PART - I GENERAL MICROBIOLOGY

1. Write a note on Louis Pasteur.

Louis Pasteur was born on December 27, 1822 in Dole, in France. He is called as Father of Microbiology. His discovery that most infectious diseases are caused by germs, known as the "germ theory of disease," is one of the most important in medical history. He disproved the theory of spontaneous generation. He proved that the process of fermentation is due to microorganisms. He was the inventor of the process of pasteurisation He developed vaccines for several diseases including rabies. The discovery of the vaccine for rabies led to the founding of the Pasteur Institute in Paris in 1888.

2. Write a short note on Robert Koch.

Robert Koch is a German Physician (born on 1843) well known to the world of microbiology for his significant contributions. He introduced aniline dyes for staining bacteria. Used agar-agar and gelatin to prepare solid culture media. Stressed the need for pure culture to study microbes in details Confirmed germ theory of disease. Laid down Koch's postulates to test the pathogenecity of causative agents. He also discovered the causal organisms of anthrax disease of cattle (Bacillus anthracis) and tuberculosis (Mycobacterium tuberculosis). He was awarded the Nobel Prize in Physiology or Medicine for his tuberculosis findings in 1905

3. Write the Kochs postulate.

In 1890 the German physician and bacteriologist Robert Koch set out criteria for finding whether a given bacteria is the causative agent of a given disease. It is called as Koch's postulate. Koch's postulates are as follows:

The bacteria must be present in every case of the disease.

Page 1

The bacteria must be isolated from the host with the disease and grown in pure culture. The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host. The bacteria must be recoverable from the experimentally infected host.

4. Write a detailed note on anatomy of bacterial cell.

Bacterial cell structure Bacterium is a prokaryote and contains a well developed cell structure which is responsible for many of their unique biological properties. Cell morphology Bacteria come in a wide variety of shapes. They are:

coccus (spherical) bacillus (rod-like) spirillum (spiral) filamentous

Cell shape is generally characteristic of a given bacterial species, but can vary depending on growth conditions. Cell size The most obvious structural characteristic of bacteria is their small size. They are microscopic in nature. For example, Escherichia coli cells, an "average" sized bacterium, are about 2 micrometres (m) long and 0.5 m in diameter. The bacterial cell wall The vast majority of bacteria have a cell wall containing a special polymer called peptidoglycan. The cell wall lies outside the cell membrane, and the rigid peptidoglycan is important in defining the shape of the cell, and giving the cell mechanical strength. The structure of peptidoglycan Peptidoglycan, also called murein, is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers. A peptidoglycan monomer consists of two joined amino sugars, Nacetylglucosamine (NAG) and N-acetylmuramic acid (NAM) is responsible for the

Page 2

rigidity of the bacterial cell wall and for the determination of cell shape. The Gram positive cell wall The Gram positive cell wall is characterized by the presence of a very thick peptidoglycan layer. Embedded in the Gram positive cell wall are polyalcohols called teichoic acids. They are responsible for linking the peptidoglycan to the cytoplasmic membrane. Teichoic acids give the Gram positive cell wall an overall negative charge due to the presence of phosphodiester bonds between teichoic acid monomers. The Gram negative cell wall Unlike the Gram positive cell wall, the Gram negative cell wall contains a thin peptidoglycan layer adjacent to the cytoplasmic membrane. In addition to the peptidoglycan layer, the Gram negative cell wall also contains an outer membrane composed by phospholipids and lipopolysaccharides, which face into the external environment. As the lipopolysaccharides are highlycharged, the Gram negative cell wall has an overall negative charge. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains.

The bacterial cytoplasmic membrane The bacterial cytoplasmic membrane is composed of a phospholipid bilayer and thus has all of the general functions of a cell membrane such as acting as a permeability barrier for most molecules and serving as the location for the transport of molecules into the cell. Other bacterial surface structures Fimbrae and Pili Fimbrae are protein tubes that extend out from the cell surface of the bacteria. They are generally short in length and present in high numbers about the entire bacterial cell surface. Fimbrae usually function to facilitate the attachment of a bacterium to a surface (e.g. to form a biofilm) or to other cells (e.g. animal cells during pathogenesis). Sex pili are similar in structure to fimbrae but are much longer and present on the bacterial cell in low numbers. Sex pili are involved in the process of bacterial conjugation. Non-sex pili also aid bacteria in gripping surfaces.

Page 3

Capsules and Slime Layers Many bacteria secrete extracellular polymers outside of their cell walls. These polymers are usually composed of polysaccharides and sometimes protein. They are structures that help to protect bacteria from phagocytosis and desiccation. Slime layers involved in attachment of bacteria to other cells or inanimate surfaces to form biofilms. Slime layers can also be used as a food reserve for the cell. Flagella Perhaps the most recognizable extracellular bacterial cell structures are flagella. Flagella are whip-like structures protruding from the bacterial cell wall and are responsible for bacterial motility (i.e. movement). The arrangement of flagella about the bacterial cell is unique to the species observed. Common forms include as follows:

A-Monotrichous; B-Lophotrichous; C-Amphitrichous; D-Peritrichous;

Intracellular bacterial cell structures The bacterium is a typical prokaryote and contains the features of it as follows: The bacterial chromosome and plasmids Unlike eukaryotes, the bacterial chromosome is not enclosed inside of a membranebound nucleus but instead resides inside the bacterial cytoplasm in the form of a structure called as nucleoid. Along with chromosomal DNA, most bacteria also contain small independent pieces of DNA called plasmids. Ribosomes and other multiprotein complexes In most bacteria the most numerous intracellular structure is the ribosome, the site
Page 4

of protein synthesis in all living organisms. All bacteria have 70S (where S=Svedberg units) ribosomes which is made up of a 50S and 30S subunits.

Bacterial cell structure

5. Write a short account on bacterial cell wall.

The vast majority of bacteria have a cell wall containing a special polymer called peptidoglycan. The cell wall lies outside the cell membrane and the rigid peptidoglycan is important in defining the shape of the cell, and giving the cell mechanical strength. The Gram-Positive Cell Wall The gram-positive bacteria are those that retain the initial dye crystal violet during the Gram stain procedure and appear purple when observed through the microscope. Common gram-positive bacteria of medical importance include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus

Page 5

faecalis, and Clostridium species.

Structure and Composition of the Gram-Positive Cell Wall In electron micrographs, the gram-positive cell wall appears as a broad, dense wall 20-80 nm thick and consisting of numerous interconnecting layers of peptidoglycan. Chemically, 60 to 90% of the gram-positive cell wall is peptidoglycan. Interwoven in the cell wall of gram-positive are teichoic acids and lipoteichoic acids. Teichoic acids extend through and beyond the rest of the cell wall.

Gram positive cell wall The outer surface of the peptidoglycan is studded with surface proteins that differ with the strain and species of the bacterium. The periplasm is the gelatinous material between the peptidoglycan and the cytoplasmic membrane.

The Gram-Negative Cell Wall The gram-negative bacteria are those that decolourize during the Gram stain procedure, pick up the counterstain carbol fuchsin , and appear pink. Common Gram-negative bacteria of medical importance

Page 6

include Salmonella species, Shigella species, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Proteus species, and Pseudomonas aeruginosa.

Structure and Composition of the Gram-Negative Cell Wall It consists of: 1. A thin, inner wall composed of peptidoglycan The peptidoglycan portion of the gram-negative cell wall is generally 2-3 nanometers (nm) thick and contains just 2-3 layers of peptidoglycan. Chemically, only 10 to 20% of the gram-negative cell wall is peptidoglycan. 2. An outer membrane The outer membrane of the gram-negative cell wall appears as a lipid bilayer about 7 nm thick. It is composed of phospholipids, lipoproteins, lipopolysaccharides (LPS), and proteins. Phospholipids are located mainly in the inner layer of the outer membrane, as are the lipoproteins that connect the outer membrane to the peptidoglycan. The lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid portion called lipid A embedded in the membrane and a polysaccharide portion extending outward from the bacterial surface. The LPS portion of the outer membrane is also known as endotoxin. In addition, pore-forming proteins called porins span the outer membrane.

Gram negative cell wall

Page 7

3. The outer membrane of the gram-negative cell wall is studded with surface proteins that differ with the strain and species of the bacterium. 4. The periplasm is the gelatinous material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane.

Functions of cell wall Maintaining the cell's characteristic shape Countering the effects of osmotic pressure Providing attachment sites for bacteriophages Providing a rigid platform for surface appendages- flagella, fimbriae, and pili all emanate from the wall and extend beyond it Play an essential role in cell division Be the sites of major antigenic determinants of the cell surface Possesses target site for antibiotics, lysozymes etc.

6. Classify bacteria depending on their shape.

Bacteria occur in three main shapes, spherical, rod-like and spiral Cocci: Spherical bacteria are called cocci (singular coccus). The cells may occur in pairs (diplococci), in groups of four (tetracocci), in bunches (staphylococci), in a beadlike chain (streptococci) or in a cubical arrangement of four (tetrad) or eight (sarcina). Bacilli: Rod-like bacteria are called bacilli (singular bacillus). They generally occur singly, but may occasionally be found in pairs (diplobacilli) or chains (streptobacilli). Spiral shaped: Spiral-shaped bacilli are called spirilla (singular, spirillum). Short incomplete spirals are called vibrios or comma bacteria.

Page 8

Other shapes: Actinomycetes- branched filamentatous bacteria Mycoplasmas- do not possess a stable shape due to lack of cell wall

7. Write an account on bacterial flagella

It is a whip-like organelle present in bacteria specialized for locomotion. The flagellum is composed of the protein flagellin and is a hollow tube 20 nm thick. It is helical, and has a sharp bend just outside the outer membrane

Page 9

called the hook which allows the helix to point directly away from the cell. A shaft runs between the hook and the basal body, passing through protein rings in the cell's membranes that act as bearings. Gram-positive organisms have 2 basal body rings, one in the peptidoglycan layer and one in the cell membrane.

Structure of flagellum Gram-negative organisms have 4 rings: L ring associates with the lipopolysaccharides, P ring associates with peptidoglycan layer, M ring is imbedded in the cell membrane, and the S ring is directly attached to the cell membrane. The filament ends with a capping protein.

Flagellar arrangement in bacteria: Monotrichous: a single flagellum, usually at one pole Lophotrichous: two or more flagella at one or both poles Amphitrichous: a single flagellum at both ends of the organism Peritrichous: flagella over the entire surface

Page 10

8. Write a short note on bacterial capsule.

Some bacteria are enclosed within a capsule. It is a layer that lies outside the cell wall of bacteria. It is usually composed of polysaccharides. This protects the bacterium, even within phagocytes, helping to prevent the cell from being killed. Encapsulated bacteria grow as 'smooth' colonies, whereas colonies of bacteria that have lost their capsules appear rough. Suspending bacteria in India ink is an easy way of demonstrating capsules. Ink particles cannot penetrate the capsular material and encapsulated cells appear to have a halo around them. This is called as negative staining.

Functions: It protects bacteria from phagocytosis. Due to the fact that the capsule helps to protect bacteria against phagocytosis, it is considered as a virulence factor. Capsules also contain water which protects bacteria against desiccation. They also exclude bacterial viruses and most hydrophobic toxic materials such as detergents. Capsules allow bacteria to adhere to surfaces and other cells.

9. Write a note on bacterial fimbriae.

Fimbrae are protein tubes that extend out from the cell surface of the bacteria. They are generally short in length and present in high numbers about the entire bacterial cell surface. Fimbrae usually function to facilitate the attachment of a bacterium to a surface (e.g. to form a biofilm) or to other cells (e.g. animal cells during pathogenesis).

10.

Write a note on bacterial spore.

Endospores are dormant alternate life forms produced by the certain bacteria like Bacillus, Clostridium etc. Structure The completed endospore consists of multiple layers of resistant coats (including
Page 11

a cortex, a spore coat, and sometimes an exosporium) surrounding a nucleoid, some ribosomes, RNA molecules, and enzymes.

Location: The position of the endospore differs among bacterial species The endospore may be: Terminal Subterminal Central Lateral

Variations in endospore morphology: (1, 4) central endospore; (2, 3, 5) terminal endospore; (6) lateral endospore

Functions: An endospore is not a reproductive structure but rather a resistant, dormant survival form of the organism. Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, etc. The endospore can survive possibly thousands of years until a variety of environmental

Page 12

stimuli trigger germination allowing outgrowth of a single vegetative bacterium. They are resistant to antibiotics, most disinfectants, and physical agents such as radiation, boiling, and drying. The impermeability of the spore coat is thought to be responsible for the endospore's resistance to chemicals.

11. . Write a short account on gram staining. The gram stain is the most widely used staining procedure in bacteriology. It is called a differential stain since it differentiates between gram-positive and gramnegative bacteria. Bacteria that stain purple/violet with the gram staining procedure are termed gram-positive; those that stain pink are said to be gramnegative. The terms positive and negative have nothing to do with electrical charge, but simply designate two distinct morphological groups of bacteria. The gram staining procedure involves four basic steps: 1. The bacteria are first stained with the basic dye methyl violet. Both grampositive and gram-negative bacteria become directly stained and appear purple after this step. 2. The bacteria are then treated with gram's iodine solution. This allows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both gram-positive and gram-negative bacteria remain purple after this step. 3. Gram's decolourizer, acetone, is then added. This is the differential step. Gram-positive bacteria retain the crystal violet-iodine complex while gram-negative are decolourized. 4. Finally, the counterstain carbol fuchsin (also a basic dye) is applied. Since the gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, which are now colourless, become directly stained by the safranin. Thus, gram-positive appear purple, and gram-negative appear pink.

Principle behind the Gram staining It is thought that in gram-positive bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within the cell. In gram positive bacteria, the alcohol/acetone mixture causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. In the case of gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain
Page 13

the crystal violet-iodine complex and the cell is decolourized.

12.

Write a short note on acid fast staining.

The acid fast staining or Ziehl-Neelsen technique is a differential staining method. It is used to stain mycobacteria, which do not take gram or methylene blue stains due to the thick, waxy outer cell wall. Since mycobacteria cannot be destained with acid, they are called acid-fast bacilli. The organisms are exposed to hot, concentrated carbol fuchsin for about 5 min, decolourized with acid and alcohol (hence the term acid fast bacilli), and finally counterstained with methylene blue. The mycobacteria are stained pink and background blue.

13.
i. ii. iii. iv.

Write a note on bacterial growth curve.

The bacterial growth curve describes various stages of growth of a pure culture of bacteria beginning with the addition of cells to sterile media and ending with the death of all of the cells present. The phases of growth typically observed include: lag phase exponential (log, logarithmic) phase stationary phase death phase (decline phase)

Lag Phase: Bacteria are becoming "acclimated" to the new environmental conditions to which they have been introduced (pH, temperature, nutrients, etc.). There is no significant increase in numbers with time. Exponential Growth Phase: The living bacteria population increases rapidly with time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth. Stationary Phase: With the exhaustion of nutrients and build-up of waste and secondary metabolic
Page 14

products, the growth rate has slowed to the point where the growth rate equals the death rate. Effectively, there is no net growth in the bacteria population.

Death Phase: The living bacteria population decreases with time, due to a lack of nutrients and toxic metabolic by-products.

14.

Write a note on bacterial toxins.

A bacterial toxin is a type of toxin that is generated by bacteria. Toxinosis is pathogenesis caused by the bacterial toxin eg. Enterotoxin of S. aureus causing food poisoning. One primary classification of bacterial toxin is exotoxin and endotoxin Exotoxins:.

Exotoxins are generated by the bacteria and actively secreted. They are mostly proteinaceous and antigenic in nature.

Endotoxin:

Endotoxins are part of the bacteria itself. Usually, endotoxin is part of the bacterial outer membrane, and it is not
Page 15

released until the bacteria are killed by the immune system. They are mostly carbohydrate in nature and are poorly antigenic.

Properties of endotoxin and exotoxin

PROPERTY Chemical nature Relationship to cell Denatured by boiling Antigenic Form toxoid Potency Specificity Enzymatic activity Pyrogenicity

ENDOTOXIN EXOTOXIN Lipopolysaccharide (mw Protein (mw = 50= 10kDa) 1000kDa) Extracellular, Part of outer membrane diffusible No Yes No Relatively low (>100ug) Low degree No Yes Usually Yes Yes Relatively high (1 ug) High degree Often Occasionally

15.

Write a short note on exotoxin.

An exotoxin is a toxin that is produced by a bacterium and then released from the cell into the surrounding environment. The damage caused by an exotoxin can only occur upon release. Exotoxins are generally heat labile (destroyed by heat), and are protein in nature. They are antigenic. Many can be detoxified with retention of antigenicity by treatment with formaldehyde (toxoid). Many are important virulence factors in pathogenic bacteria. Some examples of exotoxins include the botulinum toxin produced by Clostridium botulinum, the Corynebacterium diphtheriae exotoxin which is produced during life threatening symptoms of diphtheria.

Page 16

16.

Write a short note on endotoxin.

Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Endotoxin is invariably associated with Gram-negative bacteria whether the organisms are pathogenic or not. It is the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens such as Escherichia coli, Salmonella, Shigella, Vibrio cholera etc. The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in an animal and it activates complement by the alternative pathway, so it may be a part of the pathology of Gramnegative bacterial infections. Compared to the classic exotoxins of bacteria, endotoxins are less potent and less specific in their action, since they do not act enzymatically. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain powerful oxidizing agents such as superoxide, peroxide and hypochlorite, have been reported to neutralize them. Endotoxins cannot be converted to toxoids.

