Microbiology for nurses

 MICROBIOLOGY
 COURSE DESCRIPTION
 This course introduces the learner to the basic concepts of microbiology. The course covers the major groups of micro-organisms, describes their size, morphology, structure and biological adaptations.

 Knowledge of the relation of microbiology to health and disease provides the student with essential principles of the prevention of spreading infections and the protection of patients in hospital, families and communities
 RELATION OF COURSE TO THE PROFILE OF THE REGISTERD NURSE
The role of the health care promoter, health care giver and health care facilitator, the nurse will use the principles of microbiology in all areas of care.
 COURSE OBJECTIVES
 By the end of this course the students should be able to:
1.describe the basic concepts of microbiology and its relation to health and diseases.
2. apply principles of microbiology for the protection of self, the patient and the community.
3. plan and give health teaching based on the principles o f microbiology.
 METHOD OF TEACHING AND LEARNING
 Lecture/discussions
 Demonstrations
 Large and small group work
 Study visit of laboratories
 METHOD OF EVALUATION
 Final examination 100%
 REFERENCES FOR STUDENT NURSES
 Ananthanarayan, R,& Jayaram Panukar,C.H. (latest edition). Text book of microbiology. Orient Longman
 Bryman, N. & Stafford,C.(Latest edition).An atlas of medical microbiology common human pathogens. London: Blackwell Scientific publications.

 Collee, J.G. (latest edition). Applied medical microbiology. London: Blackwell Scientific publications.
 Guduid, J.P.,Marmion, B.P., & Swain, R.H.A. (latest edition). Medical microbiology. English language book society.

 Gwendolyn, R.& Burton, R.W. (latest edition). Microbiology for health science, New York: J.B. Lippincott
 Parker, D.C., Porter, J.A., Duerden, B.I.& Reid, T.M.S (latest edition). Short textbook of medical microbiology. English language Book society.

 Number of hours: 60 hours
 Teaching personnel: tutors, Microbiologist
 CONTENT TOPICS
 Unit I-  introduction
 Unit II- laboratory study of microbiology
 Unit III- beneficial activities of non- pathogenic micro-organisms
 Unit-IV- preventive aspects of disease in relation to microbiology
 Unit V- infection, immunity and allergy
 Unit VI- pathogenic micro-organisms
 Unit I- introduction
 Learning objectives
 By the end of this unit, the students should be able to:
1.Define microbiology and its relationship to nursing.
2.Identify the important discoveries in microbiology.

3. Describe the major group of the micro-organisms.
4. Explain the factors related to growth of  micro-organisms.

 Definition of microbiology
 Micro – small
 Bio    - life
 Logy - science
 The science of microorganisms
 Microbiology relation to nursing
 Knowledge of microbiology is indispensably linked with nursing practice. It is one of the fundamental bases of knowledge that governs how every nurse interacts with patients in many settings.

 . Nurses apply knowledge of microbiology in methods of infection control, keeping instruments clean and free of contamination, in how to dress wounds safely in a way that minimizes possibility of infection, and in recognizing types of infections.


 Wound Care

 Microorganisms are the means by which disease spreads in open wounds. Antibiotic administration is not a substitute for properly cleansing wounds. Wound cleansing, then, is a duty of significant importance for emergency room and other nurses.
 Wound irrigation, cleansing the wound with water, can be accomplished in two ways, through low pressure and high pressure. Nurses need to know which syringes generate high and low pressure and use the accomplished syringe. Knowledge of microbiology forms the foundation of this application, because the cleansing of the wound thoroughly extends beyond what the human eye can see and helps nurses to understand why high pressure irrigation is of importance.


 Aseptic technique

 The Encyclopedia of Surgery describes aseptic technique as, "a set of specific practices and procedures performed under carefully controlled conditions with the goal of minimizing contamination by pathogens." Pathogens are micro-bacterial contaminants that can cause disease and infection.
 The goal of the nurse in microbiology is to employ techniques that minimize contamination and keep aseptic all medical equipment in a clinical setting. Cleaning, sanitizing and disinfecting are a part of those duties. The Center for Disease Control estimates that there are 27 million surgical procedures every year.


 Surgical site infections account for longer hospital stays and increased costs. The operating room is the place where aseptic technique must be applied most stringently. Proper hand-washing, known as a "surgical scrub," is an essential psychomotor skill that nurses need to acquire in their duties.


 Biomedical Waste

 Properly disposing of biomedical waste is an important duty. Certain types of liquid waste, such as that generated from hand-washing, cleaning and housekeeping activities, are disposed of through hospital drainage systems. Other types of waste however, must be handled with microbiological infection in mind, using proper procedures.
 Wastes, such as items contaminated with blood or body fluids including cotton, dressings, soiled plaster casts, linens and bedding, as well as catheters and intravenous sets, are disposed of properly in color-coded packaging. This narrows opportunity for the spread of disease.

