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    Practical Bacteriology
    Practical Bacteriology
    Practical Bacteriology
    Ebook169 pages2 hours

    Practical Bacteriology

    By Choon Weng Lee

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    About this ebook

    Practical Bacteriology covers the essential skills in Bacteriology. Here you will learn how to optimize isolation and culture of bacteria. You will also learn the standard guidelines on the enumeration of culturable bacteria via spread plating, pour plating, membrane filtration method, most probable number (MPN) and other culture-independent techniques. Finally, learn how to carry out the phenotypic approach to bacterial identification whether using the dichotomous key or blunderbuss approach, and the 16S rRNA gene sequencing genotypic approach.
    LanguageEnglish
    PublisherLulu.com
    Release dateDec 31, 2021
    ISBN9781716142376
    Practical Bacteriology

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      Book preview

      Practical Bacteriology - Choon Weng Lee

      Table of Contents

      Table of Contents

      Preface

      About the Author

      Chapter 1: Isolation and growth of bacteria

      Important beginnings in practical bacteriology

      Introduction to practical bacteriology

      Environmental factors that affect the growth of bacteria

      Temperature

      Atmospheric gas

      Nutrients

      pH

      Osmolarity

      Pressure and Light

      Radiation

      Types of media for bacterial isolation

      General media

      Defined media

      Selective media

      Differential media

      Special culture techniques

      Chapter 2: Enumeration

      Culture-dependent approach

      Pour plating method

      Spread plating method

      Membrane filtration method

      Most probable number (MPN) method

      Culture-independent approach

      Hemocytometer

      Epifluorescence microscopy

      Flowcytometry

      Proxy method

      Chapter 3: Identification

      Phenotypic tests

      Cell wall and morphology for identification

      Selected biochemical tests

      Serotyping

      Alternative types of biotyping

      Genotypic bacterial identification

      Selected references for further reading

      Preface

      Microbiology is the study of microorganisms, either unicellular or multicellular microscopic organisms. Microorganisms include eukaryotes such as fungi and protists, prokaryotes such as bacteria, archaea and viruses too. The study of bacteria alone is known as bacteriology, and is an essential component of a microbiology course. Bacteriology includes bacterial genetics, biochemistry, physiology and ecology. As we know more about bacteria, it has become impossible to cover every knowledge about bacteria in a single book.

      Therefore in this book, I will cover what I consider to be the ‘bread and butter’ of a bacteriologist i.e., the isolation, enumeration and identification of bacteria. I hope it will be a good companion to the general microbiology textbooks that are widely available. I also assume that the readers are already familiar with some bacteriological terms, and I hope the information in this book will help the readers to improve their microbiological knowledge and skills. With a good understanding of these topics, you should be able to handle the job scope of any bacteriologist whether in clinical, food or environmental settings.

      Finally, I dedicate this book to my parents; my father, Mr Lee Ah Chye and my mother, Mdm Fong Ah Yok. Without whom, I will not be. I would also like to thank you for purchasing this ebook. Please email me (leechoonweng@yahoo.com) with your proof of purchase and a valid email address to obtain free updates to this ebook.

      About the Author

      Dr Lee Choon Weng, Ph.D graduated in 1993 with a 1st Class Honors degree in Microbiology from the Department of Genetics and Cellular Biology, Faculty of Science, University of Malaya, Malaysia, and then worked as a microbiologist at the BP Clinical Lab Pte Ltd, Penang, Malaysia. Throughout his studies and job as a microbiologist, he had the opportunity to carry out internships as a microbiologist at Unilever (Malaysia) Holdings Pte Ltd (then Lever Brothers Pte Ltd) and the Institute of Medical Research, Kuala Lumpur, Malaysia.

      Two years after his graduation, he started his Master of Science (M.Sc) in the field of environmental microbiology at University of Malaya. Upon completion of his M.Sc, he worked as an environmental consultant at the Perunding Utama Pte Ltd, Subang Jaya, Malaysia where he worked on a water quality modelling system and learned environmental impact assessment reporting and procedures in Malaysia.

      He was later offered a Monbugakusho scholarship from the government of Japan to further his studies. He did his doctoral degree (Ph.D) in marine microbial ecology at the Graduate School of Fisheries, Hokkaido University, Japan. Upon successful completion of his Ph.D, he worked as a lecturer at the Kolej Universiti Sains dan Teknologi Malaysia (now Universiti Malaysia Terengganu) at Kuala Terengganu, Malaysia before returning to University of Malaya. He continued his research and has taught bacteriology, microbial ecology and marine microbiology for over twenty years.

      Chapter 1: Isolation and growth of bacteria

      Important beginnings in practical bacteriology

      Historically the first media to grow bacteria, are rich meat or bone soups or broth. Broths are clarified by filtration, and bacterial growth is detected when the broth turns turbid and cloudy. However, bacteria growing in broth are difficult to isolate. Robert Koch, considered as the father of medical microbiology, observed how it is easier to work with fungi growing on potato slices. Later, he used gelatin to solidify the broth that supports bacterial growth. As each bacterium multiplies and grows on the solid medium, a colony is formed. This bacterial colony is now termed as a colony forming unit or cfu in short. 

      It is easier to isolate a single cfu on a solid medium. However solid medium from the addition of gelatin begins to liquefy at 37⁰C which is the optimum temperature for most pathogens. Therefore, agar which liquefies at 85⁰C was later used to prepare solid media. Walther and Fannie Hesse who were working in Robert Koch’s laboratory, began using agar to create solid media similar to the preparation of fruit jams. This quickly became a mainstream procedure for bacterial isolation. Furthermore, the incorporation of the petri dish in microbiological laboratories made it easier to work with bacterial culture in a safe manner and with reduced contamination. Petri dish was attributed to Richard Julius Petri who was also working in Robert Koch’s laboratory.

      With these microbiological techniques and procedures established in his laboratory, Robert Koch was able to isolate the causative agent for tuberculosis, Mycobacterium tuberculosis in 1882. These techniques remain in practice even until now albeit with minor improvements. Petri dishes are now made from plastics, and sterilized using ethylene oxide gas. These steps allow for safer decontamination and disposal of materials and microorganisms with autoclaving.

      Introduction to practical bacteriology

      Firstly, we have to understand the concept of aseptic or ‘clean’ techniques. These are essential skills for a bacteriologist in order to reduce contamination. This is because microorganisms are everywhere e.g., in the air that we breath, on the surface of tables and laboratory apparatus, on our skin, and even from our nostrils etc. Therefore, we need to clean our workspace (or workbench) before and after carrying out bacteriological work. We usually clean the workspace with commercial disinfectants (e.g., Savlon or Clorox brands) or home-made disinfectants (usually 70% v/v ethanol). We also work near a flame that is usually provided by a Bunsen burner. However sometimes we use spirit lamps to provide the flame as the main function of the flame is to create an updraft near the source of the flame. Therefore, even a

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