Introduction To Microbiology PDF

GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 INTRODUCTION TO MICROBIOLOGY MICROBIOLOGY - A branch of biology that...

GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 INTRODUCTION TO MICROBIOLOGY MICROBIOLOGY - A branch of biology that primarily researches on microorganisms that are too small to be seen with naked eyes. - Microbiology is mainly a study of microbial morphology, structure, physiology and biochemistry, heredity and variation, classification, distribution and its relationship with nature. Microorganisms - are microscopic creatures that are invisible with naked eyes - Microorganisms are classified into two: acellular and cellular. Bacteriology - Study of bacteria. - Absorbs nutrients from their environment and some make their own nutrients by photosynthesis or other synthetic processes. - Occupy space on land and can live in aquatic environment and in decaying matter. - Some are pathogenic. - Bacillus antharacis. Bacteria that cause anthrax. Mycology - Study of fungi. - Fungi are eukaryotic. - Absorbs nutrients from external environment. - Fungi are not photosynthetic. - Tinea pedis. It is better known as athlete’s foot. It is caused by fungus. Virology - Study of viruses. - A virus is a submicroscopic, parasitic entity composed of nucleic acid core surrounded by a protein coat. - Parasitic means that a virus receives food and shelter from another microorganism and is not divided into cells. - Varicella-zoster virus. A virus that causes chickenpox in humans. - Variola virus. A virus that causes small pox. “Cowpox” in cattle. Veterinary Microbiology - Branch of microbiology that studies microorganism associated with infectious disease of animals. Immunology - Study of how an organism defends itself against infection by microorganism. - When a pathogen invades the body, leukocytes engulf the microorganism and digest it as an immune response called “phagocytosis”. 1 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Phagocytosis is the ability of a cell to engulf and digest solid materials by the use of “pseupods” or “false feet”. Veterinary Immunology - Study of host (animals) responses to infectious agent. - Distinct but cognate. Protozoology - The study of protozoa, animal-like single cell microorganisms that can be found in aquatic and terrestrial environment. - Many of these protozoa obtain their food by engulfing smaller organisms. - Amoeba proteus. Phycology - The study of algae. - Algae are eukaryotic photosynthetic organisms that transform sunlight into nutrients using photosynthesis. - Eukaryotic photosynthetic microorganism has cells containing nucleus, nuclear envelope, cytoplasm and organelles and is able to carry out photosynthesis. TWO TYPES OF MICROORGANISM 1. Pathogenic - Are microorganisms that infect animals, plants and humans. They have the ability to cause infection by infiltration which leads to the production of disease. - Example: Yersinia pestis is the microorganism that caused the Black Plague. Yersinia pestis first infected fleas that were carried into populated areas on the backs of rats. These rodents then traveled on ships and then over land in search of food. Fleas jumped from the rodents and bit people, transmitting Yersinia pestis into the person’s blood stream. 2. Non-Pathogenic - They do not cause disease to an individual but rather help to maintain homeostasis in our bodies and used in the production of food and other commercial products. - Example: Flora are microorganisms found in our intestine that assist in the digestion of food and play critical role in the formation of vitamins such as vitamin B and vitamin K. They help by breaking down large molecules into smaller ones. MAJOR CATEGORY OF MICROORGANISM 1. Cellular Microorganism - These are microorganism that contains cellular contents and is involved in metabolism. - Cellular microorganism includes protozoa, bacteria, fungi and parasites. - Cellular microorganism is further divided into: a. Prokaryotic cell type/organism 2 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 o Are organism whose cell lacks nucleus and other membrane-bound organelles. o Bacteria, spyrochaeta, mycoplasma, rickettsia, and chlamydiae. b. Eukaryotic cell type o Are organisms whose cells contain nucleus and other membrane-bound organelles. o Yeast and mold. 2. Acellular Microorganism - are microorganisms that doesn’t have cellular structures. - Made out of proteins. - Includes virus, viroids and prions. - Usually contains an infectious part – the ability of an organism to invade system causing disease and transform the microorganism infectious. What is the importance of studying microbiology? 1. Biological factors - Life forms - Provide accessible tools that could explain nature and life processes. - Why do we need to use disinfectants. - In fermentation, microorganism is responsible to produce the enzymes needed. 2. Applied Biological Science - Apply theories to come up in an invention and discoveries. - Applied in agriculture (pesticide, resistance and probiotics), medicine (antibiotics, prophylaxis, vaccine and insulin) and food industry (beverage and pastries using yeast). HISTORY OF MICROBIOLOGY 1. Lucretius and Girolamo Fracastoro - Lucretius is a Roman philosopher while Girolami us a physician. - They believed that invisible creatures were responsible for disease – “animate agents” - Di pa napatunayan due to lack of equipments. 2. Francesco Stelluti - He approached Galileo Galilei and used the lenses of Galileo to observe organism. - He observed bees and weevils (small insects that infect crops) using a microscope in the early 1600s. 3 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 3. Antonie van Leeuwenhoek - Dutch merchant. - Hooke’s experiments with a crude microscope inspired Leeuwenhoek to further explore the micro world. - He was an amateur lens grinder and he improved Hooke’s microscope by grinding the lenses to achieve magnification. His microscope required one lens and used this to observe bacteria and protozoa. - He observed the pus first and he discovered that there are small animal/life/ microorganism. - He was the first to discover and described these microorganisms in which he called as “animalcules”. - He opened the door of the microbiology. - He is regarded as “The Father of Microbiology”. Emergence of Experimental Science - You do trial examinations that leads to new discoveries. - Naturalist – people who rely on nature. Life is dependent to external force. ORIGIN OF ORGANISMS 1. Spontaneous Generation - Invisible creatures/living organisms come from non-living things or inanimate objects. - Many people wanted to oppose this claim and conducted an experiment on their own to prove their points which includes: a. Francisco Redi - Father of Parasitology - Italian Physician who developed an experiment that demonstrated that an organism did not spontaneously appear. - Experimented using maggots and decaying meat. He prepared a jars filled with meat and he sealed some jars and he left the others opened. The unsealed jars contained maggots while the others did not have maggots because flies could not enter the jar to lay eggs. - His critics stated that air was the ingredient required for spontaneous generation of an organism. Air was absent from the sealed jar and therefore, no spontaneous generation could occur. - Redi repeated the experiment except this time, he placed a screen over the opened jars. This prevented flies from entering the jar. There wasn’t any presence of maggots in the rotting meat. - The discovery of Redi gave scientists an idea, including Louis Pastuer, that killing the microorganisms that caused a disease could prevent the disease from occurring. A new organism could only be generated by another microorganism when it underwent a reproductive process. Kill that microorganism and you will not have new microorganisms. This idea leads to the theory of biogenesis. ❖ Theory of Biogenesis - The theory states that a living cell is generated from another living cell. - You could stop the spread of disease by killing the microorganism who caused the disease. 