 Gram positive and negative testing

 Another duty in microbiology and nursing is administering smears for gram positive and negative testing. These are then analyzed to determine types of bacterial contamination. Specimens are taken with a wood applicator across a prepared slide. The smear needs to be thin, and the sample properly labeled, dried and cooled. It is then ready for staining.

 History of microbiology
 Disease and death have always fascinated humans, early time, people believed that disease was caused by supernatural forces and divine wrath. Later, it was believed that the environment, bodily constitution (for example, weight and height) and diet contributed to disease.
 In the first century BC, Varo and Columella suggested that diseases were caused by inhaled or ingested invisible beings and Von plenciz (1762) suggested that each disease was caused by a separate agent.
 As microbes are not visible to the unaided eye, knowledge about them had to await the development of microscope. Microbes were firs observed in 1675 by Antonie van Leeuwenhoek in Holland, whose hobby was grinding glass to make lenses and observing diverse material through them.
 In 1683, his accurate description of ‘little animalcules’ were presented to the Royal society of London, but the significance of these observations was not realized at the time.
 The debate on the origin of organisms ended with a series of classic experiments by the French chemist, Louis Pasteur (1822-1895), who proved that all forms of life arose from their like and not spontaneously, further, his studies on fermentation of wine established that fermentation was the result of microbial activity and that different type of fermentation were associated with different type of microorganisms.
 In the course of these studies, he introduced techniques of sterilization and developed the steam sterilizer, hot air oven and autoclave. Pasteur also established the differing growth needs of bacteria and studied the causes of anthrax, chicken cholera and rabies
 An accidental observation was made by Pasteur that chicken cholera bacillus culture left on the bench for several weeks lost their ability to cause disease, but could still protect the birds against subsequent infection by them. This is the process of attenuation and its discovery led to the development of live, attenuated vaccines (1881)
 Prior to this discovery, it had been noticed that persons who survived an attack of smallpox did not develop the disease when exposed to the infection again. thus, a mild form of smallpox was produced intentionally to prevent the disease from occurring (variolation).
 Edward Jenner (1796) observed that milkmaid who had suffered from cowpox infection were protected when they came in contact with smallpox. Thus, he introduced the technique of vaccination, where a similar but attenuated organism is given to individual, and coined the word vaccination (from vacca meaning cow)
 One of Pasteur’s greatest contributions was the development of a vaccine for rabies. For his many contributions to the field of microbiology, he is the father of the microbiology.
 An immediate application of Pasteur’s work was he introduction of antiseptic techniques in surgery by Joseph Lister(1867), a Scottish surgeon. Lister suggested that microbes caused surgical wound infections which could be prevented by “antisepsis”, whereby he sprayed the patients and operating field with carbolic acid.
 This resulted in a drop in the number of infections and deaths. Though a hazardous procedure, it was effective and was a milestone in surgical practice.
 While Pasteur in France laid the foundation of microbiology, Robert Koch (1843-1910) in Germany perfected bacteriological techniques during his studies on the culture and life cycle of the anthrax bacillus (1876).
 He introduced staining techniques, method of obtaining bacteria in pure culture using solid media and discovered the bacillus of tuberculosis (1882) and the cholera vibrio (1883)
 Golden era of microbiology
 1874 Hansen described the leprosy bacillus
 1879 Neisser described the gonococcus
 1881 Ogston discovered the staphylococci
 1884 Loeffler isolated the diphtheria bacillus
 1884 Nicolaier observed the tetanus bacillus
 1886 Fraenkel described the pnumococcus
 1887 Bruce identified the causative agent of Malta fever
 1905 Schaudinn and Hoffmann discovered the spirochaete of syphlis
 One ounce of practical better than one ton of theory – thank you
 CLASSIFICATIONS OF MICROORGANISM
 From the beginning of scientific study, organisms have been sorted and classified.

     1. Ancient Greeks on to 1700’s---2 kingdoms, plant and animal, not many groups besides this

2. 1735---Linnaeus also used 2 kingdom system, but established other groups and classified most known organisms into all his groups. Microorganisms didn’t get a clear place in this system, but their importance was not recognized at this time. Microbes including bacteria were placed in the plant kingdom.
 3. In 1866, Haeckel proposed adding the kingdom Protista for all microorganisms. Some accepted this idea but others continued to place bacteria in the plant kingdom.
4. Beginning in late 1930’s---the next step was the development of the electron microscope, which allowed detailed study of the tiny structures within cells for the first time.
 It was then realized that there were 2 basic types of cells---the eukaryotic cells with a distinct, membrane-bound nucleus; and the prokaryotic
 and the prokaryotic cells (bacteria all go in this group) which did not have this separate nucleus.  Although it did not happen immediately, it was realized that these cells were so different that a new kingdom was needed.

