SCOPE OF MICROBIOLOGY
WHAT IS MICROBIOLOGY?
Microbiology is a specialized area of biology that studies living things ordinarily too small to be seen with the naked eye. Such microscopic organisms are collectively referred to as Microorganism or microbes. Microbiology is one of the largest and most complex disciplines of the biological sciences, in addition to studying the natural history of microbes, it also deals with every aspect of microbe-human and microbe-environmental interaction, including infection, disease, drug therapy, immunology, genetic engineering, industry, agriculture and ecology.
Microorganisms in most cases are said to be unicellular, but we may find some that are made up to aggregate of cells that exist as a complete unit performing the same function as a single cell. Microorganisms that are study under the discipline of microbiology are
1. Bacteria
2. Fungi (yeast and mold)
3. Protozoa
4. Viruses
5. Rickettsia
WHY STUDY MICROBIOLOGY
There are several reasons that an exposure to microbiology can be useful and meaningful. Perhaps the most obvious is that it unlocks an invisible world that many human never learn about. Its unique perspective so increases our awareness of microorganisms and their roles in illness, industry and ecology that it has the power to change people’s lives.
For most persons, a background in microbiology is also valuable because of its widely practical and sensible nature. Its applications become immediately pertinent to every day situation involving preparations and preservations, soil fertility, waste disposal, and the prevention and treatment of infectious diseases.
Microbiology also leads irrevocably to the study of humans and the human conditions. Health workers who are involve in daily hands on care, assessment, and intervention must think from the microbiological point of perspective. This is a current challenge to meet technologies that promise to become a commonplace feature of patient care.
HISTORICAL FOUNDATIONS OF MICROBIOLOGY
From early times a few people realized that diseases could be spread by contact, and even before microorganisms were seen, some investigations suspected their existence and responsibility for coursing diseases.
Among them were the ROMAN PHILOSOPHER LUCRETIUS and the physician HEIRANYMUS FRACASTURIUS (1483 – 1553); they suggested that invisible living creatures cause disease. Fracasturius further presented that; the fundamental concept of epidemic diseases is due to the transmission of an agent from one individual to another.
THE DISCOVERY OF MICROOGNISMS
Until the development of magnifying lenses and microscopes that could sufficiently magnify microorganisms to allow them to be visualized, the discovery of disease-causing agents was impossible. The actual discovery of microorganisms takes place in 17th century when Anthony van leeuwenhoek (1632-1723) constructed a primitive microscope. He used his microscope to observe and described various minute organisms in specimen of pepper water; tooth scraping, gutter water, semen, blood, urine and forces. Hence he described the three shapes of bacteria as well as protozoa, sperm, and blood cells. He however did not speculate on the origin of these microbes nor did he associate them with the cause of disease. However the study of viruses did not go with the discovery of microorganisms. The discovery of viruses and their role in disease causation was made known later when Charles Chamberland constructed a percelain bacteria filter in 1884. The first viral pathogen to be studied was the tobacco mosaic disease virus.
SPONTANEOUS GENERATION OF MICROBES
Initially there was this feeling that microorganisms were spontaneously generated, hence many scientist were searching for an explanation of the spontaneous generation of living creatures in decaying meat, stagnating ponds, fermenting grain, and infected wounds. On the basis of observation, many of the so called scientists of that time believed that life could develop spontaneously from decomposing non-living material. This is the theory of spontaneous generation or abiogenesis. For over two centuries, this theory was debated and tested, until Louses Pasteur and John Tyndall finally disproved the theory of spontaneous generation and prove that life must arise from preexisting life; this is called the theory of biogenesis
The experiments devised by Louis Pasteur and others resulted in several major advances in the field of microbiology.
1. The concept that life must arise from preexisting life.
2. The techniques of sterilization and pasteurization.
3. Understanding of the biological process of fermentation,
4. The germ theory of disease
5. The development of vaccines from killed bacteria for anthrax and from attenuated or weakened viruses.
The discovery of bacterial endospores by Tyndall after first boiling a specimen led to development of the fractional sterilization process, often called typndallization, in which the endospores of bacteria are destroyed by boiling and cooling three times, allowing the spores to germinate between boiling.
The biological theory of fermentation was developed when Pasteur was investigating the reasons for the spoilage of beer and wine. This theory states that a specific microbe produces a specific change in the substance on which it grows or a specific microorganism produces a specific fermentation process.
By extending the biological theory of fermentation to animals and humans, Pasteur developed the germ theory of disease, which states that a specific disease is caused by a specific type of microorganism.
RECOGNITION OF MICROBIAL ROLE IN DISEASE CAUSATION
The possible medical significance of microbes was largely overlooked in the 200 years of Leeuwenhock discovery.
Agostino Bossi was the first person to prove that a microbe could cause disease, but the complete establishment of germ theory depended on the work of the German physician Robert Koch of Germany
Another development is that, Alaxander Ogston, a Scottish surgeon, showed that cocci produced inflammation and suppuration and is the main cause of acute abscesses. He also discovered and named staphylococcus and distinguished them from streptococcus.
A British physician introduced the practice of vaccination, the process of inoculating healthy people with attenuated or weakened orgasms so as to make the recipient develop immunity. Sir Ronald rose discovered the malaria parasite as the cause of malaria and that the organism is carried by the anopheles mosquito.
BENEFITS OF MICROBES TO MAN
1. Indigenous micro flora that live on and within our bodies such as in our mouths, intestine and on our skin inhibit the growth of pathogens in those areas by occupying space, using food supply and secreting materials (waste products, toxins, and antibiotics) that prevent or reduces the growth and multiplication of pathogens.
2. Saprophytic bacteria aid in fertilization by returning inorganic nutrients to the soil, the process is achieved by breaking down dead organic material (plants and animals) into nitrates, phosphates, carbon dioxide, water, and other chemicals necessary for plant growth.
3. Production of antibiotics, vaccines, vitamins, enzymes and hormones.
4. Nitrogen-fixing bacteria in legumes (Peas, Peanuts) are able to return nitrogen from the air to the soil in the form of nitrates to improve fertility.
5. Purification of wastewater is partially accomplished by bacteria in the holding tanks of sewage disposal plants, where faeces, garbage and other organic materials are collected and reduced to harmful waste.
6. Other nonpathogenic microbes make it possible to produce yogurt, cheese, bread, beer, wine and many other food and drink
CONDITIONS THAT INFLUENCE MICROBIAL GROWTH
(1) TEMPERATURE.
Microbes like suitable temperature for their growth and multiplication. Extremes of temperatures are harmfully to microbes. High temperatures kill them. Freezing temperatures do not kill them but rather arrest their growth and multiplication.
For each microbial species there is a definite temperature range within which growth takes place. The limits are the minimum and maximum temperature with an intermediate optimum temperature. Minimum growth temperature is the temperature at which microbial cells will grow, an optimum temperature is the temperature at which microbial cells will grow best and a maximum temperature is the temperature at which growth is possible. Above the maximum temperature the rate of growth drops.
Base on temperature requirements, bacteria are classified as follows:
i) Psychrophiles (cold loving microbes)
ii) Mesophiles (moderate temperature-loving microbes)
iii) Thermopiles (heat loving microbes).
(i) Psychrophiles are group of soil and water bacteria. They grow best at temperatures below 20 degree Celsius, usually quite well at O degree Celsius and in some cases down to about –7 degree Celsius on unfrozen media. Their importance lies in their ability to cause spoilage of refrigerated and frozen food, though none is pathogenic. Another group of psychrophiles that grow at a temperature of 0 degree Celsius and has a highest growth temperature usually between 20-30 degree Celsius are refer to psychotrophs.
Those that can be preserved in the frozen state are known as psychroduric.
(ii) Mesophiles are the most common types of microbes; they include most of the common food spoilage and disease-causing organism (pathogens). They have an optimum growth temperature of 25-40 degree Celsius and therefore are adopted to live in the bodies of animals e.g. Neisseria gonorrheae.
(iii) Thermopiles grow best at high temperatures of 55 to 80 degree Celsius and have a minimum growth temperature ranging from 20 to 40 degree Celsius. Most of these organisms live in hot springs
Thermal death point-The thermal death point of a particular organism may be defined as the lowest temperature that kills it under standard conditions, and within a given time. Under moist conditions, the thermal death point lies between 50 and 65 degree Celsius for most non-sporing mesophilic bacteria, and between 100 and 120 degree Celsius for spores of most spores forming species e.g. it is about 105 degree Celsius for clostridium tetani and 115 degree Celsius for clostridium botulinum
With dry heat, the 10 minutes thermal death point of the different spore forming bacteria are mostly between 140 and 180 degree Celsius.
(2) INFLUNCE OF MOISTURE AND DESICCATION
Living organisms require water for their normal metabolic processes. However some can survive desiccation, a process of preventing spoilage by removing moisture thereby increasing the ionic concentration so that bacterial cells suffer plasmolysis. Different species very widely in their ability to survive when dried under natural conditions, e.g. the gonococcus and the common cold virus die quickly in dry conditions, where as the tubercle bacillus, staphylococcus and the small pox virus may survive for weeks or months under dry conditions. Also, bacterial endospores survive drying, for instance the spores of bacillus anthraces dried on threads, have survived for over sixty years.
(3) INFLUENCE OF OSMOTIC PRESSURE
Microorganisms, particularly bacteria are constantly subjected to osmotic phenomena. This is as a result of the presence of a semi-permeable cytoplasmic membrane. Bacteria obtain nutrients from the surrounding water or solution through osmosis and diffusion. Survival occurs in isotonic solution. However, if a microbial cell is kept in a hypertonic solution, the cell experiences plasmolysis, i.e. temporary shrinkage of the protoplasm and its retraction from the cell wall due to the osmotic withdrawal of water. This occurs much more readily in Gram-negative than in Gram-positive bacteria.
Bacteria from saline waters, as the Dead Sea can live in high salt concentrations. These group of bacteria are refer to as extreme halophiles e.g. obligate halopliles require nearly 30% salt concentration to survive, and facultative halophiles grow in 2% salt concentration.
Hypotonic solution such as distilled water tends to enter the bacterial cell, this can cause lyses (plasmoptysis) i.e. swelling and busting of the cell as a result of excessive intake of water.
(4) INFLUENCE OF HYDROGEN ION CONCENTRATION (PH)
The term pH refers to the acidity or alkalinity of a solution.
A suitable environmental pH is an essential factor in microbial metabolism and growth. The majority of commensals and pathogenic bacteria grow best at a pH rang of 6.5 to 7.5. Some bacteria flourish in the presence of a considerable degree of acidity such as pH 4.These are termed acidophilic e.g. lactobacillus. Some bacteria survive at a pH of 1.These are called chemoautotropic
Others are very sensitive to acid, but tolerant of alkali e.g. vibrio cholerae. However, strong acid or alkaline solutions, are lethal to most bacteria, the mycobacterium tuberculosis is exceptional.
(6) LIGHT: Light must be partially or completely excluded for the proper growth and multiplication of microbes because excessive light kills them
(7) CARBON
Besides water, one of the most important requirements for microbial growth is carbon. It is the structural backbone of living matter. It is required for the skeletal or backbone of all organic molecules. Most bacteria, such as chemoheterotrophs obtain carbon from inorganic material e.g. carbohydrate, lipid, while Chemoautotrophs and photoautotrophs derive their carbon from Carbon dioxide.
(8) OXYGEN
Every living organism requires oxygen for survival. Bacteria can be classified into three groups based on their oxygen requirement.
a) Obligate aerobes grow only in the presence of oxygen, e.g. mycobacterium tuberculosis.
b) Strict or obligate anaerobes grow only in the absence of oxygen. Strict anaerobes include all species of clostridium, anaerobic staphylococci, and the Genus veillonella.
c) Facultative anaerobes grow under both aerobic and anaerobic conditions e.g. E.coli. The term microaerophilic is given to organism which are not very strict anaerobes but which grow best at reduced oxygen tension e.g.
Campylobacter species, some strains of streptococci and most mycoplasmas.
Some anaerobes are rapidly killed by oxygen. It has been suggested that they produce toxic amounts of hydrogen peroxide under aerobic conditions and lack catalase and other enzymes, which would remove it.
