be distinguished from Entamoeba coli; the differences between the encysted forms are not sufficiently great or constant to be diagnostic. The serous fluid of a primary chancre collected in a capillary tube can also be examined unstained by microscopy for spirochaetes. Stained material from leprosy lesions usually shows abundant organisms which, as yet, defy the usual cultural methods.

It is usually unrewarding to stain blood films for bacteria, but in hot countries bacteria are not the only causes of fever; malaria parasites and other bloodborne protozoa should therefore always be sought. Pus, cerebrospinal fluid (centrifuged deposit), pleural effusions, and other transudates may show bacteria or other microorganisms when stained by Gram's method; if none is seen, a film stained by the Ziehl–Neelsen (ZN) method may reveal acid-fast rods.

The demand for a new edition of this Manual has been enormous. We hope that we have done justice to it and that it will prove a fitting tribute to the late Sam Cowan. He not only obtained every paper he cited in the references but personally perused and annotated each one. We cannot alas say the same. It is now beyond the scope of one person or even of two persons to cover the entire and seemingly everchanging fields of bacterial classification, nomenclature and taxonomy, especially with the range of ‘medical’ bacteria expanding with the advancement of biotechnology and modern medicine to include many environmental organisms. For this third edition we have therefore sought the help of the experts listed on pages xv and xvi for various groups of organisms and we gratefully acknowledge all their contributions to this Manual. The opinions expressed are mostly theirs though the final responsibility is ours. We hope that together they will provide enlightenment and understanding of a subject which, though not everyone's ‘cup of tea’, is nevertheless at the heart of diagnostic medical bacteriology.

In outline, this edition follows that of the two previous ones. We have received numerous suggestions for change but have resisted many of them, preferring to regard continuity as more important.

REAGENTS

All staining reagents should be kept in well-closed glassstoppered bottles (except Loeffler's methylene blue) and protected from direct sunlight. For frequent use, flexible plastic bottles with tube outlets may be used but they must be washed out thoroughly before refilling. They should not be stored in close proximity to concentrated acids or ammonia. Distilled water for reagents should be freshly prepared and neutral in reaction.

Formulae of staining reagents are listed in alphabetical order.

Acetone – iodine solution for decolorization

Strong iodine solution

Dissolve the iodine and potassium iodide in the water and adjust to volume with the ethanol.

Mix well before use

Acid-alcohol

Mix well before use.

Albert's stain

Dissolve the dyes in the ethanol. Mix the acid with the water and add to the dye solution. Allow to stand for 24 h and then filter.

Ammoniacal silver nitrate solution

Dissolve the nitrate in the water; to 90 ml of this solution add strong ammonia solution (sp. gr. 0.880) drop by drop until the precipitate which forms just dissolves; add sufficient of the remaining AgNO3 solution drop by drop until the reagent remains faintly turbid even after shaking. When protected from light, this reagent is stable for several weeks.

Ammonium oxalate – crystal violet stain

Mix and dissolve.

For use, mix 20 ml of solution A and 80 ml of solution B.

Aqueous solutions

Simple aqueous solutions of each of the following are used in staining.

In the introduction to a Good Food Guide for bacteria, Miles (1965) described most culture media formulations as ‘kitchen recipes, written … by increasingly sophisticated cooks’. There are two entirely different kinds of media: those of defined composition and those of undefined composition, usually containing peptone. Defined media have disadvantages for identification because the characters of organisms grown in them may differ from those developed in undefined media (Meynell & Meynell, 1965). In general, published descriptions of bacteria refer to those characters found after growth in complex, undefined media, and because of this the results of biochemical tests do not always correspond with those obtained in defined media.

Media preparation seldom receives the attention it deserves; moreover, the media room is often overcrowded and understaffed, and the conditions in which media-makers work are often among the worst in the laboratory. Complaints about media, whether commercial or home-prepared, are still common and many laboratories have therefore set up internal quality control and assessment procedures (see Chapter 11); others have also made formal arrangements for supervision of media preparation.

In this chapter we discuss the general aspects of media making; formulae for the different media will be found in Appendix A. The majority of the commonly used culture media are now available commercially as dehydrated products, in either powder or tablet form, which are reconstituted by the addition of distilled water and then sterilized in the conventional manner. The manufacturers' directions for reconstitution should aways be followed for the best results.

The first edition of this Manual, judged by its spread around the world, seems to have been useful to hospital bacteriologists. It was translated into Japanese by Dr Riichi Sakazaki, who will also translate this edition.

It has not been easy to prepare a worthy successor; not only have I been unable to discuss and argue every sentence with my colleague, but I have missed the ready access to libraries that one has when working in a large research institution. However, I have been greatly helped by the Librarians at Colindale (Miss B. H. Whyte) and the Royal Society of Medicine (Mr P. Wade) and their staffs.

In this edition Chapter 2 and Appendices A, B, C and E, originally written mainly by Dr Steel, are little changed; most of the other chapters have been completely rewritten. Chapters 8 and 9 are entirely new, as are Appendices D, F, G and H. I must thank Mr A. Waltho, of the Medical Research Council's Central Store, who gave me great help in preparing the list of firms which supply media and chemicals (Appendix H) and, together with Dr O. M. Lidwell, suggested and drafted what became Table 2.1.

From the beginning, this Manual has preached consistently not only about the importance of pure cultures for identification but also of the need for standardization of test methods both within and between laboratories. We reinforce these views here with a brief outline of the current concepts of quality control and laboratory proficiency which must be understood and put into practice for good identification procedures to succeed. As before, we emphasize the importance of controlling characterization tests with organisms known to give positive and negative results; the test organisms recommended are given in Appendix D together with media and conditions suitable for their propagation short-term and storage.

