A TECHNICAL REPORT OF STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES) CARRIED OUT AT DEPARTMENT OF MICROBIOLOGY AND PARASITOLOGY, LAGOS UNIVERSITY TEACHING HOSPITALS, IDI ARABA, LAGOS. SUBMITTED BY DOGO FEMI OHIREME 19/5800 TO THE DEPARTMENT OF MICROBIOLOGY, CALEB UNIVERSITY, IMOTA, LAGOS STATE IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR OF SCIENCE DEGREE B.sc (Hons) IN MICROBIOLOGY SEPTEMBER 2022 Department of Microbiology, Faculty of Science, Caleb University, Imota, Lagos State. 22 December, 2021. The coordinator, Students’ Industrial Work Experience Scheme, Department of Microbiology, Caleb University, Imota, Lagos State. Dear Ma, LETTER OF TRANSMITTAL I Dogo Femi Ohireme submit the report of the student industrial work experience scheme (SIWES) done at the department of medical microbiology and parasitology laboratory Lagos University Teaching Hospital (LUTH) from 9th August to 28th October 2022, which contains the summary of all experience gathered, in partial fulfilment of the requirement for the award of the Bachelor of Science Degree B.sc (Hons) with the jurisdiction of the Nigeria University Commission (NUC). Yours faithfully, Dogo Femi Ohireme (19/5800) CERTIFICATION This is to certify that the industrial training was done by DOGO FEMI OHIREME (19/5800), of the department of microbiology, Caleb University, Imota Lagos under my supervision, according to the partial fulfilment of the requirement for the award of the Bachelor of Science Degree B.sc (Hons) in the department of microbiology. ____________________ ______________________ SUPERVISOR SIGNATURE AND DATE DEDICATION ACKNOWLEDGEMENT TABLE OF CONTENT Letter of transmittal Certification Dedication Acknowledgment Table of content CHAPTER ONE: INTRODUCTION 1.0. History of SIWES 1.1. General objectives of SIWES 1.2. Importance of SIWES 1.3. Duration of SIWES CHAPTER TWO: MEDICAL MICROBIOLOGY AND PARASITOLOGY 2.1. Microorganisms of medical importance CHAPTER THREE: MEDICAL MICROBIOLOGY AND PARASITOLOGY LAB (LUTH) 3.1. Organizational chart 3.2. Vision statement 3.3. Laboratory equipment 3.4. Laboratory Safety and Precaution 3.5. Laboratory routine CHAPTER FOUR: ISOLATION OF MICROORGANISMS 4.1. Culture Techniques 4.1.1. Manual 4.1.2. Automated 4.2. Identification of microorganisms 4.2.1. Colony morphology 4.2.2. Staining Techniques 4.2.3. Microscopy 4.2.4. Biochemical Tests CHAPTER FIVE: ANTIMICROBIAL SUSCEPTIBILITY TESTING AST CHAPTER SIX: CHALLENGES AND RECOMMENDATION CHAPTER ONE: INTRODUCTION 1.0. HISTORY OF SIWES The five capitalized letters ‘SIWES’ means the “Student Industrial Work Experience Scheme” SIWES was established by ITF (Industrial Training Funds) in 1973 to solve the lack of adequate proper skills for employment of tertiary institution graduates by Nigerian Industries. The Students Industrial Work Experience Scheme (SIWES) was founded as a skill training program to help expose and prepare students of universities, polytechnics, and colleges of education for the industrial work situation to be met after graduation. This scheme serves as a smooth transition from the classroom to the world of work and further helps in applying knowledge. The scheme provides students with the opportunity to acquire and expose themselves to the experience required in handling and managing equipment and machinery that are usually not made available in their institutions. Before this scheme was established, there was a growing concern and trend noticed by industrialists that graduates of higher institutions lacked sufficient practical background for employment. It used to be that students who got into Nigerian institutions to study science and technology were not trained in the practical know-how of their various fields of study. As a result, they could not easily find jobs due to their lack of working experience. Therefore, the employers thought that theoretical education in higher institutions was not responsive to the needs of the employers of labour. This was a huge problem for thousands of Nigerians until 1973. It is against this background that the fundamental reason for initiating and designing the scheme by the fund in 1973/74 was introduced. The ITF organization (Industrial Training Fund) decided to help all interested Nigerian students and established the SIWES program. It was officially approved and presented by the Federal Government in 1974. The scheme was solely funded by the ITF during its formative years, but as the financial involvement became unbearable to the fund, it withdrew from the scheme in 1978. In 1979, the federal government handed over the management of the scheme to both the National Universities Commission (NUC) and the National Board for Technical Education (NBTE). Later, in November 1984, the federal government reverted the management and implementation of the scheme to ITF. In July 1985, it was taken over by the Industrial Training Fund (ITF) while the funding was solely borne by the federal government. (Culled from Job Specifications on Students Industrial Work Experience Scheme). 1.1. GENERAL OBJECTIVES OF SIWES SIWES is strategized for skill acquisition. It is in fact designed to prepare and expose students of universities, polytechnics, and colleges of education to the real-life work situation they would be engaged in after graduation. Therefore, SIWES is a key factor required to inject and help keep alive industrialization and economic development in the nation through the introduction and practical teaching of scientific and technological skills to students. (Culled from Detailed Manual on SIWES Guidelines and Operations for Tertiary Institutions). Objectives of the Students Industrial Work Experience Scheme include: 1. Provide an avenue for students to acquire industrial skills for experience during their course of study 2. Expose students to work methods and techniques that may not be available during their course of study. 