Analytical chemists in industry are frequently faced with situations where a basic understanding of microbiology would be an advantage, for instance in the analysis of bacteria in food. Microbiology for the Analytical Chemist has been written specifically for analytical chemists who have little or no knowledge of microbiology, but might be required to interpret microbiological results. This book covers a wide range of microbiological situations in analysis. It deals with the question of establishing when a sample is contaminated, the problems of counting and identifying micro-organisms and establishing what effect they will have on the sample. The book examines the microbial contents of water and food. It also looks at the procedures for disinfecting and preservative testing. Traditional laboratory methods are discussed, and new rapid techniques are also considered. Microbiology for the Analytical Chemist is unusual in that it pulls together those aspects of microbiology which are of interest to analytical chemists and explains them at a basic level using practical situations as examples. This book will also be of interest to analytical chemists in academic or industrial laboratories, where there is no fund of microbiological experience to draw on.

Microbiology for the Analytical Chemist

By R.K. Dart

The Royal Society of Chemistry

Copyright © 1996 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-524-2

Contents

Chapter 1 Introduction, 1,
Chapter 2 Biodeterioration, 16,
Chapter 3 Equipment and Methods, 25,
Chapter 4 Microscopy and Staining, 34,
Chapter 5 Microbial Detection and Counting, 41,
Chapter 6 Biochemical Testing, 57,
Chapter 7 Practical Identification of Bacteria, 70,
Chapter 8 Microbiology of Food, 98,
Chapter 9 Microbiological Analysis of Water, 109,
Chapter 10 Sterility, Sterility Testing, Disinfectants and Preservatives, 123,
Chapter 11 Microbiological Assay, 133,
Subject Index, 151,

CHAPTER 1

Introduction

1 THE MICROBIAL CELL

Microbiology can be defined as the study of organisms that are too small to be clearly visible to the naked eye. This will include all organisms with a diameter of less than approximately 1 mm.

Micro-organisms are widely distributed between different taxonomic groups and include bacteria, protozoa, fungi, and algae. Viruses, although frequently considered in microbiology text books, are not cells.

The discovery of micro-organisms caused numerous problems relating to the placing of these species into the traditional plant and animal kingdoms, and in the mid-19th century a third kingdom, the Protista was suggested to include the protozoans, fungi, algae, and bacteria.

The development of electron microscopy showed that there was a major dichotomy between the various groups relating to the internal structure and organisation of cells. Two very different types of cells were discovered, the small, relatively simple procaryotic cell, and the more complex eucaryotic cell which is usually considerably larger.

1.1 Procaryotic Cells

The bacteria are procaryotic cells. This group also includes the organisms which used to be known as the ‘Blue–green Algae’ or Cyanophyceae, and are now known as the Cyanobacteria.

The vast majority of procaryotic organisms are unicellular, although multi-cellular bacteria forming filaments are also found. A few types are more complex structurally and may produce fruiting bodies, appendages, or stalks, although these would not generally be found in a typical analytical laboratory. Bacteria forming filaments frequently show a marked tendency to break up into individual cells. Figure 1.1 shows a typical procaryotic cell.

Procaryotic cells are surrounded by a cell membrane, a thin flexible sheet composed of protein and lipid. This has an important function in regulating the molecules which can pass into and out of the cell.

In the great majority of cases, there is a rigid cell wall outside the membrane. This cell wall has a chemistry which is unique to the procaryotic cell, and contains a number of compounds which are not found in the eucaryotes. These include peptidoglycan (murein), which is a repeating disaccharide containing the amino sugar, N-acetylmuramic acid, linked to a peptide containing D-amino acids, and diamino acids such as diaminopimelic acid. This cell wall chemistry is an important linking factor between the true bacteria and the blue–green algae. A few groups such as Mycoplasma do not possess a cell wall.

In the procaryotes, there is no discrete nucleus surrounded by a nuclear membrane, and the deoxyribonucleic acid (DNA) is not associated with the basic proteins known as histones. The DNA consists of covalently closed circles.

There are no organelles such as mitochondria present within the cell. Flagella are present in the case of motile cells; the position of these varies from species to species and may be used diagnostically. Certain species may contain spores which are highly resistant to heat and a variety of chemicals. A number of species are capable of photosynthesis, that is they can convert solar energy into chemical energy. Those that do carry out photosynthesis have chromatophores which are extensions of the cell membrane, and which are morphologically distinct from the chloroplast of the eucaryotic higher plant (see later).

There is also a considerable difference in the ribosome structure of procaryotic and eucaryotic cells. Ribosomes are the structures upon which proteins are synthesised. Procaryotic cells possess ribosomes which are 70S in size, whereas eucaryotes have 80 S ribosomes.

1.2 Eucaryotic Cells

Organisms containing eucaryotic cells include animals, plants, fungi, algae, and protozoa. Figure 1.2 shows typical animal and plant eucaryotic cells.

A cell membrane consisting of a protein and lipid mixture is present, but the eucaryotic cell membrane contains significant quantities of steroids, which are not found in most procaryotic cells. There may or may not be a cell wall outside the membrane depending on the type of cell.