4 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 b. John Needham - An English naturalist who experimented a mutton broth. - He boiled a mutton broth and sealed the jar. After few days, Needham observed that the broth had become cloudy and a single drop contained numerous microscopic creatures. He argued that the new microbes must have arisen spontaneously. However, the presence of microorganism from the broth was likely caused by not boiling the broth enough to kill all preexisting microbes. - Basically, the presence of microorganism from the broth of sealed flask or needham was caused by the organism from meat. c. Lazaro Spallanzani - He reattempted Needhams’s experiment using a sealed flask. - He prepared a flask with a seed inside and a water and heated it. There are no signs of spontaneous growth, unless the flasks were subsequently opened to air. - This suggests that microbes were introduced into these flasks from the air. - In response to Spallanzani’s discovery, Needham argued that life originates from a “life force” that was destroyed during Spallanzani’s extended boiling. Any subsequent sealing of the flasks then prevented new life force from entering and causing spontaneous generation. d. Franz Schulze and Theodore Schwann - Support the work of Spallanzani. - They viewed that air was the source of microbes. - They allowed boiled broth to come into contact with air that was either heated or passed through solutions of toxic chemicals. No microscopic organism grew in their broth. However, many vitalists discredited this work because they said that the drastic treatment of the air had rendered it inactive. e. George Friedrich Schroder and Theodor Von Dusch - Allowed air to enter a flask of heat-sterilized medium after it had passed through sterile cotton wool. No growth occurred in the medium even though the air had not been heated. f. Felix Pouchet - In 1859, he wanted to prove that microbial growth could occur without air contamination. g. Louis Pasteur - Father of microbiology and pasteurization. - He experimented on Schwann neck flask. - He proved the theory of spontaneous generation not true and the presence of microorganisms were in the air. - He proved that sterilized medical instruments became contaminated once they were exposed to air. 5 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - To settle the matter of spontaneous generation, he boiled a beef broth to destroy living microorganisms that may have been present in broth then put it in a S-shaped neck flasks. The neck of the flask trapped airborne organism. He then tried to cut the neck of the flasks for easier air exposure and a growth of organism occurred. He also tried to tilt the flask without cutting the neck for easier exposure to air of some of the solution which eventually leads to microbial growth. - His experiment proved that air is needed to support life. Microorganism are not spontaneously generated but it arises from other microorganisms. This also proved that air and other vital forces are needed for growth. ❖ Pasteurization - Apply 120-degree Fahrenheit to milk (49-60 degree Celsius) to kill pathogenic bacteria while still retaining the important contents of milk. h. John Tyndall and Ferdinand Cohn - Their discoveries led to one of the most important discoveries in sterilization. - Tyndall demonstrated that dust carry germs and that if dust was absent, broth remained sterile even if directly exposed to air. This idea of Tyndall opposes the idea of Pouche. - Tyndall also provided evidence for the existence of exceptionally heat-resistant bacteria. - Working independently, Cohn discovered that the heat-resistant bacteria recognized by Tyndall were species capable of producing bacterial endospores. Cohn later played an instrumental role in establishing a classification system for bacteria based on their morphology and physiology. 4. IgnazSemmelwees - Great studies were made during the late 1800s in the development of antiseptic techniques. It began with a report by Hungarian physician IgnazSemmelweis on a dramatic decline in childbirth fever when physicians used antiseptic techniques when delivering babies. Infections become preventable through the use of antiseptic techniques. 5. Joseph Lister - Father of modern surgery. - ASEPTIC TECHNIQUE – a technique which literally means “without dirt”. A technique for the prevention of infection in would during and after surgery. - During surgery, he sprayed carbolic acid over the patient and then bandaged the patient’s wound with a carbolic acid-soaked bandages. Infection following surgery dropped when compared with surgery performed without the carbolic acid. - Carbolic acid/phenol – one of the first surgical antiseptic. 6. Paul Ehrlich - A German chemist. - He blended chemical elements into a convocation that when inserted into an infected area, killed microorganisms without affecting the patient. - This mixture or experimentation of Ehrlich is now what we call drug. - Ehrlich’s innovation led to chemotherapy using drugs that are produced by chemical synthesis. 6 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 7. Alexander Fleming - He discovered Penicillium notatum, a microorganism that synthesizes penicillin. It became one of the first antibiotic, a substance that kills bacteria. - Penicillium notatum is a fungus that kills Staphylococcus aureus and similar microorganisms. - Before he came up with this discovery, Fleming grew cultures of Staphyloccus aureus, a bacterium, in the laboratory. He was also conducting experiments with Penicillium notatum, a mold. By accident the Staphyloccous aureus was contaminated with the Penicillium notatum, causing the Staphyloccocus to stop reproducing and die. 8. Robert Koch - Contagious disease- a diseased that is spread by being exposed to infection. - Best known for his achievement to the germ theory of disease that specific pathogenic microorganisms caused specific disease. - He proposed a criterion, namely the “Kochs Postulate” for affirming that a kind of microorganism is the reason for a unique infectious disease. The content of Koch’s postulate is: i. The microorganism should exist abundantly in all cases but not in healthy animals. ii. The microorganisms should be isolated from sick cases and grow in culture medium. iii. The cultured microorganism should cause the same disease after entering into a healthy animal iv. The microorganism should be re-isolated from the experimental animal and identified as same with the original microorganism. - Kock’s work with anthrax also developed techniques for growing culture of microorganism. He eventually used a gelatin surface to cultivate microorganisms. These gelatins inhibited the movement of microorganisms. As microorganism reproduced, the remained together, forming a colony that made them visible without a microscope. The gelatin was replaced with agar that is derived from seaweed and is still used today. - The reproduction of microorganism is called colonizing. 9. Richard Petri - Developed petri dish. - Further developed the technique of agar culture to purify or clone bacterial colonies derived from single cells. This advance made it possible to rigorously identify bacteria responsible for disease. 10. Zacharias Janssen - Developed the first compound microscope during 1590 in Middleburg, Holland. - Janssen’s microscope consisted of three tubes. One served as the outer casing and contained the other two tubes. At either ends of the inner tubes were lenses used for magnification. - Jannsen’s design enabled scientists to enlarge the image of a specimen three and nine times the specimen’s actual image. 11. Robert Hooke - In 1665, he popularized the use of compound microscope when he placed lenses over slices of cork and viewed little boxes that he called “cells” - His discovery led to the development of cell theory in the 17th century by Mathias Scleiden, Theodor Schwann and Rudolf Virchow. - The cell theory states that “all living things are composed of cells”. 7 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 12. Elie Metchnikoff - one of the first scientists who studied immunology. - He was the one who discovered phagocytosis in 1880. He discovered that leukocytes defended the body by engulfing and eating the invading microorganism. 13. Edward Jenner - An English physician who discovered something interesting about small pox and cow pox in 1796. - He discovered that those of survived smallpox never contracted smallpox again, even when they were later exposed to someone who was infected with smallpox. - To prove his theory, he took a scraping from a cowpox blister and scrapped it into the arm of James Phipps, an 8-year-old. Phipps became slightly ill when the scratch turned bumpy. Phipps recovered and was then exposed to small pox. He did not contract small pox again because his immune system developed antibodies that could fight off the virus. - Jenner’s experiment discovered how to use our body’s own defense mechanism to prevent disease by inoculating a healthy person with a tiny amount of the disease-causing organism which he later then called – vaccination. - The person or animal who receives the vaccination became immune to the disease-causing microorganism. 14. Carl Linnaeus - Developed the system for naming organisms in 1735 known as Binomial nomenclature. - Each organism is assigned two latinized named because latin or Greek was the traditional language used by scholars. a. The first name is called genus. b. The second name is called specific epithet, the name of species. c. The genus and epithet appear italicized. - Example: Staphylococcus aureus, Escherichia coli 15. Carl Woese - Developed a system that arranged organisms according to their molecular and cellular characteristics. This classification was based on the similarities in the 16s rRNA sequencing. - Ribosomes: Eukaryote – 18s small subunit, 80s Large Prokaryotes – 16s small subunit, 70 - He also devised three classification groups called domain, that is larger than a kingdom. 8 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 MICROBIAL GROWTH BINARY FISSION The most common mechanism of cell replication in bacteria is through binary fission. Process of Binary Fission 1. Before dividing, the cell grows and increases its number of cellular components. 