3. Bridging the gap between theory and practice by providing a platform to apply knowledge learned in school to real work situations 4. Enabling the easier and smoother transition from school by equipping students with better contact for future work placement 5. Introduce students to a real work atmosphere to know what they would most likely meet once they graduate. 1.2. IMPORTANCE OF SIWES All Nigerian students who study technology and science must know about SIWES. Partaking in SIWES has become a prerequisite for awarding diploma and degree certificates in many Nigerian Institutions, according to the Nigerian government’s educational policy. Undergraduate students of the following disciplines are expected to participate inf the scheme: Natural sciences, Engineering and Technology, Education, Agriculture, Medical Sciences, Environmental, and pure and applied sciences. The duration is for four months and one year for polytechnics and colleges of education students respectively and six months for university students. 1.3. DURATION OF SIWES The duration of SIWES varies across Institutions Universities: At the end of 200, 300, 400 level of a degree program for about 3-6 months Polytechnics, Monotechnic, Colleges of technology: At the end of the 1st year of the 2-year ND program, for 4 months 2.0. CHAPTER TWO: MEDICAL MICROBIOLOGY Medical microbiology is a branch of microbiology that deals with the clinical application of microbiology. It is concerned with the prevention, diagnosis, and treatment of infectious diseases (Baron, 1996). Medical microbiology is widely distributed to bacteriology, virology, mycology, and parasitology (Collegedunia, 2022). Parasitology is of emphasis because of its prevalence in this tropical part of the world (Daryani et al., 2012). Medical parasitology deals with the study of parasitic protozoa, parasitic helminths, and pathogens from various vectors such as arthropods (Mathison et al., 2019). Bacteriology is the study of bacteria like Escherichia coli, their medical importance, and their harmful effects (Matthew and Tainter, 2022). Bacteriology seems more pronounced because of its predominance as normal flora and opportunistic pathogens in human hosts (Raheema, 2021). Virology is the study of viruses (White et al., 2015). This is a silent but salient aspect of medical microbiology. It is silent because only the prevalence of a viral disease is of public health importance, just like the Ebola virus. It is salient because the mortality rate is always high and difficult to treat. Mycology is the study of fungi (Wickes and Wiederhold, 2018). Medically important fungi like Candida spp have become a treat for patients as they are opportunistic pathogens (Kumamoto et al., 2020), eukaryotes like hosts, so drug development is difficult. Yet, there is a notable resistance to antifungals among them (Sanguinetti et al., 2015). 2.1. Microorganisms of medical importance Microorganisms are tiny living entities that are not visible with the human eyes but with the aid of a microscope (Shapiro, 2007). Microorganisms are ubiquitous, found almost everywhere; on surfaces, animal and plant hosts, food, and soil. Everywhere and anywhere (Barberán et al., 2014). There are some sites in the human host that are termed sterile, that is, free from microorganisms (Knight et al., 2011). Sites like these include; blood and cerebrospinal fluid (CSF) (Johnson et al., 2016). The presence of microorganisms in these sites may lead to disease. Fatal one (Knight et al., 2011; Johnson et al., 2016). Microorganisms of medical importance can either be normal flora or pathogens (Houldcroft et al., 2017). Normal floras are microorganisms that support the usual function of the host by either preventing the attachment of pathogen to the site of infection, providing the essential nutrient needed by the host, such as vitamin c, and aiding food digestion (Panthee et al., 2022). With all of these, when there is no balance in the equilibrium of the host-microbe relationship, the normal flora could be opportunistic in their approach to causing disease. Hence the term opportunistic pathogen (Houldcroft et al., 2017). Pathogens are microorganisms capable of causing disease. They usually have virulence factors that could be determined by various means (Panthee et al., 2022). Some of them are cell wall composition (Orner et al., 2019) and the presence of a capsule which usually produces a slimy appearance (Sarkar et al., 2014). Pathogens are either Gram positive or Gram negative based on their reaction to a stain called Gram stain, which is dependent on their cell wall composition (Tripathi and Sapra, 2021). 3.0. CHAPTER THREE: MEDICAL MICROBIOLOGY AND PARASITOLOGY LAB (LUTH) 3.1. ORGANIZATIONAL CHART Head of Department Consultant Deputy Director Science Lab Technologists Medical Lab Scientists Medical Lab Technicians Clerical officers Cleaners 3.2. Vision of the laboratory To be the world’s leader in medical microbiology with a strong commitment to quality service and excellent customer care. We will achieve this by making each patient our best friend, committing ourself to professionalism and teamwork, ensuring quality in our service delivery and setting the pace for future trend in medical microbiology practice through innovative leadership, continuous research and training. 3.3. Laboratory Equipment The laboratory has various equipment that will be highlighted with their functions. Microscope The microscope is used to view microorganisms. Weighing balance It is used majorly in the media preparatory unit to measure the mass of dehydrated agar powder before dilution with water and other solvents. Autoclave An autoclave is a pressurized chamber used to sterilize laboratory tools like bottles and test tubes, even agar to avoid contamination. The autoclave uses high pressure and temperature of the steam to kill microorganisms. Bunsen burner Bunsen burner is a gas-fuel open flame used in the laboratory for intermittent sterilization of tools like the wire loop and bottleneck. Centrifuge A centrifuge is a machine that separates a mixture such as blood, cerebrospinal fluid, plasma, and platelet into their components by rotating them at high speed to subject them to gravity. So that the heaviest component