9) NITROGEN, SULPHUR
These are needed for the synthesis of cellular materials e.g. nitrogen and sulphur are required for the synthesis of protein. The synthesis of DNA and RNA require nitrogen and some phosphorus, as does the synthesis of ATP. Sulphur
Nitrogen make up about 14% of the dry weight of a bacterial cell and sulphur and phosphorus together constitute about 4%.
10) TRACE ELEMENTS
These include iron, copper, zinc, etc. They are essential for the function of certain enzyme.
11) ORGANIC GROWTH FACTORS
Organic growth factors are organic compounds or essential cells components that an organism is unable to synthesize. There are three main classes of organic growth factors:
Amino acids.
Purines and pyrimidines
Vitamins.
PRINCIPAL GROUPS OF MICROBES
ANATOMY OF PROKARYOTIC AND EUKARYOTIC CELLS.
A cell is the fundamental living unit of any organism. Cells exhibit basic characteristics of life. Anatomically, cells are grouped into
(a) Prokaryotic cells (The less complex cells)
(b) Eukaryotic cells (The more complex cells).
PROKARYOTIC CELLS.
The word prokaryotic comes from a Greek word meaning prenucleus. Member of the prokaryotic world make up a vast heterogeneous groups of vary small unicellular organisms. Prokaryotes include bacteria, blue green algae, as well as the photosynthesizing cyanobacteria.
BASIC STRUCTURE OF THE BACTERIUM
(A typical prokaryotic cell)
The bacterium is a microorganism with prokaryotic form of cellular organization. All bacteria invariably have a cell envelop, cell membrane, capsule, slime or layer, protoplasm, ribosome, and a nucleoid. The majority has a cell wall. However flagella, pili, fimbriae, capsules, slime layers, and granules are not universal components of all bacteria.
THE FLAGELLA: These are long, delicate, whip like processes attached to the bacterial cell. Some species of bacteria do not possess flagella; others simply have a single flagellum or several flagella with various arrangements. The flagella of bacteria arise from a basal body in the cell membrane and project outwardly through the cell wall and the capsule. a flagellum consist of three, four or more threads of protein twisted like a rope.
Flagella are organs of motility; they also claim a protective role. There is good evidence that flagella permit movement of the organism towards localities favourable for growth and away from unfavorable regions. Thus, bacteria tend to migrate toward regions where there is a higher concentration of nutrient solutes and away from regions containing higher concentration of toxic material.
It is said that the power of active locomotion assist pathogenic bacteria in penetrating through viscid mucous secretions and epithelial barriers, and in spreading through out the body fluids and tissues, but it must be noted that many non-motile pathogens, e.g. streptococci are not any less invasive than motile ones.
ARRANGEMENT OF BACTERAIL FLAGELLA:
PILLUS/FIMBRIA: The term fimbria refers to the smaller, shorter, numerous bristle- like fibers sprouting off the surface of many bacterial cells. They arise from the cytoplasm and extend through the plasma membrane, cell wall, and capsule. They are mush smaller than flagella, have a rigid structure, and are not associated with motility. Fimbriae have an inherent tendency to stick to each other and to surfaces. For example, they are often responsible for the mutual clinging of cells on the surface of liquids, and for the microbial colonization of inanimate solids such as rocks and glass.
Pilli are short hair-like structures attached to the cell walls of certain bacteria, e.g. Gram-negative bacilli such as E-coli, shigella and salmonella species. Functions of pili depend on the bacteria species, however they are believed to perform the following.
(a) They are use by bacteria to attach themselves to other bacteria or to membrane surfaces such as intestinal lining and red blood cells.
(b) Pilus also provides a site for attachment of bacterial virus.
(c) Bacteria that have sex pilus are able to transfer genetic material across a pilus bridge from one bacterium to another by a process known as conjugation
THE CAPSULE: Basically some bacteria have a layer of material outside the cell wall. This layer is called the capsule or the slime layer. It is a thick layer of slimy gelatinous material produced by the plasma membrane and secreted outside the cell wall. Bacterial capsule may serve any of the following functions:
(1) If the capsule is thin, the cell may be able to glide, slide or move on the surfaces of solid materials.
(2) Capsule also enable the bacterium to attach itself to mucous membrane and tooth surfaces
(3) Capsulated bacteria are not easily digested by phagocytes, therefore they stay longer in the body i.e. they resist phargocytoses. Many species including streptococcus pyogens, Haemophilus influenza, clostridium perfringens, and Bacillus Anthraces have capsule.
THE CELL WALL. The cell wall is a complex semi-rigid structure responsible for the shape of the cell. It accounts for up to 20% of the total weight of the cell. Its main function is to enable the delicate cytoplasm membrane to withstand the high osmotic pressure within the bacteria. Removal of the cell wall will result in bursting of the cell membrane. The cell wall is permeable to wide variety of solutes. It also contributes to pathogenicity of some species. The main constituent of bacterial cell wall is a complex macromolecular polymer known as peptidoglycan (murein), consisting of many polysaccharide chains linked together by peptide (small protein) chain.
THE CELL MEMBRANE: Surrounding the bacteria cytoplasm is a fine lipoprotein sheet, about 5 to 10micrometer thick called the plasma or cell membrane. It is been described as a lipid bi- layer material with protein embedded to a varying degree. The cell membrane contains primarily phospholipids making up to about 30-40% of the membrane and proteins contributing to 60%-70%. Major exceptions to this description are mycoplasmal membrane that contain a high level of lipids called sterol (rigid molecules that function in membrane stabilization)
In some locations the cell membrane folds up into fingers-like passage or pockets in the cytoplasm called mesosomes. These are prominent in gram-negative bacteria.
FUNCTIONS OF THE CELL MEMBRANE
1. The membrane is selectively permeable, that is to say, it allows particular ions and molecules to pass either into or out of the cell while preventing the movement of others.
2. The plasma membrane also serves as the location of variety of crucial metabolic processes-respiration, photosynthesis, the synthesis of lipids and cell wall constituents.
3. The membrane also contains special receptor molecules that help bacteria to detect and respond to chemicals in the surroundings.
4.The cell membrane is also involved in the discharge of a metabolic product into the extra cellular environment.
CYTOPLASM (Internal content of the cell)
The cytoplasm of a bacterial cell is a viscous watery solution, or soft gel, containing a variety of organic and inorganic solutes, and numerous small granules called ribosomes.
Its major component, water (70%-80%), serves as a solvent for a complex mixture of nutrients, including sugar, amino acids and salts. It also contains other low molecular weight compounds primarily protein, enzymes, lipids inorganic and other particles such as ribosome, mesosomes and granules.
NUCLEOID/ CHROMATIN BOBY/PLASMID
The nucleoid may be considered a primitive nucleus; it is not surrounded by a nuclear membrane, does not have a definite shape, and has little or no protein material. It usually consists of a single, circular chromosome. It is capable of duplicating itself, of guiding cell division, and of directing cellular activities.
The bacterial DNA is not enclosed by a nuclear membrane. Therefore, by definition bacteria do not have a true nucleus.
Beside the chromatin body that serves as genetic requirement for bacterial survival many of them contain other disposal pieces of DNA called plasmids. These tiny circular extra chromosomal strands often present protective traits such as resisting drugs and radiation and producing toxins and enzymes.
RIBOSOMES
These are tiny discrete units shown up as fine spherical sparks dispersed through out the cytoplasm. Chemically, a ribosome composed of two subunits each consists of a special type of RNA called ribosomal RNA -rRNA (60%) and a protein (40%). The ribosome gives the cytoplasm a granular appearance. They function as a site for protein synthesis.
GRANULES OR INCLUSIVE BODIES
Granules contain crystals of inorganic compound and are not enclosed in a membrane, e.g. sulphur granules of photosynthetic bacteria, polyphosphate granules of corynebacterium and mycobacterium. Inclusive are composed of condensed, energy-rich organic substances, including glycogen enclosed within special membranes. Bacteria fall on granules and inclusions to mobilize energy and other nutrients in terms of depleted environment sources.
MICROSCOPIC MORPHOLOGY OF BACTERIA.
Bacteria vary widely in size, ranging from spheres that measure 0.2micrometer to spiral that measures 10.0micrometer long.
Four fundamentals forms of bacteria are known. These are:
1. THE COCCUS: The coccus type of bacteria are spherical or oval in shape
TYPES
a. Micrococcus- the microcosms exist as a single cell
b. Diplococci- they remain in pairs after dividing
c. Streptococci-those that divide and remain attached in chainlike pattern
d. Staphylococci-they exists in clusters (undefined shape).
2. THE BACCILUS: The bacillus is basically a rod-shape organism; they are the largest group of bacteria. They are relatively long and thin or short and fat, most of them are spore-forming organism e.g. E-coli, diphtheria bacilli, mycobacterium leprae, tubercle bacilli etc.
TYPES
a. Diplobacilli-they appear in pairs
b. Streptobacilli-occurs in chains
c. Coccobacilli-are oval in shape
3. THE SPIRILLIUM/SPIROCHAETE: The spirillum bacteria are long slender organism, which has spiral rod shape structure; they tend to have flagella, which makes them motile. Spirochaetes resemble a spring or a stretched sling. E.g Treponema palladium, Teponema Pertonue which causes Yaws
3. VIBRIO: Vibrio bacteria are comma in shape. Eg Vibrio cholerae
RUDIMENTARY FORMS OF BACTERIA
1. CHLAMYDIAE: These organisms are small, non-motile, weakly Gram-negative obligate intracellular parasites. They grow in the cytoplasm of their host cell forming characteristic micro-colonies. One species of chlamydia that carry the greatest medical impact is chlamydia trochomatis, the cause of both a severe eye infection (trachoma) and the most common sexually transmitted diseases called lymphogranuloma venereum (LGV).
2. RICKETTSIAE: Basically, they are obligate intracellular parasites because they appear to have leaky cell membrane. Most of them are pathogens that alternate between a mammalian host and blood- sucking arthropods such as fleas, lice, mites, birds and ticks.
Human rickettsial infection results mainly from exposure to the various species of infected arthropod. Man gets inoculated through the mouthparts of the arthropods after a period of feeding, or through inhalation of aerosols. Some diseases cause by rickettsia includes Rocky Mountain spotted fever, cause by rickettsia rickettsii (transmitted by ticks); epidemic typhus fever cause by rickettsia prowazekii; trench fever cause by rickettsial Quitana.
3. SPIROCHAETES:
The spirochaetes of medical importance are Treponema, Barrelia and leptospira. The treponema pallidium is the causative organism of syphilis, a notorious sexually transmitted infection that has persecuted mankind since it was recognized in epidemic in 15 century. Other diseases cause by spirochaete include Yaws, Treponema pertenue, relapsing fever- borrelia duttonii, vencent’s angina- Borrelia Vincenttii
4. MYCOPLASMAS AND OTHER CELL-WALL-DEFICIENT
BACTERIA
The mycoplasmas are the smallest of cellular microbes. Naturally they lack cell wall, for that matter the mycoplasmal cell membrane is stabilized and can resist lyses that could occur in those bacteria with cell wall if the wall is taken off. Generally, mycoplasmas are extremely tiny and pleomorphic in nature with a minimum size of 0.1 micrometer. They also rang in shape from filamentous to coccus. They are found in many habitats, including plants, soil, and animals.
The most important medical species is mycoplasma pneumonias, which causes an atypical form of pneumonia in humans. Some bacteria may also lose their characteristic shape due to adverse growth conditions. These cell wall deficient bacteria are called L-forms.
UNUSUAL BACTERIA NOT INVOLVE IN CAUSING HUMAN DISEASE.
1. PHOTOSYNTHETIC BACTERIA: Photosynthetic bacteria are independent cells that can synthesize all require nutrients from simple inorganic compound in the presence of sunlight.
2. CYNANOBACTERIA OR BLUR GREEN ALGAE: These are gram-negative bacteria that rang in size from 1to 5 micrometers. They can be unicellular, colonial, or filamentous. Cynanobacteria grow profusely in fresh water and seawater. A major contribution of this group of bacteria is the addition of oxygen to the atmosphere and the conversion of gaseous nitrogen into a form usable by plants.