2. Proceeds to replication of DNA at the origin of replication (circular chromosome), where the chromosome is attached to the inner cell membrane. 3. Replication continues in opposite directions along the chromosome until the terminus is reached. 4. Cytokinesis (division of cytoplasm) takes place. Protein FtsZ assembles into a Z-ring on the cytoplasmic membrane. The Z- ring is anchored by the FtsZ-binding proteins and defines the division plane between the two daughter cells. 5. Additional proteins required for cell division are added to the Z- ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. 6. The daughter cells are now separated where all the cell’s outer layers (cell wall and outer membranes) must be re-modeled to complete cell division. 7. Two daughter cells of similar size form and separate, receiving a copy of the original chromosome. GENERATION TIME In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas, Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but there are exceptions. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease (leprosy), grows much more slowly, with a doubling time of 14 days. 9 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 CALCULATION OF NUMBER OF CELLS It is possible to predict the number of cells in a population when they divide by binary fission at a constant rate. As an example, consider what happens if a single cell divides every 30 minutes for 24 hours. The number of cells increases exponentially and can be expressed as 2n, where n is the number of generations. For example, if cells divide every 30 minutes, after 24 hours, 48 divisions would have taken place. If we apply the formula 2n, where n is equal to 48, the single cell would give rise to 248 or 281,474,976,710,656 cells at 48 generations (24 hours). When dealing with such huge numbers, it is more practical to use scientific notation. Therefore, we express the number of cells as 2.8 × 1014 cells. The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn, divides, giving a total of four cells in the second generation and eight cells in the third generation. Each division doubles the number of cells. GROWTH CURVE 1. Lag Phase - Represents a small number of cells “inoculum” that are added to a fresh culture media. - First stage of bacterial reproduction. - Unaware what to do. - No reproduction dahil nag-aadjust palang. - Checks if environment is good area to reproduce. - Period of adaptation. - Cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. - Its length varies depending on how different conditions are from the conditions that the bacteria came from, a s well as the condition of the bacteria cell themselves. - Typically, cells in the lag period are synthesizing RNA, enzymes, and essential metabolites that might be missing from their new environment. They can also be undertaking any necessary repair of injured cells. 2. Log Phase - Once cells have accumulated all that they need for growth, they proceed into cell division (binary fission). - Bacteria divides exponentially (12), one cell becomes 2 cells, becomes 4, becomes 8 etc. - The generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate. - Conditions that are optimal for the cells will result in rapid growth, while less than ideal will result in slow growth. 10 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Assimilation of nutrients from media and production of waste products which decreases the pH level. - Cells in the log phase is the healthiest and most uniform. They are also preferentially used for industrial applications and research work. - The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis. 3. Stationary Phase - During this phase, bacterial population runs out of essential nutrients, accumulation of waste products and gradual depletion of oxygen begins to limit aerobic cell growth causing the new cells to enter into stationary phase. - Reproduction had stopped and bacteria reached its peak in bacterial reproduction or “plateu”. - In this phase, the number of new cells produced is equal to the number of cells dying off. - Cells switch to a survival mode of metabolism. - As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids. - Stationary cultures are less susceptible to antibiotics that disrupt these processes. - In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. - Secondary metabolites, including antibiotics, are synthesized in the stationary phase. - In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. - For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful. 4. Death/Decline Phase - The number of viable cells decreases in an exponential manner. The steepness of the slope corresponds to how fats cells are losing viability. - Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. - Persisters are cells characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment. - It is though that the culture conditions have deteriorated to a point where the cells are irreparably harmed, since cells collected from this phase fail to show growth when transferred to fresh medium. SUSTAINING MICROBIAL GROWTH One example is in industries that harvest microbial products. A chemostat (culture vessel) is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment (logarithmic phase). 11 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 ALTERNATIVE PATTERNS OF CELL DIVISION Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments. In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of many new cells at once. The new cells often split from the parent filament and float away in a process called fragmentation. Fragmentation is commonly observed in the Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant bacterium Epulopiscium. Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment. Other species may form a long narrow extension at one pole in a process called budding. The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast, but it is also observed in prosthecate bacteria and some cyanobacteria. The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments, elongated cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise to new colonies. Culture Media - the medium that provides nutrient required by organism to grow. BASIC COMPONENTS 1. Beef Extract – protein requirements, vitamin and mineral. 2. Peptone – solution for transport and source of carbohydrate. 3. Yeast extract – aids in fermentation of components of media. 4. Agar – solidifying agent. Ex. Broth – beef + peptone +yeast + agar for solidification. PREPARATION OF AND COMPUTATION OF CULTURE MEDIA Recommended: 20ml/plate ; 3g/1000 ml 12 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 Components Broth Agar (g) Beef 3g 3g Peptone 5g 5g Yeast Varies if needed by the bacteria Distilled Water 1000ml 1000ml Agar None 15g Formula: Solution: X/100 ml = 3g/1000ml X/100ml = 3g/1000ml (cross multiply X = unknown weight of agar to be added during media X (1000 ml) = 3g (100 ml) preparation X = 300g/ml/ 1000 ml 100ml = volume of distilled water to be used X=0.3g 3g/1000 ml = weight of beef extract per 1000ml distilled water TYPES OF CULTURE MEDIA A. Based on Consistency 1. Liquid - is used for profuse growth (blood culture in liquid media) - Mixed organisms cannot be separated. - Nutrient broth 2. Solid - is used for the isolation of bacteria as pure culture. - Agar is most commonly used to prepare solid media. It is a polysaccharide obtained from seaweed. Agar is an ideal solidifying agent because it is bacteriologically inert, has no influence on bacterial growth, remains solid at 37 degree-Celsius and transparent. - Some of the advantages of using solid media: a. Bacteria may be identified by studying the colony character. b. Mixed bacteria can be separated. 3. Semi-solid – Gelatin B. Based on Chemical Composition I. Routine Laboratory Media 1. Basal Medium - Common nutrients needed by microorganism. - Supports growth. - Nutrient broth, nutrient agar and peptone water. Staphylococcus and Enterobacteriaceae grow in these media. 2. Enriched Media - Basal medium + enriched nutrient (blood or serum). - Needed by bacteria to survive especially fastidious bacteria that has a particular nutritional requirement. - Blood agar and Lowenstein-Jensen media. Streptococci grow in blood agar. 3. Enrichment Bacteria - A medium that increases the number of target bacteria. 13 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Alters the environment - Eg. Cold medium. 4. Selective Media - A medium that consists an inhibitory substance (inhibit the growth of another microorganism). - It favors the growth of particular bacterium by inhibiting the growth of undesired bacteria and allowing the growth of desired bacteria. - Antibiotic may be added to a medium for inhibition. - Example: MS4 – Manitol Salt Agar – inhibit gram negative bacteria. MacConkey Agar – inhibit positive and pinapatubo ang gram negative bacteria. 5. Differential Media - An indicator is included in the medium - A particular organism causes change in the indicator (blood, neutral red, tellurite) - Differentiates specie media and determine which species lack a specific biochemical process. - Allows the growth of selective bacteria. - MacConkey Agar - Differentiates lactose fermenting bacteria from non-lactose fermenting bacteria. 