will be pulled to the bottom of the container. Deep Freezer The deep freezer works with extremely low temperatures to minimize microbial and enzymatic activities. It is used as a storage medium to preserve isolates. Dry oven The dry oven is used in the media preparatory unit to cool agar medium and make them solidify well. It prevents melting by running at a temperature of about 370C. Vortex mixture The vortex mixture is a device that vibrates to mix samples in glass tubes or flasks Incubator It is used to grow and maintain microorganisms and cultures. It provides a suitable external environment such as temperature, pressure, and moisture. Water Bath It is used to boil samples, transferring water temperature to the sample in order to increase their temperature and break some bonds. Water distiller The water distiller is used in the media preparatory unit to purify water. It allows the water to boil and then condense. Bactek blood culture This is an automated blood culture used to identify microorganisms in the blood. It has a special bottle that is filled with about 8ml of blood and loaded into the well of the machine. After some hours and sometimes days. The machine alerts a positive or negative response. Vitek machine This is used for the identification and antimicrobial susceptibility testing of microorganisms. Microorganisms are diluted serially into a McFarland standard of 0.5, and the sugar disk and antibiotic disk are inserted into the tube containing the diluted microorganism. It is then loaded into the machine. 3.4. laboratory Safety precautions 1. Remember that all bacteria are potential pathogens that may cause harm under unexpected or unusual circumstances. If you as a student have a compromised immune system or a recent extended illness, you should share those personal circumstances with your lab instructor. 2. Know where specific safety equipment is located in the laboratory, such as the fire extinguisher and the eyewash station. 3. Recognize the international symbol for biohazards, and know where and how to dispose of all waste materials, particularly biohazard waste. Note that all biohazard waste must be sterilized by autoclave before it can be included in the waste stream. 4. Keep everything other than the cultures and tools you need OFF the lab bench. 5. All of the equipment and supplies used in experiments involving bacterial cultures should be sterilized. This includes the media you use and also the tools used for transferring media or bacteria, such as the inoculating instruments (loops and needles) and pipettes for liquid transfer. 6. Transfer of liquid cultures by pipette should NEVER involve suction provided by your mouth. 7. Disinfect your work area both BEFORE and AFTER working with bacterial cultures. 8. In the event of an accidental spill involving a bacterial culture, completely saturate the spill area with disinfectant, then cover with paper towels and allow the spill to sit for 10 minutes. Then carefully remove the saturated paper towels, dispose of them in the biohazard waste, and clean the area again with disinfectant. 9. Wear gloves when working with cultures, and when your work is completed, dispose of the gloves in the biohazard garbage. Safety glasses or goggles are also recommended. 10. Long hair should be pulled back to keep it away from bacterial cultures and open flame. 11. Make sure that lab benches are completely cleared (everything either thrown away or returned to storage area) before you leave the lab. 3.5. laboratory routine Each student should provide himself with a clean, short-sleeved white overall which, if possible, should be used for aseptic and bacteriological work. Before commencing any aseptic work, scrub the hands and arms to the elbow thoroughly with soap and water Swab the work bench before and after use. Used materials that are already contaminated should be disposed in a safe manner a. Instrument like the wire loop is intermittently flamed to avoid contamination b. Pipettes, slides and cover slips must be placed in the recovery container immediately after use. c. Culture and incubated materials should be disposed of after examination, and a diagnostic report has been dispatched. Proper specimen receipt should be ensured. All patients sample should be well documented and properly stored prior to processing (Though it is best to process all samples immediately). Used uncontaminated apparatus must be placed in trays provided for the purpose. Nothing should be disposed directly on the floor of the laboratory. All accidents should be reported at once. 4.0. CHAPTER FOUR: ISOLATION OF MICROORGANISMS The isolation of microorganisms is the process of obtaining a pure strain of microorganisms from a mixed culture or environment. The same process is used for the isolation of causative agents, and human pathogens from suspected infections. Suspected infections mean that we understand the kind of microorganism capable of causing the kind of disease. So that we do not isolate normal flora of the microbiota and place unnecessary emphasis. In order to isolate microorganisms of medical importance, the site of infection is usually processed and tested for the presence of pathogen. Specimen or sample collection is the process of obtaining the sample of the infected area. It could be the skin scrapping, nail clippings, blood sample, sputum sample, CSF, high vaginal swab (HVS), endocervical swab (ECS), and so on. Highly trained health personnel are allowed to collect the specimen. We were not taught hands-on how to collect specimen due to our work specification as IT students and how delicate sample collection is. Some samples like tuberculosis are labelled high risk when they can be harmful to the scientist processing them or any laboratory personnel. Specimen processing is done almost immediately after collection. And if not, for any reason, the sample should be stored in the refrigerator to reduce enzymatic activity in the sample and also preserve the viability of the suspected pathogen. In order to process specimens, a culture medium is needed. 