 5.  1969---Whittaker’s Five-Kingdom system was proposed and became widely accepted.
          a. Kingdom Monera or Prokaryotae---all prokaryocytes, which means all bacteria
          b. Kingdom Protista---unicellular eukaryocytes---protozoa, unicellular algae, slime molds
        
          c. Kingdom Fungi---unicellular yeasts, multicellular (but still microscopic)  molds, macroscopic mushrooms. Plantlike but no chlorophyll, so no photosynthesis.
          d. Kingdom Plantae---plants. All multicellular and all carry on photosynthesis.
          e. Kingdom Animalia---animals
 BACTERIA
 Bacteria are prokaryotic microorganisms that do not contain chlorophyll. they are unicellular and do not show true branching.
 Size of bacteria
 The unit of measurement used in bacteriology is the micron (micrometer,   )
 1micron ( ) or micrometre  (m )= one thousandth of a millimetre
 1 millimicron (m ) or nanometre (nm) = one thousandth of a micron or one millionth of a milimetre
 1 angstrom unit ( Å) = one tenth of a nanometre
 The limit of resolution with the unaided eye is about 200 microns, bacteria, being much smaller, can be visualized only under magnification. Bacteria of medical importance generally measure 0.2-1.5 mm in diameter and about 3-5 mm in length.
 Shape of bacteria
 Bacteria are classified according  to their shape
1. cocci (from kokkos meaning berry) are spherical or oval cell.
2. bacilli (from baculus meaning rod) are rod- shaped cells.

3. Vibrios are comma-shaped, curved rods and derive their name from their characteristic vibratory motility.
4. Spirilla are rigid spiral forms.
5. Spirochaetes (from speira meaning coil and chaite meaning hair) are flexuous spiral forms.
 6. actinomycetes are branching filamentous bacteria, so called because of a resemblance to the radiating rays of the sun when seen in tissue lesions (from actis   meaning ray and mykes meaning fungus)
7. Mycoplasmas are bacteria that do not have a cell wall and hence do not possess a fixed shape.They occur as round or oval bodies and as interlacing filaments.
 Bacteria sometimes show characteristic cellular arrangement or grouping. Thus, cocci may be arranged in pairs (diplococci), chains (streptococci), group of four (tetrads) or eight (sarcina), or as grape like clusters ( staphylococci). Some bacilli are arranged at angles to each other, presenting a Chinese letter pattern (corynebacteria)
 bacteria

