Biology 144 Microbial Diversity Study Guide & Practicals 2024 PDF

Biology 144 Microbial Diversity Study guide & Practicals - 2024 Textbook: Biology: The Dynamic Science (5th Edition). Russell, Hertz, McMillan & Benington, 2021. CENGAGE Learning. DEPARTMENT OF MICROBIOLOGY UNIVERSIT...

Biology 144 Microbial Diversity Study guide & Practicals - 2024 Textbook: Biology: The Dynamic Science (5th Edition). Russell, Hertz, McMillan & Benington, 2021. CENGAGE Learning. DEPARTMENT OF MICROBIOLOGY UNIVERSITY OF STELLENBOSCH JC Smuts Building ROOM A302 TEL: (021) 808-5845/7  GENERAL INFORMATION AVAILABLE ON SUNLEARN PowerPoint presentations and links to useful video-clips Instructions for the practical assignments and links to videos. PRACTICAL SESSIONS Two practicals will be presented: Prac 1 – Lecture on laboratory techniques and a short laboratory session Prac 2 – full laboratory session Completion and submission of the reports are compulsory. The information presented in the Practicals will be examined in Test 1. ASSESSMENTS Lectures 1-8 of this submodule will be assessed in A1E1 on 12 August 2024 and will contribute 20% of your final mark. Lectures 9 & 10 will be assessed as part of Test A1T2 (19 September 2024). The two practical assessments (combined) will contribute 5% of your final mark. Self-test questions at the end of each chapter in the Study Guide will help you to test your understanding of the key concepts. GENERAL Use the Forum on SUNLearn to interact with your classmates if you have any queries or observations to share. Contact us via SUNLearn Discussion Forum or Chat Room or by email for urgent matters. Biology 144 2 © Department of Microbiology 2024 Stellenbosch University  MODULE OUTLINE AND STUDY OBJECTIVES The module aims to introduce you to the various groups of microorganisms (viruses, bacteria, fungi and yeasts), their cell structures, life cycles and nutritional requirements. The practical importance of these microorganisms is discussed, with examples of products that are industrially produced. The intention of this module is not to give you detailed information on each group of microorganisms, but to brief you on certain basic aspects as a necessary background for a study in biology and to stimulate you to continue with further studies (and research) in microbiology. However, Microbiology also includes protozoa (Protista) and algae, which will not be covered in this module. Some of the sections are also covered in the prescribed textbook (Russell et al., 2021); the relevant chapters or pages are shown in brackets. MODULE OUTLINE Lecture 1 INTRODUCTION TO MICROBIOLOGY: Characteristics, importance and classification; microscopy Lecture 2 VIRUSES: Brief history, characteristics, morphology, classification, replication, bacteriophages, HIV, emerging viruses (Chapter 17) Lecture 3 & 4 MICROBIAL GROWTH: Environmental factors, microbial growth curves, controlling microbes Lecture 5 & 6 BACTERIA: Morphology, reproduction, diversity & classification, metabolism, environmental importance, horizontal gene transfer (Chapter 26) Lecture 7 FUNGI: Morphology, classification, reproduction, metabolism and environmental importance (Chapter 30) Lecture 8 YEASTS: Saccharomyces cerevisiae: morphology, reproduction, metabolism, alcohol production, model for eukaryotic genetics; examples of other yeasts (Chapter 30) Lecture 9 & 10 MICROBES IN ACTION: Genetic engineering, medical, industrial and environmental microbiology, Research at Dept. Microbiology, US Practical 1 (Week 2) Isolating, handling, counting and storage of microbes Practical 2 (Week 3) Microbial diversity and growth Biology 144 3 © Department of Microbiology 2024 Stellenbosch University  STUDY OBJECTIVES 1. Understand the importance of microbial organisms. 2. Understand the classification of microbial organisms. 3. Be able to identify the most important differences between Eukaryotes, Eubacteria & Archaea. 4. Morphology and replication of viruses 5. Be able to name a few diseases caused by viruses 6. Be able to discuss the replication of bacteriophages (lysogenic versus lytic cycle) 7. Discuss HIV as an example of a retrovirus, and understand important differences with coronaviruses 8. Understand how microbes were first discovered and isolated 9. Understand Koch’s postulates to prove a direct microbe-disease relationship. 10. Be able to discuss the various environmental factors that can influence microbial growth. 11. Know the groupings of microbial organisms based on their environmental growth requirements (e.g. phototrophs, acidophiles, anaerobes and halophiles) 12. Understand how microbes grow, and how we observe and measure their growth 13. Know the types of growth media and how we prepare them 14. Understand the characteristics of the growth phases of a culture in a batch system 15. Be able to discuss different methods for the control of microbial organisms 16. Know the general morphology of bacterial cells and the function of different cellular components 17. Know the different bacterial cell shapes and groupings (also refer to Practical lectures) 18. Recognise the different nutritional requirements of bacteria (autotrophy, heterotrophy, phototrophy, chemotrophy) 19. Discuss the environmental importance of bacteria 20. Understand the different forms of gene transfer 21. Know the most important aspects regarding the morphology and reproduction of fungi 22. Known the five main taxonomic groups and their most important characteristics 23. Discuss the reproductive cycle of the Basidiomycetes 24. Understand the environmental importance of fungi 25. Discuss the basic morphology of S. cerevisiae, S. pombe and C. albicans 26. Discuss the reproductive cycle of Saccharomyces cerevisiae 27. Discuss metabolism in Saccharomyces cerevisiae and its importance in alcoholic fermentations 28. Understand the principles of genetic engineering and some of its applications. 29. Name a few medical applications of microbial organisms. 30. Name a few industrial applications of microbial organisms. 31. Understand the role of microbes in environmental management. Practicals: Understand sterilization techniques and “aseptic technique” Know the use of the streak-, spread- and pour-plate techniques Know the methods used to count microbial cells (living and/or dead) Understand the basic differences between yeasts, fungi and bacteria when viewed under a microscope (cell morphology) and when seen on agar in a Petri dish (colony morphology) Biology 144 4 © Department of Microbiology 2024 Stellenbosch University  CONTENTS INTRODUCTION TO MICROBIOLOGY.................................................................................................................................. 7 GENERAL CHARACTERISTICS OF MICROORGANISMS 7 IMPORTANCE OF MICROBIAL ORGANISMS 7 CLASSIFICATION OF MICROORGANISMS (RUSSELL ET AL., P. 10-14) 8 TAXONOMIC HIERARCHY (RUSSELL ET AL., P. 565-570) 8 Prokaryotes versus Eukaryotes..........................................................................................................................................9 OBSERVING MICROBES: MICROSCOPY (RUSSELL ET AL., P. 73-76) 10 Bright-Field Microscopy...................................................................................................................................................10 Electron Microscopy........................................................................................................................................................10 SELF-TEST 10 VIRUSES........................................................................................................................................................................... 