3. GLIDING BACTERIA: An example of a gliding bacterium is myxobacteria or slime bacteria. They have no flagella, and therefore glide over moist surfaces.
EUKARYOTIC CELL STRUCTURE
Eukaryotes (ue= true, -karyo= nucleus) are complex cells with a true nucleus and much larger than prokaryotic cells. The cell wall consists mainly of cellulose but may also contain rectin or lignin. The nucleus is spherical or oval in shape surrounded by nuclear membrane; it contains the cell genetic information (DNA). The DNA is also consistently associated with chromosomal protein called histones.
Eukaryotes also have a mitotic apparatus and a number of organelles, e.g. Golgi apparatus, endoplasmic reticulum, mitochondrion and chromosome Eukaryotic cells are found in animal and plant cells, protozoa, fungi, algae and helminthes.
THE PROTOZOA
These are non-photosynthetic unicellular organisms with protoplasm clearly differentiated into nucleus and cytoplasm. They are relatively large with transverse diameters mainly in the range of 2-100um.
The cytoplasm is divided into a clear outer layer called the ectoplasm and a granular inner region called the endoplasm. Ectoplasm is involved in locomotion, feeding and protection. The endoplasm houses nucleus. Mitochondria, food particles and contractile vacuoles. The outer boundary of protozoan cell is a cell membrane that regulates the movement of food, wastes and secretions
Their surface membranes vary in complexity and rigidity from a thin, flexible membrane in amoebae, which allows major changes in cell shape and the protrusion of pseudopodia for locomotion and ingestion, to a relatively stiff pellicle in ciliate protozoa.
The size of most protozoan cells fall within the range of 3 to 300 cm, except some giant amebas and ciliates that are larger enough (13-4m in length) to be seen swinging in pond water.
NUTRITIONAL AND HABITAT RANGE
Predominantly, the protozoan habitats are fresh and marine water, soil, plant and animals.
All protozoa are heterotrophic. They usually require their food in a complex organic form. Some parasitic species have the mode of nutrition typical of animals called holozoic; they capture, ingest and digest internally solid masses or particles of food material.
Some also have special feeding structures such as oral groves, which sweep food particles into a passage way or gullet that package the captured food into vacuoles for digestion. Others are saprophytic, absorbing soluble nutrient substances derived from dead plant or animal material.
CLASSIFICATION AND DESCRIPTION OF PROTOZOA.
Classification of protozoan species is based on their motility or method of locomotion, the presents or absence of cilia.
1. Flagellates:-The flagellate type of protozoa one typically spindle in shaped with flagella projecting from one end. They have an outer tough flexible membrane and an oral groove for ingestion of food particles. An example is Trichomonas vaginalis found in the vagina and in the male urinary tract. It causes persistent infections of male and female genital tracts;Giardia Lamblia causes persistent intestinal infection. Trypanosoma gambiense is carry by tsetse fly and causes African sleeping sickness.
They move by way of whip like action of the flagella that pulls the flagellated cells through their environment.
2. Ciliates: - Ciliates protozoa swim freely, and this is facilitated by the present of cilia, e.g. paramecium, Balantidium coli that causes a rare type of dysentery,Trypanosoma.
3. Amebas (phylum sarcodina): This group of protozoa lack cilia and flagella. Movement is by the use of pseudopodia, i.e. by pushing forward a bit of cytoplasm and slowly flowing into it. This process is called amoeboid movement.
A pathogenic example is entamoeba histolytica that causes amoebic dysentery.
5. Sporozoa: These are obligate intracellular parasites, they are generally non-motile and have a complex life cycle that involves transmission between several host. Examples include plasmodium, the causative agent of malaria, toxoplasma gondii, which causes congenital infection in uterus.
FUNGI
Fungi are eucaryotic organisms that include Mushrooms, molds, and yeast. Fungi are found almost everywhere on earth. As saprophytes, their main source of food is dead, decaying organic matter. Fungi are the “garbage disposers” of nature, the vultures of the microbial world. By secreting digestive enzymes into dead plant and animal matter, they decompose this material into absorbable nutrients for themselves and other living organisms thus they are the originals “recyclers”
Fungi are often referred to as plants only because they have cellulose or chitin cell walls. However they differ from plant because they have no chlorophyll or other photosynthetic pigments.
Although many fungi are unicellular during some phases of their life cycle e.g. yeasts, many species grow as filaments called hyphen, which form a mass called mycelium.
Fungi can reproduce asexually by budding or by hyphen extension, or by the formation of spores. Sexual reproduction in many fungi involves the nuclear fusion of two gametes and their subsequent division into many sexual spores.
Morphologically, four-distinct groups of fungi are identified, each of which includes some pathogenic varieties.
1. The mould (Filamentous, mycelia fungi): This type grows long filaments or hyphae, which branch and interlace to form a meshwork or mycelium. Reproduction is by the formation of various kinds of spores. The major part of the mycelium (the vegetative mycelium) penetrates into substrate and it absorbs nutrients for growth. Other hyphae constitute the aerial mycelium, this protrudes from the vegetative mycelium into the air; they form and disseminate into the air various kinds of spores for propagation.
When grown to a large size, the mycelium is seen as a filamentous mould colony; this may become powdery on its surface due to the abundant formation of spores. The ringworm fungus is an example.
2. Yeast: Yeast is a unicellular fungus, which occur mainly as single spherical cell. Reproduction is by budding. On artificial media they form compact colonies with a creamy mucous or pasty consistence. e.g. cryptococcus neoformans.
3. The Yeast - Like Fungi: Grow partly as yeasts and partly as long filamentous cell joined end to end, forming a pseudo-mycelium, e.g. candida albicans
4. Dimorphic fungi: Grow either as filaments or as yeast according to the cultural conditions. Growth usually takes place in the mycelia form on culture media at 22OC and in the soil. It grows into yeast form on media at 37OC and on animal body.
YEAST OF MEDICAL SIGNIFICANCE
A. Candida.
B. Torulopsis.
C. Cryptococcus.
THE Candida
This is a genus of “Yeast-like fungi”, several species of which occur naturally in man and can cause disease; about 90% yeast infections are due to candida albicans. This organism is normally present in the mouth and vagina. It is responsible for infections in these sites and elsewhere when there is a disturbance of local conditions or impairment of the defense mechanisms. Candida albicans grows normally as thin-walled, non-capsulated oval yeast, 2.5 - 4.0cm in diameter, but can give rise to pseudomycelium in the body and also in cultures when aeration is poor. Candida is tolerant of acid and not sensitive to any of the antibacterial antibiotics; it thrives in its normal sites in the body when broad-spectrum antibiotics restrain the growth of the normal bacterial flora in this areas. It is uniformly sensitive to the polyene antibiotics and clostrimazole including 5-fluorocytosine.
INFECTIONS DUE TO CANDIDA ALBECANS
Candida albicans is part of the normal flora of the month, intestine and vagina. It is also an opportunistic pathogen, giving rise to local inflammation under a wide variety of circumstances.
The opportunistic candida infections may occur in pregnancy, neonatal debility, senility, minor trauma, continued exposure of the skin to moister or when the patient is debilitated by diabetes or alcoholism.
The commonest candida infections are
1. Vaginitis or Vaginal Thrush: This condition is characterized by whitish discharge with a PH below 3-2. Pus cell and yeast may be seen microscopically.
2. Oral Thrush: Common in bottle-fed infants. Creamy white patches are found covering red, raw areas of mucous membrane and tongue.
It also occurs in adults with angular cheilitis, and in ‘sore mouth. Infection of the skin is common in people with diabetes, also on vulva and gland penis and in napkin area in infants. Systemic infection may occur when the organism is inoculated directly into the tissues as it occurs in drug addicts, after heart valve operations, and in several generalized disease such as leukaemia.
TORULOPSIS
This genus is very similar to canadida, but produces no pseudomycelium. One species T. glabrata, is found in the same body sites as C. albicians and has been shown to be an opportunistic pathogen capable of establishing itself in the blood, lungs and urinary tract of debilitated patients.
CRYPTOCOCCUS
This genus of yeast produces no pseudomycelium and differs from both candida and torulopsis in possessing urease. There is one pathogenic species, C. neoformans, which is widespread in nature and present particularly in large numbers in pigeon faeces. It has been isolated from human faeces and skin. It causes sporadic, chronic and of other fatal disease in man and in a variety of domestic animals.
Infection occurs in man probably by inhalation of contaminated dust. Blood born dissemination may occur to lymph nodes, skin, bones or meninges. Meningeal infection normally leads to a slow and irregularly progressive meningoencephalitis, which is fatal if untreated.
THE VIRUS
Viruses are acellular biological entities, they are said to be obligate intracecullar parasites i.e. they require absolutely a living host cell to remain active and multiple. They have no true nucleus, cytoplasm, cell membrane or cell wall and are best described as infectious particles rather than organism and as either active or inactive rather than alive or dead. Their dependence on cells for reproduction argues against the consideration of viruses as primitive in the evolutionary sense, i.e. as the ancestors of cellular life. They range in size from 0.02 to 0.30 um in diameter. The smallest is about the size of the large hemoglobin molecule of a red blood cell. Some viruses produce diseases or genetic changes in animals, plants algae, fungi, protozoa, and bacterial cells.
Recently, virions have been described as having five specific properties that distinguish them from living cells:
(1) They possess either DNA or RNA, never both;
(2) Their replication (duplication) is directed by the viral nucleic acid within a host cell;
(3) They do not divide by binary fission or mitosis;
(4) They lack the genes and enzymes necessary for energy production, and
(5) They depend on the ribosomes, enzymes and nutrients of the infected cells for protein production.
2. Some viral coats have different specifications in terms of the cells to which they can attach and the particular surface components to which they bind.
3.The coat can interact with the host cell surface so as to permit direct penetration of the viral core across the plasma membrane.
4.It is also believe that, membrane penetration occurs through endocytosis as some viruses are seen to be endocytosed after attachment to the cell surface. However the events of membrane penetration is not yet understood.
Morphologically, viruses are known to have both symmetrical and geometrical forms, they can be spherical, oval or cylindrical.
TYPES OF VIRUS
1. BACTERIOPHAGE:
These are viruses that infect bacteria. They attach themselves to the bacteria through the use of fibers. These fibers are fastened to specific receptor sites. The central core is then pushed through the bacterial cell wall; finally, the DNA is extruded from the head, through the tail tube, and into the host cell by unknown mechanism.
Soon after the injection of the viral DNA, synthesis of host DNA, RNA, and protein is halted and the bacterial cell is force to produce viral constituents. These constituents or components will then be assembled to produce fully developed virion. With time the bacterial membrane breaks down to release viral particles capable of infecting other bacteria.
2. ANIMAL VIRUS:
Animal viruses are either DNA or RNA viruses; they cause various diseases in animals including man. They are classified by their function. Few examples are
a. Neurotrophic Virus, Attacks the brain, spinal cord and peripheral nerves e.g. poliovirus, herpes virus.
b. Respiratory Virus: They attack the respiratory tract e.g. influenza virus.
c. Viscerotrophic Virus: Attacks the liver and other internal organs. Hepatitis B Virus
d Enteric Viruses: Attacks the intestinal tract causing epidermic diarrhoea, nausea and vomiting
e. Dermotropic viruses: These viruses have affinity for the tissue of the mucous membrane of the nose and mouth and the common disease produced are: Herpes Zoster or shingles, measles, warts etc
3. PLANT VIRUS:
Plant virus has RNA as the nucleic acid; they are referred to as Ribnucleoprotein. Arthropods, nematodes and certain plant fungi transmit them.
VIRAL REPLICATION
The process of viral replication is an extraordinary biological phenomena which occurs in series of stages as
1. Adsorption: Is the process that occurs between the virus and the host cell that result in the virus attaching itself to the external surface of the host cell.