 Bacterial structure and functions
 The outer layer of the cell envelop consists of two components- a rigid cell wall and beneath it a cytoplasmic or plasma membrane. The cell envelop encloses the protoplasm, comprising the  cytoplasm, cytoplasmic inckusions such as ribosomes, granules, vacuoles and the nuclear body.
 The cell may be enclosed in a sticky layer, which may be a loose slime layer or organized as a capsule. Some bacteria carry filamentous appendages protruding from the cell surface the flagella, which are organs of locomotion, and the fimbriae, which appear to be organs for attachment.
 Cell wall
 The cell wall gives the bacterial cell its shape and also makes it rigid. The cell wall cannot be seen y direct light microscopy and does not stain with simple stains. Bacterial cell walls are composed of a mucopeptide framework. The walls of Gram-positve bacteria have a simple chemical nature than those of Gram- negative bacteria.
 Cytoplasmic membrane
 The cytoplasmic membrane is a thin layer lining the inner surface of the cell wall, separating it from the cytoplasm. It acts as a semipermiable membrane, controlling the flow of metabolic compounds to and from the protoplasm. Chemically, the membrane consists of lipoprotein with small amounts of carbohydrates. Sterols are absent, except in mycoplasma
 cytoplasm
 The bacterial cytoplasm is a suspension of and inorganic solutes in a thick watery solution. It differs from the eukaryotic cytoplasm by the absence of endoplasmic reticulum or mitochondria. The cytoplasm stains uniformly with basic dyes in young cultures but becomes increasingly granular with age.
 ribosomes
 Ribosomes are centres of protein synthesis. they are slightly smaller than the ribosome of eukaryotic cells.
 Mesosomes (condroids)
 Mesosomes are seen as vesicular folds within the plasma membrane, protruding into the cytoplasm. They are more prominent in Gram-positive bacteria. They are the principal sites of respiratory enzymes in bacteria and are like the mitochondria of eukaryotes in function.
 nucleus
 Bacterial nuclei may be seen by electron microscopy. They appear as oval or elongated bodies, generally one per cell. Bacterial nuclei have no nuclear membrane or nucleolus.
 flagella
 Motile bacteria, except spirochetes, possess one or more unbranched, long filaments called flagella, which are the organs of locomotion.. Flagella are made up of a protein (flagellin) similar to keratin or myosin,and flagella of different genera are antigeniclly diferent.
 fimbriae
 some Gram-negative bacilli carry very fine hair-like surface structures called fimbriae or pili. They are shorter and thinner than flagella (about 0.5µm long and less than 10 nm thick) and project from the cell surface as straight filaments. They arise from the cell membrane. Fimbriae can be seen only under the electron microscope
 spores
 Some bacteria, particularly members of the genera Bacillus and Clostridium have the ability to form highly resistant resting stages called spores. Sporulation (formation of spores) helps bacteria survive for long periods under unfavorable conditions. Each bacterium forms one spore, which on germination forms a single vegetative cell. sporulation is a not a method of reproduction. As bacterial spores are formed inside the parent cell, they are called endosporses.
 The fully developed spore has at is core the nuclear body, surrounded by the spore wall, a delicate membrane from which the cell wall of the future vegetative bacterium will develop. Outside this is the thick spore cortex, which in turn is enclosed by a multilayered tough sore coat. Some spores have an additional outer covering called exosporium, which may have distinctive ridges and grooves, for example in Bacillus anthracis.
 Bacterial spores may remain viable for several years. They are extremely resistant to drying and relatively resistant to chemicals and heat. Though some spores may resist boiling for prolonged periods, spores of all medically important species are destroyed by autoclaving at 120 c for 1 minutes. Methods of sterilization and disinfection should ensure that spores are destroyed in addition to vegetative cells.
 Growth and multification of bacteria
 Bacteria divide by binary fission. When a bacterial cell reaches a certain size, it divides to form two daughter cells. The cell divides by he inward growth of a septum across the cell ( transverse septum)
 Bacterial nutrition
 For growth and multification of bacteria,  the minimum nutritional requirement are water, a source of carbon, a source of nitrogen and some inorganic salts. Water is the route of enter for all nutrients into the cell as well as the elimination of all waste products. It participates in metabolic reactions and also forms an essential part of the protoplasm.
 Oxygen requirement and metabolism
 Depending on the influence of oxygen on growth and viability, bacteria are divided into aerobes and anaerobes. Aerobic bacteria require oxygen for growth. they may be obligate aerobes such as Vibrio cholera, which will grow only in the presence of oxygen, or facultative anaerobes which are ordinarily aerobic but can also grow in absence of oxygen, though less successfully.
 Carbon dioxide
 All bacteria require small amounts of carbon dioxide for growth. This requirement is usually met by the carbon dioxide present in the atmosphere, or produced within the cell (endogenously) by cellular metabolism. Some bacteria like Brucella abortus require much higher levels of carbon dioxide for growth
 temperature
 Different classes of bacteria grow at different temperature at which growth occurs best is known as the optimum temperature, which in the case of most pathogenic bacteria is 37 c
 Mesophilic bacteria grow best at temperatures of 20-40 C. all pathogens of warm- blooded animals are mesophilic.
 Psychrophilic bacteria grow best a temperatures below 20 C. they are soil and water saprophytes and, though not of direct medical importance. May cause spoilage of refrigerated food.
 Thermophilic bacteria grow best at high temperatures, 55-80 C. many of these are non-pathogenic bacteria that cause spoilage of under processed canned food. some thermophiles form spores that are exceptionally heat-resistant. Extremely therophilic bateria have been identified  which can grow at temperatures as high as 20 C.
 Moisture and drying
 Water is an essential ingredient of bacterial protoplasm, and hence, drying (desiccation) is lethal to cell. Spores are resistant to desiccation and may survive in the dry state for several decades.
 pH
 Each species of bacteria has an optimum pH at which it grows best. The majority of pathogenic bacteria grow best at neutral or slightly alkaline pH ( pH 7.2-7.6) some bacteria such as lactobacilli row best under acidic conditions. Others, such as the Vibrio cholera, are very sensitive to acid but tolerate high degree of alkalinity. strong solution of acid or alkali readily kill most bacteria, though mycobacterium are exceptionally resistant to them
 light
 Bacteria (except the phototrophic species) grow well in the dark. They are sensitive to ultraviolet light and other radiation. Cultures die if exposed to sunlight. Exposure to light may influence pigment production. Some mycobacterium form a pigment only on exposure to light and not when incubated in the dark
 Osmotic effect
 Sudden exposure to hypertonic solution may caudle osmotic withdrawal of water and shrinkage of protoplasm- plasmolysis. This occurs more commonly in Gram- negative than Gram-positive bacteria sudden transfer from a concentrated solution to distilled water may cause swelling and rupture of the cell.
 VIRUSES
 the simplest of microorganisms are cells enclosed within a cell  wall, containing both types of nucleic acid (DNA and RNA),  synthesizing their own constituents and multiplying by binary fission.
 Viruses do not have cellular organization and contain only one type of nucleic acid, either DNA or RNA, but never both. They are obligate intracellular parasites. They are completely dependent on the host cells for replication. They are not affected by antibacterial antibiotics.
 viruses