6. Maintenance Media - Maintain viability of bacterial cell. - Allows growth of bacteria and supply maintenance media. - Examples include nutrient broth and nutrient agar. 7. Reduced/Anaerobic Media - Oxygen is removed. - Contains reducing agents. - Cysteine, thioglycolate, sodium, ascorbate, cooked meat. 8. Special Media - Media used for the cultivation of specific organisms. 9. Transport Media - A media to prevent drying of specimen, maintain the pathogen to commensal ratio and inhibit overgrowth of unwanted bacteria during transfer. - Cary-Blair medium, amies medium and stuart medium. 10. Storage Media - Media used for storing bacteria for a long period of time. - Egg saline medium, chalk cooked meat broth. II. Common Media in Routine Use 1. Nutrient Broth a. Components: - 500g meat is minced and mixed with 1 liter water. - 10g peptone and 5g sodium chloride are added, pH is adjusted to 7.3 b. Uses - As a basal media for the preparation of other media. 14 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - To study soluble products of bacteria. 2. Nutrient Agar - It is solid at 37 degree-Celsius. 2.5% agar is added in nutrient broth. - It is heated at 100 degree-Celsius to melt the agar and then cooled. 3. Peptone Water a. Components - Peptone 1% and sodium chloride 0.5%. b. Uses - It is used as base for sugar media and to test indole formation. 4. Blood Agar a. Components - 5-10% defibrinated sheep or horse blood is added to melted agar at 45-50 degree-Celsius. b. Uses - Most commonly used medium. - Blood act as an enrichment material and also as an indicator. - Certain bacteria when grown in blood agar produce haemolysis around their colonies. - Certain bacteria produce no haemolysis. c. Types of Changes (Haemolysis) a) Beta (p) Haemolysis - The colony is surrounded by a clear zone of complete haemolysis. - Example: Streptococcus pyogenes is a beta haemolytic streptococcus. b) Alpha (a) Haemolysis - The colony is surrounded by a zone of greenish discoloration due to formation of biliverdin. - Example: Viridans streptococci c) Gamma (y) Haemolysis - There is no change in the medium surrounding the colony. No haemolysis. 5. Chocolate Agar or Heated Blood Agar - Prepared by heating blood agar. - Heating the blood inactivates inhibitor of growths. - It is used for culture of Pneumococcus, Gonococcus, Meningococcus and Haemophilus. 6. MacConkey Agar a. Components: - Contains agar, peptone, sodium chloride, bile salt, lactose and neutral red. b. Uses: - Most commonly used for Enterobacteriaceae - It is a selective and indicator medium a) Selective Medium - As bile salt does not inhibit the growth of Enterobacteriaceae but inhibits growth of many other bacteria. b) Differential Medium 15 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - As the colonies of bacteria that ferment lactose take a pink color due to production of acid. c. Results a) Acid turns the indicator neutral red to pink. Escherichia coli. These bacteria are called “Lactose Fermenter” b) Colorless colony indicates that lactose is not fermented. Salmonella, Shigella, and Vibrio. The bacterium is “Non-lactose Fermenter” 7. Mueller Hinton Agar - Disc diffusion sensitivity tests for antimicrobial drugs should be carried out on this media as per WHO recommendation to promote reproducibility and comparability of results. 8. Hiss’s Serum Water Medium - This medium is used to study the fermentation reactions of bacteria which cannot grow in peptone water sugar media. Pneumococcus, Neisseria, Corynebacterium. 9. Lowenstein-Jensen Medium a. Components: a) Egg – an enrichment material which stimulates the growth of Tubercle bacilli. b) Malachite green – inhibits growth of organisms other than Mycobacteria. c) Glycerol – promotes the growth of Mycobacterium tuberculosis but not Mycobacterium bovis. b. Uses - Used to culture Tubercle bacilli. 10. Dubos medium – drug sensitivity of tubercle bacilli can be carried out. a. Components - It contains “tween 80”, bovine serum albumin, casein hydrolysate, asparagine and salts. b. Uses - This liquid medium is used for Tubercle bacilli. - Tween 80 causes dispersed growth and bovine albumin causes rapid growth. 11. Loeffler Serum - Diphtheria bacilli grow in this medium in 6 hours when the secondary bacteria do not grow. a. Components - It contains sheep, ox or horse serum used for enrichment. b. Uses - Used for rapid diagnosis of diphtheria and to demonstrate volutin granules. 12. Tellurite Blood Agar a. Components - Tellurite inhibits the growth of most secondary bacteria without any inhibitory effect on diphtheria bacilli. - It is also an indicator medium as the diphtheria bacilli produce black colonies. - Tellurite metabolized to tellbrism, which has black color. b. Uses - it is used as a selective medium for isolation of Corynebacterium diphtheriae. 16 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 13. Eosin-Methylene Blue Agar (EMB Agar) - A selective and differential medium for enteric Gram-negative rods. Lactose- fermenting colonies are colored. Non-lactose fermenting colonies are non- pigmented. 14. Xylose Lysine Deoxycholate (XLD Agar) - A selective media used to isolate Salmonella and Shigella species from stool specimens. 15. Salmonella-Shigella Agar (SS Agar) - A selective medium used to isolate Salmonella and Shigella species. SS agar with additional bile salt is used if Yersinia enterocolitica is suspected. 16. Deoxycholate Citrate Agar (DCA) - The other enteric bacteria are mostly inhibited (a selective medium) - It is also a differential medium due to presence of lactose and neutral red. - It is used for isolation of Salmonella and Shigella. 17. Tetrathionate Broth - it acts as a selective medium. - It inhibits normal intestinal bacteria and permits multiplication of Salmonella. - This medium is used for isolating Salmonella from stool. 18. Selenite F Broth - Same as tetrathionate broth. For isolation of salmonella from stool. 19. Thiosulphate-Citrate-Bile-Sucrose (TCBS) Agar - A selective medium used to isolate Vibrio cholerae and other Vibrio species from stool. 20. Charcoal-Yeast Agar a. Components - Increased concentration of iron and cysteine allows growth b.Uses - Used for Legionella pneumophila 21. Tellurite- Gelatin Agar Medium (TGAM) - used as transport, selective and indicator medium. 22. Campylobacter Medium - A selective medium used to isolate Campylobacter jejuni and Campylobacter coli from stool. 23. Cary-Blair Medium - Is used as a transport medium for feces that may contain Salmonella, Shigella, Vibrio or Campylobacter species. 24. Amies Medium - used for Gonococci and other pathogens. 25. Peptone Water Sugar Media - Colorless media - After culture, change of a medium to red color indicates acid production. - A small inverted Durham tube is placed in the medium. - Gas, if produced collects Durham tube. a. Components 17 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - 1% solution of a sugar (lactose, glucose, mannitol, etc) is added to peptone water containg Andrade’s indicator in a test tube. b. Uses - These indicator media are used to study sugar fermentation. 26. Motility Indole Urea (MIU) Medium - Is used to differentiate Enterobacteria species by their motility, urease, and indole reactions. 27. Kingler Iron Agar (KIA) - A differential slope medium. - The reactions are based on the fermentation of lactose and glucose and the production of hydrogen sulfide. - Used in the identification of Enteric bacteria. 28. Christensen’s Urea Medium - This is used to identify splitting organisms like Proteus. - When used, a purple pink color indicates urea splitting. 29. Bordet-Gengou Medium a. Components - It contains agar, potato, sodium chloride, glycerol, peptone and 50% horse blood. Increased concentration of blood allows growth. - Penicillin may be added to it. b. Uses - Used for culture of Bordetella pertussis. MICROSCOPY - The technical field of using microscope to view samples and objects that cannot be seen with naked eye. - Magnification – ability of lens to enlarge specific sample. UNITS OF MEASUREMENT 1. Mm - Millimeter - Bacterial and Fungal Colony (0.5-5mm) 2. Um - Micrometer or microns - Fungal and bacterial (0.5-5um) 3. Nm - Nanometer - Viruses (20nm-300nm) TYPES OF MICROSCOPES 1. Compound Light Microscope - Examining stained or unstained particles or the cellular structures of tissues that have been cut into sections and stained. 18 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - It has a resolving power (measure of the distance of one thing from one point to another) 0.2um. 2. Electron Microscope - Used to investigate the ultra-structure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals and crystals. - Uses an electron beam of a very short wavelength as source of light. - Has a resolving power of 2nm that is 100 times greater than the light microscope. TYPES OF COMPOUND LIGHT MICROSCOPE 1. Bright Field - Observe morphology of microorganisms (bacteria/fungi) - Cannot observe microbes less than 0.2 um in diameter or thickness such as spirochetes and viruses. - 1000x magnification. 2. Dark Field - 1000x magnification - Examining thin spirochetes - Gives color to field of specimen to highlight shape of microorganism. 