2. Penetration: Penetration occurs when the virus or its nucleic acid crosses the plasma membrane into the host cell.
3. Uncoating: During uncoating, the viral nucleic acid is released from the capsid into the host cell. Inside the host cell, the viral DNA or RNA takes control of the cells metabolism and directs it to produce viral components (protomers, capsomers, and nucleic acid).
4. Assembly or viral maturation: This involves assembling the individual viral components into a whole intact virion. During this process, the capsid can first be constructed and then the viral nucleic acid packed into the capsid, or the capsid may be build around the nucleic acid.
5. Release: This occurs when the viral particle escape from the host cell. At this stage, they are active infectious viral particles.
Examples of Viruses
DNA Viruses RNA Viruses
1. Hepatitis B Virus 1. Polio virus (Enterovirus)
A Virus 2.Morbillivirus (MeaslesVirus)
C Virus 3. HIV
2. Human papilloma virus 4. Rabies virus
3. Herpes virus 5. Rubella virus
4. Variola virus (small pox) 6. Influenza virus
5. Human adenovirus 7. Pneumovirus
6. Varicella Zoster 8. Human T cell Leukemia
virus Virus (HTLV-1&2)
CULTIVATION OF MICROBES
Knowledge of environmental and nutritional factors suitable for microbial growth makes it possible to cultivate microbes in the laboratories. The cultivation is possible by the use of nutrient material called culture Medium prepared for the growth of microbes. A culture medium normally contains the necessary nutrients to support nutritional requirements of culture organisms.
Initially, meat broth was used as the first culture medium used to support the growth of microbes. Of late, a variety of media are available from commercial sources for the growth of microorganism. Liquid culture media usually turn solid when agar (A complex polysaccharide) is added. This is done when it is desirable to grow bacteria on a solid medium. For cultivation of obligate parasites (organisms that require absolutely the host cell for survival) chicken fertilize egg is usually used.
For a culture media to nutritionally support and promote the growth of microbes, it
a) must contain the right nutrients for the specific microorganism to be cultivated
b) should also contain sufficient moisture, a properly adjusted pH, and a suitable temperature.
TYPES OF CULTURE MEDIA
Culture Media are classified on three primary levels:
1. Physical form
2. Chemical form
3. Functional form
1. Physical Form
(a) Liquid Media: Liquid media are aqueous formulations that do not gel or solidify at temperatures above freezing and that tend to flow freely when the container is tilted. Examples are broths, milks, or nutrients solutions. Growth occurs throughout the container and may present a cloudy or particular appearance.
(b) Semisolid Media: Semisolid Media exhibit a gelatinous consistency, they do not liquidify at ordinary room temperature. They are used to restrict slightly the movement of motile microbes, and to localize a reaction at a specific site. When heated semisolid media liquidified
(c) Solid Media: Solid media are firm with firm surface on which cells can form discrete colonies. This type of media permits isolating and sub culturing bacteria and fungi. They are either liquefiable or non liquefiable.
2. Chemical Content Media
(a) Synthetic Media: Synthetic media contain variety of defined organic and inorganic compounds specified by means of an exact formula, to meet the exact nutritional needs of the culture organism.
(b) Nonsynthetic Media. Non synthetic media contain at least one ingredient that is not chemically defined i.e. it usually not a simple, pure compound and not also representable by an exact chemical formula. E.g. blood, serum, meat extracts.
3. Functional Media
(a) General Purpose Medium: Contain a mixture of nutrients that could support the growth of broad spectrum of microbes, e.g. broth, nutrient agar blood agar, culture agar and Soya agar.
(b) Enriched Medium: Are enriched with individual growth factors needed to culture specific species that would ordinary live on a host. Enrich Media are enriched with blood serum or hemoglobin.
(c) Selective Medium: Contain an agent or more that inhibits the growth of a certain microbe(s) but not others i.e. it selects for growth of specific microbes, e.g. mannitol salt agar, bile salt.
(d) Differential Medium: Permits the growth of several types of microbes that can be differentiated with the naked eye. The differentiation manifests itself as variations in colony size and colour, formation if gas bubbles and precipitation.
INOCULATION OF A CLINICAL SPECIMEN
Introduction of clinical specimen in/onto a culture media is called inoculation. Tools such as loops, needles, pipette and swab are used to carry out this procedure. An inoculated container of culture medium is called inoculum.
INCUBATION & INSPECTION
Once a container of culture media is inoculated, the next step is for it to be incubated. Incubation involves placing the inoculum in a temperature-controlled chamber to facilitate growth and multiplication. By using incubators other growth factors such as oxygen and carbon dioxide can be controlled.
The usual temperature for incubating microbes falls between 20 to 40oc and the period of incubation may range from a day to several weeks. During this period, the microbes grow and multiply in/on the culture medium.
Clinical observation is possible during growth or the period of incubation. Growth in a liquid medium materialized into cloudiness or sediments. And on solid media, appearance of colonies is a clear indication of microbial growth. Colonies are large masses of clinging cells, with special characteristics in terms of colour, size, rough or smooth, presentation of different characteristics gives the idea of the type of microbes that is/are growing in or on the culture media.
Each colony contains a population of a single species of bacteria. Separating a colony from all other colonies and culturing it in a fresh culture medium will result in formation of a pure culture. A pure culture therefore, is a container of culture medium containing only a single known species or type of microbe. This type of culture is most frequently use for laboratory study. It permits systematic examination and control of one microbial species.
BACTERIAL GROWTH CURVE
Once an inoculum is placed in an incubator, the inoculum bacteria need to grow. Growth occurs in phases. Identifying the phases of growth involves having a method of assessment.
In growth medium, bacteria exhibit a characteristic pattern of growth which can be studied and presented on graph into what is called bacterial growth curve, drown from the logarithm of numbers of viable microbial cells/time as shown below. The number can be obtained by counting the total number of bacterial cells, living and dead, in mls of solution or by counting only the viable cells presents. In many hospitals, electronic cells counters are incorporated into the inoculums specimen during incubation.
DIAGRAM OF THE BACTERIAL GROWTH CURVE
The population growth curve for any particular species of bacteria may be determined by growing the organism in pure cultures at a constant temperature. The graph is constructed by plotting the logarithm of the number of bacteria cells against incubation time.
The normal growth curve has four phases as shown on the diagram.
1. The Lag Phase: This is the period of cell enlargement. It is relatively a flat area on the graph, this is because the same number of cells are usually present During this phase, various intense metabolic activities exist within the microbial cells resulting in growth in size but not in numbers, therefore the bacterial population appear not to be increasing.
Duration of this period depends on conditions under which the microbes are been cultured. If the growth medium is inoculated with microbes that are already dividing, the lag phase will be short.
2. The Log Phase (Logarithmic growth phase). This is also called exponential multiplication phase; it is characterized by rapid cell division occurring at constant rate at regular intervals.
Generation time is determined during this period. Duration of this period depends on availability of growth nutrients, however one can prolong this phase by transfer active growing cells into a fresh medium.
3. The Stationary Phase: This is attained at the peak of cell population. i.e. the population reach maximum numbers (around 106 to 109 cells/ml). A balance between cell division and cell death also marks this phase because the rate of cell death is the same as the rate of cell division or multiplication. Growth at this stage or phase is limited by an increase density of cells coupled with depleted nutrients and oxygen, as well as excretion of organic acids and other biochemical pollutants into the growth medium.
An in balance phenomenon also occurs between nutrient concentration which decreases drastically and toxic concentration that increases.
4. The Death Phase: This phase will reach when nutrient supplies are completely depleted while toxic waste production is high, hence more cells die than been produced. This phase is also characterized by increasing number of dead cells. It can also be term exponential death phase.
STAINING PROCEDURES
Accurate and detail observation of microbes is made possible by the use of colour chemicals called dyes, these are used to colour tissues and other structures of microorganisms in order to make them prominent, for observation under the microscope,
TYPES OF DYES
1. Basic Dyes: Basic dyes have positively charged groups; they bind with negatively charged molecules like nucleic acid and many other proteins of most microbes. For example, bacteria are slightly negatively charged at a pH of 7, therefore, when a bacterial cell is stain or colour with a basic dye, the positive ions in the basic dye will be attracted to the negatively charged bacterial cell thereby accomplishing staining.
EXAMPLES OF BASIC DYES
- Crystal violet
- Methylene blue
- Safrani
- Malachite green
2. Acid Dyes: Acid dyes possessed negatively charged groups or ions e.g. carboxyl (-cooh). They are not attracted to most kinds of bacteria because the dye’s negative ions are normally repelled by the negatively charged bacteria cell, so that, the acid dye and up colouring or staining the background instead of the organism, examples of acid dyes India ink, Nigrosin.
STAINING
Staining is the procedure in which colour chemicals called dyes are apply to a clinical specimen to create adequate contrast between the specimen and the background so that, microbe or bacteria can be made prominent under the microscope.
Before microorganism could be stained, they must be fixed (attach) to a slide. Fixation is done by spreading a thin film of specimen on or across the surface of a slide. It is allowed to dry, and the slide pass through a flame of Bunsen burner to fix the cells. Fixation kills microbes and attaches them to slide; it also preserves various parts of microbes in their natural state for observation.
TYPES OF STAINING PROCEDURES
1 Simple staining.
2.Differential staining procedures.
Simple Staining Procedure
This procedure involves the use of single dye. Basic dyes such as crystal violet and methylene blue are frequently used in this procedure to determine the size shape and arrangement of bacteria.
Procedure
Apply a single dye to a specimen then add an additive (mordant), e.g. iodine to intensify the dye. Then keep the specimen for some seconds or few minutes, and wash off with water, dry and examine under a microscope. Under examination, all bacteria cells will appear dark purple or violet in colour.
NOTE: If a basic dye is use for this process, the dye will stick to the bacterial cells giving the dark purple or violet colour thereby rendering the microbes prominent under the microscope. This process is referred to as Positive Staining. On the other hand, if an acid dye is used, the dye will be repelled by the negatively charged bacterial cells, therefore clear unstained bacterial cells will appear against a dark background. This is referred to as negative staining. Simple staining procedure is use to determine cellular size, shape and arrangement of bacteria.
2. Differential staining Procedures
Differential staining procedure employs the use of multiple dyes that react differently with different kinds of bacteria.
USES
1. By using differential staining procedure, structural differences between microbial species can be revealed.
2. It is also use to divide bacteria into groups based on their staining properties.
Types of Differential Staining
1. Gram stain
2. Acid-fast stain (Zielh Nelson Stain)
3. Special stain.
Gram Staining
Gram staining procedure results in classification of bacteria into two main groups base on colour reaction of their cells. i.e.
1. Gram negative bacteria
2. Gram positive bacteria
Procedure
First, a heated fixed smear is stain with a basic purple dye, usually crystal violet, and then a mordent. The specimen is then rinsed using a decolorizing agent e.g. acetone. The purpose of the decolorizing agent is to wash of the basic dye. At this stage, the bacteria which cell wall does not permit decolourization will remain dark purple on examination, and are termed Gram- positive bacteria. Those that permit decolonization will appear coluorless due to the fact that the alcohol is wash off the basic dye from their cell walls; they are term Gram-negative bacteria
The colourless Gram-negative bacteria turn red-pink when the specimen is counter stain with safrani (a red dye) while the Gram-positive still remain dark purple.
Examples of Gram Positive & Negative Bacteria
Gram Positive Bacteria GramNegative Bacteria
1. Staphylococcus 1. Meisseria
2. Streptococcus 2. Veillonella
3. Pneumococcus 3. Escherichi coli
4. Bacillus Anthracis 4. Salmonella
5. Clostridium 5. Klebsiella
6. Shigella
DIFFERENTIAL QUALITIES OF GRAM POSITIVE AND NEGATIVE BACTERIA
Gram Positive Gram Negative
1.Spore forming 1.Non spore forming
2.Sensitive to cephalosporins 2.Sensitive to streptomycin
3.rod and cocci shape 3. Rod, cocci, and spiral shape
4.Produces exotoxins 4. Produces endotoxins
5.More resistance to injury 5. Less resistance
6.Have less synthetic ability. 6.Have more synthetic ability.