11 BRIEF HISTORY 11 GENERAL CHARACTERISTICS 11 MORPHOLOGY 11 CLASSIFICATION 12 REPLICATION 12 BACTERIOPHAGES 13 Lytic cycle (virulent phages; Fig. 17.7).............................................................................................................................13 Lysogenic cycle (temperate phages; Fig. 17.8)................................................................................................................13 HUMAN IMMUNODEFICIENCY VIRUS (P. 383-384) 13 SELF-TEST 14 MICROBIAL GROWTH....................................................................................................................................................... 15 FIRST EVIDENCE OF MICROBIAL GROWTH AND ITS LINK TO DISEASE 15 FACTORS THAT INFLUENCE MICROBIAL GROWTH 15 GROWTH MEDIA 17 MICROBIAL GROWTH IN BATCH SYSTEMS 18 CONTROLLING MICROBIAL GROWTH 18 SELF-TEST 19 BACTERIA......................................................................................................................................................................... 20 GENERAL MORPHOLOGY 20 REPRODUCTION 21 DIVERSITY AND CLASSIFICATION 21 METABOLISM 21 ENVIRONMENTAL IMPORTANCE 22 HORIZONTAL GENE TRANSFER (RUSSELL ET AL., P. 374-375) 23 Example of HGT...............................................................................................................................................................23 SELF-TEST 23 FUNGI.............................................................................................................................................................................. 25 GENERAL MORPHOLOGY 25 Multicellular (filamenteous) molds.................................................................................................................................25 Unicellular yeasts.............................................................................................................................................................25 Dimorphic Fungi..............................................................................................................................................................25 CLASSIFICATION 25 Biology 144 5 © Department of Microbiology 2024 Stellenbosch University  REPRODUCTION 26 Lifecycle of Basidiomycetes.............................................................................................................................................27 METABOLISM 27 ENVIRONMENTAL IMPORTANCE (P. 674-678) 27 SELF-TEST 28 YEASTS............................................................................................................................................................................. 29 INTRODUCTION 29 SACCHAROMYCES CEREVISIAE (P. 677-678) 29 Reproduction...................................................................................................................................................................29 Metabolism......................................................................................................................................................................30 Alcohol production..........................................................................................................................................................30 S. cerevisiae as a model for eukaryotic genetics..............................................................................................................31 EXAMPLES OF OTHER YEASTS 31 SELF-TEST 31 MICROBES IN ACTION...................................................................................................................................................... 32 GENETIC ENGINEERING 32 MEDICAL MICROBIOLOGY 32 INDUSTRIAL MICROBIOLOGY 33 ENVIRONMENTAL MICROBIOLOGY 34 RESEARCH @ DEPARTMENT MICROBIOLOGY, US 34 SELF-TEST 35 PRACTICAL GUIDE............................................................................................................................................................ 36 OUTLINE AND STUDY OBJECTIVES FOR PRACTICALS 36 PRACTICAL 1: ISOLATION, HANDLING, COUNTING AND STORAGE OF MICROBES............................................................. 37 ASEPTIC TECHNIQUE 37 STERILISATION OF MEDIA AND EQUIPMENT 37 MEASURING MICROBIAL GROWTH 38 Spread-plate technique..................................................................................................................................................38 Pour-plate technique......................................................................................................................................................39 ISOLATION AND STORAGE OF PURE CULTURES 39 Streak-plate technique...................................................................................................................................................39 COUNTING BACTERIOPHAGES 40 SAMPLING MICROBIAL DIVERSITY IN THE ENVIRONMENT 41 PRACTICAL 1: INSTRUCTIONS 41 PRACTICAL 2: MICROBIAL DIVERSITY AND GROWTH........................................................................................................ 42 PREPARING A WET MOUNT FOR MICROSCOPY 42 OBSERVING MICROBES: MICROSCOPY (RUSSELL ET AL., P. 73-76) 42 Bright-Field Microscopy...................................................................................................................................................42 MICROBIAL GROWTH ON SOLID MEDIA 44 PRACTICAL 2: INSTRUCTIONS 46 Biology 144 6 © Department of Microbiology 2024 Stellenbosch University  INTRODUCTION TO MICROBIOLOGY Microbiology is an exceptionally broad discipline, and diverse areas such as biochemistry, cell biology, genetics, taxonomy and even engineering form parts of medical, food, industrial and environmental microbiology. The importance of microbiology cannot be overemphasised, considering that more than 60% of all biomass is of microbial origin. Many of the products we consume daily are produced by microorganisms, e.g. bread, cheese, beer, antibiotics, vaccines, vitamins and enzymes, to name but a few. Microorganisms are indispensable components of our ecosystem, and they play an essential role in the carbon, oxygen, nitrogen, and sulphur cycles in terrestrial and aquatic systems. They also serve as a source of nutrients at the base of all food chains. GENERAL CHARACTERISTICS OF MICROORGANISMS  Diversity in size, but generally microscopic  Many are unicellular; others are multicellular  Less complex than plants and animals  Rapid growth rates  Ubiquitous  Perform complex functions in nature IMPORTANCE OF MICROBIAL ORGANISMS Prokaryotes were largely responsible for creating the atmosphere and soil properties billions of years ago. Cyanobacteria are thought to have added oxygen to the Earth’s atmosphere as a by-product of their photosynthesis. Life on Earth depends on the cycling of chemical elements between organisms and the physical environments in which they live, i.e., between ecosystems' biological and physical elements. Prokaryotes and fungi play many key roles in this chemical cycling through their involvement in decomposition and fixation. During the decomposition of biological material, carbon, nitrogen, phosphorus, sulphur, and other elements are released back into the environment by decomposers. Other prokaryotes play essential roles in the fixation of these elements from the non-living environment to return them to living organisms. The organic compounds that plants, algae, and photosynthetic prokaryotes produce from CO2 pass through the food chain to serve as building blocks for heterotrophic organisms (animals, fungi and non-photosynthetic protists). Various prokaryotes play an important role in recycling nitrogen; only certain prokaryotes can reduce free nitrogen gas (N2) to ammonia (NH3) used to build amino acids, DNA and other N-containing molecules. When the organisms that contain these molecules die, other prokaryotes, called denitrifiers, return the nitrogen to the atmosphere, completing the cycle. In aquatic environments, nitrogen fixation is carried out mainly by cyanobacteria such as Anabaena, which contain specialised