4.1. Culture Techniques Culture is the process of growing microorganisms in an environment called a medium which contains the needed growth nutrient and growth factors. The growth nutrients are the major substance needed by the microorganism to grow and multiply. They are needed in large quantities. They include carbon sources and nitrogen. Added to these is the availability of water. The growth factor is an essential part of a medium which is relatively in the small quantity needed to grow some additional functions just like folic acid for DNA synthesis. Cultural techniques can either be manual or automated. The culture media used in the lab include MacConkey agar which is used to differentiate the lactose fermenting organisms from the non-lactose fermenting organisms. The chocolate agar is made of lysed red blood cells, it is used in cultivating fastidious pathogens that are capable of growing in the human host environment. But it has recently been changed to soluble hemoglobin agar in order to avoid contaminants usually present in the blood used. Blood agar is also used to cultivate fastidious organisms in order to better understand their ability to lyse the red blood cell. There are three types of hemolysis which include the α, β, and γ hemolysis. These are better understood on the blood agar. The Sabaroud Dextrose Agar is used in the mycology lab for cultivating fungi of different types. The broths used include the selenite F broth, which serves as an enrichment medium for the growth of salmonella and shigella majorly in stool specimens. 4.1.1. Manual culture techniques The manual culture techniques involve the conventional method of Petri dish, medium (liquid broth or solid agar), and the inoculum. This method also involves streaking of the inoculum on the surface of the solid medium or stabbing in a liquid broth or semi-solid medium like agar slant. The streaking methods include the pure plate method and the streak plate method. The streak plate method is used in our lab. This is done by placing a flamed loop into the specimen and streaking on the medium. The pattern of streaking could be quadrant, or for colony count (images). 4.1.2. Automated culture techniques In the medical microbiology and parasitology lab, LUTH, we make use of automated machines like the bactek and bactalert for blood culture, and also the vitek machine for identification and antimicrobial susceptibility. The blood culture is done in the sample bottle placed into the machine, and it is scanned through by the machine and is reported to have either flagged positive or negative. 4.2. IDENTIFICATION OF MICROORGANISMS The identification of microorganisms involves the use of series of tests which include biochemical, physical, molecular, and serological to relatively compare an unknown organism to a known one with similar features. 4.2.1. Colony morphology Colony morphology is a physical means of identification of microorganisms while on the culture plate or liquid broth. Some of this which includes their size, colour and motility. Characteristics of a colony such as shape, edge, elevation, color and texture. When recording colony morphology, it is important to record color, optical properties (translucence, sheen), and texture (moist, mucoid, or dry). However, remember that color is often influenced by the environment. Shape: they could be circular, irregular, punctiform Circular Irregular Punctiform Margin (edges): they could be entirely smooth, undulated, rhizoid, lobate or filamentous Entirely smooth undulate rhizoids Lobate filamentous Elevation: they could be flat, convex, or umbonate turbidity The cloudy appearance of a liquid medium due to the presence of bacteria. You can "estimate" the number of bacteria per mL by using the table below. Turbidity # Bacteria per mL Example None 0 – 106 Light 107 Moderate 108 *Heavy 109 4.2.2. Staining Techniques Simple stain Basic dyes, such as methylene blue or basic fuchsin, are used as simple stains. They produce color contrast but impart the same color to all the bacteria in the smear. Negative staining A drop of bacterial suspension is mixed with dyes, such as India ink or nigrosin. The background gets stained black, whereas the unstained bacterial or yeast capsule stands out in contrast. This is useful in demonstrating capsules that do not take up simple stains. India ink preparation Negative stains are used when a specimen or a part of it, such as the capsule, resists taking up the stain. India Ink preparation is recommended for use in the identification of Cryptococcus neoformans. Impregnation methods Bacterial cells and structures that are too thin to be seen under the light microscope are thickened by the impregnation of silver salts on their surface to make them visible, e.g., for demonstration of bacterial flagella and spirochetes. Flagella stain Demonstrate the presence and arrangement of flagella. Flagellar stains are painstakingly prepared to coat the surface of the flagella with dye or a metal such as silver. The number and arrangements of flagella are critical in identifying species of motile bacteria. Differential staining Staphylococcus in Gram Stain Here, two stains are used, which impart different colors to different bacteria or bacterial structures, which help in differentiating bacteria. The most commonly used differential stains are: Gram staining Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology. Gram staining helps to identify bacterial pathogens in specimens and cultures by their Gram reaction (Gram-positive and Gram-negative) and morphology (cocci/rod). Acid-fast stain (Ziehl-Neelsen technique) It distinguishes acid-fast bacteria such as Mycobacterium spp from non-acid fast bacteria; which do not stain well by the Gram staining. It is used to stain Mycobacterium species (Mycobacterium tuberculosis, M. ulcerans, and M. leprae) Acid fast bacillus Endospore stain It demonstrates spore structure in bacteria as well as free spores. Few species of bacteria produce endospores, so a positive result from endospore staining methods is an important clue in bacterial identification. Bacillus spp and Clostridium spp are the main endospores producing bacterial genera. Spore of Clostridium botulinum Source: ASM Capsule stain It helps to demonstrate the presence of capsules in bacteria or yeasts. Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae are common capsulated bacteria. Giemsa stain Giemsa stain is a Romanowsky stain. It is widely used in the microbiology laboratory for the staining of: Malaria and other blood parasites, Chlamydia trachomatis inclusion bodies, Borrelia species, Yersinia pestis, Histoplasma species, Pneumocystis jiroveci cysts (formerly Pneumocystis carinii). 4.2.3. MICROSCOPY Microbiology deals with studying microorganisms that cannot be seen distinctly with the unaided eye. Observation of microorganisms is an integral part of Microbiology. Considering the nature of the objects to be studied, the microscope becomes an instrument of paramount importance. Microorganisms are observed and studied with the help of microscopes. The unit of measurement used to measure microorganisms is the Metric System. The size of the specimen determines which microscopes can be used to view the specimen effectively. Modern microscopes produce images with great clarity, magnifications that range from ten to thousands of times. Types of Microscopes Simple Microscopes Simple microscopes have only one lens, like a magnifying glass. It has a double convex lens with a short focal length. Examples of this kind of instrument include the hand lens and reading lens. When an object is kept near the lens, its principal focus with an image is produced, which is erect and bigger than the original object. Leeuwenhoeck’s simple microscopes allowed him to magnify images from 100 to 300 X. Compound Light Microscopy These are the most basic type of microscopes used in microbiology. It consists of a series of lenses that utilizes visible light as its illumination source. Various small specimens can be studied to find details with a compound light microscope. In a compound light microscope, light originates from an illuminator and passes through condenser lenses, which direct light onto the specimen. The light then enters the objective lenses, which further magnifies the image. Components of a Compound Microscope The major components of a compound microscope are: Framework: The basic frame structure is made up of metal, which includes the arm and base to which the whole of the magnification and optical components are attached. The metallic arm is connected to a U-shaped strong and heavy base that provides stability to the instrument. Stage: this is the flat horizontal platform positioned at about halfway through the length of the microscope with a hole at the center that allows the passage of light to illuminate the sample. Focus knobs: Two pairs of knobs are attached to the arm that helps in the up and down movement of the stage and adjustment and focusing of specimens of different thickness. Lens Systems: All microscopes employ different lens systems: the oculars, the objectives, and the condenser, which have different focusing power and contribute to the complete magnification system. Nose piece: A revolving nosepiece that holds the objectives is attached to the curved upper part of the arm of the microscope. The nosepiece can be rotated to position the objective with the required magnification in the path of the magnification system, beneath the body assembly and the eyepiece. Eyepiece (ocular lens): The eyepiece or ocular lens is a set of lenses held in a cylindrical tube inserted in a tubular structure on the curved upper part of the arm, above the nose piece. It consists of two or more lenses that focus the image into the eye. The newest microscopes consist of a pair of eyepieces that allows the observer to use both eyes to observe the specimen in the microscope. Such microscopes are called binocular microscopes. The normally used eyepieces have 2X, 50X, and 10X magnifications. Objective: The objectives are usually small cylindrical objects containing a single or a set of lenses attached to the nosepiece. The nosepiece holds three to five objectives, which contain lenses of varying magnifying power (2X-400 X). The total arrangement of the lenses is parfocal, which means that the sample stays in focus even when the lenses are changed from one to another in a microscope. Condenser: A condenser is also a lens that is fixed below the stage, and it focuses the beam of light coming from the light source onto the slide. The condenser is usually aided with a diaphragm and/or filters to control and manage the quality and intensity of the light passing through the sample. Light Source: The light source is mounted at the microscope’s base. The light source may be daylight, a halogen light, or LEDs and lasers, as used in the latest microscopes. The microscopes have some provision for reducing light intensity with a neutral density filter. Types of Compound Microscopes The Bright-Field Microscope – It is the simplest of all optical microscopy illumination. It helps to see the dark objects against a bright background. Dark Field Microscope – This is used to examine live or unstained microorganisms and other specimens like light-sensitive organisms or specimens that lack contrast with their background. Phase-Contrast Microscope – It is useful to examine live specimens. It does not require fixing or staining, as it can kill or discomfort the living microorganism and will make the observation inaccurate. The Differential Interference Contrast Microscope – This type of microscopy takes advantage of differences in the light refraction by different parts of living cells and transparent specimens to become visible for microscopic evaluation. The Fluorescence Microscope – This microscope uses UV light to magnify fluorescent substances. They can absorb UV light and emit visible light. Sometimes cells are also stained with fluorescent chemicals (fluorochromes) to be studied under this microscope. Confocal Microscope – Confocal microscope is used to study the detailed structure of specific objects within the cells. Two-Photon Microscope – Also known as two-photon laser scanning microscopy, it is a further refinement of precision fluorescence microscopy. Electron Microscopes – It uses electrons, electromagnetic lenses, and fluorescent screens. Electron wavelengths are 100,000 x smaller than visible light wavelengths which helps to magnify the specimen. Here the specimens are stained with heavy metal salts to be observed. All these microscopes yield a distinctive image and are used for different observations of microorganisms. 