ACID FAST STAINING (ZIELH NELSON STAIN)
Another differential staining procedure is that of Zielh Nelsons, use specially for staining organisms of the genus mycobacterium. This group consists of acid fast, non-motile, non-spore forming, and strictly aerobic rods. They possess complex fatty substances, which make them difficult to stain by normal staining procedures. All acid-fast organisms are gram-positive organism.
Procedure: A heated fixed film is flooded with a red dye (carbon fuchsin). It is then gently heated to a temperature of about 900c for several minutes to enhance penetration and retention of the dye. The slide is allowed to cool, and then wash with water and treat with acid alcohol for 10 minutes i.e. 95% alcohol containing 10% hydrochloric acid. This process removes the red stain from bacteria that are not acid fast and therefore renders them colourless while those that are acid fast retain the red colour on examination. The specimen is then counter stain with safrani, a red basic dye. The counter stain procedure will turn the non acid-fast bacteria pink.
SPECIAL STAINING
Special stain isolates specific parts of bacteria, such as endospores, flagella, and capsule. One example of special staining is negative staining, a technique that revel the presence of the defuse capsule surrounding many bacteria.
Negative staining mechanism involves staining the background. This is due to the fact that, the dye fails to penetrate the organism and therefore leaves them unstained to appear as light areas in the darkened field. Only the outline of the organisms is made apparent by this method.
CONTROL OF MICROBIAL GROWTH
There are various ways one can utilize the inability of microbes to control their growth. Rates of microbial growth and death are greatly influence by several environmental and growth factors. By altering these factors, one can either cause microbial death or inhibit their multiplication.
Effectiveness of any particular method of microbial control depends on the following factors.
1. The types and number of microbes, e.g. a higher load of contaminants requires more time to destroy.
2. Duration of treatment.
3. Concentration (dosage, intensity) of the agent. Most disinfectant is more active at higher concentration.
4. Temperature and pH of the environment.
MEASURES USED TO CONTROL MICROBIAL GROWTH
DEFINITION OF TERMS
1. Sterilization
The complete destruction of all living organisms, including cells, viable spores, and viruses, is called sterilization. Sterilization of objects can be accomplished by heat, autoclaving (heat and steam or ethylene oxide under pressure), various chemicals (such as formaldehyde), and certain levels of radiation with ultraviolet or gamma rays.
2. Disinfection
Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. The chemicals used to disinfect inanimate objects, such as bedside equipment and operating rooms, are called disinfectants. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.
Examples of various groups of agents use to achieve disinfection are:
A. Those agents that will injure or destroy the cell membrane, these include
a. Soap (these are alkaline compounds made by combining fatty acid with oil)
b. Detergents e.g. omo, aircomic detergents
c. Acids e.g. acetic acid (in the farm of vinegar)
d. Alkaline e.g. hydrogen peroxide (use to cleanse skin, wounds, and disinfection of instruments
B. Those agents that will normally inhibit enzyme activities and denature proteins. E.g.
a. Alcohol such as ethanol
b. Phenol (carbolic acid) e.g. Lysol, creolin, anerosal hexachlorophene.
c. Halogens e.g. fluorine, bromine, chlorine and iodine
d. Heavy metal e.g. silver, mercury, formaldehyde sulphanimide
C. Those that will affect the nucleus and alter genes. E.g.
a. Dyes such as acriflavine, proflavine.
3. Microbicidal Agents
The suffix-cide or –cidal refers to killing. Thus, a microbicidal agent is one that kills microbes. A bactericidal agent (bactericide) kills bacteria, but not necessarily endospores of bacteria. A disinfectant that kills fungi is a fungicide, and similarly, an agent that destroys viruses is a virucide. The general term germicide refers to any agent that destroys germs or harmful microorganisms; this agent might be used in sanitization procedures.
4. Microbistatic Agent
The suffix-stasis or –static means the inhibition or cessation of growth and reproduction of microorganisms. A bacteriostatic agent is one that inhibits the metabolism and reproduction of bacteria, causing them to degenerate and die or be destroyed. Important microbistatic agents and processes include desiccation (drying), freezing temperatures, concentrated sugar and salt solutions, and some chemotherapeutic drugs (including the antibiotics).
5. Asepsis
Because sepsis refers to the growth of infectious microbes on living tissues, asepsis means the absence of infectious microorganisms on living tissues. Thus, aseptic technique is a procedure designed to eliminate and exclude all infectious microbes by sterilization of equipment, disinfection of environment, and cleansing of the body tissues with antiseptics.
Anti microbial Methods
The methods used to destroy or inhibit microbial life are either physical or chemical, and sometimes both types are used. The effectiveness of any antimicrobial procedure depends on:
(1) length of time it is applied,
(2) temperature,
(3) concentration,
(4) nature and number of microbes and spores present, and
(5) presence of protective materials, such as the proteins in feces, blood, vomitus, and pus.
Physical Antimicrobial Methods
The physical methods commonly used in hospitals, clinics, and laboratories to destroy or control pathogens are heat, pressure, drying, radiation, and filtration.
Filtration:
Filtration is the best method of sterilization. It is use to remove microorganisms from liquids and gases. The process involves straining fluid (air or liquid) through a filter with 0-2 to 0.5cm diameter pores or through a layer of High Efficiency Particular Air (HEPA) filter with about 0.3um diameter pores large enough to allow fluid to pass through but too small for microorganism to pass through.
This method is an efficient means of removing airborne contaminants that are common source of infection. In the medical field, this process is employ to flow sterile air into operating theatres, and rooms where drugs are produced or in laboratories to prevent contamination during cultivation of microorganisms, it is also use during preparation of injection fluids. Examples of molecular filters use for this process are
1.Unglazed porcelain
2. Sintered glass
2. Cellulose
3. Plastic films
4. Asbestos.
Radiation:
Radiation produces either lethal or mutagenic effect on microorganism. Lethal effect is produce when the radiation kills the organisms on contact. This effect occurs due to occurrence of chemical changes in the organelles of the cells. Mutaganic effects refer to the ability of the radiation to cause organic disruption leading to the death of the organism.
Types of Radiations
1 . Ionizing radiations: These include gamma rays, x-ray and cathode rays. They are germicidal; this is because they are able to penetrate solid and liquids. With particular reference to bacteria, they cause ionization of the DNA molecule, thereby causing it to sustain mutation. Ionizing radiation can be use to sterilized heat sensitive articles including bone graft, sutures, plastic syringe, rubber gloves and many pharmaceutical products such as antibiotics.
2. Non Ionizing Radiation
Refers to ultraviolet rays, they cause abnormal linkage of essential components within molecules of the DNA of microbes. Sunlight is the natural source of ultraviolet radiation. Sunlight is used in everyday life practice in sterilizing materials. The other source of non ionizing radiation is the germicidal lamp use in hospital rooms, operating theatres, food preparation areas, nursing and maternity homes to destroy fungi cells, spores of bacterial vegetative cells, protozoa and virus.
Heat: Elevated temperatures tend to kill microbes because it denatures protein resulting in inactivation of enzymes. Heat also tends to make the cell wall of microbes increasingly permeable to toxic substances and loss of cellular nutrient from the cell. Forms of heat use in microbial control are moist and dry heat.
1. Moist Heat. Applied in the presence of moisture, such as by boiling or steaming, is more effective than dry heat because moist heat causes the proteins to coagulate and also cellular enzymes becomes inactivated. Also moist heat sterilization is faster than dry heart sterilization and can be done at a lower temperature; thus it is less destructive to many materials that otherwise would be damaged by higher temperatures. Moist heat comes in the form of hot water or boiling water and steam under pressure. In hospitals, sterilizers are use to obtain hot water for this process. Materials are usually exposed to boiling water for 30 minutes e.g. surgical instrument. In practice the temperature of moist heat used to achieve sterilization ranges from 60cc to 135OC. Boiling for 30mins will kill most viable bacteria, fungi, and viruses.
Steam Under Pressure: is obtained by the use of an autoclave. This device produces steam at temperature of 100OC measured at 15 pounds per square inch (PS1) or I atmosphere. The steam produced is then compressed in a close chamber to increase its temperature (This phenomenon explains the physical principles that governs the behaviour of gases under pressure, i.e. when a gas is compressed, its temperature rises in direct relation to the amount of gas). The To can be increase to about 121OC and it is this increased temperature that kills the microbes not the pressure.
This method is use to sterile heat-resistant material such as glassware, surgical dressing and wear, rubber gloves, metallic instruments, liquids, culture media and plastics.
2. Dry Heat: This is not often used. It is employ in hospital for complete destruction and disposal of infectious items, such as used disposable needles dressings, beddings. A typical example is incineration (Destruction by fine). However, the hot air oven provides another means of dry heat sterilization in most hospitals. The dry oven is usually electric or gas, it has coils that radiate heat within an enclosed compartment. Heated, circulated air comes in contact with the items, transferring its heat in the process. Sterilization is accomplished by exposure of the items to 150oc - 180oc for 2 to 4 hours.
Chemical Antimicrobial Methods
Chemical disinfection means the use of chemical agents to inhibit the growth of microorganisms, either temporarily or permanently. The effectiveness of a chemical disinfectant depends on many factors: the concentration of the chemical; the time allowed for the chemical to work; the pH, and temperature must be maintained for the specified time period to ensure the best results. The items to be disinfected must first be washed to remove any material in which pathogens may be hidden. Although the washed article may then be clean, it is not safe to use until it has been properly disinfected.
Almost all bacteria in the vegetative growing state as well as fungi, protozoa, and most viruses are susceptible to many disinfectants. The mycobacterium of tuberculosis and leprosy, the endospores of bacteria, fungal spores, and the hepatitis viruses are notably resistant. Therefore, chemical disinfection should never be attempted when it is possible to use proper physical sterilization techniques.
AntisepTiCS
Most antimicrobial chemical agents are too irritating and destructive to be applied to the mucous membranes and the skin. Those that may be safely used on human tissues are called antiseptics. An antiseptic merely reduces the number of organisms on a surface but does not penetrate the pores and hair follicles to destroy microorganisms residing there. Also, an antiseptic is applied at the site of the surgical incision to destroy local microorganisms.
CHEMOTHERAPY
Basically, chemotherapy is the treatment of diseases through the use of chemical preparation known as drugs.
Action of Chemotherapeutic Agents
a. They will either kill or inhibit the growth of microbial activities but will not harm the host, that is to say the dosage is usually such that it is low enough not to cause damage or harm to the host but high enough to kill or inhibit the activities of the offending agent.
b. They function to block the metabolic pathway of microbes without harming the host.
c. The drug may also be such that, the microorganism may have high affinity for it and hence tend to concentrate the drug within itself and therefore end up been killed by the drug. e.g. when CHQ is administered on an individual with malaria, the plasmodium tend to concentrate the drug to itself and end up been killed by the drug.
FORMS OF CHEMOTHERAPEUTIC AGENTS
Chemotherapeutic agents comes in two forms
1. Synthetic form.
2. Antibiotics.
1) Synthetic form: The synthetic chemotherapeutic agents are such that they are structural or metabolic analogs of molecules or materials use by microbes for their metabolic activities. Therefore when such drugs are administered, the microbes cannot distinguish between the analog and the natural molecules use by them. Hence they mistakenly take up the analog as the natural molecules.
Once the microbe takes up the analog, it tends to inhibit enzyme activity of the microbe that may lead to death. Some microbes may be able to utilize the analog, but at the end, produces a faulty product that kills them. For example, sulphur drugs are similar to the natural metabolic compound Para-amino benzoic acid (PABA)-An essential metabolite for many bacteria for the synthesis of folic acid. PABA also plays a part in the synthesis of Purine and certain amino acid by co-enzyme tetrahydrofolic acid. Sulfonamides molecules for instant has extreme affinity for the PABA sites of the enzyme that synthesize folic acid, thus sulfonamide causes an inadequate supply of folic acid for purine which invariably stop nuclei acid synthesis and prevent bacterial cell from multiplying.
NOTE
Although human beings require folic acid for nucleic acid synthesis as much as bacteria do, human beings cannot synthesize folic acid, it is an essential nutrient (vitamin) that must come from diet.