 Louis Pasteur was the fist to suspect that organisms smaller than bacteria existed, but it was Ivanovsky in 1892 who proved that there were tiny living organisms which could pass through filters that were able to remove all bacteria and that these agents were able to produce infection

 Beijerinck confirmed the same and coined the term virus. Although viruses could not be seen, Goodpasture in 1931, developed technique to cultivate them in chicken eggs, a technique that is still used today. In 1934, Ruska developed the electron microscope, enabling direct visualization of the virus.
 Viral diseases range from minor ailment such as the common cold to terrifying disease such as rabies and AIDS. They may cause sporadic outbreaks, endemic disease or even epidemics and pandemic influenza A that the world has experienced in the recent past
 morphology
 Size-
viruses are much smaller than bacteria. It was their small size and ‘filterability’ for which they were known as ‘filterable viruses’ and as most of them were too small to be seen under the light microscope, they were called ‘ultramicroscopic’  the extracellular infectious virus particle is called the virion
 Structure and shape
the virion consists of nucleic acid surrounded by a protein coat (capsid). The capsid together with the enclosed nucleic acid is known as the nucleocapsid. The function of the capsid is to protect the nucleic acid from agents in the environment and for sticking or absorbing to host cells. The capsid is composed of individual units called capsomeres.
 Chemical properties
viruses contain only one type of nucleic acid, either single or double stranded DNA or RNA. Their capsids are made of protein.
 Resistance
most viruses are killed easily by heat. They are generally inactivated within seconds at 56 C, within minute at 37 C and in days at 4 C. they are stable at low temperatures.










   Viruses are inactivated by sunlight, UV rays and ionizing radiation. Yet, in general, they are more resistant than bacteria to chemicals disinfections, probably because they lack enzymes. Chemicals like hydrogen peroxide, potassium permanganate, iodine and hypochlorite, formaldehyde and beta propiolactone are all able to kill viruses (virucidal) and are therefore used in vcacine preparation. Chlorination of drinking water kills most viruses (except the hepatitis virus and polioviruses)
 Viral multiplication
 The genetic information necessary for viral replication is contained in the viral nucleic acid, but as they lack the enzymes required for multiplication, the virus depends on the synthetic machinery of the host cell for replication.
 The viral multiplication cycle can be divided into six steps
1.absorption, or attachment to the host cell.
2.Penetration which occurs by the engulfment of the viral particle known as ‘viropexis’.
3.Uncoating which allows the nucleic acid to be released and incorporated into he host cell.
4.Biosynthesis whereby enzymes, capsids, nucleic acids are produce.
5.Maturation, this is the assembly of the viral particles.
6.Release whereby most virus progeny/ daughter virions are releases by destruction/ lysis of the host cell.
 Cultivation of viruses
 As viruses are obligate intracellular parasites, they cannot be grown on any artificial culture medium. Three methods are used for the cultivation of viruses;
1.Inoculation into animals
2.Embryonaed eggs
3.Tissue cultures
 DNA viruses
 Poxviridae family- skin infection, smallpox
 Herpeviridae family-herpes, chickenpox
 Hepadnaviridae family-hepatitis B infection
 Adenoviridae family-respiratory tract infections, conjunctivitis and diarrhea.
 Papoviridae family- skin and genital warts
 Parvoviridae-
 RNA viruses
 Picornaviridae-poliomyelitis and the common cold
 Orthomyxoviridae- avian influenza and pandemic influenza A and the seasonal flu.
 Paramyxoviridae- respiratory infection, measles and mumps
 Togaviridae- chikungunya, rubella is also part of this group
 Flaviviidae- dengue and yellow fever. These are called arboviruses.
 Bunyaviridae- this is the largest group of insect born infections
 Rhabdoviridae- rabies
 Reoviridae- contains the rotavirus, the number one cause of diarrhea and diarrhea-related childhood deaths worldwide.
 caliciviridae- outbreaks of diarrhea

 Coronaviridae- SARS
 Retroviridae- HIV
 Filoviridae- Ebola virus, a highly fatal haemorhagic infection
 PROTOZOA
 Parasites are eukaryotic. They may be
 Unicellular, which can only be visualized with a microscope,- entamoeba histolytica
 Multicellular and can be seen with the naked eye,- helminthes such as roundworm
 Parasites are living organisms which are fully depend on another living organisms (host) for survival. They derive the benefit of nutrition and shelter from the host they live and are not capable if independent life.
 Protozoa obtain their nutrients through ingestion, they may be motile or no motile. If they are motile, their motility can be using pseudopodia, cilia and flagella.
 Classification of medically important protozoa
 1. amoebae
 Entamoeba histolytica
 Entamoeba coli
 acanthamoeba culbertsoni
 naegleria fowleri
 Balmuthia mandrillaris
2.Flagellates
Giardia lamblia
Trichomonas vaginalis
Leishmania donovnani
Trypanosoma gambiense
3. Sporozoa
Plasmodium spp
Toxoplasma gondii
4.Ciliates
Balantidium coli
 protozoa