4.2.4. Biochemical Tests Biochemical tests are processes used for further identification of microorganisms. it involves the physical result of microorganisms reacting with reagents and complex molecules like sugar and enzymes. Biochemical tests are one of the traditional methods for identifying microorganisms, usually performed with phenotypic identification. For many years these methods were employed extensively, and they continue to be used nowadays, especially in some laboratory routines where a particular type of microorganism has to be identified rapidly. The ability of microorganisms to utilize certain biomolecules, resulting in useful organic compounds for themselves forms the basis of various biochemical tests. Biochemical tests are of different types, where the identification or distinction between different microorganisms is made on various bases. One of the traditional methods commonly used is a simple visual detection of the organism’s growth in the presence of essential nutrients by increased turbidity in the liquid medium. In other tests, however, the results are based on the change in color of the medium as a result of the change in the pH of the medium. Microorganisms can be classified into different groups based on their reaction to such tests. Some tests even allow the distinction of microorganisms to the species level. Biochemical tests are thus, essential as they are inexpensive and relatively simple to perform. The physiology of bacteria and other microorganisms differs from one another, which allows for the differentiation of such microorganisms. Biochemical tests, however, have some disadvantages. Despite being inexpensive and allowing both quantitative and qualitative information about the diversity of microorganisms present in a sample, these methods are laborious and time-consuming, and results are only observed after several days. In some cases, false positives are obtained, especially when considering similar microbial species. Catalase test The catalase test is a test to demonstrate the presence of catalase enzyme by breaking down hydrogen peroxide into oxygen and water. A small number of bacteria is added to a drop of hydrogen peroxide (3%) on the slide. The catalase test is a simple test used by microbiologists to help identify species of bacteria and to determine the ability of some microbes to break down hydrogen peroxide by producing the enzyme catalase. If bubbles of oxygen are observed, it means that the bacteria have the enzyme catalase, because it catalyzes the decomposition of hydrogen peroxide into oxygen and water. The organism is then said to be catalase positive (for example: Staphylococcus aureus). Oxidase test This test is used to identify microorganisms that contain the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between the Enterobacteriaceae and Pseudomadaceae families. Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. Artificial electron donor and acceptor molecules are provided in the oxidase test. When the electron donor is oxidized by the action of cytochrome oxidase, the medium turns dark purple and is considered a positive result. The microorganism Pseudomonas aeruginosa it is an example of an oxidase positive bacterium. Coagulase test Coagulase is an enzyme that helps blood plasma clot. This test is performed on Gram-positive and catalase-positive bacteria species to identify Staphylococcus aureus (coagulase positive). Coagulase is a virulence factor of this bacterial species. Clot formation around an infection caused by this bacterium probably protects it from phagocytosis. This test is very useful when you want to differentiate between Staphylococcus aureus of other species of Staphylococcus which are coagulase negative. Urease test This test is used to identify bacteria capable of hydrolyzing urea, using the enzyme urease. It is commonly used to distinguish gender Proteus from other enteric bacteria. The hydrolysis of urea produces ammonia as one of its products. This weak base increases the pH of the medium above 8.4, and the pH indicator (phenol red) changes from yellow to pink. An example of a urease-positive bacteria is Proteus mirabilis. Citrate test Citrate testing is used to determine the ability of the bacteria to use sodium citrate as the only source of carbon and inorganic ammonium hydrogen phosphate (NH4H2PO4) as a source of nitrogen. The citrate utilization test is possible only if the organisms are capable of fermenting citrate. The process takes place via the enzymes is called citrase. The API (Analytical Profile Index) API identification products are test kits for identification of Gram positive and Gram-negative bacteria and yeast. API strips give accurate identifications based on extensive databases and are standardized, easyto-use test systems. The kits include strips that contain up to 20 miniature biochemical tests which are all quick, safe and easy to perform. API (Analytical Profile Index) 20E is a biochemical panel for identifying and differentiating members of the family Enterobacteriaceae. It is hence a well-established method for manual microorganism identification to the species level. Objective To identify and differentiate members of family Enterobacteriaceae. Principle The API range provides a standardized, miniaturized version of existing identification techniques, which up until now were complicated to perform and difficult to read. In the API 20E, the plastic strip holds twenty mini-test chambers containing dehydrated media having chemically-defined compositions for each test. They