Eucaryotic cells possess a nucleus, surrounded by a nuclear membrane which delineates the nucleus from the rest of the cell. This nucleus contains the majority of the cell DNA and is associated with histones. The nuclear DNA is linear. The nucleus is also the centre for the synthesis of ribonucleic acid (RNA).

All material inside the cell membrane, but outside the nuclear membrane, is known as the cytoplasm. Other organelles are found in the cytoplasm of the eucaryotic cell. These include the mitochondria, which are the powerhouse of the eucaryotic cell producing much of the energy in the form of adenosine triphosphate (ATP). Chloroplasts are found in photosynthetic species such as plants and algae. Neither of these structures is found in procaryotes. Various other organelles such as a nucleolus, centrioles, and a Golgi apparatus are also found. There may be one or more cilia or flagella in the case of motile cells, but these differ considerably from the flagella of bacterial cells.

Eucaryotic micro-organisms of importance to the analytical chemist include the fungi, protozoa and algae.

1.3 Nomenclature and Taxonomy

Micro-organisms are named using a binomial system. Every species has two latinised names, the first being the generic name and the second the specific name which identifies the organism within the genus. The first letter of the generic name is in capitals and the whole name is either italicised or underlined. In situations where no ambiguity can arise only the first letter or a shortened version of the generic name may be used, although the full name should always be used the first time it arises.

Nomenclature may cause problems. Occasionally, newly isolated organisms may not be recognised for what they are, and are given a new name. This can cause considerable confusion when the organism is finally correctly identified.

Taxonomy is the science of classification and has several functions. The first is to describe the species which is the basic taxonomic unit. The second is to catalogue these species into some arrangement enabling the relationships between species to be recognised. A third practical aspect is identification, that is, the matching of an unknown organism with a known species.

The division between species at the plant and animal levels is generally relatively easy on the basis of their morphology (appearance), although problems can arise in distinguishing between the simpler algae and protozoa. This however is not true with bacteria as the range of distinguishable shapes and sizes is too small.

Bacteria are therefore classified and identified into strains on the basis of a number of tests (e.g. morphological, staining, biochemical, serological). A species may then be defined as a cluster of strains showing a high degree of similarity, but differing from other clusters of strains (species) in a number of characteristics.

In taxonomy, the strains are grouped into individual species which are then grouped into progressively higher series of categories. In order, these are the genus, which is a collection of species, the family, the order, the class and the phylum. This is known as a hierarchical classification, as each ascending category unites a larger number of taxonomic groups on the basis of a smaller number of common properties. This hierarchical type of approach reflects to a considerable extent the evolutionary relationships of the organisms involved. In bacteriology, the two important groupings are the species and the genus. The various other hierarchical groups are not of great practical significance.

The use of various characteristics carries no guarantee that the tests being used are significant. The classification of bacteria therefore tends to be heavily biased towards the practical aspects of microbiology, such as the identification of medically and industrially important species.

The hierarchical type of approach to practical bacterial identification makes use of a number of characteristics which are given differential or weighted values. Tests are carried out in a pre-determined order designed to confirm or eliminate various possibilities. By following this pre-determined order, the experienced microbiologist is frequently able to reduce the possibilities to a small number of groups relatively quickly. In the majority of cases, characteristics would be examined in the following approximate order: source of the organism, colonial appearance on the growth medium, staining followed by microscopic examination, a variety of biochemical tests, and finally serological tests.

Other types of taxonomy are also found. Numerical classification or Adansonian classification is based on the quantification of similarities and differences, with no weighting being given to any one character. It should therefore be possible to quantify the relationships between cells on the basis of the number of similarities they share, relative to the number examined. The method is basically a cluster analysis in n-dimensional space, where n is the number of variables examined. The clusters can then be related to each other by means of a dendrogram. In this type of classification, individual characters gain an importance not given to them in hierarchical taxonomy. The great advantage of this method is that it requires the determination of a large number of characteristics. The method also requires the use of a computerised data base and is now used as the basis of a number of rapid identification systems, e.g. the API system.

Irrespective of the type of classification system used, practical classification and identification on a day to day basis must, of necessity, be based on characteristics which are easy and simple to observe and measure. One obvious example is motility, which depends on flagella. Motility/non-motility is a useful differential character, which is easy to observe using simple techniques and is therefore widely used in classification. However, the number and position of the flagella, which may be polar or peritrichous (all round the cell), is also a differential character. To observe these reliably requires an electron microscope, which is not a generally available laboratory tool, and therefore this character is not used in routine identification.

Other classification methods, such as the analysis of guanine and cytosine (GC) ratios in DNA, and DNA homology have no place as yet in the routine testing of the analytical laboratory.

From the practical point, the most important classification relates to safety, and several categories of micro-organisms can be defined depending on how dangerous they are and the level of containment required.

Group 1: Organisms unlikely to cause disease in humans.

Group 2: Organisms that may cause human disease and may be a hazard to laboratory workers, but are unlikely to spread in the community. Laboratory exposure rarely causes infection and effective prophylaxis and/or treatment is usually available.