 FUNGI (MYCOLOGY)
 Medical mycology is the study of the fungi that affect human and the diseases that they cause. Candida albicans was first described as early as 1839. fungal infections, however, are extremely common and some of them cause serious illness or even death. Most fungi are found in the soil.
 Modern advances in treatment, such as the widespread use antibiotics, steroid and immunosuppressive agent led to as increase in patients at risk of fungal infections.
 Depending on cell morphology, fungi can divided into two main groups
 1.yeast
 2.moulds
 A third group, the dimorphic fungi, are those that can exit as either yeast or moulds
 Yeast are unicellular fungi which occur as spherical or oval cell and reproduce by simple budding. On culture they form smooth, cream colonies.
 A more complex, multicellular growth pattern used by some fungi is the production of a tubular, thread-like structure called hypha. A tangled mass of hyphae is called the mycelium. Fungi and they reproduce by forming different types of spores.
 Dimorphic fungi can occur a moulds or as yeasts, depending on the temperature of growth. In host tissue or cultures at 37 C they occur as yeasts, while in the environment and in cultures at 22 C they occur in moulds.
 The old classification system of fungi was based mainly on the morphology of the fungi and the spores they produced. it recognized four classes
1.ascomycetes- for spores within a sac or ascus, -yeast and filamentous fungi
2.Basidiomycetes – form spores on a basidium or base.
3.Zygomycete – have aseptate hyphae and produce spores within sac-like structures called sporangia
4.Deuteromcetes – or fungi imperfecti was an artificial group, which included many fungi of medical importance. Recent molecular studies have accepted the ascomycete and basidiomycete classes, but removed the zygomycete and deuteromycete classes and replaced them with smaller group of fungi.
 Mycoses (fungal infections)
 Human fungal infections are classified into three types
 Superficial infections- skin, hair , nails and mucous membrane.
 Subcutaneous mycoses
 Systemic mycoses-organ of the  body, although the lung is usually the first site of infection.
 yeast
 fungal

 UNIT –II LABORAORY STDY OF MICROBIOLGY
 Identified the main part of the microscope.
 Describe procedures of staining.
 Describe animal inoculation.
 MICROSCOPY
 Microscope are require to study the morphology of bacteria. It was Antonie  van Leeuwenhoek who first observed bacteria using hand-ground  lenses over three hundred years ago. Since then microscopes have undergone many changes.
 Types of microscopes
 Optical or light microscope
 Fluorescence microscope
 Phase contrast microscope
 Dark field/dark ground microscope
 Electron microscope
 Optical or light microscope
 Bacteria may be examined under the compound microscope, either in the living state or after fixation and staining. Examination of wet films or ‘hanging drop’ indicate the shape, arrangement, motility and approximate size of the cells. The disadvantage of this type of microscopy is that due to lack of contrast, details cannot be easily seen.
 Components