usually detect enzymatic activity, mostly related to fermentation of carbohydrates or the catabolism of proteins or amino acids by the inoculated organisms. A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation, metabolism produces color changes that are either spontaneous or revealed by adding reagents. All positive and negative test results are compiled to obtain a profile number, then compared with profile numbers in a commercial codebook (or online) to identify the bacterial species. The Test Kit The test kit enables the following tests: ONPG: test for β-galactosidase enzyme by hydrolysis of the substrate o-nitrophenyl-b-Dgalactopyranoside ADH: decarboxylation of the amino acid arginine by arginine dihydrolase LDC: decarboxylation of the amino acid lysine by lysine decarboxylase ODC: decarboxylation of the amino acid ornithine by ornithine decarboxylase CIT: utilization of citrate as only carbon source H2S: production of hydrogen sulfide URE: test for the enzyme urease TDA (Tryptophan deaminase): detection of the enzyme tryptophan deaminase: Reagent- Ferric Chloride. IND: Indole Test-production of indole from tryptophan by the enzyme tryptophanase . ReagentIndole is detected by addition of Kovac’s reagent. VP: the Voges-Proskauer test for the detection of acetoin (acetyl methylcarbinol) produced by fermentation of glucose by bacteria utilizing the butylene glycol pathway GEL: test for the production of the enzyme gelatinase which liquefies gelatin GLU: fermentation of glucose (hexose sugar) MAN: fermentation of mannose (hexose sugar) INO: fermentation of inositol (cyclic polyalcohol) SOR: fermentation of sorbitol (alcohol sugar) RHA: fermentation of rhamnose (methyl pentose sugar) SAC: fermentation of sucrose (disaccharide) MEL: fermentation of melibiose (disaccharide) AMY: fermentation of amygdalin (glycoside) ARA: fermentation of arabinose (pentose sugar) Method Confirm the culture is of an Enterobacteriaceae. To test this, a quick oxidase test for cytochrome c oxidase may be performed. Pick a single isolated colony (from a pure culture) and make a suspension of it in sterile distilled water. Take the API20E Biochemical Test Strip which contains dehydrated bacterial media/bio-chemical reagents in 20 separate compartments. Using a Pasteur pipette, fill up (up to the brim) the compartments with the bacterial suspension. Add sterile oil into the ADH, LDC, ODC, H2S, and URE compartments. Put some drops of water in the tray and put the API Test strip and close the tray. Mark the tray with an identification number (Patient ID or Organism ID), date and your initials. Incubate the tray at 37oC for 18 to 24 hours. Result Interpretation For some of the compartments, the color change can be read straightway after 24 hours but for some reagents must be added to them before interpretation. Add the following reagents to these specific compartments: TDA: Put one drop of Ferric Chloride IND: Put one drop of Kovacs reagent VP: Put one drop of 40 % KOH (VP reagent 1) & One drop of VP Reagent 2 (α-Naphthol) Get the API Reading Scale (color chart) by marking each test as positive or negative on the tray’s lid. The wells are marked off into triplets by black triangles, for which scores are allocated reading scale Add up the scores for the positive wells only in each triplet. Three test reactions are added together at a time to give a 7-digit number, which can then be looked up in the codebook. The highest score possible for a triplet is 7 (the sum of 1, 2 and 4) and the lowest is 0. Identify the organism by using an API catalog or apiweb (online) 5.0. CHAPTER FIVE: ANTIMICROBIAL SUSCEPTIBILITY TEST AST It is not enough to just identify your organism. You also need to know what antimicrobial agents your organism is susceptible to. There are several methods to determine this. Dilution testing is used to quantitatively determine the minimal concentration (in mg/ml) of antimicrobial agent to inhibit or kill the bacteria. This is done by adding two-fold dilutions of the antimicrobial agent directly to an agar pour, a broth tube, or a micro-broth panel. The lowest level that inhibits the visible growth of the organism is considered the Minimum Inhibitory Concentration (MIC). The agar pour method is considered the reference test procedure in Europe. The broth dilution method is more widely accepted in North America. The E test (AB Biodisk) is a plastic strip with a gradient concentration of antimicrobial agents impregnated in it. The strip is placed directly on the surface of an inoculated plate. The MIC is read from the strip where the growth inhibition intercepts the disk. These strips are relatively expensive. Many physicians however do not need no know the exact MIC, but just which antibiotics the pathogen is susceptible, intermediate, or resistant to. The Kirby-Bauer agar diffusion method is well documented and is the standardized method for determining antimicrobial susceptibility. White filter paper disks (6 mm in diameter) are impregnated with known amounts of antimicrobial agents. Each disk is coded with the name and concentration of the agent. For example, 10 µg of Ampicillin is indicated on the disk by AM-10. The code is listed on the Disk Zone Diffusion Diameter Chart. Pseudomonas Aeruginosa Image Pseudomonas aeruginosa on MHA incubated at 37°C for 24 hours. The impregnated disks are placed on an inoculated Mueller Hinton Agar (MHA) plate. The drug diffuses through the agar. The plates are incubated for 16-24 hours. The agar may be supplemented with blood or you may use blood agar for fastidious organisms. The diameter of the visible zone of inhibition is measured and compared to reference values. There should be sufficient bacteria to form a visible lawn of growth where it is not inhibited by the drug. The results are interpreted qualitatively as resistant, intermediate, or susceptible. The standard protocol must be followed exactly for you, or any clinical lab, to interpret the results reliably. There may be some inhibition of growth and the organism could still be considered resistant to that antimicrobial