17.

Define and classify sterilisation with examples.

Definitions Sterilization: Sterilization is a term that refers to the complete killing or elimination of living organisms including the spores in the sample being treated. Disinfection Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases.

Page 17

Types of sterilization i. Physical method a. Heat Moist heat sterilization Dry heat sterilisation

b. Filtration c. Radiation ii. Chemical method PHYSICAL METHOD: HEAT Moist heat sterilization Kills microorganisms by coagulating their proteins.

Methods in moist heat sterilization Temp below 100oC: Pasteurisation, Inspissator. Temperature at 100oC: Boiling. Steam at atmospheric pressure: Koch/Arnolds steamer. Steam under pressure: Autoclave.

a. Pasteurisation Process of killing of pathogens in the milk but does not sterilize it. Milk is heated at 63oC for 30 mins. (HOLDER METHOD) At 72oC for 15-20 Sec. Rapid cooling to 13oC (FLASH PROCESS)

b. Inspissation Sterilizes by heating at 80-85oC for half an hour for 3 successive days Used to sterilize media such as Lowenstein-Jensen media & Loefllers serum slope.

c. Boiling Temperature at 1000C. Kills only vegetative forms of bacterial pathogens.

Page 18

d. Steam at atmospheric pressure Steam is generated using a steamer (Koch/ Arnold) Consists of a Tin cabinet Has a conical lid to enable the drainage of condensed steam Perforated tray above ensures materials are surrounded by steam. For routine sterilization exposure of 90 mins is used

e. Tyndallisation /Intermittent Sterilization For media containing sugar and gelatin exposure of 100 oC for 20 min for 3 successive days is used On first exposure it will kill bacterial cells, but not bacterial spores, allowing it to cool and allowing the spores to germinate, and then re-heating to kill the bacteria. The process is termed as Tyndallisation /Intermittent Sterilization

f. Autoclave Works on the principle of Steam under pressure Invented by Charles Chamberland in 1879. Autoclave consists of a vertical or a horizontal cylinder. One end has an opening which is meant for keeping materials to be sterilised. The lid is provided with a Pressure gauge, to measure the pressure A safety valve is present to permit the escape of steam from the chamber Articles to be sterilised are placed in the basket provided Sterilisation is carried out under pressure at 121 C for 15 minutes.

Dry heat sterilization: a. Hot air oven Kills by oxidation effects The hot air oven utilizes dry heat to sterilize articles Operated between 50oC to 250/300oC. A holding period of 160oC for 1 hr is desirable. There is a thermostat controlling the temperature. Double walled insulation keeps the heat in and conserves energy Uses: To sterilise Forceps, Scissors, Scalpels, Swabs. Pharmaceuticals products like Liquid paraffin, dusting powder, fats and grease.

Page 19

b. Flaming Inoculation loop or Wire, the tip of Forceps and spatulas are held in a bunsen flame till they are red hot.

c. Incineration Microorganisms are burned by exposing them to an open flame This is an excellent method of destroying materials such as contaminated cloth, animal carcasses and pathological materials.

FILTRATION Filtration helps to remove bacteria from heat labile liquids such as sera and solutions of sugar, Antibiotics. The following filters are used RADIATIONS Two types of radiations are used NON IONISING IONISING Candle filters Asbestos filters Sintered glass filter Membrane filters

Non ionising radiation: Electromagnetic rays with longer wavelength Absorbed as heat Can be considered as hot air sterilisation Used in rapid mass sterilisation of prepacked Syringes and catheters Eg: UV rays Ionising radiation X- rays, gamma rays & cosmic rays. High penetrative power No appreciable increase in the temperature COLD STERILISATION Sterilise plastics Syringes, catheters, grease fabrics, metal foils
Page 20

CHEMICAL METHODS Chemical agents act by Protein coagulation Disruption of the cell membrane Removal of Sulphydryl groups Substrate competition

a. Alcohol Ethanol /Isopropyl alcohol are frequently used No action on spores Concentration recommended 60-90% in water Uses Disinfection of clinical thermometer. Disinfection of the skin Venupuncture

b. Aldehydes Formaldehyde & Glutaraldehyde are frequently used Formaldehyde is bactericidal, sporicidal & has a lethal effect on viruses. Glutaraldehyde is effective against Tubercle bacilli, fungi and viruses Uses To preserve anatomical specimens Destroying Anthrax spores in hair and wool 10% Formalin+0.5% Sodium tetra borate is used to sterilise metal instruments

c. Halogens Iodine in aqueous and alcoholic solution has been used widely as a skin disinfectant Actively bactericidal with moderate against spores Chlorine and its compounds have been used as disinfectants in water supplies & swimming pools

d. Phenols Obtained by distillation of coal tar Phenols are powerful microbicidal substances Phenolic derivatives have been widely used as disinfectants for various purposes in hospitals
Page 21

Eg: Lysol, cresol

e. Gases Ethylene Oxide Uses Specially used for sterilising heart-lung machines,respirators,sutures,dental equipments, books and clothing. Also used to sterilise Glass, metal and paper surfaces ,plastics, oil,some foods and tobacco. Colourless ,Highly penetrating gas with a sweet ethereal smell. Effective against all types of microorganisms including viruses and spores

Formaldehyde gas Widely employed for fumigation of operation theatres and other rooms

Beta propiolactone Used in fumigation For sterilisation 0.2% BPL is used Has a rapid biocidal activity Very effective against viruses

f. Metallic salts The salts of silver, copper and mercury are used as disinfectants. Act by coagulating proteins Marked bacteriostatic, weak bactericidal and limited fungicidal activity

18.

Give an account on Hot air oven.

Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles (Kills by oxidation effects). Generally, they can be operated from 50 to 300 C. A holding period of 160oC for 1 hr is desirable. There is a thermostat controlling the temperature. These are digitally controlled to maintain the temperature. Their double walled insulation keeps the heat in and conserves energy, the inner layer being a poor conductor and outer layer being metallic. There is also an air filled space in between to aid insulation. An air circulating fan helps in uniform distribution of the heat. These are fitted with the adjustable wire mesh plated trays or aluminium trays and may have an on/off rocker
Page 22

switch, as well as indicators and controls for temperature and holding time. The capacities of these ovens vary.

Diagram of a Hot air oven

19.

Write a note on autoclave.

Works on the principle of Steam under pressure Invented by Charles Chamberland in 1879. Autoclave consists of a vertical or a horizontal cylinder. One end has an opening which is meant for keeping materials to be sterilised. The lid is provided with a Pressure gauge, to measure the pressure. A safety valve is present to permit the escape of steam from the chamber. Articles to be sterilised are placed in the basket provided. Sterilisation is carried out under pressure at 121 C for 15 minutes.

Page 23

Diagram of an autoclave

20.

Write a note on bacterial filters.

Filtration helps to remove bacteria from heat labile liquids such as sera and solutions of sugar, Antibiotics. The following filters are used: Candle filters Asbestos filters Sintered glass filter Membrane filters

CANDLE FILTERS Widely used for purification of water Two types (a) Unglazed ceramic filter Chamberland filter (b) Diatomaceous earth filters Berkefeld filter

Page 24

ASBESTOS FILTER Disposable single use discs High adsorbing tendency Carcinogenic Eg: Seitz filter SINTERED GLASS FILTER Prepared by heat fusing powdered glass particles of graded size Cleaned easily, brittle, expensive.

MEMBRANE FILTERS Uses Made of cellulose esters or other polymers Water purification & analysis Sterilization & sterility testing Preparation of solutions for parenteral use

AIR FILTERS Air can be filtered using HEPA (High Efficiency Particle Air) filters. They are usually used in biological safety cabinets. HEPA filters are at least 99.97% efficient for removing particles >0.3 m in diameter.

21. Write a note on ionising radiation as sterilising agents.

Ionizing rays: Ionizing rays are of two types. They are particulate and electromagnetic rays.

Particulate: Electron beams are particulate in nature. High speed electrons are produced from a heated cathode. Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals.

Page 25

Advantages: Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the instruments can be switched off.

Disadvantage: Disadvantage includes poor penetrative power and requirement of sophisticated equipment. Electromagnetic rays: Electromagnetic rays such as gamma rays are produced from nuclear disintegration of certain radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but require longer time of exposure. These high-energy radiations damage the nucleic acid of the microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics. Advantage: High penetrating power Disadvantage: Disadvantages include; unlike electron beams, they cant be switched off, glasswares tend to become brownish, loss of tensile strength in fabric. Gamma irradiation impairs the flavour of certain foods..

22.

Write a note on cold sterilisation.

Use of radiations is one of the important methods of sterilisation. Since radiation does not generate heat, it is termed "cold sterilization. Two types of radiation are used. They are: i. Ionising radiations ii. Non-ionising radiations Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative power.

Page 26

Non-ionizing rays: Rays of wavelength longer than the visible light are non-ionizing. Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective. UV rays are generated using a high-pressure mercury vapour lamp. It is at this wavelength that the absorption by the microorganisms is at its maximum, which results in the germicidal effect. UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA replication. UV readily induces mutations in cells irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to the effective UV radiation are inactivated within seconds. Since UV rays dont kill spores, they are considered to be of use in surface disinfection. UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories, corridors, etc. Advantages: Very effective surface sterilant and easy to use. Disadvantage: Disadvantages of using UV rays include low penetrative power, limited life of the UV bulb, some bacteria have DNA repair enzymes that can overcome damage caused by UV rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't penetrate glass, paper or plastic.

Ionizing rays: Ionizing rays are of two types. They are particulate and electromagnetic rays. Particulate: Electron beams are particulate in nature. Highspeed electrons are produced from a heated cathode. Electron beams are employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals. Advantages: Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the instruments can be switched off. Disadvantage: Disadvantage includes poor penetrative power and requirement of sophisticated
Page 27

equipment. Electromagnetic rays: Electromagnetic rays such as gamma rays are produced from nuclear disintegration of certain radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but require longer time of exposure. These high-energy radiations damage the nucleic acid of the microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares and fabrics. Advantage: High penetrating power Disadvantage: Disadvantages include; unlike electron beams, they cant be switched off, glasswares tend to become brownish, loss of tensile strength in fabric. Gamma irradiation impairs the flavour of certain foods..

23.

Classify culture media.

Classification of culture media: I. Based on their consistency a) solid medium b) liquid medium c) semi solid medium

Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media Special media Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media
Page 28

II.

Media for biochemical reactions

Based on Oxygen requirement - Aerobic media - Anaerobic media

24. Write a short account on anaerobic cultivation of bacteria/anaerobic culture methods.

An anaerobic bacteria culture is a method used to grow anaerobes from a clinical specimen. Anaerobic bacterial cultures are performed to identify bacteria that grow only in the absence of oxygen and which may cause human infection. Anaerobic bacteria differ in their requirement and sensitivity to oxygen. Cl.tetani is a strict anaerobe grows at an oxygen tension < 2 mm Hg. Production of vacuum Displacement of oxygen with other gases Chemical method Biological method Reduction of medium

Methods:

a. Production of vacuum: Incubate the cultures in a vacuum desiccator. b. Displacement of oxygen with other gases Displacement of oxygen with hydrogen, nitrogen, helium or CO2. Eg: Candle jar c. Chemical method Alkaline pyrogallol absorbs oxygen. McIntosh Fildes anaerobic jar Consists of a metal jar or glass jar with a metal lid which can be clamped air tight. The lid has 2 tubes gas inlet and gas outlet The lid has two terminals connected to electrical supply. Under the lid small grooved porcelain spool, wrapped with a layer of palladinised asbestos.

Page 29

Working: Inoculated plates are placed inside the jar and the lid clamped air tight. The outlet tube is connected to a vacuum pump and the air inside is evacuated. The outlet tap is then closed and the inlet tube is connected to a hydrogen supply. After the jar is filled with hydrogen, the electric terminals are connected to a current supply, so that the palladinised asbestos is heated. Act as a catalyst for the combination of hydrogen with residual oxygen.

Gaspak Commercially available disposable envelope. Contains chemicals which generate H2 and CO2 on addition of water. Cold catalyst in the envelope Indicator is used reduced methylene blue. Colourless anaerobically Blue colour on exposure to oxygen

d. Biological method Absorption of oxygen by incubation with aerobic bacteria, germinating seeds or chopped vegetables.

e. Reduction of oxygen By using reducing agents 1% glucose, 0.1% Thioglycolate

25.

Write a short note on blood culture.

When the infectious agent is circulating in blood (e.g. in septicaemia, endocarditis, pneumonia) then it has to be aseptically withdrawn by venepuncture and cultured. Blood culture has to be performed on special liquid media, under both aerobic and anaerobic conditions. The blood is aseptically transferred to a rich growth medium (e.g. brain-heart infusion broth) containing anticoagulants. Cultures are checked for turbidity and gas production daily, up to a week. Positive cultures are sampled and the organism(s) isolated and identified.

Page 30

26.

Write a note on selective media.

Selective media contain specific chemicals which do not affect the growth of the bacterium to be isolated but discourage the growth of other bacteria in the mixture. For example MacConkey (MAC) Agar: A selective media used to isolate Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective only for Gram negative bacteria. Mannitol salt agar or MSA It contains a high concentration (~7.5%-10%) of salt (NaCl), making it selective for Staphylococci since this level of NaCl is inhibitory to most other bacteria.

27.

Write a short note on transport medium.

It is a medium for temporary storage of specimens being transported to the laboratory for isolation of bacteria. It maintains the viability of all organisms in the specimen without altering their concentration. It contains only buffers and salt and lacks carbon, nitrogen, and organic growth factors and thus prevents microbial multiplication. The transport media used in the isolation of anaerobes must be free of molecular oxygen.

Examples: Thioglycollate broth for strict anaerobes Stuart transport medium-a non-nutrient soft agar gel containing a reducing agent to prevent oxidation, and charcoal to neutralise certain bacterial inhibitors for gonococci. Buffered glycerol saline for enteric bacilli. Venkat-Ramakrishnan(VR) medium for V. cholerae

Page 31

28.

What is an enriched media?

Enriched media contain the nutrients required to support the growth of a wide variety of organisms, mostly the fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45C), which turns brown and gives the medium the colour for which it is named.

29.

Write a note on blood agar.

Blood agar is an enriched, differential media used to isolate fastidious organisms and to detect haemolytic activity. It contains mammalian blood (usually sheep or horse), typically at a concentration of 510%. When streaked on Blood Agar, many species of bacteria cause haemolysis i.e., destruction of the erythrocytes in the medium. Haemolytic reactions are generally classified as alpha, beta or gamma according to the appearance of zones around isolated colonies growing on or in the medium: Alpha haemolysis is a greenish discoloration that surrounds a bacterial colony growing on the agar. This type of haemolysis represents a partial decomposition of the haemoglobin of the red blood cells. Alpha haemolysis is characteristic of Streptococcus pneumonia and so can be used as one of the diagnostic feature in the identification of the bacterial strain. Beta haemolysis represents a complete breakdown of the haemoglobin of the red blood cells in the vicinity of a bacterial colony. There is a clearing of the agar around a colony. Beta haemolysis is characteristic of Streptococcus pyogenes and some strains of Staphylococcus aureus. Gamma haemolysis is a lack of haemolysis in the area around a bacterial colony. A blood agar plate displaying gamma haemolysis actually appears brownish. This is a normal reaction of the blood to the growth conditions used (37 C in the presence of carbon dioxide). Gamma haemolysis is a characteristic of Enterococcus faecalis.

30.

Write a note on enrichment media.

The enrichment media is a liquid media which promotes the growth of a particular organism by providing it with the essential nutrients and rarely contains certain inhibitory substance to prevent the growth of normal competitors. e.g. Selenite F
Page 32

broth- this media favours the growth of Salmonella also prevents the growth of normal competitors like E. coli but E.coli do not perish (die) in the medium but they do not flourish like Salmonella

31.

What is an indicator media or differential media?

Differential media or indicator media distinguish one microorganism type from another that grows on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. Examples of differential media include:

Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation MacConkey (MCK), which is differential for lactose fermentation Mannitol salt agar (MSA), which is differential for mannitol fermentation

32. Write a note on Robertson cooked meat medium or write a note on an anaerobic medium.
In 1916, Robertson developed a cooked meat medium for use in the cultivation of certain anaerobes isolated from wounds. It is now popularly called as Robertson cooked meat medium. Nutritional requirements needed by most bacteria are provided by beef heart, peptone and dextrose. Dextrose, yeast extract, haemin and vitamin K are added to enhance the growth of anaerobic microorganisms. Amino acids and other nutrients are supplied by the muscle protein in the heart tissue granules. Reducing substances, which permit the growth of strict anaerobes, are supplied by the muscle tissue and the iron filings. It is thought that the meat particles act as a reducing and detoxifying substance, thereby disabling harmful by products that may be produced by the replicating organism. Because reducing substances are more available in denatured protein, the meat particles are cooked before use in the medium. Robertson Cooked Meat Medium is used for the cultivation and maintenance of clostridia and for determining proteolytic activity of anaerobes. It supports the growth of most spore forming and non-spore forming obligate anaerobes and may be used for a variety of purposes including the maintenance of stock cultures. This medium is also useful as an enrichment broth as backup to plated media or for
Page 33

cultivating anaerobes that may be present in small numbers in a population and as a subculture medium for determination of proteolysis (meat digestion) and spore formation by Clostridium species.