cells (heterocysts) impermeable to oxygen. In soil, nitrogen fixation occurs in the roots of plants that harbour symbiotic colonies of nitrogen-fixing bacteria, e.g. Rhizobium in legumes, Frankia in many woody shrubs, and Anabaena in water ferns. Microorganisms are responsible for various human, animal and plant diseases and have played a significant role in history. In 1347, Europe was struck by the Plague or “Black Death”, caused by the bacterium Yersinia pestis, which killed more than a third of the population (about 25 million people) by 1351. During the following century, the disease struck again, eventually wiping out 75% of the European population. Some historians believe this disaster changed European culture and prepared the way for the Renaissance. Biology 144 7 © Department of Microbiology 2024 Stellenbosch University CLASSIFICATION OF MICROORGANISMS (RUSSELL ET AL., P. 10-14) Although all organisms share some characteristics, several vital differences divide organisms into groups. The most important groupings are based on their evolutionary relationships, while molecular techniques have shed new insight into microbial diversity. Taxonomy – or the classification of organisms - is a necessary part of biology that provides a reference point for further discussion of various organisms, their habitats, interactions, genetic relationships, biochemistry, etc. Organisms are classified into different “levels” and “sub-levels” based on shared characteristics. It is important to remember that the classification system is not fool-proof and that organisms get reclassified as we learn more about their characteristics. The relevant grouping also tells us more about their genotypic characteristics (chromosomes or genomes, gene structures, DNA sequences, etc.). All of this determines the phylogenetic position of a single organism (species) or a group of organisms, in other words, where they fit into the “tree of life”. This is of particular importance when a new isolate needs to be identified, and it tells us more about the evolution of organisms. If the adaptation resulted in significant genetic changes, it may represent a new species. If more than one species with the same characteristics is identified, they are grouped into the same genus. More than one genus (plural: genera) are grouped into the same family, etc., as described below. TAXONOMIC HIERARCHY (RUSSELL ET AL., P. 565-570) The most widely accepted classification schemes are hierarchical, with each subunit encompassing a smaller but more similar group of organisms. Genera (e.g. Escherichia) with similar characteristics are grouped into a Family (e.g. Enterobacteriaceae); Families with similar characteristics are grouped into Orders (e.g. Enterobacteriales); Orders with similar characteristics are grouped into Classes (e.g. Gammaproteobacteria), and Classes with similar characteristics are grouped into a Phylum (e.g. Proteobacteria). Another two levels of classification, known as Kingdom and Domain, are also sometimes used (e.g. Kingdom Animalia, Domain Eukarya). The first classification systems were based on only two kingdoms, i.e. animals and plants, followed by Carl Woese’s six-kingdom system (Archaebacteria, Eubacteria, Protista, Fungi, Plantae and Animalia). Biologists currently use a classification system that groups all living organisms into three domains, i.e. Archaea, Bacteria and Eukarya. Viruses can only replicate in association with prokaryote or eukaryote host cells; they are thus not considered living organisms and don’t feature in any domains. Carl Linnaeus developed the binomial nomenclature system to enable taxonomists to name and classify organisms more precisely (Russell et al., p. 534). The Latinized name in italics consists of two parts: the first part, which is capitalised, is the generic name (e.g. Escherichia). The second part, non-capitalised, is the specific epithet (coli). The full species name is Escherichia coli. The generic name is often abbreviated, e.g. E. for Escherichia. The specific epithet is always written fully and does not change upon reclassification. Each species consists of several strains (a population of organisms that descended from a single organism or pure culture isolate). One strain of a species, usually the one which is the most representative of the species, is named the type strain. The type strain is used as a reference strain in taxonomical studies to classify a microbe at least to the species level. Domain Eukarya The Domain Eukarya (or Eukaryota) includes the kingdoms Protista, Fungi, Plantae and Animalia. Protista (unicellular, colonial or multicellular protists) display various reproductive cycles, energy and carbon derivation. Fungi are chemoheterotrophic, mostly multicellular decomposers with a unique reproductive cycle. Plantae are multicellular photoautotrophs with rigid cell walls, whilst Animalia are multicellular chemoheterotrophs with cell walls. It is generally believed that the Domain Eukarya arose from the first prokaryotic organisms more than 1.7 billion years ago. It includes all organisms with eukaryotic cells, i.e. those with membranous organelles (including mitochondria and chloroplasts). The organisms in this domain represent most organisms we see each day and share the following characteristics:  Eukaryotic cell structure Biology 144 8 © Department of Microbiology 2024 Stellenbosch University   Unicellular (some Protists and yeasts), colonial (some Protists) or multicellular (most Fungi, Plantae, and Animalia) organisms  Asexual reproduction via mitosis; sexual reproduction that allows the exchange of genetic information  A variety of modes of access to energy and carbon, vary between kingdoms The evolution of the domain Eukarya is believed to have occurred from early prokaryotes by two primary processes: 1. All membranous organelles - except for the mitochondria and chloroplasts - are ultimately continuous with the plasma membrane and each other. They represent the endomembrane system that includes the nucleus (surrounded by a double membrane), rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, vacuoles and transport vesicles (that move between the components). The eukaryotic endomembrane system arose by a process known as membrane in-folding: the plasma membranes of the original prokaryotic cells that gave rise to the first eukaryote ones folded inward and developed further to give rise to various membrane structures. 2. The mitochondria and chloroplasts of eukaryotic cells are proposed to have arisen from small prokaryotic cells that established residence inside larger ones by endosymbiosis, deriving their outer membranes from the host cell's plasma membrane. Mitochondria arose from small heterotrophic prokaryotes that had excellent efficiency in aerobic respiration, and chloroplasts originated from small photosynthetic autotrophs. They eventually evolved to assume a closely interdependent relationship with the cell and cannot even survive today. Domain Bacteria Organisms in the domain Bacteria lack membrane-bound organelles such as the nucleus and endoplasmic reticulum. All members of domain Bacteria are prokaryotes, with their small size, ability to rapidly reproduce, and diverse habitats, making them the most abundant and diversified group of organisms on Earth. There are many kinds of eubacteria (true bacteria), and their evolutionary links are poorly understood. Comparisons of the nucleotide sequences of ribosomal RNA (rRNA) molecules are beginning to reveal how these groups are related to one another and the other two domains. One view of our current understanding is that the root of the ‘tree of life’ is within the eubacterial domain, with the archaebacteria and eukaryotes being more closely related to each other than to eubacteria. Domain Archaea The most primitive group, the archaebacteria, are