Human cells lack this special enzymatic system for incorporating PABA into folic acid, therefore human cells metabolism cannot be inhibited by sulfa drugs.
Sulfonamide derivatives serve as synthetic analogs of bacteria, they are very affective for a wide range of microbes e.g. streptococci, staphylococci, pneumococci, meningococci
Other synthetic analogs are
- Trimethoprim
- Aminoghycosides e.g. streptomycin, gentimycin
- Tetracycline
- Chloramphenicol.
ANTIBIOTICS
Antibiotic are metabolic products of aerobic spore forming bacteria and fungi that can inhibit or destroy other organism. The use of this category of drugs became popular in 1929 when Flemy discovered penicillin. Since then many antibiotics have been identified for combating diseases. Examples of antibiotic are tetracycline, erythromycin, penicillin, refampicin chloramphenicol, etc.
Antibiotics fall into three main categories. Those that are
1. Active mainly against gram-positive bacteria e.g. penicillin, erythromycin, lincomycin.
2. active mainly against gram-negative e.g. polymycin, streptomycin nalidixic acid
3. Active against both gram-positive and gram-negative bacteria (broad spectrum antibiotics). e. g. Tetracycline, Ampicillin, cephalosporins.
PREVENTION OF HOSPITAL INFECTIONS
General Control Measures
Prevention of Airborne Contamination:- Respiratory infections are most often transmitted through the air. Observe the following measures to decrease the number of pathogens transmitted by this means:
· Cover the mouth and nose when coughing or sneezing.
· Limit the number of persons in a room.
· Remove the dirt and dust from floor and furniture by damp dusting.
· Open the room to fresh air and sunlight whenever possible.
· Roll linens together carefully to prevent dispersal of microbes in the air.
· Remove bacteria from the air by a filtered air-conditioning system.
· Prevent the Spread of Communicable Diseases
Handling food and dishes
Regulations for safe handling of food and dishes are not difficult to follow.
They include
· Using high quality fresh food
· Properly refrigerating and storing food
· Properly washing, preparing, and cooking food
· Properly disposing of uneaten food
· Thoroughly washing hands and fingernails before handling food and after visiting a restroom
· Properly disposing of nasal and oral secretions in tissues
· Covering hair and wearing clean clothes and aprons
· Providing periodic health examinations for kitchen workers
· Prohibiting anyone with an infection or intestinal upset from handling food or dishes.
· Keeping all kitchen equipment and all other equipment scrupulously clean
· Rinsing and then washing dishes in a dishwasher in which the water temperature is above 80oC (176o F)
Handling of Fomites
Fomites are any articles or substances other than food that may harbor and transmit microbes. Examples of fomites are dishes, bedpans, urinals, thermometers, washbasins, bed linen, and clothing and other personal patient items. Observing the following rules may prevent transmission of pathogens by these items:
· Use disposable equipment and supplies wherever possible.
· Disinfect or sterilize equipment as soon as possible after use.
· Use individual equipment for each patient.
· Use an individual thermometer for each patient and store each thermometer in a disinfectant solution.
· Empty bedpans and urinals, wash them in hot water, and store them in a clean cabinet between uses.
· Place bed linen and soiled clothing in bags to be sent to the laundry.
Hand-washing
Hand Washing is the single most important aseptic precaution. Hands should be kept clean at all times and should be washed before and after each patient contact to preclude carrying pathogens from one patient to another. Common-sense rules that one should always follow include.
· Use of gloves or tongs to handle contaminated materials
· Wash hand with disinfectant soap before and after contact with a patient
· Rinse hands under running water
· Dry hands and apply antiseptic lotion to prevent capping
INFECTION CONTROL PROCEDURES
1. Medical and Surgical Asepsis
The techniques used to achieve asepsis depend on the site, the circumstances, and the environment.
Once basic cleanliness is achieved, it is not difficult to maintain asepsis. The goal of medical asepsis is to exclude all pathogenic microorganisms from the immediate environment. Medical asepsis includes all the precautionary measures necessary to prevent direct transfer of pathogens from person to person and indirect transfer of pathogens through the air or from instruments, bedding, equipment, and other inanimate objects (fomites).
In the hospital, medical asepsis is practiced using sterile equipment, dressings, medications, and any items that could transfer microorganisms to susceptible sites. Invasive procedures, such as drawing blood, giving injections, catheter insertion, cardiac catheterization, and lumbar punctures, must be performed under strict aseptic precautions. The most controlled and strict aseptic procedures must be applied in the operating room and surgical area. The goal of surgical asepsis is to exclude all microorganisms from the immediate environment. Surgical aseptic techniques include those practices that make and keep all objects and the area itself sterile. These practices are necessary during all surgical procedures, as well as any other procedure that involves exposure of the deep body tissues, to prevent the entry of any microorganisms into those tissues.
The surgical area of the patient’s skin must be shaved and thoroughly cleansed and scrubbed with soap and antiseptic. If the surgery is to be extensive, the surrounding area is covered with a plastic film or sterile cloth drapes so that a sterile surgical field is established. The surgeon and all surgical assistants must scrub for 10 minutes with a disinfectant soap and cover their clothes, mouth, and hair, because these might shed microorganisms onto the operative site.
2. Universal Precautions
Universal precautions include but are not limited to the following procedures:
· Hands should always be washed before and after contact with patients. Hands should be washed even when gloves are used. If hands come into contact with blood, body fluids, or human tissue, they should be washed with soap and water.
· Gloves should be worn when contact with blood, body fluid, tissues, or contaminated surfaces is anticipated.
· Masks and protective goggles should be worn if aerosolization or splattering is likely to occur such as in certain surgical procedures, wound irrigations, and bronchoscope.
· The need for emergency mouth-to-mouth resuscitation and mouthpieces should be minimized by strategically locating resuscitation bags or other ventilation devices where they are available for use in areas where they may be needed.
· Sharp objects should be handled cautiously to prevent accidental cuts or punctures. Used needles should not be bent, broken, reinserted into their original sheath, or unnecessarily handled. They should be discarded intact immediately after use into an easily accessible, impervious needle disposal container. All needle stick accidents, mucosal splashes, or contamination of open wounds with blood or body fluids should be reported immediately.
· Blood spills should be cleaned up promptly with a disinfectant solution, such as a 1:10 dilution of chlorine bleach.
· All patients’ blood specimens should be considered biohazardous
·
3. Isolation of Patients
Patients are placed in isolation for one of two reasons: (1) to prevent the spread of pathogens to other susceptible people, or (2) to protect a very susceptible patient from exposure to pathogens.
Reverse Isolation
Certain patients are especially vulnerable to infection; among them are patients with severe burns, those who have leukemia, patients who have received a transplant, immunodeficient persons, and those receiving radiation treatments. Premature babies are also highly susceptible. All such patients are protected through isolation procedures. This type of isolation is referred to as reverse isolation. The room must be thoroughly cleaned and disinfected before the patient is admitted. Those entering the room must wear sterile gowns and masks to prevent depositing microorganisms into the room from their clothes or their respiratory tract. Proper hand washing procedures must also be followed before entering the room.
Source Isolation
Whenever possible, patients with contagious diseases should be isolated in private air-conditioned rooms with a private bath. In this manner infectious agents are isolated within a definite area, and thereby spread of the pathogens to other patients is prevented. Isolation procedures should be determined by
· The mode of transmission of the disease from one person to another
· The location of the pathogen causing the infection, its portal of exit (intestinal, respiratory, or wound drainage), and its portal of entry
· The virulence of the pathogen and its susceptibility to antibiotics.
Isolation Techniques
Isolation techniques are designed to immediately destroy the pathogenic organisms in the infectious discharge of the patient. Non disposable items are disinfected, with a minimum of handling, then cleaned thoroughly and resterilized. If the patient’s infection involves spore-forming bacteria such as those found in gas gangrene and tetanus, then contaminated equipment must be autoclaved to destroy the spores before the item can be reused. Disposable equipment must be incinerated or thoroughly autoclaved before disposal.
Hand-Washing
Doctors, nurses, and other hospital personnel entering the room must be particularly careful to wash their hands before and after caring for a patient. Hand contact with doorknobs, telephones, elevator buttons, and furniture easily transmits pathogens to and form the patient. Fingernails should be kept short and clean, and jewelry should not be worn in an isolation room.
Gowns
In every isolation room, gowns should be used. A clean fresh gown should be available for each person who enters the isolation room on each occasion and discarded after each use. To be protective, gowns should be large enough to be fastened securely. They must be kept dry, remember that a moist environment is required for growth of pathogens, so if an area is moist, the pathogens may gain access to clothing worn under the gown.
4. Medical Waste Disposal
General Regulations
According to the Occupational Health and Safety Administration Standards, medical wastes must be disposed off properly. These standards include the following:
1. Any receptacle used for decomposable solid or liquid waste or refuse must be constructed so that it does not leak and must be maintained in a sanitary condition. This receptacle must be equipped with a solid, tight-fitting cover, unless it can be maintained in a sanitary condition without a cover.
2. All sweepings, solid or liquid wastes, refuse, and garbage shall be removed to avoid creating a menace to health and shall be removed as often as necessary to maintain the place of employment in a sanitary condition.
3. The infection control program must address the handling and disposal of potentially contaminated items.
Sharp Instruments: Reusable and Disposables
These items must be disposed off in the following manner:
1. Needles shall not be recapped, purposely bent or broken by hand, removed from disposable syringes, or otherwise manipulated by hand.
2. After use, disposable syringes, scalpel blades, and other sharp items must be placed in puncture-resistant containers for disposal.
3. These containers must be easily accessible to all personnel needing them and must be located in all areas where needles are commonly used, as in areas where blood is drawn, including patient rooms, emergency rooms, intensive care units, and surgical suites.
4. The containers must be constructed so that the contents will not spill if knocked over and will not cause injuries.
Laboratory specimens
All specimens of body fluids must be put in a well-constructed container with a secure lid to prevent leaking during transport and must be disposed off in an approved manner. Contaminated materials used in laboratory tests should be decontaminated before processing or be placed in bags and disposed off in accordance with institutional policies for disposal of infectious waste.
1. Use sterile gloves for procedures involving contact with normally sterile areas of the body.
2. Use examination gloves for procedures involving contact with mucous membranes and for other patient care or diagnostic procedures not requiring sterile gloves, unless otherwise indicated
3. Change gloves between patient contacts.
4. Do not wash or disinfect surgical or examination gloves for reuse. Disinfectants may cause deterioration and washing with disinfectants may allow liquids to penetrate the barrier through undetected holes in the glove.
5. Use general-purpose utility gloves for housekeeping chores involving potential blood contact and for instrument cleaning and decontamination procedures. Utility gloves may be decontaminated and reused but should be discarded if there is evidence of deterioration.
Masks
Masks generally are not worn outside the operating room. However, if a patient is isolated because of a respiratory infection, the patient should be masked whenever it is necessary to go for treatment in other parts of the hospital. Personnel working closely with an isolated respiratory patient may wish to wear masks for self-protection. Remember that wet masks are not effective. Used masks should be promptly discarded or laundered and autoclaved. Hands should be washed again after touching a used mask.
Gloves
The type of latex or vinyl gloves selected should be appropriate for the task being performed.
INSTRUMENT PROCESSING
Proper instrument processing is critical for reducing infection transmission during clinical or surgical procedures.
Objectives of instruments processing
1. To remove as many microorganism as possible from instruments and other items so that microorganism are not transmitted to client during clinical procedures.
2. To reduce the risk of infection to staff by eliminating harmful microorganisms and viruses that may be present on medical waste and used instruments and other items that come into contact with a client’s fluid or tissues during clinical procedures.
Steps of instrument processing
There are four steps to processing instruments and other items used during clinical and surgical procedures. These are
1. Decontamination.
2. Cleaning.
3. Sterilization.
4. Storage.
DECONTAMINATION
Decontamination kills viruses (such as hepatitis B, and HIV) and many other microbes, thereby making items safer to handle. To decontaminate items, the concentration of whatever solution you intend to use should be such that it is in the require strength to kill microbes but not high to increase supply.
STEPS OF DECONTAMINATION
Step 1. A solution for decontamination is usually kept in each unit. The most common solution used in our hospitals is 0.5% chlorine.