Robertson cooked meat medium

33.

Write a note on antibiotic sensitivity test.

The antibiotic sensitivity test is an in vitro test of the effectiveness of selected antibacterial agents against bacteria recovered from a patient. A microbe is considered sensitive (or susceptible) to an antimicrobial agent if it is inhibited by an antibiotic of a standard therapeutic dose. The reverse is true for a resistant organism. Organisms are considered intermediate in susceptibility if the inhibiting concentration of the antimicrobial agent is slightly higher than that obtained with a therapeutic dose. Laboratory testing for antimicrobial sensitivity The action of an antimicrobial drug against an organism can be measured by: qualitatively (disc diffusion tests) quantitatively (minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) tests). Disc diffusion test The disc diffusion test is the most commonly used method of testing the sensitivity of a microorganism to an antimicrobial agent. A common application of this method is the Kirby Bauer test, developed in the 1960s. Here, the isolate to be tested is seeded over the entire surface of an agar plate and drug-impregnated filter paper discs are applied. After overnight incubation at 37C, zones of growth inhibition are observed around each disc, depending on the sensitivity of a particular organism to a given agent.

Page 34

Assessment of MIC and MBC Determining the MIC and MBC gives a quantitative assessment of the potency of an antibiotic. The minimum concentration of the drug that inhibits the growth of the test organism in the tube is recorded as the MIC, i.e. the lowest concentration that will inhibit the visible growth in vitro. The MBC is defined as the minimum concentration of drug that kills 99.9% of the test microorganisms.

34.

Write a short essay on genetic transfer in bacteria.

Genetic transfer is the mechanism by which DNA is transferred from one bacterium to another.

Once donar DNA is inside the recipient, crossing over can occur. The result is a recombinant cell that has a genome different from either the donar or the recipient.

In bacteria genetic transfer can happen three ways: a. Transformation b. Transduction c. Conjucation

Page 35

Transformation

After death or cell lyses, some bacteria release their DNA into the environment. Other bacteria, generally of the same species, can come into contact with these fragments, take them up and incorporate them into their DNA by recombination. This method of transfer is the process of transformation. Any DNA that is not integrated into he chromosome will be degraded. The genetically transformed cell is called a recombinant cell because it has a different genetic makeup than the donar and the recipient.

Transduction

Another method of genetic transfer and recombination is transduction. This method involves the transfer of DNA from one bacterium to another with the use of a bacteriophage (phage). o A phage is a virus that infects bacteria. o The phage T4 and the phage lambda, for example, both infect E. coli.

Conjugation

A third mechanism by which genetic transfer takes place is conjugation. This mechanism requires the presence of a special plasmid called the F plasmid. Plasmids are small, circular pieces of DNA that are separate and replicate indepentently from the bacterial chromosome. A conjugation event occurs when the male cell extends his sex pili and attaches to the female. This attached pilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred from the male to the female. When transfer is complete, the result is two male cells.

Page 36

PART II IMMUNOLOGY
1. Define and classify immunity with examples.
The, immunity is defined as the resistance to infectious disease causing agents by virtue of the immune system. Immunity can be classified as a. Innate immunity b. Adaptive or acquired immunity d. Innate Immunity Innate immunity is resistance that is pre-existing. It is nonspecific and includes barriers to infectious agentseg, skin and mucous membranes, phagocytic cells, inflammatory mediators, and complement components. It may vary with age and with hormonal or metabolic activity. e. Adaptive Immunity Adaptive immunity, which occurs after exposure to an antigen (eg, an infectious agent) is specific and is mediated by either antibody or lymphoid cells. It can be passive or active. i. Passive Immunity Passive immunity is the immunity acquired by the transfer of antibodies to a person from another individual, as through injection or placental transfer to a foetus. Natural passive immunity It is a form of acquired immunity resulting from antibodies that are transmitted naturally through the placenta by mother to a foetus or through the colostrums (first formed breast milk after delivery) to an infant.

Artificial passive immunity It is the injection of antiserum (antibodies) or lymphocytes preformed in another

Page 37

host for treatment or prophylaxis.

Advantage: The main advantage of passive immunization is the prompt availability of large amounts of antibodies. Disadvantage: The disadvantages are the short life span of these antibodies and possible hypersensitivity reactions if antibodies from another species are administered. Active immunity Active immunity is the immunity resulting from the development of antibodies in response to the presence of an antigen, as from vaccination or exposure to an infectious disease. When B cells and T cells are activated by a pathogen/antigen, memory B-cells and Tcells develop. Throughout the lifetime of an animal these memory cells will remember each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. This type of immunity is both active and adaptive because the body's immune system prepares itself for future challenges. Active immunity often involves both the cell-mediated and humoral immunity. The active immunity may be either naturally acquired active immunity or artificially acquired active immunity Naturally acquired active immunity Naturally acquired active immunity occurs when a person is exposed to a live pathogen, and develops a primary immune response, which leads to immunological memory. This type of immunity is natural because it is not induced by deliberate exposure.

Artificially acquired active immunity Artificially acquired active immunity can be induced by a vaccine, a substance that contains antigen. A vaccine stimulates a primary response against the antigen without causing symptoms of the disease. There are four types of traditional vaccines:

Inactivated vaccines are composed of micro-organisms that have been killed with chemicals and/or heat and are no longer infectious. Most vaccines
Page 38

of this type are likely to require booster shots. Live, attenuated vaccines are composed of micro-organisms that have been cultivated under conditions which disable their ability to induce disease. These responses are more durable and do not generally require booster shots. Toxoids are inactivated toxic compounds (toxins) from micro-organisms which develop the protective antibodies against the toxins. Subunit -vaccines are composed of small fragments of disease causing organisms

2. Classify immunity and define on active immunity.

Following is the classification of immunity

Active immunity Active immunity is the immunity resulting from the development of antibodies in response to the presence of an antigen, as from vaccination or exposure to an infectious disease. When B cells and T cells are activated by a pathogen/antigen, memory B-cells and Tcells develop. Throughout the lifetime of an animal these memory cells will remember each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. This type of immunity is both active and adaptive because the body's immune system prepares itself for future challenges. Active immunity often involves both the cell-mediated and humoral immunity. The active immunity may be either naturally acquired active immunity or artificially

Page 39

acquired active immunity Naturally acquired active immunity Naturally acquired active immunity occurs when a person is exposed to a live pathogen, and develops a primary immune response, which leads to immunological memory. This type of immunity is natural because it is not induced by deliberate exposure. Artificially acquired active immunity Artificially acquired active immunity can be induced by a vaccine, a substance that contains antigen. A vaccine stimulates a primary response against the antigen without causing symptoms of the disease. There are four types of traditional vaccines:

Inactivated vaccines are composed of micro-organisms that have been killed with chemicals and/or heat and are no longer infectious. Most vaccines of this type are likely to require booster shots. Live, attenuated vaccines are composed of micro-organisms that have been cultivated under conditions which disable their ability to induce disease. These responses are more durable and do not generally require booster shots. Toxoids are inactivated toxic compounds (toxins) from micro-organisms which develop the protective antibodies against the toxins. Subunit -vaccines are composed of small fragments of disease causing organisms

3. Write a short note on passive immunity.

Passive immunity is the immunity acquired by the transfer of antibodies to a person from another individual, as through injection or placental transfer to a foetus. Natural passive immunity It is a form of acquired immunity resulting from antibodies that are transmitted naturally through the placenta by mother to a foetus or through the colostrum to an infant.

Artificial passive immunity It is the injection of antiserum (antibodies) or lymphocytes preformed in another host for treatment or prophylaxis.

Page 40

Advantage: The main advantage of passive immunization is the prompt availability of large amounts of antibodies. Disadvantage: The disadvantages are the short life span of these antibodies and possible hypersensitivity reactions if antibodies from another species are administered.

4. Write a short note on complement.

Complement is a group of serum proteins which work with (complement with) antibody to eliminate pathogens. Complement is NOT antigen-specific and it is activated immediately in the presence of pathogen, so it is considered as a part of innate immunity. However, antibody activates some complement proteins, so complement activation is also part of humoral immunity. Complement stimulates inflammation, facilitates antigen phagocytosis, and lyses some cells directly. Because it is such a powerful inflammatory agent, its activity is tightly regulated. Complement components are normally present in body fluids as inactive precursors. The alternative pathway of complement activation can be stimulated directly by microorganisms and is important in the early stages of the infection before the production of antibody. It is part of the innate immune system. The classical pathway requires antibody, which may take weeks to develop. Both pathways can lead to the lytic or membrane attack pathway. During the course of complement activation, numerous split products of complement components, with important biological effects, are produced.

5. Write a note on functions of complement.

Complement is a group of serum proteins which work with (complement with) antibody to eliminate pathogens. Complement is NOT antigen-specific and it is activated immediately in the presence of pathogen, so it is considered part of innate immunity. However, antibody activates some complement proteins, so complement activation is also part of humoral immunity. Complement stimulates inflammation, facilitates antigen phagocytosis, and lyses some cells directly.

Page 41

Following are the important functions of complement: Complement activation stimulates several antimicrobial activities. The endpoint is formation of a membrane attack complex (MAC), which inserts into lipid membranes of bacteria and causes osmotic lysis. Complement fragments (C3b) called opsonins adhere to microorganisms and promote leukocyte chemoattraction, antigen binding and phagocytosis, and activation of macrophage and neutrophil killing mechanisms. Complement fragments (C3a and C5a) called anaphylatoxins promote an inflammatory response by binding to complement receptors on mast cells and triggering release of histamine, which increases blood vessel permeability and smooth muscle contraction. Complement is also important for virus neutralization and immune complex removal. Many bacteria have mechanisms that allow them to evade complement-mediated damage.

6. Write a short note on immunoglobulin.

Immunoglobulins are glycoprotein molecules that are produced by plasma cells (activated B cell) in response to an antigen and which function as antibodies. Structure of Immunoglobulins: Immunoglobulins are composed of four polypeptide chains: two "light" chains (lambda or kappa), and two "heavy" chains (alpha, delta, gamma, epsilon or mu). The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, IgM, respectively). Light chains are composed of 220 amino acid residues while heavy chains are composed of 440-550 amino acids. Each chain has "constant" and "variable" regions. "Hypervariable" regions, or "Complementarity Determining Regions" (CDRs) are found within the variable regions of both the heavy and light chains. These regions serve to recognize and bind specifically to antigen. The four polypeptide chains are held together by covalent disulfide (-S-S-) bonds.

Page 42

The five major types of immunoglobulin are:

IgG. IgG antibodies are found in all body fluids. They are the smallest but most common antibody (75% to 80%) of all the antibodies in the body. IgG antibodies are very important in fighting bacterial and viral infections. IgG antibodies are the only type of antibody that can cross the placenta in a pregnant woman to help protect her baby. IgA. IgA antibodies are found in secretions. Hence they are called as secretary immunoglobulin. They are found in saliva, tears, and blood. About 10% to 15% of the antibodies present in the body are IgA antibodies. IgM. IgM antibodies are the largest antibody. They are found in blood and lymph fluid and are the first type of antibody made in response to an infection. They also cause other immune system cells to destroy foreign substances. IgM antibodies are about 5% to 10% of all the antibodies in the body. IgE. IgE antibodies are found in the lungs, skin, and mucous membranes. They cause the body to react against foreign substances such as pollen, fungus spores, and animal dander. IgE antibody levels are often high in people with allergies. IgD. IgD antibodies are found in small amounts in the tissues that line the belly or chest. How they work is not clear.

7. Write a note on secretary immunoglobulin.

Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. IgA has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA). In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum and secretions from the genitoPage 43

urinary tract, gastrointestinal tract, prostate and respiratory epithelium. The two IgA monomers are linked by two additional chains - J chain (joining chain) and the secretory component. The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes, thus sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions.

8. Write a note on Immunoglobulin A.

Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. IgA has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA). In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum and secretions from the genitourinary tract, gastrointestinal tract, prostate and respiratory epithelium. It is also found in small amounts in blood as a monomeric IgA (non secretary IgA). The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes, thus sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions. IgA is a poor activator of the complement system, and opsonises only weakly. Its heavy chains are of the type .

9. Write a note on anaphylaxis.

It belongs to Type I Hypersensitivity reaction. Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death. Anaphylaxis is triggered when the immune system overreacts (hypersensitivity) to a usually harmless substance (an allergen such as peanut or penicillin) causing mild to severe symptoms that affect various parts of the body. Symptoms usually appear within minutes to a few hours.

Symptoms of anaphylaxis may include:

Breathing: wheezing, shortness of breath, throat tightness, cough, hoarse voice, chest pain/tightness, trouble swallowing, itchy mouth/throat, nasal stuffiness/congestion Circulation: pale/blue colour, low pulse, dizziness, low blood pressure, shock, loss of consciousness Skin: hives, swelling, itch, warmth, redness, rash Stomach: nausea, pain/cramps, vomiting, diarrhoea
Page 44

Other: anxiety, itchy/red/watery eyes, headache

10.
i. ii. iii. iv. v.

Type I hypersensitivity give a note.

Type I hypersensitivity is also known as immediate or anaphylactic hypersensitivity. Immediate hypersensitivity is mediated by IgE. The primary cellular component in this hypersensitivity is the mast cell or basophil. IgE has very high affinity for its receptor on mast cells and basophils. A subsequent exposure to the same allergen cross links the cell-bound IgE and triggers the release of various pharmacologically active substances These pharmcologicaly active substances will lead to various allergic reactions. The reaction may involve: skin (urticaria and eczema), eyes (conjunctivitis), nasopharynx (rhinorrhea, rhinitis), bronchopulmonary tissues (asthma) and gastrointestinal tract (gastroenteritis).

11.

Write a note on serum sickness.

Serum sickness is a type III hypersensitivity reaction that results from the injection of foreign protein or serum. Mechanism: After an appropriate antigen is introduced, an individual's immune system responds by synthesizing antibodies after 4-10 days. The antibody reacts with the antigen, forming soluble circulating immune complexes that may diffuse into the vascular walls, where they may initiate fixation and activation of complement. Complement-containing immune complexes generate an influx of polymorphonuclear leukocytes into the vessel wall, where proteolytic enzymes that can mediate tissue damage are released. Immune complex deposition and the subsequent inflammatory response are responsible for the widespread vasculitic lesions seen in serum sickness.

Page 45

Symptoms:

Fever General ill feeling Hives Itching Joint pain Rash Swollen lymph nodes

Symptoms usually do not develop until 7 - 21 days after the first dose of antiserum or exposure to the medication. However, some people may develop symptoms in 1 - 3 days if they have previously been exposed to the substance. Treatment: Corticosteroid creams or ointments or other soothing skin medications may relieve discomfort from itching and rash. Antihistamines may shorten the length of illness and help ease rash and itching. Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen or naproxen, may relieve joint pain. Corticosteroids taken by mouth (such as prednisone) may be prescribed for severe cases. Medications causing the problem should be stopped, and future use of the medication or antiserum should be avoided.

12.

Write a short note on type III hypersensitivity.

Type III or Immune Complex hypersensitivity involves circulating antibody that reacts with free antigen. These circulating complexes can then become deposited on tissues. Tissue deposition may lead to reaction with complement, causing tissue damage. This type of hypersensitivity develops as a result of systematic exposure to an antigen and is dependent on i) the type of antigen and antibody and ii) the size of the resulting complex. More specifically, complexes that are too small remain in circulation; complexes too large are removed by the glomerulus; intermediate complexes may become lodged in the glomerulus leading to kidney damage. One example of a Type III hypersensitivity is serum sickness, a condition that may develop when a patient is injected with a large amount of serum e.g. antitoxin that was produced in an animal. After about 10 days, anti-antitoxin antibodies react with the antitoxin forming immune complexes that deposit in tissues. Type III hypersensitivities can be ascertained by intradermal injection of the antigen, followed by the observance of an "Arthus" reaction (swelling and redness at site of injection) after a few hours.

Page 46

13. Write a note on delayed hypersensitivity/Type IV hypersensitivity

Type IV hypersensitivity is also known as cell mediated or delayed type hypersensitivity. Delayed hypersensitivity reactions are inflammatory reactions initiated by mononuclear leukocytes. The term delayed is used to differentiate a secondary cellular response, which appears 48-72 hours after antigen exposure, from an immediate hypersensitivity response, which generally appears within 12 minutes of an antigen challenge. These reactions are mediated by T cells and monocytes/macrophages rather than by antibodies. They are also termed type IV hypersensitivity reactions. The classical example of this hypersensitivity is tuberculin (Montoux) reaction which peaks 48 hours after the injection of antigen (PPD or old tuberculin). The lesion is characterized by induration and erythema. Diagnostic tests in vivo include delayed cutaneous reaction (e.g. Montoux test and patch test (for contact dermatitis). In vitro tests for delayed hypersensitivity include mitogenic response, lympho-cytotoxicity and IL-2 production. Corticosteroids and other immunosuppressive agents are used in treatment.

14.

Write a short note on agglutination.