restricted to marginal habitats such as hot springs or areas of low oxygen concentration. Archaebacteria (now more commonly referred to as the Archaea) have significant differences in their cell walls and biochemistry compared to the Bacteria. Their gene translation machinery relates more to that of Eukarya than Bacteria, and they have some genes with introns, which are absent in Bacteria. Archaea includes bacteria that grow under extreme conditions, e.g. methanogenic bacteria (grow anaerobically, producing methane), halophiles (grow at high salt concentrations) and thermophiles (grow at temperatures of 80 - 100 °C). Since Bacteria and Archaea inhabit some environments thought by paleontologists to resemble what the early Earth was like, it is believed that both descended from a common ancestor and that the Eukarya later split from the Archaea. Prokaryotes versus Eukaryotes Prokaryotes and Eukaryotes share some characteristics, such as containing DNA as genetic material; their cytoplasm is membrane-bound; they use ribosomes for protein synthesis and have similar metabolic pathways. The three key characteristics unique to eukaryotes are compartmentalisation, multicellularity and sexual reproduction. Eukaryotes have a nucleus and membrane-bound organelles. The DNA of prokaryotes floats freely around the cell; the DNA of eukaryotes is held within its nucleus. The organelles of eukaryotes allow them to exhibit much higher levels of intracellular division of labour than is possible in prokaryotic cells. Other apparent differences between prokaryotes and eukaryotes include:  Size: Eukaryotic cells are, on average, 10-fold bigger than prokaryotic cells. Biology 144 9 © Department of Microbiology 2024 Stellenbosch University  Genomic composition: The DNA of eukaryotes is much more complex and extensive than the DNA of prokaryotes. Whereas prokaryotic DNA is ‘naked’, eukaryotic DNA is compacted around histones.  Cell Wall: Most prokaryotes have a cell wall composed of peptidoglycan, a single large polymer of amino acids and sugar. Eukaryotic cell walls don’t contain peptidoglycan. OBSERVING MICROBES: MICROSCOPY (RUSSELL ET AL., P. 73-76) (Note: This section will be discussed as part of the Practical lectures) The first person to observe and describe microorganisms accurately was the amateur microscopist Antonie van Leeuwenhoek (1632 - 1723) of Delft, Holland. Microorganisms vary in size, generally ranging between 30 - 300 nm (viruses), 1 - 4 µm (bacteria) and >10 µm (yeasts, moulds, protozoa and algae). Microscopy is thus the only means to observe and study individual cells. Two types of microscopes are routinely used, i.e. the light microscope (bright- field, dark-field, phase-contrast, fluorescent microscopy, and confocal laser microscopy) and the electron microscope (transmission- or scanning microscope). Bright-Field Microscopy An ordinary bright-field microscope consists of an eyepiece (ocular) with a 10x magnification power and two or three objectives with lenses of 10x, 40x and 100x magnifying power. The total magnification is calculated by multiplying the objective and ocular magnifications—for example, a 40x objective and 10x ocular yields an overall magnification of 400x. Electron Microscopy Electron microscopes operate with electron beams instead of light. The resolution of electron microscopes can be as high as 0.1 nm (1 Å), depending on the type of microscope. The resolution of a modern transmission electron microscope (TEM) is roughly 1000 times better than that of a light microscope, and objects 0.5 nm apart can be differentiated. In a TEM, the electrons are passed through the specimen. Thin sections of a specimen must be prepared and often stained with metal ions. The TEM is useful in studying internal organelles or virus particles. The scanning electron microscope (SEM), on the other hand, produces images from electrons emitted (“bounced” off) by the object's surface and helps study the surface structures of cells. The resolution of an SEM is approximately 7 nm. SELF-TEST 1. When we are ill and have to take a course of antibiotics, the drugs can kill bacteria without harming our cells. List the differences between Bacteria and Eukarya that YOU think would make it possible to target drugs against bacteria alone. (3) 2. Match the definition/description with the correct term: (4) Definition/description Choice of terms Answer Taxonomic domain associated with Prokaryotes, peptidoglycan, Archaea, protists, methanogenesis Eukarya Basic taxon of classification system Species, strain, phylum, prototype, phenotype Kingdom that Saccharomyces Bacteria, Saccharomyces, Eukarya, protista, cerevisiae belongs to fungi DNA-associated feature that ONLY rRNA gene, intron, nucleus, codon, mitosis occurs in Eukarya Biology 144 10 © Department of Microbiology 2024 Stellenbosch University  VIRUSES (Russell et al., Chapter 17, Section 17.2) BRIEF HISTORY Epidemics caused by viruses began when humans developed more densely populated communities that allowed viruses to spread rapidly and become endemic. Viral infections of plants and livestock also increased, with devastating consequences as humans became dependent on agriculture. Smallpox and measles viruses are among the oldest in humans, having evolved from viruses that infected other animals. Influenza pandemics have been recorded since 1580 and have occurred with increasing frequency in subsequent centuries. The influenza pandemic of 1918–1919 killed millions of people, making it one of the most devastating in history. The nature of viruses remained unknown until the invention of the electron microscope in the 1930s when the science of virology gained momentum. In the 20th century, many diseases were found to be caused by viruses. AIDS (Acquired Immune Deficiency Syndrome), caused by HIV (Human Immunodeficiency Virus), is the first great pandemic of the second half of the 20th century. In 2017, there were about 1.8 million new cases of HIV worldwide, with an estimated 940 000 people dying from AIDS-related illnesses. An estimated 36.9 million people worldwide live with HIV, and 21.7 million people are receiving antiviral therapy for HIV. Although there is a decline in the HIV infection and death rate, it remains one of the most significant health problems in Southern Africa. New viruses may emerge due to mutations, spreading from a previously isolated community or changing hosts. Examples of emerging (new) viruses include SARS (Severe Acute Respiratory Syndrome), Ebola, Zika and variants of Influenza viruses. The SARS-CoV2 virus, responsible for the COVID-19 pandemic, is believed to be either a man- made virus or one that crossed over from animals. GENERAL CHARACTERISTICS Viruses cannot replicate independently of their host cells; they can’t grow or metabolise and are thus not considered living cells. Virologists refer to the number of infectious particles, or plaque-forming particles, rather than the number of living particles. However, viruses have shown a remarkable ability to mutate the glycoproteins in the capsids or envelope to allow entry into their host cells. Viruses are metabolically inactive outside the host cell and thus depend on the host cells for replication. Most viruses are host-specific (Tables 17.1 and 17.2); although some have a broader host range, none are known to cross the eukaryotic/prokaryotic boundary. Factors that affect the host range include:  Correct attachment: viral protein needs to bind to a receptor on the cell surface (e.g. HIV is primarily restricted to cells with the CD4 antigen on their surface).  After entry, the appropriate cellular machinery for virus replication must be available (e.g., some DNA viruses can only replicate in dividing cells with high levels of dNTPs for viral DNA synthesis).  