agent if the zone diameter is smaller than the reference values listed on the chart. Also note that different antimicrobial agents have different measurements for resistant, intermediate, and susceptible. A zone of inhibition may be considered susceptible for one antimicrobial agent and not for another. For example, in order for ampicillin (AM-10) to be an effective antimicrobial agent, the zone of inhibition for enterics and most streptococcus must be greater than 16 mm while for staphs it must be greater than 28 mm. E Coli Image E. coli on MHA incubated at 37°C for 24 hours. When determining which antimicrobial agents are best for treatment when multiple zones of inhibition are present, be sure to look at the relative zone of inhibition for that particular antimicrobial agent and compare your measurements to that. For example, let’s say that your enteric organism has a zone of inhibition around the Polymixin B disk of 20 mm and a zone of inhibition around the Tetracycline disk of 20 mm. Because these measurements are larger than the susceptibility zones listed on the Disk Zone Diffusion Diameter Chart, both of these antibiotics would be considered as possibilities for treatment. However, when we look more closely, we see that a 20 mm zone of inhibition for Tetracycline is only 1 mm larger than what is required to be susceptible while a 20mm zone of inhibition for Polymixin B is 8mm larger than the minimum susceptibility measurement needed. In this particular case then, Polymixin B and Tetracycline would both be adequate for treatment, but the Polymixin B would be the best choice. Materials 1 actively growing broth or streaked plate of a single organism (pure culture) Gram stain materials (optional) 1 Mueller Hinton Agar (MHA) plate (use BHI plate for all streps) 1 jar sterile saline 1 sterile test tube 1 sterile 5 mL pipette (with pipette bulb) 1 sterile swab 0.5 McFarland test standard McFarland reference card Spectrophotometer (optional) 8 disk dispenser or individual disk dispensers Antibiotic disk cartridges Ampicillin (AM-10) Chloramphenicol (C-30) Nalidixic Acid (NA-30) Penicillin G (P-10) Polymixin B (PB-300) Streptomycin (S-10) Tetracycline (Te-30) Trimethoprim (TMP-5) Procedure Procedures were taken from HardyDisk® Antimicrobial Sensitivity Test (AST) Disks, 2001. 1. Perform a Gram Stain to confirm culture purity from your subculture plate. 2. Using a sterile 5 mL pipette, add 5mL of sterile saline to a sterile test tube. Alternatively, a tube of sterile water or a tube of sterile tryptic soy broth (TSB) can be used. 3. Using an inoculating loop or needle, select several colonies from your subculture plate and transfer to a tube of sterile saline. Select several colonies so you don’t inadvertently pick a non-representative colony. 4. Dilute your organism to obtain a turbidity equivalent to the 0.5 McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard against the black-lined McFarland reference card to accurately rate the turbidity. This could also be measured in a spectrophotometer (87% transmittance at 686nm). 5. Within 15 minutes of diluting your organism, dip a sterile swab into the properly adjusted inoculum. Lift it slightly out of the suspension and firmly rotate the swab several times against the upper inside wall of the tube to express excess fluid. If your swab is too wet, your agar surface will not dry correctly, and the antimicrobial agents in the disk will diffuse through the wet surface and not into the agar. 6. Streak the entire surface three times with the swab, turning the plate 60 degrees between streaking (turn the swab too) to obtain an even inoculation. streaking figure 7. Close the lid and let sit for 3-5 minutes before applying the drug impregnated disks. 8. Apply the disks by means of a dispenser using aseptic technique. Deposit disks so that the centers are at least 24 mm apart; up to 12 disks may be placed on a 150 mm plate, 5 disks on a 100 mm plate. We usually apply more than the recommended disk number to conserve plates and our recommendations are not used to treat any patients! 9. Lightly press the disk down with a sterile swab to make contact with the surface. You don't want to smush it into the agar itself! 10. Place your plate agar side up (inverted) in a 37°C incubator. Streptococcus organisms should be on BHI instead of MHA plates. Streptococcus organisms should be incubated in an atmosphere enriched with 5-10% CO2. 11. Examine the plate after 16-24 hours incubation. 12. Measure (in mm) only zones showing complete inhibition by gross visual inspection. Hold the measuring device (ruler or calipers) over the back of the inverted plate over a black non-reflective surface and illuminate from above. 13. Compare the values you obtained with those on the Disk Diffusion Zone Diameter Chart to determine the susceptibility level to the antibiotics used. 14. Report the values as: Resistant - indicates that clinical efficacy has not been reliable in treatment studies. Intermediate - implies clinical applicability in body sites where the drug is physiologically concentrated or when a high dosage can be used. Susceptible - implies that an infection due to the organism may be treated with the concentration of antimicrobial agent used unless otherwise contraindicated. CHAPTER SIX: CHALLENGES AND RECOMMENDATIONS The challenges encountered include; the absence of payment for the training which made feeding and transportation a burden. The observable dominance of the medical laboratory science staff over the microbiologist which suggests a low probability of being employed in the public health sector. Recommendation Students should be paid a certain amount as a means of encouragement, to also sort transport and feeding. The microbiology society of Nigeria should ensure the employability of microbiology graduates. REFERENCES 1. Barberán, A., Ramirez, K. S., Leff, J. W., Bradford, M. A., Wall, D. H., & Fierer, N. (2014). Why are some microbes more ubiquitous than others? Predicting the habitat breadth of soil bacteria. 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