Agglutination is the clumping of cells such as bacteria or red blood cells in the presence of an antibody. The antibody or other molecule binds multiple particles and joins them, creating a large complex. Agglutination is commonly used as a method of identifying specific bacterial antigens, and in turn, the identity of such bacteria. Because the clumping reaction occurs quickly and is easy to produce, agglutination is an important technique in diagnosis. French physician Fernand Widal in 1896, used the agglutination reaction as the basis for a test for typhoid fever. Widal found that blood serum from a typhoid carrier caused a culture of typhoid bacteria to clump, whereas serum from a typhoid-free person did not. This Widal test was the first example of serum diagnosis. Austrian physician Karl Landsteiner found another important practical application of the agglutination reaction in 1900. Landsteiner's agglutination tests and his discovery of ABO blood groups was the start of the science of blood transfusion and

Page 47

serology which had made transfusion possible and safe.

15.

Write a short note on precipitation.

When soluble homologous antigen and antibody comes into contact in appropriate pH and temperature there will be a formation precipitate at the zone of equivalence. The precipitation reaction can be performed in two ways: i. In solutions ii. In gels The following are the important types of precipitation reactions in gel: Immunodiffusion Immunoelectrophoresis Serum electrophoresis.

Clinical applications: i. Used to detect antibodies against various infectious diseases ii. Used to identify antigens of various microorganisms eg. Identification of HBsAg. iii. Used to find the rise in titre of the antibody in certain infectious diseases. iv. Serum immunoelectrophoresis is used to find the immunoglobulin deficiency diseases.

16.

Define and classify hypersensitivity reactions.

Hypersensitivity (also called hypersensitivity reaction) refers to undesirable reactions produced by the normal immune system. Hypersensitivity reactions require a pre-sensitized (immune) state of the host. There are four types of hypersentivity reactions: Type 1: Immediate Hypersensitivity Reaction 1. Mediated by IgE antibody to specific antigens 1. Mast cells stimulated and release histamine 2. Reaction within seconds to one hour of exposure 3. Examples i. Anaphylaxis ii. Urticaria

Page 48

Antibody dependent Cell Cytotoxic Antibody Reaction 1. Mediated by IgG and IgM to specific antigens 2. Examples i. Transfusion Reaction ii. Rhesus Incompatibility (Rh Incompatibility) Type 3: Immune Complex mediated hypersentivity 1. Antigen-Antibody complexes deposit in tissue 2. Reaction within 1-3 weeks after exposure 3. Examples i. Systemic Lupus Erythematosus ii. Arthus Reaction (e.g. Farmer's Lung) iii. Rheumatoid Arthritis iv. Serum Sickness Type 4: Delayed-Type Hypersensitivity/cell mediated hypersentivity 1. Mediated by T-Lymphocytes to specific antigens i. Involves major histocompatibility complex (MHC) ii. Reaction within 2-7 days after exposure 2. Examples i. Mantaux Test ii. Allergic contact dermatitis (e.g. Nickel allergy)

17. Write a note on innate immunity. Innate (natural) immunity is so named because it is present at birth and does not have to be learned through exposure to an invader. It thus provides an immediate response to foreign cells. However, its components treat all foreign substances in much the same way. They recognize only a limited number of identifying substances (antigens) on foreign cells, although these antigens are present on many different cells. Innate immunity has no memory of the encounters and does not provide any lasting protection against future infection. The important innate immune mechanisms are:

i. Anatomical barrier like skin, mucous membrane etc. ii. Blood proteins like complement, acute phase proteins etc iii. Cells like natural killer cells, macrophages etc

Page 49

PART III SYSTEMATIC BACTERIOLOGY

1. Classify Staphylococcus. Outline the lesions produced by staphylococcus and describe the laboratory diagnosis of infections caused by staphylococcus. Add a note on its morphology, staining characteristics and its pathogenecity.

The main classification of staphylococci is based on their ability to produce coagulase, an enzyme that causes blood clot formation. Thus the staphylococci may be:
i.

Coagulase positive staphylococci Staphylococcus aureus saphrophyticus

ii. Coagulase negative staphylococci - S. epidermidis and S.

Morphology: The staphylococci are gram-positive cocci, usually arranged in grape-like irregular clusters. Staphylococci are non-motile and do not form spores. Pathogenecity A variety of enzymes and toxins are produced by Staphylococcus aureus. Staphylococcus aureus causes a variety of suppurative (pus-forming) infections and toxinoses in humans. It causes superficial skin lesions such as boils, styes and furuncules; more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections; and deep-seated infections, such as osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of superantigens into the blood stream.
Page 50

S. aureus expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues; (2) invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase); (3) surface factors that inhibit phagocytic engulfment (capsule, Protein A); (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production); (5) immunological disguises (Protein A, coagulase); (6) membrane-damaging toxins that lyse eucaryotic cell membranes (hemolysins, leukotoxin, leukocidin; 7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G, TSST, ET); and (8) inherent and acquired resistance to antimicrobial agents.

Diseases caused by S. aureus Laboratory diagnosis: Specimens Surface swab pus Blood Tracheal aspirate Spinal fluid
Page 51

depending upon the localization of the infection.

Smears Typical staphylococci appear as gram positive cocci in clusters in Gram-stained smears of pus or sputum. Culture Culture plate Blood agar - Grows as golden yellow colour colonies Mannitol salt agar - Grows as bright yellow colour colonies Catalase Test Staphylococci are catalase positive and the streptococci are catalase negative. Coagulase Test S. aureus coagulates dilute human serum or rabbit plasma (i.e. it is coagulasepositive), whereas S. epidermidis does not (coagulase-negative). This test could be done either in a test tube (the tube test), which requires overnight incubation or on a slide (the slide test), which is a rapid test.

Serologic and Typing Tests Protein A - latex agglutination test. S. aureus - agglutination S. epidermidis No agglutination

Page 52

2. Classify Streptococcus. Outline the lesions produced and the laboratory diagnosis of infections caused by Streptococcus pyogenes.

Classification of streptococcus:

Lesions produced by S. pyogenes: Infections of the skin can be superficial (impetigo) or deep (cellulitis and Erysipelas). Invasive streptococci cause joint or bone infections, destructive wound infections (necrotizing fasciitis) and myositis, meningitis and endocarditis. Impetigo is a highly contagious bacterial skin infection most common among preschool children. It is also known as school sores. Erysipelas is an acute streptococcus bacterial infection of the dermis, resulting in inflammation. This disease is most common among the elderly, infants, and children.

Page 53

Cellulitis is a diffuse inflammation of connective tissue with severe inflammation of dermal and subcutaneous layers of the skin. The typical symptom of cellulitis is an area which is red, hot, and tender. Necrotizing fasciitis (NF), commonly known as flesh-eating disease or Flesheating bacteria syndrome, is a rare infection of the deeper layers of skin and subcutaneous tissues, easily spreading across the fascial plane within the subcutaneous tissue.

Diagnostic Laboratory Tests Specimens Specimens to be obtained depend upon the nature of the streptococcal infection. A throat swab, pus, or blood is obtained for culture. Serum is obtained for antibody determinations. Smears Smears from pus often show single cocci or pairs rather than definite chains. Culture Specimens suspected of containing streptococci are cultured on blood agar plates. Blood cultures will grow haemolytic group A streptococci within hours or a few days. Certain alpha-haemolytic streptococci and enterococci may grow slowly. The degree and kind of haemolysis (and colonial appearance) may help place an organism in a definite group. S pyogenes can be identified by rapid tests specific for the presence of the group Aspecific antigen and by the PYR test. Streptococci belonging to group A may be presumptively identified by inhibition of growth by bacitracin, but this should be used only when more definitive tests are not available.

Antigen Detection Tests Several commercial kits are available for rapid detection of group A streptococcal antigen from throat swabs. These kits use enzymatic or chemical methods to extract the antigen from the swab, then use EIA or agglutination tests to demonstrate the presence of the antigen. The tests can be completed minutes to hours after the

Page 54

specimen is obtained. Serologic Tests A rise in the titer of antibodies to many group A streptococcal antigens can be estimated. Such antibodies include antistreptolysin O (ASO), particularly in respiratory disease; anti-DNase and antihyaluronidase, particularly in skin infections; antistreptokinase; anti-M type-specific antibodies; and others. Of these, the anti-ASO titer is most widely used.

3. Write a note on toxins and enzymes produced by streptococcus.

The following are some of the important toxins and enzymes produced by Group A streptococci: (1) M protein, fibronectin-binding protein (Protein F) and lipoteichoic acid for adherence; (2) hyaluronic acid capsule as an immunological disguise and to inhibit phagocytosis; M-protein to inhibit phagocytosis (3) invasins such as streptokinase, streptodornase (DNase B), hyaluronidase, and streptolysins; (4) exotoxins, such as pyrogenic (erythrogenic) toxin which causes the rash of scarlet fever and systemic toxic shock syndrome.

4. What is sore throat?

The most common infection due to beta-haemolytic S pyogenes is streptococcal sore throat or pharyngitis. S pyogenes adhere to the pharyngeal epithelium by means of lipoteichoic acidcovered surface pili. In infants and small children, the sore throat occurs as a subacute nasopharyngitis with a thin serous discharge and mild fever but with a tendency of the infection to extend to the middle ear and the mastoid. The cervical lymph nodes are usually enlarged. The illness may persist for weeks. In older children and adults, the disease is more acute and is characterized by intense
Page 55

nasopharyngitis, tonsillitis, and intense redness and oedema of the mucous membranes, with purulent exudate, enlarged, tender cervical lymph nodes, and (usually) a high fever. Twenty percent of infections are asymptomatic.

5. Write a note on streptococcal infections.

Name Streptococcus pyogenes Streptococcus agalactiae Enterococcus faecalis (and other enterococci) Common and Important Diseases Pharyngitis, impetigo, rheumatic fever, glomerulonephritis Neonatal sepsis and meningitis

Abdominal abscess, urinary tract infection, endocarditis

Viridans streptococci (many species) Streptococcus pneumoniae

Dental caries (S mutans), endocarditis, abscesses (with many other bacterial species) Pneumonia, meningitis, endocarditis

6. Write a note on viridans streptococcus.

The viridans streptococci include S mitis, S mutans, S salivarius, S sanguis, and others. Typically they are alpha-haemolytic, but they may be non-haemolytic. Their growth is not inhibited by Optochin, and colonies are not soluble in bile (deoxycholate). The viridans streptococci are the most prevalent members of the normal flora of the upper respiratory tract and are important for the healthy state of the mucous membranes there. They may reach the bloodstream as a result of trauma and are a principal cause of endocarditis on abnormal heart valves. Some viridans streptococci (eg, S mutans) synthesize large polysaccharides such as
Page 56

dextrans or levans from sucrose and contribute importantly to the genesis of dental caries. In the course of bacteremia, viridans streptococci, pneumococci, or enterococci may settle on normal or previously deformed heart valves, producing acute endocarditis. Rapid destruction of the valves frequently leads to fatal cardiac failure in days or weeks unless a prosthesis can be inserted during antimicrobial therapy.

7. Write a brief account on non-suppurative lesions of streptococci.

The nonsuppurative sequelae of Group A streptococci infections include the following:

Acute rheumatic fever Acute glomerulonephritis

Acute Rheumatic Fever This is the most serious sequela of S pyogenes because it results in damage to heart muscle and valves. The onset of rheumatic fever is often preceded by S pyogenes infection 14 weeks earlier, although the infection may be mild and may not be detected. In general, however, patients with more severe streptococcal sore throats have a greater chance of developing rheumatic fever Typical symptoms and signs of rheumatic fever include fever, malaise, a migratory nonsuppurative polyarthritis, and evidence of inflammation of all parts of the heart (endocardium, myocardium, pericardium). The carditis characteristically leads to thickened and deformed valves and to small perivascular granulomas in the myocardium (Aschoff bodies) that are finally replaced by scar tissue. Erythrocyte sedimentation rates, serum transaminase levels, electrocardiograms, and other tests are used to estimate rheumatic activity. Acute Glomerulonephritis This sometimes develops 3 weeks after S pyogenes skin infection (pyoderma, impetigo). Glomerulonephritis may be initiated by antigen-antibody complexes on the
Page 57

glomerular basement membrane. The most important antigen is probably in the streptococcal protoplast membrane. In acute nephritis, there is blood and protein in the urine, oedema, high blood pressure, and urea nitrogen retention; serum complement levels are also low. A few patients die; some develop chronic glomerulonephritis with ultimate kidney failure; and the majority recover completely.

8. Describe the morphology, staining characteristics, cultural characteristics, pathogenesis, laboratory diagnosis and prophylaxis of C. diptheriae.

Morphology Corynebacteria are 0.51 m in diameter and several micrometers long. Diphtheria bacteria are Gram-positive, pleomorphic, often club-shaped rods. The individual cells tend to group in V, Y, or palisade arrangements.

Culture characteristics C diphtheriae grow aerobically on most ordinary laboratory media. Loeffler's serum medium, corynebacteria grow much more readily than other respiratory organisms, and the organisms show typical morphology in smears. On blood agar, the C diphtheriae colonies are small, granular, and grey, with irregular edges, and may have small zones of haemolysis. On agar containing potassium tellurite, the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly.

Pathogenesis: Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response. The necrotic epithelium becomes embedded in exuding fibrin and red and white cells, so that a grayish "pseudomembrane" is formedcommonly over the tonsils, pharynx, or larynx. The regional lymph nodes in the neck enlarge, and there may be marked oedema of the entire neck. The diphtheria bacilli within the membrane continue to produce toxin actively. This is absorbed and results in distant toxic damage, particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle, liver, kidneys, and adrenals, sometimes accompanied by gross haemorrhage.
Page 58

The toxin also produces nerve damage, resulting often in paralysis of the soft palate, eye muscles, or extremities.

Laboratory diagnosis Specimen: The swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered. Swabs should be collected from beneath any visible membrane. The swab should then be placed in semisolid transport media such as Amies. Gram staining: Smears stained Gram stain show beaded rods in typical arrangement. Culture medium: Inoculate a blood agar plate (to rule out haemolytic streptococci), a Loeffler slant, and a tellurite plate (eg, cystine-tellurite agar) and incubate all at 37 C. In 1218 hours, the Loeffler slant may yield organisms of typical "diphtherialike" morphology. In 3648 hours, the colonies on tellurite medium are sufficiently definite for recognition of C diphtheriae.

A presumptive C diphtheriae isolate should be subjected to testing for toxigenicity. (1) A filter paper disk containing antitoxin is placed on an agar plate. The cultures to be tested for toxigenicity are spot innoculated 7 to 9 mm away from the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitate bands between the disk and the bacterial growth. This is the modified Elek method described by the WHO Diphtheria Reference Unit. (2) Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C diphtheriae isolates. (3) An immunochromographic strip assay allows detection of diphtheria toxin in a matter of hours. This assay is highly sensitive. Prophylaxis Protective immunization with diphtheria toxoid is the most important preventive measure. Such toxoids are commonly combined with tetanus toxoid (Td) and sometimes with pertussis vaccine (DPT) as a single injection to be used in
Page 59

initial immunization of children. For booster injection of adults, only Td toxoids are used.

9. Write a short note on Elek method?

This method is used to test the toxigenicity of the C. diphtheria that has been isolated from the clinical sample. Method: A filter paper disk containing antitoxin (antibody to toxin) is placed on an agar plate. The cultures to be tested for toxigenicity are spot innoculated 7 to 9 mm away from the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitate bands between the disk and the bacterial growth.

10.

Write a note on DPT vaccine.

DPT (also DTP and DTwP) refers to a class of combination vaccines against three infectious diseases in humans: diphtheria, pertussis (whooping cough) and tetanus. The vaccine components include diphtheria and tetanus toxoids, and killed whole cells of the organism that causes pertussis (wP). Five doses are commonly given to children between the ages of two months to five years old. They provide lifelong immunity, in most cases to diphtheria and pertussis, but do not provide lifelong immunity to tetanus. Tetanus vaccinations need to be repeated every 8-10 years in order to remain effective. Most children have mild reactions to the DPT vaccine. These include fever for a few days after receiving the DPT vaccine, and soreness at the shot site, which is usually the thigh in infants and the arms in older children.

Page 60

11. Classify spirochaetes and describe the laboratory diagnosis of syphilis.

Classification The spirochaetes are a large, heterogeneous group of spiral, motile bacteria. One family (Spirochaetaceae) of the order Spirochaetales consists of three genera of free-living, large spiral organisms. The other family (Treponemataceae) includes three genera whose members are human pathogens: (1) treponema, (2) borrelia, and (3) leptospira.

Laboratory diagnosis of syphilis Specimens The tissue fluid expressed from early surface lesions for demonstration of spirochaetes Blood for serologic tests.

Dark-field Examination A drop of tissue fluid or exudate is placed on a slide and a coverslip pressed over it to make a thin layer. The preparation is then examined under oil immersion with dark-field illumination for typical motile spirochetes.

Immunofluorescence Tissue fluid or exudate is spread on a glass slide, air dried, and sent to the laboratory. It is fixed, stained with a fluorescein-labeled antitreponeme serum, and examined by means of immunofluorescence microscopy for typical fluorescent spirochetes.