 All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path, listed here in the order the light travels through them: In addition the vast majority of microscopes have the same 'structural' components:
 Ocular lens (eyepiece) (1)
 Objective turret or Revolver or Revolving nose piece (to hold multiple objective lenses) (2)
 Objective (3)
 Focus wheel to move the stage (4 – coarse adjustment, 5 – fine adjustment)
 Frame (6)
 Light source, a light or a mirror (7)
 Diaphragm and condenser lens (8)
 Stage (to hold the sample) (9)
 Fluorescence microscope
 fluorescence microscopy makes use of fluorescence labels, which when illuminated by the correct wavelength of light, appear florescent. This property can be used to highlight features of microbes and makes this technique easier for the microscopist.
 Phase contrast microscope
 This improves the contrast and makes the structures within the cells that differ in thickness or refractive index clearer. In addition, the differences in refractive indices between bacterial cells and the surrounding medium make them clearly visible.
 the rays light that pass through the object are inhibited compared to those that pass through the surrounding medium, producing ‘phase’ differences between the two types of rays. Thus, ‘phase’ differences are converted into differences in intensity of light, producing light and dark contrast in image. This type of microscopy is most useful for unstained preparation
 Dark field/dark ground microscope
 Another method of improving the contras is the dark field (dark ground) microscope in which reflected light is used instead of the transmitted light of the ordinary microscope. The contrast gives an illusion of increased resolution, so that very slender organisms such as spirochetes can be clearly seen against a dark background.
 Dark field microscopy is very useful for detecting the darting motility of vibrio cholerae from stool samples, where it is seen as a ‘shooting star’ against the dark background.
 Electron microscope
 In the electron microscope, a beam of electrons is used instead of the beam of light used in the optical microscope. the electron beam is focused by circular electromagnets, which correspond to the lenses in he light microscope. The object which in the path of the beam scatters the electrons and produces an image which is  focused on a fluorescent viewing screen.
 STAINING METHODS
 in preparation of live bacteria, it is not possible to see much structural detail under the light microscope due o lack of contrast. Hence, It is customary to use staining techniques to produce colour contrast. Bacteria have an affinity to basic dyes due to the acidic nature of their protoplasm.
 methods of staining techniques
 Simple stains
 Negative staining
 Impregnation methods
 Differential stains- (i) gram stain
       (ii)acid fast stain
 Simple stains
 Dyes such as methylene blue or basic fuchsin are used for simple staining. They provide colour contrast, but result in all bacteria having the same colour.
 Negative staining
 Bacteria are mixed with dyes such as India ink or nigrosin that provide a uniformly coloured background against which the unstained bacteria stand out in contrast. This is particularly useful in the demonstration of capsules which do not take up simple satins.
 Impregnation methods
 Cells and structures too thin to be see under the ordinary microscope may be made visible if they are thickened by impregnation of silver on the surface. Such methods are used for the demonstration of spirochetes and bacterial flagella.
 Differential stains
 These stains give different colours to different bacteria or bacterial structure. The two most widely used differential stains are the Gram stain and the acid fast stain.
 The gram stain was originally devised by the histologist Hans Christian Gram (1884) as a method of staining
 Gram staining technique
 Primary staining with crystal violet, methyl violet or gentian violet.
 Application of a dilute solution of iodine.
 Decolourisation with an organic solvent such as ethanol, acetone or aniline.
 Counterstaining with a dye of contrasting colour, such as carbol fuchsin, safranine or neutral red
 The Gram stain differentiates bacteria into two broad groups: gram- positive which are those that resist decolourisation, appearing violet; and Gram- negative bacteria which are decolourised by organic solvents and therefore take up the counter stain, appearing red.
 Gram staining is an essential procedure used in he identification of bacteria. Gram reactivity is of considerable importance as Gram-positive and Gram-negative bacteria differ not only in staining characteristics and in structure but also in several other properties such as growth requirements, susceptibility to antibiotics and pathogenicity.
 Acid fast stain or Ziehl-Neelsen stain
 Ehrlich discovered the acid fast stain in which mycobacterium tuberculosis after being stained with aniline dyes resisted decolourisation with acids. The method, as modified by Ziehl and Neelsen, is in common use now.
1. The smear is stained with a solution of carbol fuchsin with the application of heat.
2. It is then decolourised with 20% sulphuric acid.
3. lastly, It is counterstained with a contrasting dye such as methylene blue. The acid fast bacteria retain the fuchsin (red) colour, while he others take counterstain.
 Albert’s stain
 This stain is used to demonstrate the metachromatic granule of corynebacterium diphtheriae. Using this stain, the granules appear bluish black, whereas the body of the bacilli appears green or bluish green.
 Types and preparation o culture media
 In order to identify and study bacteria, they have to be grown or ‘cultured’ in special material called media. Over the years, numerous culture media have been devised. The original media used by Louis Pasteur were liquids such as urine or meat broth.
 Liquid media are useful for obtaining large quantities of bacterial growth. This may be required for preparing antigens or vaccines. Bacteria grow in liquid medium suspended throughout the liquid. However, it is difficult to isolate different types of bacteria occurring in mixed populations in liquid media.
 On solid media, bacteria produce visible growth called colonies, which are all identical to the single bacterial cell from which they originated.  These colonies have a typical morphology and may exhibits other characteristic features, such as pigmentation. The earliest solid medium was cooked cut potato used by Robert Koch.
 Later, he introduced gelatin to solidify liquid media. But it was not satisfactory, as gelatin is liquefied at 24 C and also be many proteolytic bacteria. The use of agar to solidify culture media was suggested by Frau Hess, the wife of one of the  investigators in Koch’s laboratory, who had seen her mother using agar to make jelly.