Immediately after use, contaminated instruments and other items are placed in the container of 0.5% chlorine solution for a period of 10minutes.
Step 2. After 10minutes, remove the items from the chlorine solution and either rinse with water or clean immediately. Excessive soaking in the solution can damage instruments.
NOTE Always wears utility gloves when removing instruments from decontamination solution. Special consideration must be taken when decontaminating reusable items such as needles and syringes, glove, linens, and storage containers.
Reusable needles/ syringes. Fill the assembled needle and syringe with 0.5% chlorine solution, flush several times and then soak the needle and syringe for 10minutes. Rinse by flushing several times with clean water.
Gloves. Before removing contaminated gloves, dip the glove hands into 0.5% chlorine solution to rinse the outer surfaces to remove blood, other fluid and tissue. Carefully remove the glove. If the glove is disposable, dispose of them properly. If they are to be reused, place them in a container of 0.5% chlorine solution and soak for 10minutes before cleaning. Always place gloves in a different container than the one used to decontaminate instruments.
Storage containers. Fill the container with 0.5% chlorine solution and soak for 10minutes before cleaning. Rinse or clean immediately.
Objectives of decontamination/cleaning.
1. To reduce the number of microorganisms
2. To eliminate fluid or tissue remains.
3. To remove contaminants that can collect in joints, grooves, and teeth of items.
How to make 0.5% chlorine solution
FORMULAS
(a) For liquid household bleach
% of chlorine in liquid bleach
- 1
0.5%
Example. To make 0.5% chlorine solution from 3.5% chlorine concentration.
Solution. % Chlorine in liquid bleach = 3.5%
. . 3.5%
.
0.5% = 7 –1 = 6parts of water for each chlorine.
(b) Using bleach powder
Calculate the ratio of bleach to water using the following
%Chlorine desired
* 1000 = Gram of powder for each liter of water.
%Chlorine in bleach powder
Example: To make 0.5% chlorine solution from calcium hydrochloride powder containing 35% available chlorine.
%Chlorine desired = 0.5%
%Chlorine in bleach = 35%
. . 0.5%
. * 1000 = 14.3 grammaes
35%
(c) Using chlorine releasing tablet
Follow manufacturer’s instructions.
3. CLEANING
Cleaning removes organic material, dirt and foreign matter that can interfere with sterilization or high-level disinfect ion (HLD). Cleaning also drastically reduces the number of microorganisms including bacterial endospores, on instruments and other items.
Cleaning refers to scrubbing with a brush, detergent, and water and is a crucial step in instrument processing. If items are not clean, further processing might not be effective because
1. Microorganisms trapped in organic material may be protected and survive further processing
2. Organic material and dirt can make the chemical used in some processing technique less effective.
Steps in cleaning
Step 1 Using a soft brush or old toothbrush, detergent and water, scrub instruments and other items vigorously to completely remove all blood, other body fluid, tissue and other foreign matter. Hold instruments and other items under the surface of the water while scrubbing and cleaning to avoid splashing. Disassemble instruments with multiple parts, and be sure to brush in the grooves, teeth, and joints of items where organic material can collect and stick.
Step 2 Allow items to air-dry or dry them with a clean towel
4. STERILIZATION /HIGH-LEVEL DISINFECTION.
Sterilization ensures that instruments and other items are free of all microorganism including bacterial endospores. If sterilization is not possible or feasible, high-level disinfect ion is the only acceptable alternative for these items.
Forms of sterilization
1. Steam sterilization (autoclaving). Steam sterilization in an autoclave is one of the most common forms of sterilization used in health care facilities. Steam sterilization requires moist heat under pressure.
2. Dry heat sterilization (electric oven). Dry heat sterilization requires high heat for specific period of time. For sterilization to be achieved, a constant supply of electric is necessary. Only glass and metal objects can be sterilized by dry heat. Do not use this method for other items such as surgical gloves, which may melt or burn because of the high temperatures
Time require to sterilized instruments and other items for different temperatures are
- 170degree Celsius----- 1 hour
- 160 ‘’------------------- 2 hours
- 150 ‘’ --------------------2.5 hours
- 140 ‘’ --------------------3 hours
3. Chemical (Cold) sterilization. Chemical sterilization is used for instruments and other items that are heat-sensitive, or when methods that require heat are unavailable. Items are sterilized by soaking them in a particular chemical solution follow by rinsing them in sterile water.
A. USING HIGH-LEVEL DISINFECTION
High-level disinfect ion is used when sterilization is not feasible. HLD eliminates bacteria, viruses, fungi, and parasites, but it does not reliably kill all bacterial endospores, which cause disease such as tetanus and gangrene.
Forms of High-level Disinfect ion
1. HLD by boiling.
2. Chemical HLD
3. HLD by steaming.
4. STORAGE
After sterilization or HLD items should be used or properly stored immediately. Proper storage is as important as proper decontamination, cleaning, sterilization, or HLD. If items are not stored properly, all the effort and supplies used to properly process them will have been wasted, and the items will be contaminated
METHOD OF TAKING AND LABELING OF SPECIMEN
A hospital laboratory services become more efficient if there is an intelligent co-operation between the ward staff and the laboratory staff. This can be achieve if those who work on the ward thus the nurse knows the way specimen are collected and sent to the laboratory.
The bacteriologist or the assistant collects some specimens. Others are by the Doctor. Those that are collected by the Doctor are biopsy tissues, blood specimen, cerebro-spinal fluid or and ascetic fluid or aspirations from other body cavities. The nursing staff collects other specimens and still the patients themselves can collect other specimen after they have been given the necessary instructions. However, the responsibility of correct labeling and prompt dispatch of the specimen to the laboratory is nursing staff responsibility.
LABELING OF THE SPECIMEN
The following identification should be written on a piece of paper and attached or pasted on the bottle.
1. The name of the hospital
2. The ward
3. Patient’s name
4. Kind of specimen
5. Examination required
6. Date and time collected
FILLING THE LABORATORY REQUEST FORM
Indicate the following in the laboratory request form:
1. The name of the hospital
2. The ward
3. Patients name
4. Age
5. Sex
6. Kind of specimen
7. Examination required
8. Diagnosis of the patient
9. Drug on which the patient is
10. The name and the signature of the Doctor who ordered the examination
11. Date and time the specimen was taken.
NOTATION IN THE WARD LABORATORY BOOK
1. The date and time the specimen was sent
2. The patient’s name
3. Type of specimen
4. Examination required
5. Name of the nurse
6. Leave a column for recipient name or signature
This ward laboratory book accompanies all specimens to the laboratory and the nurse must insist that the laboratory technician signs the book after receiving the specimen.
The nurse indicates all specimens taken from the patient in the nurse’s note which is among the patient’s papers and the nurse who took the specimen should write his/her name and date to indicate that the specimen has been taken.
METHOD OF COLLECTION OF SPECIMEN
1. All materials or specimen for bacteriological examination must be collected into sterile containers.
2. Materials that are to be sectioned and examined microscopically must always be fixed immediately after the organ or tissue has been removed from the patient. Fixation of a specimen is usually carried out by immersion of the tissue in 5-10% formalen solution. Fixed material are however not good for bacteriological examination because the formalin kills any microbe that might have been presence.
3. Material for chemical test of blood for the estimation of blood urea, blood sugar or glucose and blood protein should be prevented from clotting. An anti coagulant such as oxalate or citrate acid must be put in the specimen bottle before the specimen is collected into it.
4. Specimen for bacteriological test must be collected with special precaution against accidental contamination.
5. All specimen for routine examination need not be collected into sterile bottles, however the bottles must be scrupulously clean.
6. In some blood examinations only the serum is required. The blood must therefore be allowed to clot, such specimen are agglutination tests, such as Khan test and Wasserman reaction test for the diagnosis of syphilis and widal test for the detection of typhoid antibodies. Also blood for grouping and cross matching, which is a prelude to transfussion, the specimen, should be a clotted blood.
7. When ever possible, collect all specimens before antibiotic and chemotherapeutics are given unless otherwise indicated. If the drugs are given before the specimens are taken, the tendency is that the drug might kill the organisms and that the organism might not be seen during laboratory examination. Also it is better to collect blood for culture and sensitivity and blood for malaria parasites when patient’s temperature are high, because during this stage, the organisms are active in the peripheral blood and so they may be easily taken into the blood specimen.
8. Before the specimen are taken provide an explanation for the patent, this will allay his anxieties make him to cooperate and also let him know what is expected of him.
9. Provide supportive nursing activities before the specimen are taken bearing in mind that patients may be of pain, embarrassment or afraid of findings obtained from the specimen.
10. Was your hand before collecting any specimen so that you do not transmit your microbes to the patient
11. Wash your hands after the collection so that you do not contaminate yourself with microbes from the patient and specimen and subsequent transmission to other patients.
SOME BACTERIOLOGICAL INVESTIGATIONS
SPUTUM
Sputum is the mucous from the lungs, bronchi and trachea. It is very important to differentiate it from saliva, which is clear liquid secreted by the salivary glands from the mouth and sometimes referred to as spit.
Healthy individuals do not produce sputum. Patients with respiratory disorders do and they need to cough to bring sputum up from the lungs, bronchi and trachea into the mouth and then to be expectorated with the collecting container.
PURPOSE FOR SPUTUM SPECIMEN
1. To aid in the diagnosis of and treatment of respiratory illness by identifying the microbes or cells present in the sputum and then identifying the antibiotic to which they are sensitive.
2. To confirm the presence of the tubercle bacillus sometimes referred to as the acid bacilli (AFB) in the sputum.
3. To assess the effectiveness of therapeutic measures.
ASSESS THE PATIENT AND THE SPUTUM
1. Observe the appearance of the sputum obtained and the amount. Sputum can be described as liquid or thick colour white, yellow, bloodstain, green or clear. The amount can be described as copious, large, small etc.
2. Determine whether patient has difficulty in producing sputum and the time sputum is produced most readily. Many patients find it easier to produces early in the morning after it has collected in the lung during the night. It is also more likely that such early morning specimen of sputum might contain the organisms, therefore whenever possible early morning specimens are suitable for laboratory investigations.
3. Observe the patients respiration and note any abnormality or difficulty in breathing.
4. Observe the colour of the patient’s skin and mucous membrane for cyanosis, which is an indication of impaired breathing and hence defective oxygen intake.
PROCEDURE
For collection of sputum for bacteriological examinations, instruct the patient first to rinse the mouth and gargle before expectorating the sputum. Rinsing and gargling the mouth reduce contamination by microbes in the mouth. It is not a sterile specimen because the bronchi and alveoli are in direct contact with the unsterile atmospheric air. Nevertheless, the sputum should be collected into a clean container with a cover. Ensure that the patient has actually coughed up sputum and not just saliva
The specimen should be as large as possible since small specimen may exclude the organism. Label and sent to the laboratory immediately.
URINE SPECIMEN FOR ROUTINE EXAMS
Urine specimen is the most frequently collected specimen because urine provides valuable information about many of the bodies biological processes. A multitude of tests are performed in urine and many methods are used to collect the specimen.
The three common methods are:
1. Urine for routine examination
2. 24hr specimen of urine or time urine collection.
3. Urine for culture and sensitivity test (CS)
Urine for routine examination can be collected randomly but early morning specimen is good because during the night urine have accumulated in the bladder and might contain all the abnormalities. In the day the possibility of dilution by high fluid intake is present and some of the items, the routine analysis urinalysis is intended to check the following
1. The appearance of the urine (colour and the degree of cloudness)
2. The PH (acidity or alkalinity)
3. The specific gravity
4. Test for glucose, ketone bodies and proteins
5. Urine sediments such as white cells and bacteria.
PURPOSE
Abnormal characteristics or constituent of urine can reveal many disease processes. Urine RE is therefore done for the following reasons:
1. To determine the presence of urinary tract infections by elevated WBC count or findings of specific microbes.
2. To detect kidney dysfunction e.g. changes in specific gravity, the protein content and the presence of RBC’s indicates kidney damage.