Serologic Tests for Syphilis (STS) These tests use either nontreponemal or treponemal antigens. Nontreponemal Antigen Tests The VDRL (Venereal Disease Research Laboratory) and RPR (rapid plasma reagin) tests are nontreponemal antigen tests used most commonly. The toluidine red unheated serum test (TRUST) also is available. The antigens employed are cardiolipids extracted from normal mammalian tissue. Lecithin and cholesterol are added to enhance reaction with syphilitic
Page 61

"reagin" antibodies. Reagin antibodies are the mixture of IgM and IgG antibodies directed against the cardiolipin-cholesterol-lecithin complex produced due to the damage of the tissues during syphilis. The particles of the lipid antigen remain dispersed with normal serum but flocculate when combining with reagin. The VDRL test requires microscopic examination to detect flocculation, whereas the RPR and TRUST have added coloured particles and can be read without microscopic magnification. Results develop within a few minutes, particularly if the suspension is agitated.

Treponemal Antibody Tests Fluorescent Treponemal Antibody (FTA-ABS) Test This is a test employing indirect immunofluorescence (killed T pallidum + patient's serum + labeled antihuman gamma globulin). The FTA-ABS test is the first to become positive in early syphilis, is routinely positive in secondary syphilis, and usually remains positive many years after effective treatment.

Treponema pallidum-Particle Agglutination (TP-PA) Test These are the T pallidum hemagglutination (TPHA) and microhemagglutination for T pallidum(MHA-TP) tests. Particles are sensitized with T pallidum subspecies pallidum antigens. The test is performed with diluted serum. Antibodies against T pallidum react with the sensitized particles. A mat of agglutinated particles indicates a positive result.

12.

Write a short note on Hutchinsons teeth.

Hutchinson's teeth (also known as Hutchinson's incisor, Hutchinson's sign or Hutchinson-Boeck teeth) are a sign of congenital syphilis in which the incisal edge is notched and narrower than the neck area at the gums. Babies with this have teeth that are smaller and more widely spaced than normal and which have notches on their biting surfaces. It is named after Sir Jonathan Hutchinson, an English surgeon and pathologist, who first described them.

Page 62

13.

Write an account on VDRL test.

The VDRL (Venereal Disease Research Laboratory) test is nontreponemal antigen tests used most commonly to diagnose syphilis. The antigens employed are lipids extracted from normal mammalian tissue. The purified cardiolipin from beef heart is a diphosphatidylglycerol. Lecithin and cholesterol are added to enhance reaction with syphilitic "reagin" antibodies. Reagin antibodies are mixture of IgM and IgG antibodies directed against the cardiolipin-cholesterol-lecithin complex formed due to tissue destruction. The test is based on the principle that the particles of the lipid antigen flocculate when combining with regain present in the patients sera. The VDRL test requires microscopic examination to detect flocculation. Flocculation indicates a positive VDRL test.

14.

Write a note on yaws.

Yaws is endemic, particularly among children, in many humid, hot tropical countries. It is caused by T pallidum subspecies pertenue. The primary lesion, an ulcerating papule, occurs usually on the arms or legs. Transmission is by person-to-person contact in children under age 15. Transplacental, congenital infection does not occur. Scar formation of skin lesions and bone destruction are common, but visceral or nervous system complications are very rare. It has been debated whether yaws represents a variant of syphilis adapted to transmission by nonsexual means in hot climates. There appears to be cross-immunity between yaws and syphilis. Diagnostic procedures and therapy are similar to those for syphilis. The response to penicillin treatment is dramatic.

15. Write a short account on the morphology of Treponema pallidum.

They are long, slender spiral, gram-negative bacilli. Slender spirals measuring about 0.2 m in width and 515 m in length. The spiral coils are regularly spaced at a distance of 1 m from one another.
Page 63

The organisms are actively motile, rotating steadily around their endoflagella even after attaching to cells by their tapered ends. T pallidum has an outer sheath or glycosaminoglycan coating. Inside the sheath is the outer membrane, which contains peptidoglycan and maintains the structural integrity of the organisms. The spirals are so thin that they are not readily seen unless immunofluorescent stain or darkfield illumination is employed. They do not stain well with aniline dyes, but they can be seen in tissues when stained by a silver impregnation method.

16. Mention the organisms causing enteric fever. Describe the laboratory diagnosis of Enteric fever caused by Salmonella typhi.

There are four types of Salmonella that causes enteric fever. They are as follows: i. ii. iii. iv. Salmonella paratyphi A (serogroup A), Salmonella paratyphi B (serogroup B), Salmonella choleraesuis (serogroup C1), and Salmonella typhi (serogroup D).

Diagnostic Laboratory Tests Specimens Blood for culture must be taken repeatedly. In enteric fevers and septicemias, blood cultures are often positive in the first week of the disease. Bone marrow cultures may be useful. Urine cultures may be positive after the second week. Stool specimens also must be taken repeatedly. In enteric fevers, the stools yield positive results from the second or third week on

Bacteriologic Methods for Isolation of Salmonella Culture medium Differential Medium MacConkey agar permits rapid detection of Non- lactose fermenters (NLF) Bismuth sulphite medium permits rapid detection of salmonellae which form black colonies because of H2S production. Many salmonellae produce H2S.

Page 64

Selective Medium Cultures The specimen is plated on salmonella-shigella (SS) agar, Hektoen enteric agar, XLD, or deoxycholate-citrate agar, which favour growth of salmonellae and shigellae over other Enterobacteriaceae. Enrichment Cultures The specimen (usually stool) also is put into selenite F or tetrathionate broth, both of which inhibits replication of normal intestinal bacteria and permit multiplication of salmonellae. After incubation for 12 days, this is plated on differential and selective media. Final Identification Suspected colonies from solid media are identified by biochemical reaction patterns and slide agglutination tests with specific sera. Serologic Methods Agglutination Test In this test, known sera and unknown culture are mixed on a slide. Clumping, when it occurs, can be observed within a few minutes. This test is particularly useful for rapid preliminary identification of cultures. There are commercial kits available to agglutinate and serogroup salmonellae by their O antigens Tube Dilution Agglutination Test (Widal Test) Serum agglutinins rise sharply during the second and third weeks of Salmonella typhi infection. The Widal test to detect these antibodies against the O and H antigens has been in use for decades. At least two serum specimens, obtained at intervals of 710 days, are needed to prove a rise in antibody titer. Serial dilutions of unknown sera are tested against antigens from different salmonellae species. The interpretive criteria when single serum specimens are tested vary, but a titer against the O antigen of > 1:100 and against the H antigen of > 1:200 is considered positive. Results of serologic tests for salmonella infection must be interpreted cautiously because the possible presence of cross-reactive antibodies limits the use of serology.

Page 65

17.

Write a note on widal test.

Widal test is a tube agglutination test employed in the serological diagnosis of enteric fever. The test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist Serum agglutinins rise sharply during the second and third weeks of Salmonella typhi infection. The Widal test to detect these antibodies against the O and H antigens has been in use for decades. At least two serum specimens, obtained at intervals of 710 days, are needed to prove a rise in antibody titer.

Procedure: Patient serum is doubly diluted by mixing and transferring from 1:10 to 1:640 in three-four rows. First row usually comprises of Felix tubes, where somatic S.typhi O antigen is added. For all the remaining rows, Dreyers tubes are taken; where different flagellar H antigens are added. Each tube must contain 0.5ml of diluted serum. A test tube with only saline is kept in each row as control. All the tubes (including control) in a row are mixed with 0.5ml of antigen suspension. The first row is treated with S.typhi O antigen, the second row with S.typhi H antigen, the third row with S.paratyphi AH antigen and the fourth row with S.paratyphi BH antigen. Since infections by S.paratyphi B are rare, this antigen is usually omitted in the test. After all the tubes have been treated with specific antigen\ suspensions, the widal rack is placed in a thermostatically controlled water bath maintained at 37oC for overnight incubation

Reading the results: The control tubes must be examined first, where they should give no agglutination. The agglutination of O antigen appears as a matt or carpet at the bottom. Agglutination of H antigens appears loose, wooly or cottony. The highest dilution of serum that produces a positive agglutination is taken as titre. The titres for all the antigens are noted.

Page 66

Interpretation of results: The interpretive criteria when single serum specimens are tested vary, but a titer against the O antigen of > 1:100 and against the H antigen of > 1:200 is considered positive. Results of serologic tests for salmonella infection must be interpreted cautiously because the possible presence of cross-reactive antibodies limits the use of serology.

18. Mention the causative agents of gas gangrene. Describe the laboratory diagnosis and prophylaxis of gas gangrene.

The most frequent causative pathogen in gas gangrene is Clostridium perfringens. Laboratory diagnosis: Specimens: Specimens consist of material from wounds, pus, and tissue. Gram staining: The presence of large gram-positive rods in Gram-stained smears suggests gas gangrene clostridia; spores are not regularly present. Culture: Material is inoculated into chopped meat-glucose medium and thioglycolate medium and onto blood agar plates incubated anaerobically. The growth from one of the media is transferred into milk. A clot torn by gas in 24 hours is suggestive of C perfringens. Lecithinase activity is evaluated by the precipitate formed around colonies on egg yolk media. Final identification rests on toxin production and neutralization by specific antitoxin.

Prophylaxis: Penetrating abdominal wounds should be surgically explored and drained, any tears in the intestinal walls closed, and antibiotic treatment begun early. Patients undergoing elective intestinal surgery should receive preventive antibiotic therapy. Use of antibiotics prior to and directly following surgery has been shown to
Page 67

significantly reduce the rate of infection from 2030% to 48%. Any abrasion, break in the skin, or infection tissue should be cared for immediately. Any dying or infected skin must be removed promptly to prevent the spread of bacteria. No vaccine is available.

19.

Write a short note on gas gangrene.

Gas gangrene is a bacterial infection that produces gas within tissues. It is a deadly form of gangrene usually caused by Clostridium perfringens bacteria. Infection spreads rapidly as the gases produced by bacteria expand and infiltrate healthy tissue in the vicinity. Because of its ability to quickly spread to surrounding tissues, gas gangrene should be treated as a medical emergency. Gas gangrene is caused by a bacterial exotoxin-producing clostridial species, which are mostly found in soil and other anaerobes (e.g., Bacteroides and anaerobic streptococci). These environmental bacteria may enter the muscle through a wound and subsequently proliferate in necrotic tissue and secrete powerful toxins. These toxins destroy nearby tissue, generating gas at the same time. Gas gangrene can cause necrosis, gas production, and sepsis. Progression to toxemia and shock is often very rapid.

20. Describe the morphology, pathogenesis, laboratory diagnosis and immunisation of Clostridium tetani.

Morphology: The clostridia are large anaerobic, gram-positive rods. They are motile and possess peritrichous flagella Spores of clostridia are usually wider than the diameter of the rods in which they are formed.

Pathogenesis: C tetani is not an invasive organism. The infection remains strictly localized in the area of devitalized tissue (wound, burn, injury, umbilical stump, surgical suture) into which the spores have been introduced.
Page 68

Germination of the spore and development of vegetative organisms that produce toxin are aided by (1) necrotic tissue, (2) calcium salts, and (3) associated pyogenic infections, all of which aid establishment of low oxidation-reduction potential. The toxin released from vegetative cells reaches the central nervous system and rapidly becomes fixed to receptors in the spinal cord and brain stem and exerts its actions. Release of the inhibitory glycine and gamma-aminobutyric acid is blocked, and the motor neurons are not inhibited. Hyperreflexia, muscle spasms, and spastic paralysis result. Extremely small amounts of toxin can be lethal for humans.

Laboratory diagnosis: The lab diagnosis is made by demonstration of Cl. tetani by microscopy, culture or animal inoculation. Microscopy is unreliable The wound is inoculated on one half of a blood agar plate. Cl. tetani produces a swarming growth which may be detected on the opposite half of the plate after 1-2 days incubation anaerobically. The material is also incubated in cooked meat broth and further subcultured on blood agar plate for further detection. Toxigenecity test is performed in blood agar plate by incorporating the antitoxin which will neutralise the toxin and thereby the cultures will not show the haemolysis around their colonies. Toxigenecity test can also be performed in animals with the inoculation of pure culture and the antitoxin into the animals.

21. Write an account on immunisation against tetanus.

Tetanus is a totally preventable disease. Universal active immunization with tetanus toxoid should be mandatory. Tetanus toxoid is produced by detoxifying the toxin with formalin and then concentrating it. Aluminum-salt-adsorbed toxoids are employed. Three injections comprise the initial course of immunization, followed by another dose about 1 year later. Initial immunization should be carried out in all children during the first year of life. Thereafter, "boosters" can be spaced 10 years apart to maintain serum levels of more than 0.01 unit antitoxin per milliliter. In young children, tetanus toxoid is often combined with diphtheria toxoid
Page 69

and pertussis vaccine. (Note: Toxoid means inactivated toxin which can induce immunity but it is non toxic)

22. Describe the morphology, staining characteristics, cultural characteristics, pathogenesis, laboratory diagnosis and prophylaxis of pulmonary tuberculosis.

Morphology: In tissue, tubercle bacilli are thin straight rods measuring about 0.4 x 3 m. On artificial media, coccoid and filamentous forms are seen with variable morphology from one species to another.

Staining properties: Mycobacteria cannot be classified as either gram-positive or gram-negative. Once stained by basic dyes they cannot be decolorized by alcohol, regardless of treatment with iodine. True tubercle bacilli are characterized by "acidfastness"ie, 95% ethyl alcohol containing 3% hydrochloric acid (acidalcohol) quickly decolorizes all bacteria except the mycobacteria. Acid-fastness depends on the integrity of the waxy envelope. The Ziehl-Neelsen technique of staining is employed for identification of acid-fast bacteria. In smears of sputum or sections of tissue, mycobacteria can be demonstrated by yellow-orange fluorescence after staining with fluorochrome stains (eg, auramine, rhodamine).

Culture characters: Pathogenesis: Mycobacteria in droplets 15 m in diameter are inhaled and reach alveoli. The disease results from establishment and proliferation of virulent organisms and interactions with the host. In the majority of cases, the pathogens enter the lung in droplets, where they are phagocytosed by alveolar macrophages. TB bacteria are able to reproduce in these macrophages due to their ability to inhibit formation of the phagolysosome. Within 1014 days the TB bacteria move into the regional lymph nodes, where
Page 70

they reproduce and stimulate a cellular immune response. The Ghons complex (primary complex, PC) develops between six and 14 weeks after infection. At the same time, granulomas form at the primary infection site and in the affected lymph nodes, and macrophages are activated by the cytokine MAF (macrophage activating factor).

Laboratory diagnosis: It requires microscopic and culture identification of bacteria. Microscopy: Sputum, exudates, or other material is examined for acid-fast bacilli by ZiehlNeelsen staining. Fluorescence microscopy with auramine-rhodamine stain is more sensitive than acid-fast stain. If acid-fast organisms are found in an appropriate specimen, this is presumptive evidence of mycobacterial infection.

Culture: A selective agar media i.e.Lwenstein-Jensen medium should be inoculated with the specimen in parallel with broth media cultures. Incubation is at 3537 C in 510% CO2 for up to 8 weeks. If cultures are negative in the setting of a positive acid-fast stain or slowly growing atypical mycobacteria are suspected, then a set of inoculated media should be incubated at a lower temperature (eg, 2433 C) and both sets incubated for 12 weeks.

DNA Detection The polymerase chain reaction (PCR) holds great promise for the rapid and direct detection of M tuberculosis in clinical specimens.

23.

Write a note on Tuberculin/Mantoux test.

The tuberculin test is a type of skin test performed to diagnose the tuberculosis infection.
Page 71

It is also called as Mantoux test. The tuberculin proteins are isolated as purified tuberculin (PPD = purified protein derivative). Five tuberculin units (TU) are applied intracutaneously in the tuberculin test. A positive reaction appears within 48 to 72 hours as an inflammatory reaction (induration) at least 10mm in diameter at the site of antigen application. A positive reaction means that the person has either been infected with TB.

24.

Write a note on BCG.

Bacillus Calmette-Gurin (or Bacille Calmette-Gurin, BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus, Mycobacterium bovis, that has lost its virulence in humans by being specially cultured in an artificial medium for years. The bacilli have retained enough strong antigenicity to become a somewhat effective vaccine for the prevention of human tuberculosis. At best, the BCG vaccine is 80% effective in preventing tuberculosis for a duration of 15 years. BCG is given as a single intradermal injection at the insertion of the deltoid. BCG immunization leaves a characteristic raised scar that is often used as proof of prior immunization.

25.

Write a note on Acid fast bacilli.

Acid-fast bacilli (AFB) are rod-shaped bacteria that can be seen and counted under the microscope on a specially stained sample on a glass slide, called an AFB smear. The most common acid-fast bacilli are members of the genus Mycobacterium. Mycobacterial cell walls contain a waxy substance composed of mycolic acids. Hence cannot be stained by gram staining method. The most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen stain, in which the acid fast bacilli are stained bright red and stand out clearly against a blue background. Another method is the Kinyoun method, in which the bacteria are stained bright red and stand out clearly against a green background. The two important acid fast bacilli are Mycobacterium tuberculosis and Mycobacterium leprae.

Page 72

26.

Write a short account on Mycobacterium leprae.

Mycobacterium leprae (described by Hansen in 1873) is the causative pathogen of leprosy. They are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain readily, once stained they resist decolorization by acid and are therefore called "acid-fast" bacilli. It has not been cultivated on nonliving bacteriologic media. When bacilli from human leprosy (ground tissue nasal scrapings) are inoculated into footpads of mice, local granulomatous lesions develop with limited multiplication of bacilli. Inoculated in the armadillos ( a mammal) will develop extensive lepromatous leprosy. The leprosy is divided into two major types, lepromatous and tuberculoid, with several intermediate stages

27.