Laboratory quality control

Quality control can be regarded as the continual monitoring of equipment, reagents and working practices as well as the provision of training with specified details of laboratory methods and procedures to ensure proficiency. The lack of test reproducibility both within and between laboratories, discussed in previous editions of this Manual, has now been well documented (Sneath & Johnson, 1972; Lapage et al., 1973; Sneath, 1974; Sneath & Collins, 1974; Snell, DeMello & Phua, 1986). Variation in test results between laboratories may be due to differences in the sensitivities of the methods used. Such differences are of considerable practical importance as the results given in the diagnostic tables and keys may be applicable only if the methods stated are used. This applies also to micromethods and diagnostic kits.

The availability of laboratory-based computers is increasing considerably, not only because of reductions in their price and size but also because programs suitable for epidemiological and microbiological purposes are more readily available. Historically, laboratory computing developed from the work of relatively few laboratories with limited access to the large mainframe computer facilities available in the 1960s. These were the ‘first generation’ of laboratory computers. They have all been replaced now by second and even third generation computer ‘hardware’ (the collective term used to describe the computer processor, the magnetic disk or tape storage apparatus as well as the visual display terminals and printer) and the associated ‘software’ (the programs for various applications). With the development of the ‘silicon chip’ all the associated hardware and software is available now for small sized micro-computers which have memory power and data storage capacity equivalent to those of their large predecessors. Any detailed description will undoubtedly soon be out of date but computer facilities with visual display terminals, printers, and adequate magnetic disk storage for as much as three years' clinical laboratory workload together with the necessary software (and training) are currently available at a modest cost. Such computer systems are well suited not only for bacterial identification purposes but also for a broad range of other uses in the microbiology laboratory.

My colleague, Dr K. J. Steel, died suddenly on 25 September 1964, between the completion of the manuscript and the proof stage of the book. His death at the age of 34 is a great loss for he seemed destined to reach the highest branches of bacteriology. In this Manual he was responsible for the whole of Appendices A to D and F and for much of Chapter 3; and he played a big part in revising and recasting the tables that form the heart of our work. I hope that the book will serve as a fitting memorial to a great collaborator and friend.

As a subject, taxonomy is often regarded as dull and uninspiring. With understanding, however, it can be stimulating and exciting, as indeed were the personal and sometimes unorthodox views expressed by Cowan in the previous edition of this Manual. Moreover, taxonomy is fundamental to the application and use of the increasingly available commercial identification kits now used so widely for medical (and other) bacteria so that knowledge of the basic principles concerned is perhaps more than ever essential.

The application of taxonomy, as a science, to bacteriology took a long time. Bulloch's (1938) History of Bacteriology, Brock's (1961) Milestones in Microbiology and Postgate's (1969) Microbes and Man epitomise the applied and practical nature of early bacteriology. This was concerned chiefly with ‘diseases’ of wine, plants and animals, and little thought was given to theoretical aspects such as taxonomy. The main lines of investigation were to determine causes and, if possible, find cures, from which diagnostic bacteriology and preventive medicine developed. To the early medical bacteriologists, the systematic classification of bacteria was not essential. Indeed it was not until botanists who took an interest in these ‘new’ and strange organisms tried to apply variations of their own classical rules for naming them that bacterial taxonomy first began. By and large medical bacteriologists were content to let bacterial names look after themselves; they used names which were meaningful to them in relation to illness, such as ‘Bacillus typhosus’ or more generally ‘the typhoid bacillus’.

TAXONOMIC TERMS

We have been unable to avoid the use of some taxonomic terms in this Manual, and short notes on some of those more commonly used are given below. Those who would like more help or greater detail should consult A Dictionary of Microbial Taxonomy by Cowan (1978).

Accession number. The number allotted to a culture when it is accessioned (accepted) into a permanently established culture collection. Even if the classification (and the name) of the organism changes, the accession number remains the same.

Antibiogram. A record of the sensitivity or resistance of an organism to the different antibiotics listed. It is often an essential part of the bacteriological report made to the clinician who sent the specimen. Sometimes the sensitivity or resistance to particular antibiotics can aid in identification although the possibility of changes during antibiotic therapy should always be remembered. Non-therapeutic substances such as lysozyme or O/129 may also assist in the identification of an organism.

Carboxyphilic; capnophilic. Used to describe an organism whose growth is improved, or made possible, by an increase in the CO2 content of the atmosphere.

Category. Used in taxonomy to indicate RANK in a hierarchical system of classification: genus, species, and so on. Often also used with the ordinary meaning of ‘kind’.

Chemotaxonomy. A term used to describe the chemical nature of the structure and functions of organisms as applied to their taxonomy.

In characterization by stages, the first-stage table is combined with a figure and shows how, with a small number of selected characters, Gram-positive bacteria can be divided into groups that correspond to those used in orthodox classifications. Not all of the theoretically possible combinations of characters are shown in Table 6.1 because many of them do not seem to occur in nature. Each shaded square indicates the genus or genera that have the characters shown in the same column in the table above it. Equivocal characters, those difficult to determine, and characters markedly influenced by culture medium or test method can make a genus span more than one column; we have therefore tried to concentrate on the reactions given by most strains of a species in the kind of media likely to be used in routine diagnostic laboratories (majority reactions or characters) though in doing this we may perhaps have introduced a tidiness that is not warranted by the biological nature of the scheme. An example of generic spread is seen with Aerococcus, which appears in the third and fourth columns of Table 6.1; in this case, the reason for the spread is that the catalase reaction is not always easy to read and may be interpreted in different ways by different workers. Those who expect a large volume of gas to be produced may record the feeble reaction of A.