3. Phase Contrast - 1000x magnification - Observe unstained living organism - Research purposes than diagnostic purpose. 4. Fluorescence Microscope - 1000X Magnification - Detect microbes in cells, tissue and clinical specimen. - Primarily on immunodiagnostic technique (immunofluorescence) - Fluorescent dye attached to organism. - Ex. Body fluids and lymph nodes. 5. Nomarski Differential Interference Contrast Microsope - Produces 3D image of a living specimen due to prisms and 2 beams of light used. 6. Atomic Force Microscopy - Provide 3D image - Uses stylus to positioned extremely closed to the specimen. - The interaction is recorded in a computer. - A powerful technique that can image almost any type of surface, including polymers, ceramics, composites, glass, and biological samples. 7. Confocal Scanning Laser Microscope - Couples a light source to a microscope and makes use of a fluorescent dye. 19 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 TYPES OF ELECTRON MICROSCOPE 1. Scanning Electron Microscope - 100,000X - Scan the surface area - Examining surface features of cells and viruses. - Specimen is viewed on screen. - Give the illusion of depth (3D) when coated with thin film of heavy metal. 2. Transmission Electron Microscope - 200,000 X - Beams of electron is transmitted through specimen to form image. - Specimens are placed on grids and stained with electron-dense compounds such as Potassium Phosphotungstate. SPECIMEN PREPARATION 1. Smear - A preparation process where a specimen is spread on a slide. Prepare a smear using heat fixation. Fixation Uses air and heat to attach microorganism on a slide, it kills the microorganism and preserves its morphology. 2. Wet Mount - A preparation process where a live specimen in culture fluid is placed on a concave glass side or a plain glass slide. The concave portion of the glass tube forms a cup-like shape that is filled with a thick, sympy substance, such as carboxymethyl cellulose. - The microorganism is free to move about within the fluid, although the viscosity of the substance slows its movement. STAINING OF SPECIMEN Basic Dye - Cationic (positive ion) - The affinity (strong attraction) of bacteria for basic dyes is due to primarily to the large amount of nucleic acid in the cell’s protoplasm 20  GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Nucleic acid – negative - Ex. Crystal violet, methylene blue, safranin Acidic Dye - Anionic (Negative Ion) - Repelled by the negative charge of bacteria. Thus, staining the bacterial smear has the effect of coloring the background of sample. Leaving the bacterial cells clear and bright against dark background. - Ex. Nigrosin or Nigra Ink (Black), eosin and congo red. TYPES OF STAINS 1. Simple Staining - Directly staining of bacterial cell with a positively charged dye to see bacterial detail (Morphology of organism). - Use methylene blue, crystal violet or safranin. 2. Differential Staining - Uses multiple dyes. - Cells stain different colors based on properties. - Reveals basic morphology such as size and shape. - Differentiate organisms from another. - Use Gram stain (positive and negative), Acid-Fast stain (fast or non-fast). 3. Structural Staining - Highlights the structure of microorganism like the flagella (number and location), spore(location) and capsule (use nigrosin). MICROBIAL STAINING 1. Gram Staining - Christian Gram. - A method for the differential staining of bacteria. - Gram positive microorganism stain purple. Gram negative microorganism stain pink. Primary Crystal 1 minute 1 Stain Violet Secondary Saffranin 30 seconds 4 V Stain I Mordant Lugol’s 1 minute 2 iodine A Decolorizer Acetone or 10-30 3 ethyl seconds S alcohol 2. Acid-Fast Staining / Ziehl-Neelsen Method - Developed by Franz Ziehl and Friedrick Neelsen - A red dye that attraches to the waxy material in the cell walls of bacteria. 21 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Microorganisms that retain the Zichl-Neelsen acid-fast strain are called acid-fast. Those that do not retain it turn blue because the microorganism doesn’t absorb the Ziehl-Neelsen acid-fast stain - Use to determine bacteria that are able to resist decolorization by acid alcohol (acid-fast) due to the presence of lipids that bind tenaciously with the primary stain. - Procedure: Primary Stain Carbon 5 min (heat) Fuchsin Secondary Methylene 15 seconds Stain blue Wash Acid alcohol 1 minute 3. Schaeffer- Fulton Endospore Stain - A special stain to colorize endospore. TAXONOMY OF MICROORANISMS Species - is the unit classification of microorganisms. - However, it is impractical to study the characteristics of one cell, so we study culture instead. 1. Pure (axenic) culture - When it consists of a single kind of microorganism. - Microorganism grown from one cell. 2. Mixed culture - Culture with two or more kinds of microorganism. 5 MAJOR KINGDOM (by Whittaker) 1. Monera (Prokaryote) - Bacteria 2. Protista - Amoebas, diatoms, and other single-celled eukaryotes, and sometimes simple multicellular organisms, such as seaweeds. 3. Fungi - Multicellular, filamentous organisms that absorb food. 4. Plantae - Multicellular organisms that make food through photosynthesis. 5. Animalia - Multicellular organisms that ingest food. 22 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 3 DOMAIN - In 1990, Carl Woese formed a new category, called a Domain, to reflect evidence from nucleic acid studies that precisely revealed evolutionary, or family, relationships. - He proposed three domains, based largely on the type of RNA in cells.: 1. Archaea - Have distinct molecular structures and physiological characteristics. - They live in extremely hot, saline, or acidic anaerobic environments. - prokaryotes that do not have peptidoglycan cell walls. Acidophiles Acidic environment Alkalophile Alkalinic environment Thermophiles Hot environment Psychrophiles Cold environment Halophiles Salty environment Piezophiles Extreme Pressure PHYLUM UNDER ARCHAEABACTERIA a. Euryarchaeota - Methanogens – thrive in areas without oxygen such as deep water and volcano. - Halophiles – thrive in salty environment, requires oxygen, produces pink pigment and can also thrive in alkalinic environment (11pH). - Thermoacidophiles b. Crenarchaeota - can thrive in 75-degree Celsius environment. - Can thrive in environment with a pH level of 2-3. - Can be found in hot sulfur springs or volcanic vents. c. Thaumarchaeota - Are widespread in marine and terrestrial habitats. - Some organisms are known to oxidize ammonia aerobically and play an important role in global nitrogen cycling. d. Korarchaeota - Thermophiles from hot springs. - It is suggested that these organisms grow by fermentation of peptides. - This phylum is still under study (candidate) - Consists of hyperthermophiles. e. Nanoarchaeota - Members of nanoarcheaota are associated with different host organisms and environmental conditions. - Despite small size, a reduced genome and limited respiration, they have unusual metabolic features. - This phylum is still under study. - Consists of hyperthermophiles. 23 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 2. Bacteria/ Eubacteria - Have a diameter of 0.5-1um. - Due to its small size, the surface area/volume ratio is very high compared to large organisms of similar shape. This large surface facilitates easy nourishment of all areas of the cell. So, there is no circulatory mechanism to distribute the nutrients. - Have peptidoglycan cell walls. A molecular structure of the cell walls of eubacteria which consists of N-acetyglucosamine, N-acetylmuramic acid, tetrapeptide, side chain and murein. - Kingdoms that belong to domain eubacteria: Animalia Plantae Fungi Protista PHYLUM UNDER BACTERIA Phylum With Veterinary Importance a. Gram negative – Proteobacteria (all gram- a. Gram negative bacteria negative bacteria) b. Gram positive bacteria b. Gram positives c. Clamydia c. Cyanobacteria and Prochlorophytes – d. Flavobacterium produces pigments e. Spirochetes d. Chlamydia e. Planctomycetes/Perillula f. Verrucomicrobia g. Flavobacterium – bacteriodes, flavobacterium h. Cytophaga i. Green-sulfur bacteria j. Sphirochetes k. Deinococcus l. Green-non sulfur m. Hyper thermophilic bacteria n. Defferibacter o. Fusobacteria p. Thermodesulfobacteria q. Nitrospira 3. Eukarya - is the domain that is made up of organisms that contain nucleus within their cells. NOTE: 1. Viruses are not classified according to binomial system because they are not cells and they cannot reproduce. 2. Viruses are only grouped according to families based on virion morphology and nucleic acid type. 24 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 NAMING OF ORGANISM Nomenclature (Linnean Binomial System of Nomenclature) - was established by a Swedish naturalist during 18 th century. It consists of generic and specific names. - The assignment of names to the taxonomic groups following the international rules: a. There is one correct name for an organism b. Names that cause error or confusion should be rejected c. All names are in Latin regardless of origin - The first word is capitalized - The second word is not capitalized - Both words are underlined/italicized d. Correct name of a species is determined by valid publication and legitimacy. 