Write a short note on Actinomycetes.

Gram-positive branching filamentous bacteria that sporulate or fragment There are two medically important genuses. They are actinomyces and nocordia. Actinomyces: Actinomyces israelii the commonest Has branching filaments Facultative anaerobes Normal flora of oral cavity Causes Actinomycosis characterised by multiple abscess and granuloma formation Tissue destruction, fibrosis and sinus formation Other species- causes dental plaque, gingivitis and periodontitis

Nocordia: Nocardia infection is acquired by inhalation of or direct skin inoculation (traumatic) by environmental organisms

Page 73

Nocardia asteroides Pulmonary disease Gram positive, branched, strictly aerobic Environmental saprophytes (exogenous infection) Lightly acid-fast Causes pulmonary diseases- pneumonia and lung abscess

Nocardia brasiliensis Skin abscesses Nocardia brasiliensis is the most common cause of cutaneous nocardiosis. It is typically associated with traumatic inoculation of contaminated soil. Unlike N. asteroides, N. brasiliensis is more commonly a disease of the immunodeficients

28.

What is actinomycosis?

Actinomycosis is an infectious bacterial disease caused by Actinomyces species such as Actinomyces israelii. It can also be caused by Propionibacterium propionicus. Actinomycosis occurs rarely in humans but rather frequently in cattle as a disease called lumpy jaw. This name refers to the large abscesses that grow on the head and neck of the infected animal. The disease is characterized by the formation of painful abscesses in the mouth, lungs, or gastrointestinal tract. Actinomycosis abscesses grow larger as the disease progresses, often over months. In severe cases, they may penetrate the surrounding bone and muscle to the skin, where they break open and leak large amounts of pus. The purulent leakage via the sinus cavities contains "sulphur granules," not actually sulphur-containing but resembling such particles. These granules contain aggregates of progeny bacteria.

29.

Write a short account on sulphur granules.

They are seen in pus specimens obtained in actinomycosis. Pus from draining sinuses, sputum, or specimens of tissue are examined for the presence of sulphur granules (yellow colour granules). The granules are hard, lobulated, and composed of tissue and bacterial filaments, which are club-shaped at the periphery. Keep the separated granules between 2 slides
Page 74

Crush and gram stain Gram positive branching filaments are seen

30.

Write a note on bacteroids.

Bacteroides comprise a major portion of the human normal flora, predominating in the intestinal tract. These organisms are, like other anaerobes, generally opportunistic and can cause a variety of infections throughout the body. The most common infections include pleuropulmonary, intraabdominal and infections of the female urogenital tract. Bacteroides make up about one-third of the total anaerobic isolates obtained from various infections. B. fragilis is particularly important because of 1) the frequency of isolation and 2) its resistance to antibiotics. Bacteroides produce several exoenzymes including collagenase, neuraminidase, DNAse, heparinase and some proteases. These enzymes may play a role in the pathogenesis of the organism, assisting the bacteria in the invasion of host tissues following an initial trauma.

31.

Write a note on Borrelia vincentii.

Borrelia vincentii is an organism that exists normally in the human mouth in low concentrations and safe proportions. It is an anaerobic spirochete that is also Gram-negative. When allowed to grow past regular conditions many adverse effects can occur causing much pain and discomfort to the mouth and throat. The disease caused by Borrelia vincentii was discovered by French physician Henri Vincent, its common name is Vincents Disease or Vincent s Angina. It is also widely known as Trench Mouth, due to an outbreak in soldiers in trenches during World War. Vincents Disease can be passed very easily, which is why it is important to have good oral hygiene. Kissing, unwashed drinking glasses, or sharing eating utensils can transmit Vincents Disease. Thus those with poor oral hygiene are more at risk of contracting the disease. On top of that other factors such as stress, poor diet and nutrition, tobacco usage, and already having a systematic disease can all increase chances of getting the disease. Symptoms include foul breath, ulcers in the inter-dental papillae, ulcers on the gums that easily bleed, increase in the space between teeth, gums covered with white/gray
Page 75

layer of dead skin, and fever accompanied with fatigue.

32.

Name the organisms causing meningitis.

Meningitis is an inflammation of the meninges, the membranes that cover the brain and spinal cord. It is usually caused by bacteria or viruses, but it can also be caused by certain medications or illnesses. Many different types of bacteria can cause bacterial meningitis. In newborns, the most common causes are Group B streptococcus, Escherichia coli, and Listeria monocytogenes. In children, Streptococcus pneumoniae (pneumococcus) and Neisseria meningitidis (meningococcus) are more often the causes. Haemophilus influenza type b (Hib), can also cause the illness but because of widespread childhood immunization, these cases are now rarer. Similarly, many different viruses can lead to viral meningitis, including enteroviruses (such as coxsackievirus, poliovirus, and hepatitis A) and the herpesvirus.

33.

Write a short note on Lactobacilli.

Lactobacillus is a genus of Gram-positive facultative anaerobic or microaerophilic bacteria Lactobacilli are saprophytes in vegetable and animal material (e.g. milk). Lactobacilli are found as commensals in the oral cavity, gastrointestinal tract and female genital tract. In the oral cavity they constitute less than 1% of the total flora. Although considered beneficial, some Lactobacillus species have been associated with dental caries. Lactobacillus count in saliva has been used as a "caries test" for many years. Transmission routes are unknown. Lactobacilli are major constituents of the vaginal flora and help maintain its low pH equilibrium. Lactobacilli grow under microaerophilic conditions in the presence of carbon dioxide and at acidic pH (6.0). Media enriched with glucose or blood promotes growth.
Page 76

PART IV MYCOLOGY
1. Write a short note on Candida albicans.
C. albicans is a Gram-positive, budding, oval yeast with a diameter of approximately 5 m. Gram-positive pseudohyphae are observed frequently and septate mycelia occasionally. Candida albicans is the normal commensal of oral cavity, GIT, female genital tract and sometimes even on skin. Candida albicans is a dimorphic fungus, i.e. it can take two forms. At normal temperature it exists as oval, single yeast At body temperature, pH, and the presence of serum it may develop into a hyphal form. Pseudohyphae, composed of chains of cells, are also common. C. albicans can be grown on the Sabouraud dextrose agar culture medium. After 48 hours of incubation on agar mediums, round, whitish, somewhat rough-surfaced colonies form. The important diseases (Candidiasis) caused by C. albicans are Oral Thrush Oesophagitis Cutaneous Candidiasis Vaginitis Systemic Candidiasis

2. Write a short note on candidal infections/candidiasis.

It can be either superficial or systemic candidiasis. Superficial candidiasis Oral thrush Oral thrush is an infection of yeast fungus, Candida albicans, in the mucous membranes of the mouth. Pseudomembranous white/yellow colonies or clusters, appearing anywhere in the oral cavity May be discrete or extensive Can be easily removed by wiping
Page 77

The people whose dentures don't fit well can sustain breaks in the mucous membranes in their mouth, which can act as a gateway for candida. People who suffer from this problem often have moist, pale pink spots on their lips, known as angular cheilitis, which is an indication of a candida infection.

Oesophagitis Pseudomembranous lesions extend into lower pharynx and oesophagus causing difficulty swallowing, nausea, and retrosternal and epigastric pain

Cutaneous Candidiasis In cutaneous candidiasis, the skin is infected with candida fungi. Infection can involve almost any skin on the body, but most often it occurs in warm, moist, creased areas such as the armpits and groin. The fungus that most often causes cutaneous candidiasis is Candida albicans.

Vaginal candidiasis Vaginal candidiasis, called yeast infection or vaginitis, is an infection of the vulva and/or vagina. It causes a smelly, thick, white-yellow discharge that might occur with itching, burning and swelling.

Systemic candidiasis Systemic candidiasis is when Candida spreads throughout the body, and it can be life-threatening. Infection might include the brain, heart, kidneys, eyes, liver, genital tract and joints. This form occurs most often in people with low white blood cell counts (neutropenia). This type of infection is also called disseminated candidiasis.

3. Write a short note on Mycetoma.

Mycetoma (also known as "Madura foot,"and "Maduromycosis") is a granulomatous infection of dermal and subcutaneous tissue that may extend to muscle or even bone. Chronic subcutaneous infection caused by actinomycetes or fungi caused by fungi are called eumycetoma (40%) Actinomycetoma is caused by actinomycetes (60%) It is characterized by the formation of abscesses, which contain large aggregates of fungal or actinomycete filaments known as grains
Page 78

4. Write a short note on opportunistic fungal infections/opportunistic mycosis.

These are fungal infections of the body which occur almost exclusively in patients whose normal defence mechanisms are impaired. Weakened immune function may occur due to inherited immunodeficiency diseases, drugs that suppress the immune system (cancer chemotherapy, corticosteroids, drugs to prevent organ transplant rejection), radiation therapy, infections (e.g., HIV), cancer, diabetes, advanced age and malnutrition. The organisms involved are fungi which have a very low inherent virulence. The most common infections are: Aspergillosis Candidiasis Cryptococcosis Pneumocystis carinii Zygomycosis

Cryptococcus neoformans Organism is ubiquitous and infections occur worldwide C. neoformans recovered in large amounts in pigeon poop. Does not cause disease in birds. Primary site of human infection is the lungs Cryptococcal meningitis is most common disseminated manifestation. Can spread to skin, bone and prostate.

Aspergillus These molds are widely distributed in nature. They grow on decaying vegetation, producing chains of conidia. Transmission is by airborne conidia. In Aspergillosis, germination and colonization occurs commonly on ear, nasal cavity, paranasal sinus, and open pulmonary cavity surfaces, with minimal tissue invasion.

Candida albicans C. albicans is a member of the indigenous microbial flora of humans. Found in the gastrointestinal tract, upper respiratory tract, buccal cavity, and vaginal tract. Growth is normally suppressed by other microorganisms found in these areas.
Page 79

Alterations of gastrointestinal flora by broad spectrum antibiotics or mucosal injury can lead to gastrointestinal tract invasion. Skin and mucus membranes are normally an effective barrier but damage by introduction of catheters or intravascular devices can permit Candida to enter the bloodstream.

Pneumocystis carinii a small, unicellular fungus that causes pneumonia (PCP), the most prominent opportunistic infection in AIDS patients this pneumonia forms secretions in the lungs that block breathing & can be rapidly fatal if not controlled with medication

Rhizopus, Absidia, & Mucor Zygomycota are extremely abundant saprobic fungi found in soil, water, organic debris, & food Genera most often involved are Rhizopus, Absidia, & Mucor Usually harmless air contaminants invade the membranes of the nose, eyes, heart, & brain of people with diabetes, malnutrition with severe consequences (Zygomycosis).

Page 80

PART V PARASITOLOGY

1. Write a note on black water fever.

Blackwater fever is a complication of malaria characterized by intravascular haemolysis, haemoglobinuria and kidney failure. Blackwater fever is caused by heavy parasitization of red blood cells with Plasmodium falciparum. Within a few days of onset there are chills, with rigor, high fever, jaundice, vomiting, rapidly progressive anaemia and the passage of dark red or black urine. There is rapid and massive destruction of red blood cells (RBCs) with the production of haemoglobinaemia (haemoglobin in the blood, but outside the RBCs), haemoglobinuria (haemoglobin in urine), intense jaundice, anuria (passage of less than 50 milliliter of urine in a day), and finally death in the majority of cases. The most probable explanation for blackwater fever is an autoimmune reaction.

2. Write a short note on hydatid cyst.

Hydatid cyst is the larval cystic stage (called echinococcal cysts) of tapeworm Echinococcus granulosus that may cause illness in intermediate hosts, generally herbivorous animals and human who are infected accidentally. The hydatid cysts, (from hydatis = water bladder) is normally a fluid-filled cyst with one or multiple chambers, the wall of which is made up of an inner, cellular, germinative layer and an outer, acellular, laminated layer (cuticular layer), enclosed by a layer of host connective tissue. Brood capsules develop five to six months on the germinative layer, each containing up to 20 or more protoscoleces with four suckers and two rows of rostellar hooks. The thin brood capsules burst to release free protoscoleces into the hydatid fluid, which form, together with the brood capsules, their remains and calcareous corpuscles the so-called hydatid sand. The size of the cysts depends on their age and other factors. The average cyst diameter in humans is 115 cm, although it can vary between a few mm and 20 cm. Cysts in humans often contain smaller daughter cysts.
Page 81

3. Write a short account on Ascaris lumbricoides.

Ascaris lumbricoides is the giant roundworm of human, belonging to the phylum Nematoda. It is responsible for the disease ascariasis in human. It is the largest and most common parasitic worm in humans. Ascaris lumbricoides is characterized by its great size. Males are 24 mm in diameter and 1531 cm long. The males' posterior end is curved ventrally and has a bluntly pointed tail. Females are 36 mm wide and 2049 cm long. Ascaris lumbricoides, or "roundworm", infections in humans occur when an ingested infective egg releases a larval worm that penetrates the wall of the duodenum and enters the blood stream. From here, it is carried to the liver and heart, and enters pulmonary circulation to break free in the alveoli, where it grows and molts. In 3 weeks, the larvae pass from the respiratory system to be coughed up, swallowed, and thus returned to the small intestine, where they mature to adult male and female worms. Fertilization can now occur and the female produces as many as 200,000 eggs per day for a year. These fertilized eggs become infectious after 2 weeks in soil;
Page 82

they can persist in soil for 10 years or more.

4. Write an account on morphology of trophozoites of Entamoeba histolytica.

After the metacyst has been ingested by the host it will be transformed into trohozoites which are metabolically active. E. histolytica trophozoites have an amorphous shape and are generally 15-30 m in diameter. The trophozoites move by extending a finger-like pseudopodium (psd) and pulling the rest of the body forward (called ameboid movement). The pseudopodia, and sometimes the outer edge of the trophozoite, have a clear refractile appearance and is referred to as the ectoplasm (ecto). The rest of the cytoplasm has a granular appearance and is called the endoplasm (endo). Occasionally a glycogen vacuole (vac) is evident. Nuclear (Nu) morphology in stained specimens is characterized by a finely granular ring of peripheral chromatin and a centrally located karyosome (ka).

5. Write a short account on life cycle of Ascaris lumbricoides.

Ascaris lumbricoides, or "roundworm", infections in humans occur when an ingested infective egg releases a larval worm that penetrates the wall of the duodenum and enters the blood stream. From here, it is carried to the liver and heart, and enters pulmonary circulation to break free in the alveoli, where it grows and molts. In 3 weeks, the larvae pass from the respiratory system to be coughed up, swallowed, and
Page 83

thus returned to the small intestine, where they mature to adult male and female worms. Fertilization can now occur and the female produces as many as 200,000 eggs per day for a year. These fertilized eggs become infectious after 2 weeks in soil; they can persist in soil for 10 years or more.

6. Write a note on laboratory diagnosis of Hook worm infection.

During the early stages of infection, diagnosis of hookworm disease depends on clinical symptoms along with appropriate travel or exposure history. In The definitive test is examination of stool for hookworm eggs, and it takes about five months for worms to mature and begin producing eggs. In some cases it may take even longer. Diagnosis depends on finding characteristic worm eggs on microscopic examination of the stools, although this is not possible in early infection. The eggs are oval or elliptical, measuring 60 m by 40 m, colourless, not bile stained and with a thin transparent hyaline shell membrane. When released by the worm in the intestine, the
Page 84

egg contains an unsegmented ovum. During its passage down the intestine, the ovum develops and thus the eggs passed in faeces have a segmented ovum, usually with 4 to 8 blastomeres.

Adult worms are rarely seen (except via endoscopy, surgery or autopsy), but if found, would allow definitive identification of the species. Because hookworm eggs are often indistinguishable from other parasitic eggs, PCR assays could serve as a molecular approach for accurate diagnosis of hookworm in the faeces.

7. Write a note on life cycle of Entamoeba histolytica.

Life cycle of Entamoeba histolytica. Cysts with four nuclei (i.e. metacysts) are ingested orally with contaminated food or drinking water. After excysting in the small intestine, both the cytoplasm and nuclei divide to form eight small amebulae (i.e. metacystic trophozoites).

Page 85

Mature trophozoites (i.e. minuta forms) reproduce by constant binary fission. Uninucleate cyst (i.e. precyst) contains chromatoid bodies and (often) a large glycogen vacuole. Cysts with two nuclei and chromatoid bodies. Cysts with four nuclei (metacysts) are set free with the faeces and become infectious when ingested by man.

8. Write a note on benign tertian Malaria.

This malaria is caused by Plasmodium vivax. This form of malaria is less severe than Malaria caused by P. falciparum, but there is a higher probability for relapses to occur. It is characterized by febrile paroxysms (fever) that occur every 48 hours (hence the name tertian).

Page 86

It is the most common form of the disease, is rarely fatal but is the most difficult to cure.