3. To screen for diabetes mellitus which is positive if sugar and ketone bodies.
4. To determine metabolic functions of the body by changes in PH.
5. To determine hormonal function.
6. To determine liver function dysfunction by the presence of bilirubin.
TIMED SPECIMEN (URINE) OR 24-HOUR SPECIMEN
The purpose of collection of 24hr specimen of urine is for the detection of tubercle bacilli in cases of tuberculosis of the kidney and the urinary tract.
PROCEDURE
Explain the procedure to the patient and the purpose of investigation. Inform him all urine pass from a specific period say 6am in the morning will be collected and stored and the last urine pass at the end of the 24 hours will also be collected and will finish up the collection.
With information the patient can participate in the collection of the timed specimen. . g. He will inform the staff immediately after voiding urine so that it can be added to the already collected. To prevent decomposition of the urine, the container for the collection is kept in a refrigerator and urine added to whenever the patient passes urine. At the end of the 24 hours the whole urine is labeled and sent to the laboratory together with a laboratory request form.
URINE FOR CULTURE AND SENSITIVITY TEST
SPECIMEN OF URINE
This is a sterile specimen and should be collected with aseptic precautions and into sterile containers.
PROCEDURE
Explain the purpose of the specimen to the patient and to prevent the urine from contamination from skin microbes. Also inform him that the specimen is collected during the middle of the action.
Set a tray containing gauges swabs, a cleaning agent such as cetavlon, sterile normal saline, 2 pairs of gloves a sterile specimen bottle, polythene sheet for protecting the bed, a receiver for the used swabs, a urinal or a bedpan.
METHOD
Screen the bed and ensure privacy, wash your hands dry them and put the gloves on. Using the swab and the cetavlon wash the genitals. Pull back the foreskin in an uncircumcised penis and clean the glands penis properly. At the urethra meatus clean the area by circular turns using the swabs once. Then rinse or clean the area with the normal saline. In the females clean from the top vagina, then separate the labiamagora with the index and meddle fingers in and wash the area. Then separate the labia minora and wash the and then clean the vestibule of the vagina from the clitoris down to the perineum using each swab once. Then rinse the area with the normal saline.
After the cleaning, ask the patient to void into a bedpan or a receiver. If he is a man then in the middle of the act use the specimen to collect some of the urine after which the patient continues to void to a completion. Cork the container and the outside with some of the antiseptics lotion.
Label and send the specimen to the laboratory. Discard the material, wash them and put them in their proper places. Make patient comfortable. Wash your hands.
TO COLLECT URINE FOR CS FROM A CHILD
Clean the genital as described already then with a sterile forceps place a layer of gauge over the vulva. Support it with a sterile polythene rubber and tie it with a sterile t-bandage. Do the same for a male infant. After the infant has passed the urine untie the t-bandage and use 2 sterile forceps to squeeze the urine from the gauges into the specimen container. Discard and wash the equipment. Label and send the specimen to the laboratory.
COLLECTION OF STOOL SPECIMEN
Specimen or faeces are sent to laboratory for the following reasons:
1. To detect the presences of occult (hidden) blood, in this case only a small sample of stool is required. For such an examination, the patient abstains from meat, fresh vegetables and all iron drugs for three days before the stool is taken for the occult blood test. This is because, meat, iron drugs, green leafy vegetables will give the same positive readings as blood in the stool. This examination is usually done for the diagnosis of gastric and duodenal ulcer.
2. To analysis for dietary products and digestive secretions. E.g. fat content and bile content
3. To detect the presence of parasites such as amoeba, worms, or eggs or ova of worms. Usually the stools of patients with diarrhoea or dysentery are analysed for parasites. To test for parasites, a specimen needs to be sent to the laboratory while it is still warm. The parasites die when the specimen gets cold.
4. To detect the presence of bacteria or virus. For this purpose only small amount of faeces is collected into sterile container.
PROCEDURE
1. Inform the patient and explain to him/her the purpose of the specimen and what is expected of him/her during the collection of the specimen.
2. Access the faeces for colour, odour, consistency, amount and the presence of abnormal constituent such as blood, mucus or worms. Also determine from the patient whether he/she experiences any discomforts during defaecation or whether he/she feels irritation around the rectal area.
ASSEMBLING THE EQUIPMENT NECESSARY FOR THE PROCEDURE
1.Wash your hands
3. The specimen container with a led or cover or a sterile swab stick in a test are use for taking of stool for culture and sensitivity test or a wooden spatula is used to transfer the stool from the sterile bedpan into the sterile stool container.
4. A sterile bedpan
5. Urinal in case of a male
Let a female patient pass urine into another bedpan before she defaecates. The stool must not be mixed with urine. If the patient is able, instruct him/her to carry out the procedure of collecting stool specimen. If she is unable, then assist her. After the defaecation, transfer the specimen into the container. Clean the patient up as necessary.
Remove the bedpan and empty it. Wash your hands, Label the specimen and arrange for it to be sent to the laboratory.
NB// For specimen of stool for the detection of amoeba, the whole specimen of stool must be sent immediately after the patient has passed the stool because when the temperature of the stool falls the amoeba both motile and non motile or encysted forms cannot be seen.
During the collection of stool specimen, include any unusual portion in the specimen such as pus, streaks of blood, cysts ova of worms, shred of epithelium.
NOSE AND THROAT SWAB
A nose and ear specimen is collected from the mucosa of the throat or nose and is then culture and examine for the presence of pathogens. It is a sterile specimen and they are commonly ordered in streptococcal sore throat and in tonsillitis and in chronic cases of nasal catarrh. It is also ordered to evaluate the effectiveness of therapy especially when a patient is on an antibiotic for the treatment of throat or nasal conditions.
PROCEDURE
Inform the patient and explain the procedure to him so that he will cooperate during the process.
EQUIPMENTS: Wash your hands before assembling the following equipments needed for the procedure.
1. 2 sterile swab sticks in test tube
2. A tongue depressor to depress the tongue so that the pharynx can be seen
3. A head light for illumination of the throat. In the case of the nose have an otoscope with a nasal speculum to dilate the nostrils.
4. A receiver to contain the used instruments.
Instruct the patient to open his mouth and pull the tongue forward in order to expose the throat. If this is difficult depress the tongue with a tongue depressor and ask the patient to say “ ah!” to express the pharynx then use the swab mounted stick to double some of the secretion from the tonsil and pharyngeal mucosa taking care not to allow the swab to touch any part of the mouth. Draw the stick back and gently insert it into the test tube and cork.
For the nose, gentle insert a lighted nasal speculum up one nostril and using the swab-mounted stick, touch any redden area of the nasal mucosa or double the swab in any exudates present in the nose. Take care that the swab does not touch any other part or edges of the nose. Label and sent the specimen to the laboratory.
Remove and wash equipment end place them in their proper places. Make patient comfortable and wash your hands. Indicate in the nursing notes that the specimen has been taken.
OBTAINING SPECIMEN FROM WOUND DISCHARGE (WOUND SWAB)
PURPOSE: Specimen may be taken from a draining or discharging wounds to identify microbes that are causing the infection and to determine the drugs that will be sensitive. Usually, the specimen is taken when the wound is opened or about to be dressed.
ASSESSMENT OF THE WOUND
Assess the appearance of the wound and the discharge. Infection can cause redden tissue with a thick discharge which may be foul smelling, whitish or coloured. Determine the amount of discharge whether scanty or copious. Also determine the kind of discharge, e.g. whether pus, serous, sanguineous.
Also ask the patient whether the wound is painful. A patient with an infected wound may complain of burning pain. Also assess the patient for fever, chills thirst etc. this indicates the presence of infection. Report your findings
EQUIPMENT AND PROCEDURE
If the specimen is to be taken during the dressing of the patient’s wound, then the dresser prepares a dressing trolley, and then after a surgical hand wash she takes the specimen. During the process of taking the specimen let the swab absorb as much drainage as possible. Take the specimen from the part of the wound that has unusual discharge. Insert the swab back into the container, label and sent to the laboratory
If the swab is taken from a wound, which is dressed, then inform the patient about the procedure. Ensure privacy, wash hands get a few dressing and forceps for opening the wound. Obtain the specimen in the usual way after the dressing has been removed. Cover the wound with a sterile dressing or dress it completely.
OBTAINING A SPECIMEN OF URETHRAL DISCHARGE AND VAGINAL DISCHARGE
Some discharge from fro m the vagina or urethra is normal. Whether in new borns, children or adults. However, discharges that are abnormal either in amount or type are frequently examined in the laboratory to identify the presence of pathogenic microbes in the urethral or vaginal discharges.
The specimen is to be taken by nurses and they need to known the differences between abnormal and normal urethral discharge.
The normal vaginal discharge is usually minimal in quantity; it is non-purulent, whitish or clear and contains no blood.
PROCEDURE
Inform and explain the procedure to the client and gain his acceptance before under taking the procedure. This is very important because you are invading the person’s privacy.
EQUIPMENT
Wash your hands and get the following equipment ready. A sterile specimen swab in a sterile container. A pair of gloves, this is necessary for holding the penis or for the opening of the vulva so that the microbes of the patients don’t infect you and also it prevents transferring your own microbes to the patient.
URETHRAL DISCHARGE FROM THE FEMALE.
Separate the labia majora with one hand and pull the labia monora back upward from the sides to expose the urethral orifice. Using the other hand to hold the swab stick, touch the end of the sterile swab against the discharge, taking care that you don’t touch the skin or mucus membrane around.
VAGINAL DISCHARGE
Touch the end of the sterile swab against the discharge in the vagina. The specimen should be taken from as high as the vagina vault. To enable you reach and see the vault, use a vaginal speculum to separate the vaginal walls. If trichomonas vaginalis is suspected, take an additional swab and send the additional swab in normal saline.
URETHRAL DISCHARGE FROM MALE.
To obtain a specimen of urethral discharge from a male, hold the penis near the end. Retract the foreskin or prepuce in the case of an uncircumcised penis. Using the other hand to hold the specimen swab touch the end of the sterile swab against the urethral discharge. Replace the swab inside its container and cover it.
Make patient comfortable, discard equipment, wash and replace them in their proper areas. Wash hands. Label and send specimen to the Laboratory. Indicate in the nurse’s note that the specimen has been taken.
MICROBIAL INFECTION AND THE BODY’S DEFENCES
HOST-AGENT ENVIRONMENT INTERACTION (THE TRILOGY RELATION)
Microbiologists understand that disease results from complex relations among casual agent, susceptible host and environmental factors. The interaction among these three elements.
- Agent
Host and
Environment is called epidemiologic triangle or trilogy relation.
A change in one of these elements of the triangle can influence the occurrence of disease by increasing or decreasing a person’s risk for disease. The agent and the host, as well as their interaction are influenced by the environmental context in which they exist, while they also influence the environment.
Agent
Environment Host
The trilogy relation
1. THE AGENT
Agents are divided into infectious agents e.g. bacteria, fungi, and viruses etc. For an infectious agent to be able to cause disease it should be able to emerge from its habitant, reaches a new host, infect the host and multiply.
Reservoir of infections agents
A source is the location from which the pathogen is immediately transmitted to the host. The source can either be animate (e.g. humans or animals) or inanimate (e.g. water, soil or food)
ANIMATE SOURCE
Human sources serve as carries. A carrier is an effected individual who is a potential source of infection to other. Four types of carrier are recognized
- A healthy carrier- an individual who habour the pathogen but is not ill
- Convalescent carrier-an individual who has recovered from an infectious disease but continue to labour the pathogens and capable of infection others.
-Active Carrier- an individual who has an overt disease and therefore capable of infecting others
-Incubatory Carrier- an individual who is incubating pathogens is large numbers but not sick and capable of infecting others.
ii) Other Animals – Some infective agents which affect man have their reservoir in animals Infectious diseases of vertebrate animals which can be transmitted to man under natural conditions are called zoonsis e.g. rabies, viral encephalitis, plague, salmonelosis
This group also includes pathogenic microbes.
INANIMATE SOURCE
These include
(i) Chemical agent – heavy metals, toxic chemicals, pesticides
(ii) Physical agents – radiation, heat, cold, machine
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