There must be mechanisms to release virus particles to ensure the infection can spread. MORPHOLOGY Viruses are unique, simple organisms lacking cell structures and metabolic processes. They differ significantly in appearance and size, ranging from 30 nm to 300 nm. The first “giant” virus, Mimivirus, was discovered in 2003. Since then, other larger viruses, some bigger than bacteria or even eukaryotic cells, have been found in various environments. Some researchers believe that giant viruses may have evolved from an ancestral cell very different from what gave rise to bacteria, archaea, and eukaryotes. Biology 144 11 © Department of Microbiology 2024 Stellenbosch University The genetic material may consist of single- or double-stranded DNA or RNA that may be linear or circular. All viruses are composed of one or more molecules of either DNA or RNA, enclosed by a protective protein coat called a capsid. Capsids are self-assembling proteins and may be icosahedral, helical or complex (p. 394 and Fig. 17.6). Many viruses encode a few structural proteins - those that make up the mature virus particle (or virion) and sometimes an enzyme that participates in the replication of the viral genome (p. 393-394). Other viruses can encode many more proteins; most of these participate in replication but do not end up in the mature virion. General capsid structure includes:  Helical – hollow protein cylinders with capsid proteins arranged in a rod-like spiral around the genome  Icosahedral (polyhedral) – 20 equilateral triangular units that form an icosahedral structure  Complex – neither purely icosahedral nor helical Some viruses contain an additional membranous envelope outside the capsid containing complex carbohydrates, lipids, and proteins, but this is acquired from the host cell, usually by budding through a host cell membrane (Figure 17.9). Five primary structural forms of viruses in nature:  Naked icosahedral, e.g. poliovirus, adenovirus, hepatitis A virus.  Naked helical, e.g. tobacco mosaic virus. No human viruses with this structure are yet known.  Enveloped icosahedral, e.g. herpes virus, yellow fever virus, rubella virus.  Enveloped helical, e.g. rabies virus, influenza virus, mumps virus, measles virus.  Complex, e.g. pox virus, bacteriophages. CLASSIFICATION Primary characteristics used in classification include the nature of their genome and their physical structure:  Nucleic acid (RNA or DNA; single- or double-stranded; etc.)  Virion structure (icosahedral/helical/complex; naked or enveloped; etc.) Secondary characteristics include the virus's replication strategy. Occasionally, viruses grouped based on the above criteria can be classified as a subgroup with fundamentally different replication strategies. REPLICATION Since viruses depend on the host cell for propagation, viruses are cultured in the laboratory by inoculating living host cells or cell lines. Virus concentration is determined by virion counts or the number of infectious units. Viruses can exist in two phases – extracellular and intracellular. Extracellular viruses possess few or no enzymes and cannot replicate. When intracellular, viral nucleic acids start replicating, this induces the host metabolism to synthesise virion components. The host biosynthetic machinery is thus hijacked to replicate virus particles. The general sequence of the virus replication process:  Infection of the host (attachment, entry and uncoating)  Transcription of viral genes  Replication – new nucleic acid generated  Translation – protein synthesis  Assembly of new virus particles  Release of the mature virion Biology 144 12 © Department of Microbiology 2024 Stellenbosch University BACTERIOPHAGES These viruses infect bacteria, such as Escherichia coli, and exhibit lytic or lysogenic life cycles. During the lytic cycle, viral replication is followed by host cell lysis to release the virus particles. During the lysogenic cycle, the viral genetic material is incorporated into the host genome for replication sometime in the future. The infected bacterium, therefore, does not start producing new virus particles immediately; the phage is latent and is called a prophage. Bacteriophage Lambda can carry out both lifecycles: Lytic cycle (virulent phages; Fig. 17.7) Five stages of the bacteriophage lytic cycle are recognised: 1. Attachment: portions of capsid combine with bacterial cell wall receptors in a specific lock-and-key manner. 2. Penetration: viral enzyme digests part of the cell wall and injects viral DNA into the bacterial cell. 3. Biosynthesis: biosynthetic apparatus of the host is taken over for viral DNA and coat protein production. 4. Maturation: DNA and capsid proteins are assembled to produce new phage particles. 5. Release: Lysozyme, coded for by a viral gene, is produced and disrupts the bacterial cell wall, thereby releasing phage particles and causing host cell death. Lysogenic cycle (temperate phages; Fig. 17.8)  Following attachment and penetration, the viral DNA is integrated into the genome (DNA) of the bacterial cell; the virus is now called a prophage.  Prophage is replicated within the host DNA, and all bacterial daughter cells (lysogenic cells) carry a copy of the viral DNA.  Environmental factors such as UV radiation may induce the prophage to excise from the host genome and induce a lytic cycle, thus producing new virus particles. During the assembly of virus particles or excision of a prophage, host DNA may be packaged in the heads of the viral particle. Bacterial genes may then be transferred to the next host via the bacteriophage. The transfer of the host DNA by this mechanism is termed transduction. During the lysogenic lifecycle, virus genes are expressed as host genes. In some cases, the expression of these viral genes may alter the host cell in novel ways. An example of this “phage conversion” is Vibrio cholerae, which usually exists in an aquatic environment in a harmless form. Conversion to the deadly disease-causing bacterium is due to infection of the V. cholerae cell by a bacteriophage that integrates a gene coding for the cholera toxin into the bacterial genome. HUMAN IMMUNODEFICIENCY VIRUS (P. 383-384) Human Immunodeficiency Virus (HIV) is a complex retrovirus with a genome of two identical positive, single- stranded RNA molecules. The disease is thought to have originated in Western Africa via transfer from primates to humans but has since spread worldwide. HIV infects white blood cells of the human immune system with CD4+ receptors on their host cell membranes (T- helper cells), allowing the virus to enter the cytoplasm where it can replicate. The HIV genetic material includes genes encoding viral enzymes (reverse transcriptase, protease and integrase) integrated into the host’s genome. Reverse transcriptase converts the single-stranded RNA genome of HIV to double-stranded cDNA (copy DNA), which is integrated into the host genome (= provirus) through the action of the viral integrase. As normal cellular DNA transcription occurs, the HIV-derived messenger RNA (mRNA) is also produced from the HIV DNA. Translation of viral mRNA results in new virus production, i.e. biosynthesis, maturation and release. The DNA never leaves the host DNA, in contrast to a prophage that is excised. Furthermore, the release does not involve the destruction of the host cell but occurs through budding, whereby the virus carries away part of the host cell. Biology 144 13 © Department of Microbiology 2024 Stellenbosch University After release, HIV can infect more human CD4+ cells; the high decrease in CD4+ T cells causes the loss of cell- mediated immunity, thereby causing the body to succumb to attacks from opportunistic infections. The enzyme reverse transcriptase is error-prone and incorporates one mistake (mutation) per cDNA copy produced. This high mutation rate, coupled with a deteriorating immune system, results in immune system