9. Discuss in brief on laboratory diagnosis of malaria.

The diagnosis of malaria is confirmed by blood tests and can be divided into microscopic and non-microscopic tests. The microscopic tests involve staining and direct visualization of the parasite under the microscope. Direct Test: 1. Peripheral smear study Light microscopy of thick and thin stained blood smears remains the standard method for diagnosing malaria. It involves collection of a blood smear, its staining with Romanowsky stains and examination of the Red Blood Cells for intracellular malarial parasites. Thick smears are 2040 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection limit of 1050 trophozoites/l. Thin smears allow one to identify malaria species (including the diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts, gametocytes, and malarial pigment in neutrophils and monocytes. The diagnostic accuracy relies on the quality of the blood smear and experience of laboratory personnel. 2. Quantitative Buffy Coat (QBC) test It involves staining of the centrifuged and compressed red cell layer with acridine orange and its examination under UV light source. It is fast, easy and claimed to be more sensitive than the traditional thick smear examination.The fluorescing parasites can then be observed under ultraviolet light at the interface between red blood cells and buffy coat. Non Microscopic test: This includes fluorescence microscopy of parasite nuclei stained with acridine orange, rapid dipstick immunoassay, and Polymerase Chain Reaction (PCR) assays. These tests involve identification of the parasitic antigen or the antiplasmodial antibodies or the parasitic metabolic products. Nucleic acid probes and immunofluorescence for the detection of Plasmodia within the erythrocytes The gel diffusion, counter-immunoelectrophoresis, radio immunoassay, and enzyme
Page 87

immunoassay for malaria antigens in the body fluids; and haemagglutination test, indirect immunofluorescence, enzyme immunoassay, immunochromatography, and Western blotting for anti-plasmodial antibodies in the serum

10.

Write a short note on Entamoeba.

Entamoeba is a genus of Amoebozoa found as internal parasites or commensals of animals. Entamoeba cells are small, with a single nucleus and typically a single lobose pseudopod taking the form of a clear anterior bulge. They have a simple life cycle. The trophozoite (feeding-dividing form) is approximately 10-20 m in diameter and feeds primarily on bacteria. It divides by simple binary fission to form two smaller daughter cells. Almost all species form cysts, the stage involved in transmission (the exception is E. gingivalis). Depending on the species, these can have one, four or eight nuclei and are variable in size; these characteristics help in species identification. Several species are found in humans. Entamoeba histolytica is the pathogen responsible for 'amoebiasis' (which includes amoebic dysentery and amoebic liver abscesses), while others such as Entamoeba coli and E. dispar are harmless.

11. Describe the lifecycle, pathogenesis, lab diagnosis and treatment of Plasmodium falciparum.
Plasmodium falciparum is one of four distinct species of the malaria parasite that affect humans, of which two predominate as threats to public health. Plasmodium falciparum is found globally but is commonest in Africa. This gives rise to acute infections that may rapidly become life-threatening. Chronic infections also cause debilitating anaemia. Life cycle: When an female mosquito infected with Plasmodium falciparum feeds on a human, parasites in the 'sporozoite' stage enter the bloodstream, are carried to the liver and infect liver cells. Here, they develop over seven days into a large 'hepatic schizont' which contains about 30 000 tiny invasive parasites called merozoites. The liver cell ruptures and merozoites are released into the blood where they invade red blood cells. About 20 minutues after invading, the merozoite turns into a form called a 'feeding trophozoite'. This eats the contents of the cell, and breaks down haemoglobin to amino acids and haem. Later, the trophozoite divides several times to produce 12-32 merozoites, which fill
Page 88

the red blood cell as an 'erythrocytic shizont'. The red cell ruptures and merozoites are released into the blood stream, where they repeat this multiplication step by invading uninfected red blood cells. This is called the 'blood cycle', and greatly amplifies the infection. Some merozoites do not develop into trophozoites, but instead form sexual stage gametocytes. Gametocyte development takes 10-12 days for Plasmodium falciparum. Early stage gametocytes lurk in organs such as brain and bone marrow, while late stage gametocytes circulate in the blood and are taken up by a mosquito during a blood meal. The fever associated with untreated Plasmodium falciparum infection classically occurs every 48 hours when large numbers of infected red blood cells rupture at the same time, releasing pyrogens (fever-causing chemicals) into the blood. Red blood cell rupture also contributes to anaemia associated with malaria. At the peak of infection, a person will carry up to 2 million parasites per microlitre of blood. Red blood cells infected with trophozoite stage Plasmodium falciparum sequester in capillaries of the brain. This contributes to symptoms of cerebral malaria.

Page 89

Pathogenesis: Plasmodium falciparum causes severe malaria via a distinctive property not shared by any other human malaria, that of sequestration. Within the 48-hour asexual blood stage cycle, the mature forms change the surface properties of infected red blood cells causing them to stick to blood vessels (a process called cyto-adherence). This leads to obstruction of the microcirculation and results in dysfunction of multiple organs, typically the brain in cerebral malaria. These adhesions result in the sequestration of parasite-infected RBCs in vital organs (particularly the brain), where they interfere with the microcirculation and metabolism. Sequestered parasites continue to develop and avoid being cleared by host defence mechanisms. Diagnosis: Microscopic examination: Microscopic examination of peripheral blood smear (thick and thin smears). For P. faliparum infection the ring forms and gametocytes (crescent shape) can be seen. The multiple rings in individual red blood cell is the diagnostic feature. Further Maurers dots and Ziemanns dots are seen in the red blood cells. No enlargement of RBC.

Page 90

Serological test: Indirect immunofluroscence test, indirect haemagglutination assay (IHA) and ELISA are some of the serological test available. Molecular method: PCR (polymerase chain reaction) can be done. Treatment: In the past, chloroquine was effective for treating nearly all cases of malaria. In recent studies, chloroquine-resistant P. falciparum malaria has been observed with increasing frequency across the country. The treatment of P. falciparum malaria is based on areas identified as chloroquine resistant/ sensitive. Artemisinin Combination Therapy (ACT) should be given in resistant areas whereas chloroquine can be used in sensitive areas. ACT should be given only to confirmed P. falciparum casesfound positive by microscopy. ACT consists of an artemisinin derivative combined with a long acting antimalarial (amodiaquine, lumefantrine, mefloquine or sulfadoxine-pyrimethamine).

Page 91

PART VI VIROLOGY

1. Write a short note on classification of virus.

The viruses are classified based on different properties. Based on genome the viruses are classified as: DNA virus Double stranded DNA virus Single stranded DNA virus RNA virus Double stranded RNA virus Single stranded RNA virus

Based on capsid symmetry they may be: Helical virus Polyhedral virus Complex virus

Based on presence of envelope they may be: Enveloped virus Non-enveloped virus

2. Write a short note on cultivation of virus.

The viruses can be grown in three ways: Embryonated hens egg Animals Cell culture

Diagnostic laboratories attempt to recover viruses from clinical samples to establish disease causes.

Page 92

Embryonated Hens egg Embryonated eggs are among the most useful and available forms of living animal tissue for the isolation and identification of animal viruses, for titrating viruses, and for quantity cultivation in the production of viral vaccines. The embryo proper, chorioallantoic membrane, yolk sac, allantoic sac, or amniotic sac may be inoculated in hen eggs of various ages, so that a wide choice of types of tissue is available to fit the characteristics of the virus under study or for special studies. The chorioallantoic membrane is frequently used; in some infections, such as smallpox, vaccinia, and herpes simplex, characteristic lesions are produced which in some cases may resemble those in the natural host. Cell culture: The availability of cells grown in vitro has facilitated the identification and cultivation of newly isolated viruses and the characterization of previously known ones. There are three basic types of cell cultures. i. Primary cultures are made by dispersing cells (usually with trypsin) from freshly removed host tissues. In general, they are unable to grow for more than a few passages (generations) in culture. ii. Diploid cell lines are secondary cultures which have undergone a change that allows their limited culture (up to 50 passages) but which retain their normal chromosome pattern. iii. Continuous cell lines are cultures capable of more prolongedperhaps indefinitegrowth that have been derived from diploid cell lines or from malignant tissues. They invariably have altered and irregular numbers of chromosomes. The type of cell culture used for viral cultivation depends on the sensitivity of the cells to a particular virus.

3. Write a short note on viral inclusion bodies.

In the course of viral multiplication within cells, virus-specific structures called inclusion bodies may be produced. They become far larger than the individual virus particle and often have an affinity for acid dyes (eg, eosin). They may be situated in the nucleus (herpesvirus), in the cytoplasm (poxvirus), or in both (measles virus). In many viral infections, the inclusion bodies are the site of development of the virions (the viral factories). The presence of inclusion bodies may be of considerable diagnostic aid. The intracytoplasmic inclusion in nerve cellsthe Negri bodyis pathognomonic for rabies.

Page 93

4. Write a short note on polio vaccine.

Two polio vaccines are used throughout the world to combat poliomyelitis (or polio). They are inactivated polio vaccine and oral polio vaccine. Inactivated polio vaccine (IPV) IPV (Inactivated Polio Vaccine), also called the Salk vaccine, was developed by Dr. Jonas Salk in 1952. The vaccine is a clear, colourless sterile suspension for subcutaneous injection. IPV contains strains of the 3 types of polioviruses (Types 1, 2, and 3), originally grown in monkey kidney cell culture and inactivated by exposure to formaldehyde.

Oral polio vaccine (OPV) The Oral Polio Vaccine (OPV) was developed in 1958 by Dr. Albert Sabin. Sabin attenuated the wild type poliovirus by passaging the virus in monkey kidney epithelial cells. The commonly used form of the oral polio vaccine is trivalent, which means that it contains live attenuated strains of the three serotypes of poliovirus. Trivalent OPV is characterized in vivo by efficient growth properties in the intestinal tract, with unaltered immunogenic properties with respect to original type of virus. This means that an individual immunized with trivalent OPV induces longlasting (frequently life-long) protective immunity of the gastrointestinal tract to all known forms of poliovirus.

5. Write a short note on antirabies vaccine.

The antirabies vaccines are pre-prepared antibodies that are injected normally injected at the site of rabid dog bite to neutralise the rabies virus. There are two different antirabies vaccines: Human Rabies Immune Globulin (HRIG) HRIG is a gamma globulin prepared by cold ethanol fractionation from the plasma of hyperimmunized humans. There are fewer adverse reactions to human rabies immune globulin than to equine antirabies serum.

Page 94

Antirabies Serum, Equine This is concentrated serum from horses hyperimmunized with rabies virus. It has been used in countries where HRIG is not available.

6. Write a short note on Herpes Zoster.

After the symptoms of chickenpox have abated, the Varicella Zoster Virus persists in the spinal ganglia and perhaps in other tissues as well. Following reactivation, zoster (shingles) develops. It is believed that waning immunity allows viral replication to occur in a ganglion, causing intense inflammation and pain. Virus travels down the nerve to the skin and induces vesicle formation. It usually starts with severe pain in the area of skin or mucosa supplied by one or more groups of sensory nerves and ganglia. Within a few days after onset, a crop of vesicles appears over the skin supplied by the affected nerves. The trunk, head, and neck are most commonly affected.

7. Write a short note on Herpes simplex virus (Type I & II).

Herpes is the name of a group of viruses that cause painful blisters and sores. It is an enveloped DNA virus. There are two distinct herpes simplex viruses: type 1 and type 2 (HSV-1, HSV2) The herpes simplex virus is the pathogen that causes a vesicular exanthem (fever blisters, herpes labialis, or genitalis), encephalitis, and a generalized infection in newborns (herpes neonatorum). The herpes simplex viruses establish latent infections in nerve cells; recurrences are common.

HSV I: Initial infection with herpes simplex type 1 usually occurs in early childhood. The portal of entry is normally the oral mucosa and the infection usually manifests as a gingivostomatitis. The viruses then wander along axons into the CNS, where they persist in a latent state in the trigeminal (Gasseri) ganglion. Following reactivation, the viruses follow the same route back to the periphery, where they cause the familiar vesicular exanthem (fever blisters, herpes labialis).
Page 95

Despite established immunity, such reactivation can manifest repeatedly because the viruses wander within the nerve cells and do not enter the intercellular space, thus remaining beyond the reach of the immune defenses. Possible complications include keratoconjunctivitis and a highly lethal form of encephalitis.

HSV II: It causes diseases like Herpes genitalis, aseptic meningitis, and neonatal infection. TransmissionSexual contact in adults and during passage through the birth canal in neonates. PathogenesisInitial vesicular lesions occur on genitals. The virus then travels up the axon and becomes latent in sensory (lumbar or sacral) ganglion cells. Recurrences are less severe than the primary infection. HSV-2 infections in neonate can be life-threatening because neonates have reduced cell-mediated immunity. Asymptomatic shedding of HSV-2 in the female genital tract is an important contributing factor to neonatal infections. Laboratory DiagnosisVirus causes CPE in cell culture. Identify by antibody neutralization or fluorescent-antibody test. Tzanck smear reveals multinucleated giant cells but is not specific for HSV-2.

8. Write a short note on Hepatitis B virus.

Hepatitis B virus (HBV) is a member of the Hepadnavirus family. The virus particle, (virion) consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein. The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity similar to retroviruses. The outer envelope contains embedded proteins which are involved in viral binding of, and entry into, susceptible cells. The virus is one of the smallest enveloped animal viruses with a virion diameter of 42 nm, but pleomorphic forms exist, including filamentous and spherical bodies lacking a core. These particles are not infectious and are composed of the lipid and protein that forms part of the surface of the virion, which is called the surface antigen (HBsAg), and is produced in excess during the life cycle of the virus. Hepatitis B virus can cause an acute illness with symptoms that last several weeks, including yellowing of the skin and eyes (jaundice), dark urine, extreme fatigue,
Page 96

nausea, vomiting and abdominal pain. People can take several months to a year to recover from the symptoms. HBV can also cause a chronic liver infection that can later develop into cirrhosis of the liver or liver cancer.

9. Mention the viruses causing the Hepatitis. Write the following on Hepatitis B virus morphology, pathogenesis, lab diagnosis and vaccines/immunoprophylaxis.
Viral hepatitis is a systemic disease primarily involving the liver. Most cases of acute viral hepatitis in children and adults are caused by one of the following agents: hepatitis A virus (HAV), the etiologic agent of viral hepatitis type A (infectious hepatitis); hepatitis B virus (HBV), which is associated with viral hepatitis B (serum hepatitis); hepatitis C virus (HCV), the agent of hepatitis C (common cause of posttransfusion hepatitis); hepatitis E virus (HEV), the agent of enterically transmitted hepatitis. Other viruses are associated with hepatitis that cannot be ascribed to known agents, and the associated disease is designated non-AE hepatitis.

i. ii. iii. iv. v.

Morphology: The hepatitis B virus (HBV) is the main representative of the family of hepadnaviruses. Its genome consists of partially double-stranded DNA (hepadnavirus = hepatitis DNA virus). The HBV possess an envelope made up of a cellular double lipid layer in which are integrated the hepatitis B surface (HBs) antigen, a 25 kDa polypeptide. This envelope encloses the actual capsid, which consists of the hepatitis B core (HBc) antigen with 21 kDa and contains the genome together with the DNA polymerase. The complete, infectious virion, also known as a Dane particle after its discoverer, has a diameter of 42 nm, the inner structure 27 nm. The virus replicates in liver cells. The Dane particles and the HBs antigen, but not the HBc antigen, are released into the bloodstream, whereby the HBs antigen is present in two different forms, a filamentous particle approximately 22 x 100 nm and a spherical form with a diameter of about 22 nm. A further viral protein is the HBe antigen, which represents a posttranslational, truncated form of the HBc antigen and is no longer capable of spontaneous capsid formation. It is also released from the hepatic cells into the blood.
Page 97

Pathogenesis: Infection is parenterally transmitted. The virus replicates in the liver and virus particles, as well as excess viral surface protein, are shed in large amounts into the blood. Viraemia is prolonged and the blood of infected individuals is highly infectious. The incubation period of hepatitis B is four to 12weeks, followed by the acute infection phase, icteric, or anicteric course, with a variable duration of two to 12 weeks. The hepatic cell damage resulting from an HBV infection is not primarily due to cytopathic activity of the virus, but rather to a humoral and cellular immune response directed against the virus-induced membrane antigens (HBs, HBc) on the surface of the infected hepatocytes. 0.51% of those infected experience a fulminant, often lethal, hepatitis. In 8090% of cases the infection runs a benign course with complete recovery and elimination of the HBV from the body. A chronic infection develops in 510%. Three forms are differentiated, but mixed forms are possible: healthy HBV carriers, chronic persistent hepatitis (CPH) without viral replication, and finally chronic aggressive hepatitis (CAH) with viral replication and a progressive course. A chronic infection can result in development of a carcinoma (hepatocellular carcinoma, HCC) or cirrhosis of the liver, with incidence varying widely from one geographic area to another. The delta agent appears to have an unfavourable
Page 98

influence on the clinical course, usually making the disease more aggressive, increasing the number of complications and worsening the prognosis. Immunoprophylaxis of Hepatitis B virus infection Hepatitis B vaccination is the most effective measure to prevent HBV and its consequences. A vaccine for hepatitis B has been available since 1982. Active immunisation Plasma derived vaccines: The initial vaccine was prepared by purifying HBsAg associated with the 22-nm particles from the plasma of healthy HBsAg-positive carriers and treating the particles with virus-inactivating agents (formalin, urea, heat). Preparations containing intact 22-nm particles have been highly effective in reducing HBV infection. Recombinant DNA vaccine: These vaccines consist of HBsAg produced by a recombinant DNA in yeast cells or in continuous mammalian cell lines. The HBsAg expressed in yeast forms particles 15 30 nm in diameter, with the morphologic characteristics of free surface antigen in plasma though the polypeptide antigen produced by recombinant yeast is not glycosylated. The vaccine formulated using this purified material has potency similar to that of vaccine made from plasma-derived antigen. Passive immunisation The passive immunization using specific hepatitis B immune globulin (HBIG) have shown a protective effect if it is given soon after exposure. HBIG is not recommended for preexposure prophylaxis because the HBV vaccine is available and effective. Persons exposed to HBV percutaneously or by contamination of mucosal surfaces should immediately receive both HBIG and HBsAg vaccine administered simultaneously at different sites to provide immediate protection with passively acquired antibody followed by active immunity generated by the vaccine. Laboratory diagnosis of Hepatitis B virus infection: General lab findings: Liver biopsy permits a tissue diagnosis of hepatitis. Tests for abnormal liver function, such as serum alanine aminotransferase (ALT) and bilirubin, supplement the clinical, pathologic, and epidemiologic findings.