1. Based on Description Examples: a. Staphylococcus aureus – from the Greek staphyle (bunch of grapes) and kokkos (berry). Thus, staphylococcus is a cocci-shaped bacteria that tend to be arranged in cluster that are described as “grape-liked”. b. Saccharomyces cerevisiae - saccharomyces (sugar fungus) and cerevisiae (means beer making. Thus, Saccharomyces cerevisiae is a fungus that utilizes sugar to make beer. c. Penicillium notatum - penicillum (brush-liked), notatus (wind). Refers to the chains of conidia that resemble a broom. 2. In Honor of the Discoverer a. Klebsiella pneumoniae - discovered by Edwin Klebs. - A bacteria that can be found in the environment and associated with pneumonia in patient population with alcohol use disorder or diabetes mellitus. b. Salmonella typhimurium - Discovered by Daniel Salmon. - Typhi (typhoid), Muridae (rodents). - A pathogen that causes typhoid fever in mice. It is a less serious pathogen for humans but is the causative agent of a common for-borne enteric infections. 3. Based on habitat a. Massilia armeniaca – named after Marseille and Armenia. b. Bavariicoccus – Bavaria Germany. 16s rRNA Sequencing - A culture free method to identify and compare bacterial diversity from complex microbiomes or environments that are difficult to study. - Based from the size of RNA. - Svedberg unit – a measurement of particle’s size indirectly based on its sedimentation rate under acceleration. It represents the “s” in 16s rRNA. 25 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Prokaryote – 16s, small and only have 70s ribosomes. The 70s ribosome is made up of 50s and 30s subunits. The 50s subunit contains 23s and 5s rRNA while the 30s subunit contains the 16s rRNA.It can destroy small ribosome. - Eukaryote- 18s, 80s ribosomes. Larger than the prokaryote. INHIBITS CELL WALL SYNTHESIS 1. Penicillin - Have been shown to inhibit bacterial cell wall synthesis. Could kill microorganism that destroys the cell wall. - Gram positive bacteria but not Gram-negative bacteria (no cell wall) - Narrow spectrum antibiotic. PROKARYOTE VS EUKARYOTE LIFE PROCESSES OF LIVING THING 1. Metabolism - Break down nutrients for energy or extract from the environment. 2. Responsiveness - React to internal and external environmental changes. 3. Movement - Whether it is the entire organism relocating within its environment, cells within that organism or the organelles inside those cells. 4. Growing - Increase the size or number of cells 5. Differentiation - The process whereby cells that are unspecialized become specialized. - Example: A single fertilized human egg, developing into an individual (Prokaryotic cells do not differentiate). 6. Reproduction - Form new cells to create a new individual. PROKARYOTES - are single-celled organisms. - Most ancient forms of life on earth. - Characterized by the absence of true nucleus and membrane-bound organelles. - Nuclear structure is called nucleoid. It contains the cell’s genetic material and is usually a single circular molecule of DNA. - Typically have high resistance to many conditions. - Replicates through binary fission. 26 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 DOMAIN BELONGS TO PROKARYOTE 1. Bacteria - Have nucleoid (not separated by a membrane) - Lack histone proteins. - Do not have membrane-bound organelles such as mitochondria and endoplasmic reticulum. - Have simpler structures. - Have a cell membrane, in some cases cell wall and ribosomes. - Have flagella or pili. - Reproduction is through binary fission. 2. Archaea - Consists of three RNA. - Can sustain in harsh environments which are extreme such as hot spring, oceans, gut of humans, etc. - They lack peptidoglycans. EUKARYOTES - Contains eukaryotic cells (plants, animals, fungi and protists) - It is characterized by having a true nucleus containing DNA within a nuclear membrane and also containing membrane- bound organelles in its cytoplasm. - They are larger in size than prokaryotic cells. - Nucleus is separated from the cytoplasm by a nuclear membrane. - Flagella and cilia are the locomotory organs. - Cell wall is the outermost layer. - Cell divides through both asexual and sexual processes. - Photosynthesis: The process by which plants and other photoautotrophs generate carbohydrates and oxygen from carbon dioxide, water, and light energy in chloroplasts. COMPARISON BETWEEN PROKARYOTE AND EUKARYOTIC CEL 27  GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 Prokaryote Eukaryote Lysosome and Peroxisome Absent Present Microtubules Absent Present Endoplasmic reticulum Absent Present Mitochondria Absent Present Cytoskeleton Absent Present Ribosomes Smaller Larger Vesicles Present Present Cell Wall Present Not-present Cell division Binary fission Mitosis Reproduction Binary fission Sexual and Asexual Flagella Smaller Larger Complexity Generally simpler in structure More complex structurally and functionally die to and function organelles and cellular compartmentalization Example Archaea, Bacteria Plant, Fungi, Animal Cell EUKARYOTIC CELL STRUCTURE 1. Nucleus - Controls the functions of the entire cell. - Houses the cell’s DNA and directs the synthesis of proteins and ribosomes - It is where gelatinous matrix is seen. - Nucleus has three main components: a. Nucleolus - a dark area where the ribosomal RNA is formed and will exit to the nuclear membrane to become ribosome. b. Nuclear membrane - A double phospholipid layer that encloses the cell’s nucleus. - Continuous with endoplasmic reticulum. - Contains nuclear pores that allows substances to enter and exit the nucleus. c. Nucleoplasm - Substance inside the nucleus surrounded by nuclear membrane. - It is where the chromosomes are embedded. - Where ribosome synthesis happens. - Nucleoplasm + cytoplasm = protoplasm. Chromosomes - It consists of linear DNA where the genes are located and proteins (histone/non-histone). - Linear structures. Genes - Contains genetic information that enables the cell to produce one or more gene products (mostly protein). - Some gene codes for the production of RNA (rRNA and tRNA) - The collection of these genes is called “Genotype” (Genome) which is responsible for the characteristics or traits of an individual. Histone - any of various simple water-soluble proteins that are rich in the basic amino acids lysine and arginine and are complexed with DNA in the nucleosomes of eukaryotic chromatin 28 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 Chromatin - A complex of DNA, RNA, and proteins within the cell nucleus out of which chromosomes condense during cell division - Describes the material that makes up chromosomes, which are structures within the nucleus that are made up of DNA 2. Cytoplasm - The substance in which various cellular components are formed. - The location for most cellular processes, including metabolism, protein folding, and internal transportation. - Difference between eukaryotic and prokaryotic cytoplasm: a. Eukaryotes - Has complex internal structure that consist of small rods (microfilaments and intermediate filaments) and cylinders (microtubules). These structures make up the cytoskeleton. - Functions of Cytoskeleton: Provides support and shape Assist in transportation of substances through the cell. Assist in moving the entire cell (phagocytosis) Movement of the organelles (Cytoplasmic Streaming) b. Prokaryotes - Unlike eukaryotic cells, prokaryotic cells don’t have a nucleus and lack organelles. 3. Endoplasmic Reticulum - Extensive network of flattened membranous sacs/cisterns. - It synthesizes lipids and store/modifies proteins. - It also provides pathway for transport of those molecules to all parts of the cell through secretory vesicles. 4. Ribosomes - Are attached to the outer surface of the endoplasmic reticulum and can also be found as free in the cytoplasm. - It is responsible of protein synthesis in the cell. They receive their “orders” for protein synthesis from the nucleus where the DNA is transcribed into messenger RNA (mRNA). This mRNA travels to the ribosomes, which translate the code provided by the sequence of the nitrogenous bases in the mRNA into a specific order of amino acids in a protein. - Ribosomes in the eukaryotic cell is much large and denser compared to prokaryotes. - In eukaryotes, there are 80s ribosome each of which consist of large 60s subunits with 3 molecules of rRNA and small 40s subunit with 1 molecule or rRNA. 5. Golgi Complex - Generally, near the nucleus. - Similar to endoplasmic reticulum which consist of flattened sacs that are stocked on each other like a pile of dishes with expanded areas at the end. - Its function is to transport substances from and within the cell. The expanded areas detached from the cisterns to form secretory vacuoles. - Golgi complex receives proteins and lipids which will be then sorted, packaged and delivered by the secretory vesicles. Faith of Substances 29 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 a. Lipids – transported and become part of plasma membrane. b. Enzyme – will be carried by lysozyme. c. Proteins – are released to the exterior cell by fusion of secretory vesicles and plasma membrane. 