collapse, resulting in AIDS. AIDS is a collective name for several diseases emanating from a defective immune system. Without treatment, death results within 10 - 15 years following infection. CORONA VIRUS (SARS-CoV-2) The SARS-CoV-2 virus causes Severe Acute Respiratory Syndrome (SARS) in some patients. It is a single-stranded RNA virus, with a replication mechanism different from HIV. Its positive-strand genome acts directly as messenger RNA translated by the host cell’s ribosomes, producing a viral replication enzyme that can make RNA copies without needing a DNA template. You do not need to know the details of its replication strategy, but you must understand how it differs from HIV. SELF-TEST 5-min Khan Academy video – lytic vs lysogenic virus replication strategies: https://www.youtube.com/watch?v=J4BN4dARpio 23-min Khan Academy video overview of viruses: https://www.youtube.com/watch?v=0h5Jd7sgQWY 1. Explain why viruses are considered to be “non-living”. (3) 2. How does an enveloped virus obtain its envelope – does its genome code for it? (3) 3. What is the function of the reverse transcriptase enzyme and why does a retrovirus carry them inside its capsid? (2) 4. What happens when a prophage is activated by, for example, UV light? (3) Biology 144 14 © Department of Microbiology 2024 Stellenbosch University  MICROBIAL GROWTH Microorganisms are ubiquitous: they are in the air we breathe, the food we eat, and on surfaces we touch daily, but we cannot see them. Many factors influence microbial growth, and we can manipulate many of these to promote growth (e.g., study them in the laboratory) or inhibit growth (e.g., sterilize something or treat infections). FIRST EVIDENCE OF MICROBIAL GROWTH AND ITS LINK TO DISEASE The German physician Robert Koch (1843 - 1910) was the first to produce evidence that bacteria could cause disease. Koch injected healthy mice with cells of Bacillus anthracis, which he obtained from sick animals. After transferring anthrax by inoculation through a series of 20 mice, he incubated a piece of mouse spleen containing the anthrax bacillus in beef serum. The bacilli grew, reproduced and produced spores. When the isolated bacilli or spores were injected into mice, anthrax developed. His criteria for proving the relationship between a microorganism and a specific disease are known as Koch's postulates, which can be summarised as follows: 1. The respective microorganism must be present in every sick individual but absent from healthy individuals. 2. The suspected microorganism must be isolated and grown in pure culture. 3. The same disease must result when the isolated microorganism is inoculated into a healthy host. 4. The same microorganism must be isolated again from the diseased host. The first growth medium used by Robert Koch consisted of boiled potatoes, solidified with gelatine, which he used to isolate bacterial pathogens. However, gelatine is hydrolysed by many bacteria and melts at temperatures above 28 °C. The solution to this problem came from the wife of one of Koch's assistants, Fannie Hesse. She was using agar (a kelp extract) to make fruit jellies and suggested using agar instead of gelatine since microorganisms do not destroy agar, and it only melts at 100 °C. During that time, one of Koch's assistants, Richard Petri, developed the Petri dish, routinely used to isolate and study microorganisms. Koch isolated the bacillus that caused tuberculosis, later named Mycobacterium tuberculosis, in 1882. The following 30 to 40 years are the golden period during which most disease-causing (pathogenic) bacteria were isolated and identified. FACTORS THAT INFLUENCE MICROBIAL GROWTH Nutrients Microbes acquire environmental nutrients, with carbon, oxygen, hydrogen, phosphorus, potassium, nitrogen, sulphur, calcium, iron, sodium, chlorine, magnesium, and other essential elements. However, microbes vary significantly in source, chemical form, and amount of nutrients they need. Macro-nutrients are required in higher amounts for cell components and metabolism (e.g. carbon, oxygen and phosphate for synthesising carbohydrates and proteins). Micronutrients or trace elements are needed at lower amounts for enzyme function and to maintain protein structure (e.g. zinc, manganese and nickel). Depending on their carbon source, microbes are classified as either heterotrophs or autotrophs: Heterotrophs must obtain their carbon in an organic form, e.g. carbohydrates, proteins, lipids and nucleic acids. Some organic nutrients exist in a simple form for absorption (monosaccharides, amino acids), but many larger molecules must be digested before they can be absorbed. Not all heterotrophs can use the same carbon sources; some are restricted to a few substrates, whereas others can metabolise more than 100 different substrates. Autotrophs use CO2 (or other forms of inorganic carbon) as their carbon source and convert it into organic compounds. These organisms are not dependent on other living cells and form the base of the food chain that supports all other life forms. Biology 144 15 © Department of Microbiology 2024 Stellenbosch University Metabolism refers to a cell's biochemical assimilation (anabolic pathways) and nutrient dissimilation (catabolic pathways). Anabolic pathways include reductive processes leading to the production of new cellular material, whereas catabolic pathways are oxidative processes that remove electrons from substrates or intermediates used to generate energy. These processes use NADP or NAD as co-factors that can be recycled to generate energy in the form of ATP. It is important to note that microorganisms can adapt to varying growth environments, and the preferred pathways will depend on their immediate environmental conditions. Glucose is the most popular source of energy production in microbial organisms that convert glucose to pyruvate via glycolysis, whereby the production of energy (in the form of ATP) is coupled to the generation of reducing power (NADH) for biosynthetic pathways (Russell et al., p. 147-154). In the presence of oxygen (respiration), pyruvate is oxidatively decarboxylated via acetyl Co-enzyme A and the citric acid cycle to yield two molecules of CO2 and reductive equivalents in the form of NADH and FADH2. In the absence of oxygen (fermentation), pyruvate is decarboxylated to acetaldehyde, followed by a reduction to ethanol and two molecules of CO2. During this process, NAD is recycled to maintain the redox balance. Temperature Microbes have preferred temperature ranges: most bacteria and fungi prefer more moderate temperatures with an upper limit below 70 °C, whereas Archaea can survive extreme temperatures – some very cold (just above 0 °C) and others very hot (80 °C to well over 100 °C). Cold-loving microbes or psychrophiles grow optimally at 15 °C, ranging between 0 °C and 20 °C. A huge and diverse population of psychrophilic prokaryotes can be found in Antarctica. Psychrotrophs generally prefer warmer conditions (20 – 30 °C), but can survive a wide range of temperatures (0 – 35 °C) and grow on refrigerated foods. Most microbes (many human pathogens) are mesophiles, preferring a 20 – 45 °C range. Thermophiles have an optimum temperature of 55 – 65 °C, but can survive over a range of 45 – 100 °C. They can be found in deep-sea volcanoes, hot water springs or compost heaps. Hyperthermophiles (optimum greater than 80 °C) are at the upper limits of life – the archaeans Geogemma barossii and Methanopyrus kandleri hold the record for reproduction at the highest temperatures, i.e. 121 °C and 122 °C, respectively. pH Each microbial species grows within a defined pH range, with maximum growth at its optimum pH. Most bacteria are neutrophiles (grow at pH 5.5 to 8.5, but prefer pH 7); others are acidophiles (pH 1 – 5.5) or alkalophiles (pH 8.5 – 11.5). Fungi generally prefer slightly acidic conditions (pH 4 to 6) and are known as pseudophiles. Oxygen Oxygen levels in the environment can vary enormously, often determining which microbes can grow in specific niche areas. For example, a lake will have highly aerated waters at its surface, less oxygen at increasing depths, and no oxygen in the thick sediment at the bottom. Microbes that find oxygen toxic and cannot grow in its presence are obligate anaerobes (p. 573) and have to be cultured in special anaerobic cabinets in the presence of N2 and CO2. All other microbes require and/or tolerate atmospheric oxygen (20% O2) and fall into various categories:- obligate aerobes (completely dependent on O2; facultative anaerobes (do not require oxygen but grow better in the presence of oxygen); aerotolerant (not influenced by the presence or absence of oxygen; and microaerophiles (need 2% - 10% O2). Molds and fungi are aerobic, whereas yeasts are mostly facultative anaerobes. Bacteria are found in most of the groupings. Example: Microbes in fresh versus polluted river water: The type of microbial populations differs between unpolluted and polluted fresh water. Unpolluted water from mountain streams is usually low in organic nutrients, with a limited number of bacteria, dominated by autotrophic bacteria. Large amounts of organic matter from sewage and industrial activities are present in polluted waters, and the microbes are usually heterotrophic. The digestion of organic matter by these organisms is often incomplete, producing acids, alcohols and various gases. Coliform (such as E. coli) is the major type of bacteria, but non-coliform bacteria, such as Streptococcus, Proteus and Pseudomonas are usually also present. Biology 144 16 © Department of Microbiology 2024 Stellenbosch University Under certain conditions, nutrients enter the river from sources like sewage treatment plants or urban run-off, causing the polluting organisms to multiply rapidly and consume most of the available oxygen in the water. The oxygen depletion will kill most of the fish and other river inhabitants, but also results in a layer of dead organisms, mud and silt accumulating at the bottom, where anaerobic species of Clostridium, Desulfovibrio, etc., will flourish. These organisms produce gases, such as H2S, that can poison the water and kill the river ecosystem. Osmotic pressure Water activity (AW) refers to the amount of water available to a cell for growth. Pure water has an AW = 1, whereas water containing many solutes (such as salts or sugars) has a lower AW (e.g. 0.6 in honey). Bacteria obtain most nutrients from their watery environment and are sensitive to osmotic pressure. All bacterial, plant or animal cells regulate the movement of water molecules across their membranes (Fig 5.10). In a hypotonic solution, cells must prevent too much water from entering the cell as the cell may burst (lyse). Microbes have rigid cell walls to maintain their shape and accumulate glycerol and similar compounds to counteract the effect of this water pressure. In a hypertonic solution, microbes have too little water outside the cell and face the threat of dehydration. When too much water is lost from a cell, the plasma membrane pulls away from the cell wall (plasmolysis), and the cell dies. Many bacterial species surround themselves with a capsule or mucilage layer, such as glycan, to counteract dehydration. Osmotolerant organisms can grow over a wide range of water activities, e.g. microbes that cause spoilage of sweet foods (AW = 0.98). Fungi can grow at a lower AW than bacteria and cause spoilage of dry foods such as bread. Some microbes (osmophiles) are adapted to grow only under high osmotic pressures, such as halophiles that prefer high concentrations of NaCl. Archaea are generally more resistant to extreme environments than bacteria (p. 582); e.g. haloarchaea that thrive in the Dead Sea require 20-30% NaCl to survive. These organisms are adapted for this purpose: the plasma membrane and cell walls are stabilized by high sodium ion concentrations; they accumulate potassium ions in their cytoplasm to balance the ionic strength outside the cell, and their cell walls lack peptidoglycan to allow for more cell wall flexibility. GROWTH MEDIA To investigate the abundance and diversity of microbes, we need to prepare media in which most microorganisms can grow. Growth media can be divided into two main groups:  Chemically defined (synthetic) media comprises known ingredients at known concentrations.  Complex media is rich in vitamins and other nutrients; the exact chemical composition varies between suppliers (e.g. yeast extract). Various synthetic and complex media are commercially available. Preparation of these media merely requires the addition of a defined mass to a specific volume and then autoclaving for 15 min at 121 °C at 100 kPa. In addition to the compounds mentioned above, a suitable environment must be created for the microorganisms, i.e. the appropriate temperature, pH, osmotic pressure, atmospheric oxygen, etc. Media can be prepared in liquid, solid, and semi-solid form. Both the semi-solid and solid media contain a gelling agent called agar. 1. Liquid medium (or nutrient broth) is typically used for fermentation and biochemical studies or to measure biomass. Microorganisms are not visible to the naked eye, but if the growth conditions provide the appropriate nutrients, temperature and other requirements, microbial growth can be detected in liquid culture by the following methods:  Turbidity or opaqueness of the liquid media  Pellicle formation, where a mass of cells floats on the surface of the liquid media  Sediment formation, where cells deposit on the bottom of the tube  Slime production that “binds” cells together Biology 144 17 © Department of Microbiology 2024 Stellenbosch University  2. Solid media is used to examine colony properties, isolate and store pure cultures and study specific biochemical reactions. Any liquid medium can be used with the addition of a solidifying agent, with agar being the most commonly used at 1-3% (m/v). It is an unbranched polysaccharide obtained from the cell membranes of some species of red algae; it melts at 95 °C and solidifies (gels) at 42 °C. It has no nutritional properties and is not hydrolysed by most organisms. Solid media can be prepared as a slant, a stab or an agar plate (in a Petri dish). Microbes on a solid nutrient surface will grow and divide until they form a colony, which is visible on the surface of the agar with the naked eye. 3. Semi-solid media is relatively soft and contains a reduced amount of agar (0.2-0.5%). It is primarily used to test organisms' motility and oxygen requirements. MICROBIAL GROWTH IN BATCH SYSTEMS Microorganisms reproduce by budding (e.g. yeasts) or binary fission (bacteria). Microbial growth thus refers to an increase in cell numbers, not size, as in the case of eukaryotes. Microbial growth is exponential (i.e. one cell gives rise to two cells, which divide to produce four cells, etc.). Cell numbers can be expressed as “colony forming units” (CFU) on solid media or (less accurately) as turbidity in liquid media. Bacteria growing in a batch system (i.e. a closed system with limited nutrients) follow a typical “growth curve” of lag, log, stationary and death phases. Lag Phase This is the first phase, right after the inoculation of a microbial culture into the growth medium. Usually, no or very little cell division takes place. The length of such a lag phase depends on the type of organism, the culture conditions, etc. Older cells (e.g. from a culture that has been standing for a long time) or cells that have been under stress (e.g. kept at unfavourable temperatures) need a longer time to recover, with a more extended lag phase than vigorously growing cells. Furthermore, essential enzymes, cofactors and ATP must be produced before