Group 3: Organisms that may cause severe human disease and which present a serious hazard to laboratory workers. There may be a risk of spread in the community but there is usually effective prophylaxis and/or treatment available.

Group 4: Organisms that cause severe human diseases and are a severe hazard to laboratory workers. There is a high risk of spread in the community, and there is usually no effective prophylaxis and/or treatment.

For most practical purposes, the organisms found in an analytical laboratory will usually fall into Groups 2 or 3, and therefore laboratories should be of a sufficiently high standard to handle Group 3 type organisms.

2 EUCARYOTES

The eucaryotic micro-organisms include the protozoa, fungi and algae. The inclusion of some of the more highly specialised members into these three groups can be easily recognised. However, for some of the simpler ones such divisions may be difficult to recognise, and transition between algae and protozoa is common.

2.1 Algae

The algae are organisms possessing chloroplasts, carrying out photosynthesis and producing oxygen. They are a highly diverse group which can be divided into a number of classes. Many algae are unicellular micro-organisms, but they also contain giant multicellular species such as the kelps which may grow to a length of 50 m. Many of the unicellular species are motile by means of one or more flagella. Their main interest to the analytical chemist is the ability of some groups of unicellular planktonic species to cause the so-called ‘red tides’, and produce potent phytotoxins causing poisoning of shellfish and other species.

2.2 Protozoa

The protozoa are a very diverse group of unicellular organisms lacking in photosynthetic abilities. Most of them show no resemblance to algae, but some of the simpler species are obviously related to algae which have lost their photosynthetic apparatus. This loss would restrict the range of environments available to the species, and cause a series of evolutionary changes leading eventually to protozoans whose algal origins have become unrecognisable.

A number of classes of protozoans can be distinguished, and there are a number of species which parasitise man. Many of these are carried by insect vectors, causing diseases such as malaria and sleeping sickness (trypanosomiasis), and are beyond the scope of this book. Several species are water-borne or may be found on uncooked food contaminated by sewage, and when ingested can cause diseases such as amoebic dysentery. Many of these are tropical or sub -tropical, but one water-borne organism causing problems in the UK is a member of the genus Giardia.

2.3 Fungi

The fungi are also a non-photosynthetic group, the majority being highly specialised and well adapted to soil which is their main habitat. A few very primitive aquatic species, which are motile, show some resemblance to flagellated protozoans. Figure 1.3 shows a diagrammatic representation of a typical fungus.

Most fungi consist of a mycelium which is a highly branched series of tubes containing multiple nuclei. Individual branches are referred to as hyphae. Asexual reproduction usually occurs by means of spores pinched off at the tip of the hypha.

The cell wall is composed mainly of a polymer of N-acetylglucosamine known as chitin, although there may also be limited amounts of cellulose present.

The fungi are generally sub-divided into four groups based on the structure of their sexual stages, although there is some controversy over the number. These are the Phycomycetes, Ascomycetes, Basidiomycetes, and Deuteromycetes (Fungi Imperfecti).

The Phycomycetes are the simplest group, although considerable differences can be found within them depending on whether the species being studied is aquatic or terrestrial. They have two common features which distinguish them from other fungi. The first is the asexual spores which are always endogenous, i.e. they are formed inside a saclike structure, whereas in other groups the asexual spores are exogenous or formed externally on the tips of the hyphae. Secondly the mycelium in the Phycomycetes has no cross walls (septa), whereas all other groups have cross walls. Phycomycetes commonly found in spoilage situations include species from the genera Mucor and Rhizopus.

Fungi with septa and external (exogenous) asexual spores are divided into Ascomycetes and Basidiomycetes on the basis of the development of their sexual spores. The structures involved, which are morphologically very different, are known as the asci (singular ascus) or basidia (basidium). Numerous fungi from the Ascomycetes are found in spoilage situations, where they may act either by toxin production or cause chemical damage leading to physical deterioration. Species from the genera Pénicillium, Aspergillus, and Fusarium are very common in these situations.

It is obviously impossible to classify any fungus whose sexual stage is unknown, and these species are placed in the Fungi Imperfecti. This is essentially a holding group, and as the sexual stage of an organism is discovered it is transferred to one of the other groups, usually the Ascomycetes or Basidiomycetes. This can cause problems with organisms being found under two generic names, one relating to the perfect or sexual stage, and the other relating to the imperfect or asexual stage.

The basic structure amongst the Ascomycetes, Basidiomycetes and Fungi Imperfecti is the mycelium. There are, however, a number of fungi which have lost the mycelial growth characteristic and become unicellular. These unicellular fungi are known as yeasts. Typically, yeasts are oval cells which multiply by budding or fission. In yeasts that divide by budding, the parent cell forms a bud which enlarges until it is almost as big as the parent, nuclear division takes place and a cross wall is formed producing two cells (see Figure 1.4). Division by fission occurs when a cell grows, then lays down a cross wall to produce two equal sized cells (see Figure 1.5).

(Continues…)Excerpted from Microbiology for the Analytical Chemist by R.K. Dart. Copyright © 1996 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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