 Agar obtained from seaweed is now universally used to prepare solid media. Its constituents are a long chain polysaccharide , inorganic salts and small quantities of a protein- like substance. Its unique property is that it melts at 98 C and usually sets at 42 C depending on agar concentration. Approximately 2% agar is used for solid media.
 Peptone is another ingredient of common media. it is composed of a mixture of partially digested proteins, a variety of inorganic salt and growth factors
 Types of media
 Media have been classified in many ways
 Solid media, liquid media, semisolid media
 Simple media, complex media, synthetic or defined media, semi defined media. Special media. special media are further classified into enriched media, enrichment media, selective media, indicator or differential media, sugar media and transport media
 Aerobic media, anaerobic media
 Simple media (basal media)
 A simple medium like nutrient broth consists of peptone, meat extract, sodium chloride and water. Nutrient agar, made by adding 2% agar to nutrient broth, is the simplest and most common medium in routine diagnostic laboratories
 Complex media
 These media have additional special ingredients that are required for the growth of or for bringing out certain characteristics of certain bacteria
 Synthetic or defined media
 These media are prepared from pure chemical substances and exact composition of the medium is known.
 Enriched media
 In these media, substances such as blood, serum or egg are added to a basal medium. They are used to grow bacteria which are more fastidious (demanding) in their nutritional needs. Examples are blood agar, chocolate agar and egg containing media. Enriched media are always solid media.
 Enrichment media
 Liquid media into which certain substances are assed which inhibit the growth of some bacteria and selectively stimulate the growth of the others are called enrichment media. For example, in stool cultures the non- pathogenic or commensal bacteria, will normally overgrow Vibrio cholerae. However, if alkaline peptone water is used for the culture, this inhibits the commensal bacterial and allow the Vibio cholerae to grow.
 selective media
 This select out the organism of interest by adding substances which inhibits other bacteria, for example, thiosulphate citrate bile salt sucrose agar (TCBS)  medium for Vibrio cholerae. Such solid media are known as selective media.
 Indicator media
 these media contain an indicator that changes colour when a bacterium grows on them. An example is the incorporation of sucrose in TCBS medium. Vibrio cholerae ferments sucrose and so appears as yellow colonies on the medium.
 Differential media
 A medium that has special substances added to it which help to bring out differing characteristic of bacteria and thus help to distinguish between them is called a differential medium. For example, MacCoonkey’s medium which shows up lactose fermenters as pink colonies, while nonlactose fermenters are colourless or pale. This may also be termed an indicator medium.
 sugar media
 The term ‘sugar’ in microbiology denotes any fermentable substance. They may be:
 Monosaccharides –arabinose, dextrose
 Disaccharides – saccharose, lactose
 Polysaccharides – starch, inulin
 Trisacchrides – raffinose
 Alcohols – glycerol, sorbitol
 Glucosides-  salicin, esculin

 Transport media
 Transport medium is used when specimen might contain delicate organisms (for example, Neisseria gonorrhea) which may not survive the time taken for transport to the laboratory. Secondly, transport medium can be used when non- pathogens may be overgrown by the pathogen ( such as the Shigella species, which cause bacillary dysentery, being overgrown by commensals in stool samples). These are termed transport media
 Anaerobic media
 These media are used to grow anaerobic organisms, for example, Robertson’s cooked meat medium and thioglycollate medium.
 Animal inoculation
 inoculation /in•oc•u•la•tion/ (-ok″u-la´shun) introduction of microorganisms, infective material, serum, or other substances into tissues of living organisms, or culture media; introduction of a disease agent into a healthy individual to produce a mild form of the disease followed by immunity.
 medical term) the introduction of a substance (inoculum) into the body to produce or to increase immunity to the disease or condition associated with the substance. It is performed by making multiple scratches in the skin after placement of a drop of the substance on the skin, by puncture of the skin with an implement bearing multiple short tines, or by intradermal, subcutaneous, or intramuscular injection. Introduction can also be intranasal or oral. -inoculate, v.
 Unit iii
 By the end of this unit the student should be able to identify the beneficial activities of non- pathogenic micro organisms
 Purification, formulation and decay
 Purification of sewage and water
 Production of food and plants
 Uses of micro-organisms in food, industry and in the healthy human body.
 Formulation

 Formulation is a term used in various senses in various applications, both the material and the abstract or formal. Its fundamental meaning is the putting together of components in appropriate relationships or structures, according to a formula. It might help to reflect that etymologically Formula is the diminutive of the Latin Forma, meaning shape.
 Material applications

 in more material senses the concept of formulation appears in the physical sciences, such as physics, chemistry, and biology. It also is ubiquitous in industry, engineering and medicine, especially pharmaceutics.
 Gram stain

 Negative stain

 Positive stain

 Endospore stain

 Capsule stain











 Water purification

 Water purification is the process of removing undesirable chemicals, biological contaminants, suspended solids and gases from contaminated water.
 The goal is to produce water fit for a specific purpose. Most water is purified for human consumption (drinking water) but water purification may also be designed for a variety of other purposes, including meeting the requirements of medical, pharmacology, chemical and industrial applications.
 In general the methods used include physical processes such as filtration, sedimentation, and distillation, biological processes such as slow sand filters or activated sludge, chemical processes such as flocculation and chlorination and the use of electromagnetic radiation such as ultraviolet light.
 The purification process of water may reduce the concentration of particulate matter including suspended particles, parasites, bacteria, algae, viruses, fungi; and a range of dissolved and particulate material derived from the surfaces that water may have made contact with after falling as rain.