growth. Exponential phase Microorganisms grow and divide at the maximum rate possible during the exponential or log phase. The growth rate depends on the growth medium and how well the cells adapt to the new environment (temperature, pH, osmotic pressure, etc.). The growth rate is constant during the exponential phase, i.e., the cells divide regularly. The maximum cell concentration at the end of logarithmic growth can vary depending on the type of microorganism and environmental factors. Cell counts as high as 1011 per ml may be reached for bacteria. Stationary phase Eventually, population growth ceases, and the growth curve reaches a plateau. Most cells are still metabolically active, but there is no net increase in cell numbers, and the culture becomes ‘stationary’. This could be due to several factors, for example, depletion of nutrients, lack of oxygen (in the case of aerobic microorganisms), decrease in pH (as a result of organic acids produced during growth) or accumulation of toxic by-products. Although cells are not reproducing, they can produce important secondary metabolites such as pigments and antimicrobial compounds to help them survive this stressful phase. Death Phase All metabolic activity of cells stops. Detrimental environmental changes like nutrient deprivation and the build-up of toxic wastes (e.g. lactic acid produced by Streptococcus) lead to a decline in viable cells. The death of microorganisms is usually logarithmic, i.e. a constant number of cells die every hour. CONTROLLING MICROBIAL GROWTH Temperature Heat treatment is one of the most common methods to control microbial growth. Moist heat denatures and Biology 144 18 © Department of Microbiology 2024 Stellenbosch University inactivates proteins (including enzymes) and disrupts cell membranes, whereas dry heat causes oxidation of the cell constituents. Three types of heat treatment are commonly used: 1. To destroy bacterial spores, temperatures above 121 °C are required for complete sterilisation. The most common laboratory sterilisation method involves autoclaving (using an industrial pressure cooker), which combines steam and pressure (121 °C for 15 min at 2 Bar pressure). 2. Pasteurization only reduces the number and/or types of microbes but is a relatively short-term solution since the remaining microbes will eventually start growing again. Milk, for example, can be pasteurized in a batch by heating at 63 °C for 30 minutes (LTLT – Low-Temperature Long Time) or by using a flash method at 72 °C for only 15 seconds (HTST – High-Temperature Short Time). UHT (Ultra High Temperature) treatment at 141 °C is done for only 2 seconds and completely sterilizes the milk without denaturing it. 3. Tyndallisation breaks heat-labile chemical bonds and is achieved by heating with flowing steam at 100 °C for 30 minutes on three consecutive days. This process doesn’t destroy dormant spores; any spores germinating during the three days are destroyed. Glassware and medical instruments are usually sterilized by dry heat in an oven (170 °C for 2 hours). Heat-labile products (e.g., vitamins, plastics, or spices) must be sterilised by irradiation or filtration. Extremely low temperatures are also used to slow or prevent microbial growth (i.e. it is bacteriostatic) but cannot permanently sterilize a product (bactericidal). For example, we store food in a refrigerator or freezer to prevent spoilage. Rapid freezing does less damage than slow freezing; microbes kept as cultures are therefore rapidly frozen using liquid nitrogen and stored at -80 °C to keep them viable over many years. Irradiation Irradiation can be used to sterilize objects because it permanently damages living organisms’ nucleic acid. Ultraviolet (UV) light causes dimerization of the thymine residues in DNA, which leads to cell death. However, it has very low penetrating power and is best used to sterilize surfaces. Ionising radiation (X-rays / gamma rays) is highly penetrating and breaks the sugar-phosphate backbone of DNA. It sterilises plasticware, surgical instruments, spices, and other food products. Bacterial endospores are resistant to irradiation. Desiccation Inducing a hypertonic environment by dehydration or increasing a substance's sugar or salt content may inhibit microbes but does not eliminate them. Many foodstuffs are preserved by drying (desiccation) or adding salt or sugars, e.g. dried fruit, cereals, jams, honey, biltong and anchovies. Filtration Temperature-sensitive substances such as vitamins, antibiotics and protein solutions must be sterilized using a filter with a very small pore size (usually 0.22 μM) that will retain most microbes. SELF-TEST 1. The microbes in our large intestine are “mesophilic” and “anaerobic” – explain what these two terms mean. (2) 2. Name three important microbiological discoveries attributed to Robert Koch. (3) 3. Why is honey (and sugar in general) such a good preservative in food? (2) Biology 144 19 © Department of Microbiology 2024 Stellenbosch University  BACTERIA (Russell et al., Chapter 26) Bacteria are the smallest cellular organisms able to replicate independently and are typically 0.5-5 μm in size. The smallest bacteria, Rickettsia (obligate intracellular parasites), are the size of the largest viruses, whereas the largest bacterium, discovered off the coast of Namibia, is visible with the naked eye. Bacteria are the most abundant organisms on Earth, with a collective biomass that is 10 times that of eukaryotes. They comprise two of the three domains of life, are masters of adaptation and are found in almost every conceivable habitat. They are the oldest forms of life and critical to our existence and the ecological balance. Some bacteria are pathogens, but various species are used to produce commercially important products, including chemicals, food and pharmaceuticals. GENERAL MORPHOLOGY Bacteria are single-celled organisms that do not show the integration of activities between cells or specialisation of cells within a population. They have no internal compartments or membrane-bound organelles (Fig. 26.3), but have a complex membrane system formed by invaginations of the plasma membrane. The plasma membrane surrounds the cytoplasm (the internal, fluid part of the cell) and the nucleoid (nuclear DNA, RNA and some proteins e.g. transcription factors), the ribosomes (which translate mRNA into proteins), inclusion bodies (energy storage) and sometimes small, accessory “rings” of DNA called plasmids. The plasma membrane is a fluid bilayer of phospholipids, with the nonpolar (hydrophobic) tails of the lipids that face one another and the polar (hydrophilic) heads that face outwards towards the extra- or intracellular fluids. Various proteins are embedded in the plasma membrane and participate in metabolic and cellular processes. The plasma membrane is a permeability barrier to protect the cell from losing nutrients or being harmed by environmental toxins and waste. The large surface area also acts as a site for photosynthetic and/or respiratory functions. The electron transport chains of obligately aerobic bacteria use proton-motive force to produce energy (stored in ATP) via ATP synthases in the plasma membrane. Bacterial cell walls consist mainly of peptidoglycan, comprised of N-acetylmuramic acid (NAM) and N- acetylglucosamine (NAG) held together in a rigid lattice by peptide cross-bridges. This structure gives bacterial cells their rigidity and helps protect their inner components. Some bacteria, such as mycoplasmas, do not have a cell wall, whereas archaea lack peptidoglycan in their cell walls. There are two main types of bacterial cell walls, differentiated by a staining technique called the Gram stain (Fig. 26.4). Gram-positive bacteria have a thicker, single layer of more than 50% peptidoglycan and only 1-4% lipids. Gram-negative bacteria have a thinner layer of