6. Mitochondrion - Is often called as the “Powerhouse of the cell” because of their central role in ATP production. - Is a spherical or rod-shaped organelle that appears throughout the cytoplasm, - Consist of double membrane similar in structure in plasma membrane. - They have DNA of their own and machinery that allows them to replicate and reproduce on their own by growing and dividing. - Outer is smooth and the inner is arranged in series of folds called “cristae”. Because of the nature and arrangement of the cristae, it provides enormous surface area for on which chemical reaction occurs. Proteins that are needed for cellular respiration are found in cristae. - It contains enzymes that produces ATP. - At the center is a semi-fluid substance called “matrix”. Metabolic processes happen in the matrix. - The endosymbiotic hypothesis suggests mitochondria were originally prokaryotic cells, capable of implementing oxidative mechanisms. These prokaryotic cells may have been engulfed by a eukaryote and became endosymbionts living inside the eukaryote. 7. Chloroplast - A membrane-enclosed structure that contains both the pigment chlorophyll and the enzymes required for the light gathering phases of photosynthesis. - The chlorophyll is contained in flattened membrane sac called “Thylakoids” and stacks of this is called Grana. - Chloroplast can also multiply and increase their size. - Present in algae and green plants. 8. Lysozymes/ Lysosome - Are formed in the Golgi complex and look like membrane-enclosed spheres. - It only has single membrane and lack internal structure. - Contains enzyme capable of breaking down many kinds of molecules. - It functions in digestion and removal of waste. - Lysozyme – are enzymes for digestion in lysosome. It can digest bacteria. - In white blood cells, there are large number of lysozymes. 9. Vacuoles and Vesicles - Is a space or cavity in the cytoplasm that is enclosed by a membrane called Tonoplast. - This structure was derived from golgi complex and has several diverse functions: a. Temporary storage for substances. b. Storage of water which increase cell size. c. Help bring food to cell. d. Storage of metabolic waste and poison. 10. Centrioles - Are pair of cylindrical structures located near the nucleus that contains microtubules. - Play role in eukaryotic cell division. - Formation of cilia and flagella. 30 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 11. Cell Membrane - a mosaic composed of large molecules of proteins and phospholipids. - It is made up of bilayer of phospholipids with protein interspread within it. - They have the property of selective permeability. - Regulated the passage of nutrients, waste products and secretions in and out of the cells. - Only relatively small, non-polar materials can easily move through the cell membrane. - Passive transport is the movement of substances across the membrane that does not require the use of energy while active transport is the movement of substances across the membrane using energy. - Osmosis is the diffusion of water through a semi- permeable membrane down its concentration gradient; this occurs when there is an imbalance of solutes outside of a cell compared to the inside the cell. - Protects the cell. 12. Peroxisomes - Carry out oxidation reactions that break down fatty acids and amino acids. - It also detoxifies many poisons that may enter the body. ANIMAL CELL VS PLANT CELL Although both are eukaryotic cell, there are unique structural differences between animal and plant cells. Centrosomes and lysosomes are found in animal cells, but do not exist within plant cells. The lysosomes are the animal cell’s “garbage disposal”, while in plant cells the same function takes place in vacuoles. Plant cells have a cell wall, chloroplasts and other specialized plastids, and a large central vacuole, which are not found within animal cells. The cell wall is a rigid covering that protects the cell, provides structural support, and gives shape to the cell. While the central vacuole plays a key role in regulating a plant cell’s concentration of water in changing environmental conditions. 31 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 Plants are able to make their own food, like sugars, while animals (heterotrophs) must ingest their food. The chloroplasts, found in plant cells, contain a green pigment called chlorophyll, which captures the light energy that drives the reactions of plant photosynthesis. Protist - Any of the eukaryotic unicellular organisms including protozoans, slime molds and some algae; historically grouped into the kingdom Protoctista. Autotroph - Any organism that can synthesize its food from inorganic substances, using heat or light as a source of energy. Heterotroph- an organism that requires an external supply of energy in the form of food, as it cannot synthesize its own. BACTERIAL STRUCTURE Cell Wall Cell Membrane Cytoplasm Chromosome Plasmids Flagella Pili A. Flagella - Made out of protein. - Hair like, helical appendages that protrude to the cell wall and responsible for the swimming motility. - Propel microorganism away from harm and towards food in a movement known as “taxis”. Movement caused by a light stimulus is referred to as phototaxis and a chemical stimulus causes a chemotaxis movement to occur. - Has basal body, hook and filament. TYPES OF FLAGELLA a. Monotrichous- One flagellum in one polar end. b. Lophotrichous – two or more flagellum at one polar end. c. Amphitrichous – one flagellum in both polar ends. d. Amphilophotrichous – two or more flagellum in both polar ends. e. Peritrichous- flagellum surrounding the periphery of the bacterial cell. f. Atrichous- no flagella. 32 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 MOVEMENT OF FLAGELLA Temporal Sensing – flagella measure the changes in chemical and physical concentration over time as they travel along gradients. Runs – swimming forward, counterclockwise rotation. Tumbles – cells stops and jiggles, clockwise rotation of flagella. AXIAL FILAMENT - Internal flagella - Found only in the spirochetes. - Are composed of from two to over a hundred axial fibril (endofloagella) that extend from both ends of the bacterium between the outer membrane and the cell wall, often overlapping in the center of the cell. TYPES OF MOTILITIES 1. Swarming 2. Twitching 3. Gliding 4. Sliding MOTILITY OF SPIROCHETES 1. Gliding – motile when in contact with solid surface. 2. Chemotaxis – towards/away from chemical gradient (chemoreceptors) 3. Phototaxis – motility of organisms in response to intensity of light. 4. Magnetotaxis – coordinating movement in response to magnetic field(magnetosomes). 33 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 B. Pili (Fimbriae) - Hollow, non-helical, filamentous appendages that are thinner, shorter and more numerous than flagella. - Tubules that are used to transfer DNA from one cell to another cell. - Made out of protein. - F plus (sex pilus) – respiratory, urogenital, genital. This structure allows for the exchange of genes via the formation of “mating pairs). C. Capsule - a viscous substance that envelopes the cell wall. Composed mainly of polysaccharide or polypeptides. - It prevents dehydration. - Block attachment from bacteriophage. - Antiphagocytic. - Attachment and stability (repelling because of similar chargers). - Functions to protect bacteria from toxic compounds and desiccation and allowing them to adhere to surfaces and to escape the immune system of the host. D. Glycocalyx - Known as the pericellular matrix and sometime cell coat. - Amorphous fibers of polysaccharides attached to bacterial surfaces. - A glycoprotein and glycolipid covering which surrounds the cell membranes of bacteria, epithelial cells and other cells. - Dental plaque. - Hair-like structure surrounding the cell membrane. - It can be firmly attached to the cell’s surface, called capsule, or loosely attaché, called slime layer. A slime layer is water-soluble and is used by the prokaryotic to adhere to surfaces external to the cell. - Glycocalyx is used by a prokaryotic cell to protect it against attack from the body’s immune system. E. Cell Wall - A rigid structure located outside the plasma membrane and gives the cell its shape, providing structural support for the cell. - Protects the cell from its environment. It is the job of the cell wall to resist the pressure from the inside and outside environment of the cell. If pressure inside the cell becomes too great, the cell wall bursts, which is referred to as lysis. - The cell wall contains peptidoglycan(murein) which is made up of a polysaccharide backbone that is responsible for rigidity and shape of cell wall. It is Insoluble, porous and cross-linked polymer. - The polysaccharide that makes up the cell wall includes N-acetylglucosamine, N- acetylmuramic acid with amino side chain composed of L- alanine, D-alanine, D-glutamic acid, and Diaminopimelic acid. - In breaking the cell wall, lysozyme is needed. 34 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 COMPARISON OF THE CELL WALL OF GRAM POSITIVE AND GRAM-NEGATIVE BACTERIA Gram Negative - Complex cell wall. (Thin) - The inside is made of Peptidoglycan. While the outer membrane is composed of phospholipids and lipopolysaccharides. - The cell wall does not retain crystal violet dye when the cell is stained. The cell appears pink. - Contains outer membrane made out of lipopolysaccharides (LPS), endotoxin core polysaccharides(O) and disaccharide of glucosamine (lipid A). - Also contains proteins (porins- pore forming proteins, non-porin and lipoproteins). Gram Positive Bacteria - Thick cell wall – 60-100% peptidoglycan. - Has many layers of peptidoglycan that retain crystal violet dye when the cell is stained. - Contain teichoic acids. - Cell wall substances include ribitol – 5 carbon sugar and glycerol – 3 carbon sugar. - Contain lipoteichoic acids linked to the glycolipids of cell membrane. F. Cell Membrane /Cytoplasmic membrane - Primarily composed of phospholipids. - Is semipermeable in nature. - Primarily composed of: a. 20-30% phospholipid b. 60-70% protein c. Peripheral proteins. - Hydrophobic barrier. - Contains enzymes that are needed for respiratory metabolism and synthesis of capsular and cell wall components. - It prevents leakage of cytoplasmic constituents outside and allows transport of only specific nutrients in and out of the cell. - Regulates flow of molecules into the cell and removes waste from the cell by opening and closing passages called channels. Two types of transport mechanisms are used to move substances through the cytoplasmic membrane. These are passive transport and active transport. - In photosynthesis of prokaryotes, the cytoplasmic membrane functions in energy production by collecting energy in the form of light. - Finally, the cytoplasmic membrane acts as an anchor for important proteins. - Site of protonmotive force a. ATP synthesis b. Nutrient transport systems. c. Flagellar motility. 35 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 G. Cytoplasm and Cytosol - The cytosol is the intracellular fluid of a prokaryotic cell that contains proteins, liquids, enzymes, ions, waste and small molecules dissolved in water, commonly referred to as semifluid. - Substances dissolved in cytosol are involved in cell metabolism. - The cytosol also contains a region called the nucleoid, which is where the DNA of the cell is located. - Cytosol is located in the cytoplasm of the cell. - Functions for cell growth, metabolism and replication. - It is a gel-like matrix composed of water, enzymes, nutrients, wastes, and gases and contains cell structures such as ribosome, chromosome and plasmids. - Areas of cytoplasm: a. Cytoplasmic area – ribosomes are found b. Chromatic area – rich in DNA c. Fluid portion – with dissolved substances. H. Chromosomes - Singles - Nucleoid contains the DNA of a cell. - Circular molecule of double stranded DNA complexed with small amounts of protein and RNA. - The nuclear material is very long (1000 times than size of cell) as to why it need to be highly coiled. I. Plasmids (Episomes) - Extrachromosomal - Small, circular pieces of double stranded DNA. - Separately from bacterial chromosome. - Replication (antibiotic resistant) - Transferred through conjugation. CYTOPLASMIC INCLUSIONS AND VACUOLES Volutin Granules - Metachromatic granules - Reserve source of phosphates. Poly B-hydroxybutyrate (PHB) - Reserve source of energy and carbon. Elemental Sulfur - Elemental globules - Bacteria growing in environment (hydrogen sulfide) 1. Mesosome - is a convoluted membranous structure formed by the invagination of the plasma membrane. Serves as an extension. - Helps in the synthesis of the cell membrane, replication of DNA, and protein synthesis. 36 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Also helps in the equal distribution of chromosomes into the daughter cells during cytokinesis. - Also increases the surface area of the plasma membrane to carry out various enzymatic activities. - It helps in secretion processes as well as in bacterial respiration. 2. Gas Vesicle - Is a hollow structure made of protein. - It usually has the form of a cylindrical tube closed by conical end caps. - Gas vesicles occur in five phyla of the bacteria and two groups of the Archaea, but they are mostly restricted to planktonic microorganisms, in which they provide buoyancy. 3. Magnetosome - Is defined as an intracellular organelle consisting of a single- magnetic-domain crystal of a magnetic iron mineral enveloped by a lipid-bilayer membrane that contains proteins that are unique to it. 4. Carboxysome - Are intracellular structures found in many autotrophic bacteria. - Includes cyanobacteria, knallgasbacteria, nitroso-and Nitrobacteria. - They are proteinaceous structures resembling phage heads in their morphology. - They contain the enzymes of carbon dioxide fixation in these organisms. 5. Chlorosome - Is a photosynthetic antenna complex found in green sulfur bacteria and many green non-sulfur bacteria together known as “green bacteria”. - They differ from other antenna complexes by their large size and lack of protein matrix supporting the photosynthetic pigments. - Green sulfur bacteria are group of organisms that generally live in extremely low-light environments, such as at depths of 100 meters in the Black Sea. - The ability to capture light energy and rapidly deliver it to where it needs to go is essential to these bacteria, some of which see only a few photons of light per chlorophyll per day. - To achieve this, the bacteria contain chlorosome structures, which contain up to 250, 000 chlorophyll molecules. - Chlorosomes are ellipsoidal bodies, in GSB their length varies from 100 to 200 nm, width of 50- 100 nm and height of 15-30nm. In GNsB, the chlorosome are somewhat smaller. - Chlorosomes are a type of chromatophores that are found in photosynthetic bacteria. 6. Sheats - Are bacteria that grow as long filaments whose exterior is covered by a layer known as sheath. - Forms chains / Trichomes (Hollow tube) - Coated with ferric/manganese hydroxides. - Aerobic bacteria from fresh water/marine environments. 37 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Sheats – for attachment of cell to solid surfaces. - Semi-rigid extension. SPORES SPORE - Resting or dormant cell. - Metabolically inactive. - Produced during unfavorable conditions such as: a. Nutrition deficiency b. Extreme temperature and pH c. Presence of antimicrobials - Vegetative cell (activity, means can produce toxins and considered as infectious) becomes spore (inactive). - Average size ranges from 0.2um. SPORULATION - The process of formation of spores - Occurs during the late log phase or early stationary phase. - When environmental conditions become unfavorable sporulation starts. It takes about eight hours. The DNA is replicated and a membrane wall called spore septum begins to form between it and the rest of the cell. - The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA. The developing structure is now known as a forespore. - Calcium dipicolinate is incorporated into the forespore during this time. Next, the peptidoglycan cortex forms between the two layers and the bacterium add a spore coat to the outside of the forespore. - Sporulation is now complete, and the mature endospore will be released when the surrounding vegetative cell is degraded. GERMINATION - Return or favorable nutritional conditions. - Activation. - Production of single vegetative cell from spores. - During germination, endospores lose their resistance to heat and staining. - Outgrowth occurs, characterized by synthesis of new cell material and development of organism to new cell. 2 TYPES OF BACTERIAL SPORES 1. Endospore - Produced within the bacterial cell. - Bacillus, Clostridium. - Sporosarcina, Thermoactinomyces. 2. Exospore - Produced outside the cell. 38 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 - Very few bacteria produce exospore. - Methylosinus. ENDOSPORE - thick-walled bodies. - Highly refractive and resistant to staining, dessication, heat, infection and radiation. ❖ Characteristics of Endospore 1. Cannot be stained by regular staining due to the impermeability of the endospore wall to dyes and stains. Endospores can be stained by the Schaffer and Fulton stain, which stains endospores green and bacterial bodies red. 2. Resistant to dessication, heat, radiation, disinfecting agents and staining. 3. Most spores can resist 90 C for at least 10 minutes. Dehydrated state and dipicolinic acid (DPA) may be responsible for heat resistance. 4. Spores are usually produced by cells growing in rich media which are approaching the end of active growth. SHAPE AND POSITION OF BACTERIAL SPORE 5 MAJOR LAYERS OF ENDOSPORE 1. Core - Spore protoplast. - Innermost part - It is a gel-like dehydrated state (10-25% water). - Provides heat resistance to the endospore. - Consist of: a. Chromosome (nucleoid) b. All components of the protein synthesizing apparatus in the cytoplasm surrounded by a cytoplasmic membrane. c. Ribosome 39 GENERAL VETERINARY MICROBIOLOGY 1st Semester Rhoan B. Arena 2024-2025 d. Other cellular materials. - Calcium dipicolinate is present in high amount (10-15% of spore dry weight). This content also enhances the heat resistance of endospores. - Also contains high percentage of small acid soluble proteins (SASP) a. Synthesize during sporulation b. Prevent the potential damage of core DNA from UV radiation, desiccation and drying. c. Provide nutrition and energy for spore germination. 2. Spore Wall - The innermost layer surrounding the core. - Lies outside the core cytoplasmic membrane.