Page 99

Detection of viral markers: Viral markers DNA polymerase activity, HBV DNA, and HBeAg, which are representative of the viremic stage of hepatitis B, occur early in the incubation period, concurrently or shortly after the first appearance of HBsAg. HBsAg is usually detectable 26 weeks in advance of clinical and biochemical evidence of hepatitis and persists throughout the clinical course of the disease but typically disappears by the sixth month after exposure. High levels of IgM-specific anti-HBc are frequently detected at the onset of clinical illness. Because this antibody is directed against the 27-nm internal core component of HBV, its appearance in the serum is indicative of viral replication. Antibody to HBsAg is first detected at a variable period after the disappearance of HBsAg. It is present in low concentrations. Before HBsAg disappears, HBeAg is replaced by anti-HBe, signaling the start of resolution of the disease. Anti-HBe levels often are no longer detectable after 6 months. By definition, HBV chronic carriers are those in whom HBsAg persists for more than 6 months in the presence of HBeAg or anti-HBe. HBsAg may persist for years after loss of HBeAg. In contrast to the high titers of IgMspecific anti-HBc observed in acute disease, low titers of IgM anti-HBc are found in the sera of most chronic HBsAg carriers. Small amounts of HBV DNA are usually detectable in the serum as long as HBsAg is present.

Detection methods The most useful detection methods are ELISA for HBV antigens and antibodies PCR for viral DNA.

10.

Write a short note on oral manifestations of AIDS.

The earliest indicators of HIV infection may manifest in the oral cavity, and some 50 disease entities that may affect the orofacial region of HIV-infected patients have been described.
Page 100

The more common orofacial manifestations of HIV infection are: Fungal infections - oral candidiasis (erythematous and pseudomembranous variants mainly); linear gingival erythema and angular cheilitis (both are possibly due to mixed bacterial and fungal infections) Viral infections - hairy leukoplakia, Kaposi's sarcoma, herpes infections, papillomas Bacterial infections - gingivitis and periodontitis cervical lymphadenopathy and lymphomas such as non-Hodgkin's lymphomas

11.

Write a short note on transduction.

Transfer of DNA from a donor to a receptor bacteria with the help of transport bacteriophages. Bacteriophages are viruses that infect bacteria. During their replication process, DNA sequences from the host bacterial cell may replace all or part of the genome in the phage head. Such phage particles are then defective.

They can still infect the bacteria and inject their DNA, but the infected bacterial cell will then neither produce new phages nor be destroyed.

12.

Write an account on MMR vaccine.

The MMR vaccine is a "3-in-1" vaccine that protects against measles,mumps, and rubella -- all of which are potentially serious diseases of childhood. One out of 30 children with measles develops pneumonia. For every 1,000 children who get the disease, one or two will die from it.

The MMR is one of the recommended childhood immunizations. The first shot is recommended when the child is 12 to 15 months old. The timing of vaccination is important to make sure the child is properly protected. It must not be given too early.

A second MMR is recommended at 4 - 6 years, but may be given at any time thereafter.

Page 101

Women of reproductive age who have not received the MMR vaccination in the past or in whom blood tests have shown they are not immune, should receive the MMR vaccine. Women should NOT receive this vaccine if they are pregnant or planning to become pregnant within the next 1 to 3 months

Page 102

PART VII MICROBIOLOGY FOR DENTISTRY

1. Write a short account on oral microflora.

Oral flora comprises a diverse array of organisms. Bacteria are by far the predominant group of organisms The presence of nutrients, epithelial debris, and secretions makes the mouth a favourable habitat for a great variety of bacteria. Oral bacteria include streptococci, lactobacilli, staphylococci and corynebacteria, with a great number of anaerobes, especially bacteroides. At birth the oral cavity is sterile but rapidly becomes colonized from the environment, particularly from the mother in the first feeding. Streptococcus salivarius is dominant and may make up 98% of the total oral flora until the appearance of the teeth (6 - 9 months in humans). The eruption of the teeth during the first year leads to colonization by S. mutans and S. sanguis. These bacteria require a nondesquamating (nonepithelial) surface in order to colonize. They will persist as long as teeth remain. Other strains of streptococci adhere strongly to the gums and cheeks but not to the teeth. The creation of the gingival crevice area (supporting structures of the teeth) increases the habitat for the variety of anaerobic species found. The complexity of the oral flora continues to increase with time, and bacteroides and spirochetes colonize around puberty. The normal bacterial flora of the oral cavity clearly benefit from their host who provides nutrients and habitat. There may be benefits, as well, to the host. The normal flora will occupy the available colonization sites which makes it more difficult for other microorganisms (nonindigenous species) to become established. Also, the oral floras contribute to host nutrition through the synthesis of vitamins, and they contribute to immunity by inducing low levels of circulating and secretory antibodies that may cross react with pathogens. Finally, the oral bacteria exert microbial antagonism against nonindigenous species by production of inhibitory substances such as fatty acids, peroxides and bacteriocins. On the other hand, the oral flora are the usual cause of various oral diseases in humans, including abscesses, dental caries, gingivitis, and periodontal disease. If oral bacteria can gain entrance into deeper tissues, they may cause abscesses of alveolar bone, lung, brain, or the extremities. Such infections usually contain mixtures of bacteria with Bacteroides melaninogenicus often playing a dominant
Page 103

role. If oral streptococci are introduced into wounds created by dental manipulation or treatment, they may adhere to heart valves and initiate subacute bacterial endocarditis.

2. Write an account on aetiology of dental caries (microbiology of dental caries).

The dental caries or tooth decay is a destructive process causing decalcification of the tooth enamel leading to cavitation of the tooth. Dental caries formation takes place due to a process called demineralization. The term "demineralization" simply describes the fact that minerals (like calcium compounds) are leached (dissolved away) from a tooth's hard tissues (enamel, dentin, cementum). In the case of teeth, demineralization takes place as a process (over a period of time) due to the repeated exposure of a tooth's surface to acidic compounds. The acids that cause tooth demineralization (cavity formation) are produced by specific types of bacteria (mutans streptococci and lactobacilli) that live in dental plaque. The bacteria that cause tooth decay utilize sugars (glucose, sucrose, fructose, lactose, or cooked starches) as their food source. The fermentation of sugar created during the digestion of these sugars are the acids (especially lactic acid) that cause the demineralization of tooth enamel and dentin. The living part of the tooth, the pulp, becomes damaged. The bacteria invade and infect the pulp of the tooth. The blood vessels and nerves may die due to the infection. Root canal therapy is required to repair the tooth. The infection can then spread to form a tooth abscess (collection of pus) around the root tip. As the infection inside the tooth's root canal builds up, the bone around it gets infected.

3. Write a short note on dental plaque.

Dental plaque is an accumulation of thin film on the outer surface of the tooth. This mainly consists of microorganisms, most of which are bacteria of Streptococcus mutans species. The mechanisms of plaque formation include.

Absorption of proteins and bacteria to form a film on the tooth surface. Irreversible adhesion due to intermolecular interactions between cell surfaces and the pellicle. Secondary colonisers attach to primary colonisers by intermolecular
Page 104

interaction. The cells divide and generate a biofilm.

Plaque consists of microorganisms and extracellular matrix. The microorganisms that form the biofilm are mainly Streptococcus mutans and anaerobes, with the composition varying by location in the mouth. Examples of such anaerobes include fusobacterium and actinobacteria. The extracellular matrix contains proteins, long chain polysaccharides and lipids. The microorganisms present in dental plaque are all naturally present in the oral cavity, and are normally harmless. However, failure to remove plaque by regular tooth brushing means that they are allowed to build up in a thick layer. Those microorganisms nearest the tooth surface convert to anaerobic respiration; it is in this state that they start to produce acids.

Acids released from dental plaque lead to demineralization of the adjacent tooth surface, and consequently to dental caries. Saliva is also unable to penetrate the build-up of plaque and thus cannot act to neutralize the acid produced by the bacteria and remineralize the tooth surface. They also cause irritation of the gums around the teeth that could lead to gingivitis, periodontal disease and tooth loss. Plaque build up can also become mineralized and form calculus (tartar).

4. Write a brief account on nosocomial infection/hospital acquired infection.

Infections acquired during a hospital stay are called nosocomial infections. It is also called as Hospital acquired infections. Hospitals generally have a high rate of nosocomial infections and the reasons are rather obvious. Nosocomial infections may arise from inhalation of droplets in the air or spread by direct hand contact from hospital staff or visitors. Most nosocomial infections afflict patients with reduced immune response either due to age, serious disease, certain medications, or recent surgery.

Microbes involved in nosocomial infection: Like any infectious condition, nosocomial infections can be bacterial, viral, fungal, or even parasitic. The most common pathogens include staphylococci (especially staphylococcus aureus), pseudomonas, and Escherichia coli.

Page 105

However, various newer pathogens are becoming more important. Fungal conditions, mainly from candida, comprise approximately 9% of nosocomial infections. Fungal nosocomial infections: Several fungi have become more common in nosocomial infections. The most common are Candida (mostly Candida albicans), Aspergillus, Fusarium, Trichosporon, and Malassezia. Candidiasis remains the most common type of nosocomial fungal infection, particularly in the immunocompromised. Risk factors for fungal infections include antibiotic treatments, chemotherapy, intravascular catheters, neutropenia, haemodialysis, or prior fungal infection. Nosocomial Urinary tract infections: Urinary tract infections are the most common nosocomial infections. These infections can be caused by various pathogens such as E. coli, Pseudomonas, or Enterococcus. Antibiotic resistance: Many of the pathogens that cause nosocomial infections have a high level of resistance to antibiotic treatments. These emerging pathogens are the most serious concerns, because they are more difficult to treat. Some of the major concerns are methicillin resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus, and vancomycinresistant enterococci (VRE). Prevention of nosocomial infections: There are numerous preventive measures ranging from the obvious to high-tech. The goals are to avoid transmission by hand, by air, and by blood. Handwashing by medical staff after attending a patient prevents many nosocomial infections. Other measures include avoiding hand contact, especially to the conjunctiva or nasal areas. Various sterilization measures are helpful ranging from simple acts like sterilizing ventilators to full scale air filtering systems in the hospital. In some cases it may be appropriate to vaccinate certain patients against particular pathogens.

Page 106

PART VIII PRACTICALS

Study of Morphology of bacteria- Staining methods
Staining procedure can also be classified in different ways. Simple staining involves the use of only 1 dye and is used primarily as a means to study the morphology and structure of organisms. Differential staining uses more than 2 dyes and is also used to differentiate the organisms into one of two groups.

Simple staining there are two methods: positive staining - where the actual cells are themselves coloured and appear in a clear background; negative staining where the cells remain clear (uncoloured) and the background is colored to create a contrast to aid in the better visualization of the image.

Differential staining Gram staining is the most used technique in the identification of bacteria. It divides bacteria into two. They are gram positive and gram negative bacteria. Acid fast staining for Mycobacterium Spore staining

Preparation of smear: The following diagram depicts the methods of smear preparation

Page 107

GRAM STAINING

INTRODUCTION The Gram stain (named after Christian Gram,Danish scientist and physician, (18531938) is the most useful and widely employed differential stain in bacteriology. It divides bacteria into two groups gram negative and gram positive. PRINCIPLE: A gram stain is performed to distinguish between gram positive and gram negative bacteria. Both types of bacteria are stained with methyl violet. The addition of iodine forms a methyl violet-iodine complex within the cell. When decolourizer is added the thick cell wall of the Gram positive bacteria is dehydrated by the alcohol. This makes the cell wall less porous and allows the bacterial cell to retain the methyl violet-iodine complex and block out the counter stain. Gram negative bacteria have an extra lipid-rich outer layer and thin cell wall,
Page 108

which increases in porosity when the decolouriser is added. This facilitates the removal of the crystal violet-complex and allows the counter stain to permeate the cell. Further the gram positive cells have more acidic protoplasm which may account for their retaining the basic primary dye more strongly than gram negative bacteria.

Procedure: Flood slide with methyl violet (the primary stain). Allow to stand for one minute Wash off with tap water. Flood with Grams iodine (a mordant). Leave for one minute. Wash off with tap water. Decolourize with acetone until no more colour washes off (usually 5 - 10 seconds). This is a most critical step. Be careful not to over decolourize, as many gram-positive organisms may lose the violet stain easily and thus appear to be gram negative after they are counterstained. Wash off with tap water. Apply dilute carbol fuchsin (the counterstain) for one minute. Wash off with tap water. Drain and blot gently with a blotting paper. Air dry the slide thoroughly before you examine the preparation under the microscope. Observe the slide under oil immersion objective.

ACID FAST STAINING

It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB) along with some others of this genus. It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain. It can also be used to stain few other bacteria like Nocardia. The reagents used are ZiehlNeelsen carbol fuchsin, acid alcohol/dil. acid and methylene blue. Acid-fast bacilli will be bright red after staining.

Principle: Acid fast staining is another widely used differential staining procedure in bacteriology. This staining method was developed by Paul Ehrlich in 1882, during his work on etiology of tuberculosis. Some bacteria resist decolourization by both acid and alcohol and hence they are referred as acid-fast organisms. Acid alcohol is very intensive decolourizer. This staining technique divides bacteria into two groups (i)
Page 109

acid-fast and (ii) non acid-fast. This procedure is extensively used in the diagnosis of tuberculosis and leprosy. Acid-fastness property in certain Mycobacteria and some species of Nocardia is correlated with their high lipid content. Due to high lipid content of cell wall, in some cases 60% (w/w), acid-fast cells have relatively low permeability to dye and hence it is difficult to stain them. For the staining of these bacteria, penetration of primary dye is facilitated with the use of 5% aqueous phenol which acts as a chemical intensifier. In addition, heat is also applied which acts as a physical intensifier. Once these cells are stained, it is difficult to decolourize Once stained, acid fast bacteria are able to retain stains that are more easily removed in other bacteria using acidic ethanol/dilute acid. This property gives them their designation as "acid fast." The high lipid content of the mycobacterial cell wall is probably responsible for the weak initial staining and the subsequent strong retention following prolonged staining. In the classic Ziehl-Neelson technique of acid-fast staining, the smear is flooded with carbol-fuchsin dye, heated, and then decolorized. The non-acid fast organisms are then counterstained with methylene blue. Acid-fast organism will appear red due to the retention of the carbol-fuchsin while the background and any non-acid-fast organisms will appear blue due to the methylene blue counterstain. The Kinyoun method is similar but heat treatment is not necessary because a more concentrated fuchsin dye is used (cold method). A third type of acid-fast stain is the fluorochrome stain which utilizes auramine, a fluorescent dye. This stain uses the same principle as the above stains, but visualization is via fluorescent microscopy. Procedure: 1. Flood the entire slide with Carbol Fuchsin. Ensure enough stain is added to keep the slides covered throughout the entire staining step. 2. Using a Bunsen burner, heat the slides slowly until they are steaming. Maintain steaming for 5 minutes by using low or intermittent heat (i.e. by occasionally passing the flame from the Bunsen burner over the slides) Caution: Using too much flame or heat can cause the slide to break. 3. Rinse the slide with water. 4. Flood the slide with 3% acid-alcohol/20% sulphuric acid and allow to decolourise. Flood the slides until the slides are clear of stain visible to the naked eye. 5. Rinse the slide thoroughly with water and then drain any excess from the slides. 6. Flood the slide with the counterstain, Methylene Blue. Keep the counterstain on the slides for 1 minute.

Page 110

7. Rinse the slide thoroughly with water.

Expected Viva voce questions on Gram staining and Acid fast staining:
How are staining techniques classified?
Simple stain: where only one stain is used and all bacteria are stained similarly. Eg:Methylene blue, dilute carbol fuchsin Differential staining: where different bacteria stain differently to a common staining technique depending on their physiological properties. Eg: Grams stain and Acid fast staining Special stain: where structures of bacteria like spores, granules, capsule etc are demonstrated. For example: Silver impregnation technique for demonstration of spirochetes Feulgen stain for demonstration of nucleus Sudan black stain for demonstration of lipid vacuoles Ryus stain for demonstration of flagella Alberts stain for demonstration of metachromatic granules

Negative staining: where the background is stained with an acidic dye such as India ink or Nigrosin. Used for demonstration of capsules.

How are stains classified?

Stains are classified based on the pH of their chromophore (colour bearing ion) into acidic, basic and neutral. Acidic dyes have anionic chromophore eg., sodium+ eosinate-. Basic dyes have cationic chromophore eg., methylene blue+ chloride-. Neutral dyes have both acidic and basic component that nullify each other. They are Romanowskys stain and are used in staining parasitic forms. Acidic dyes combine more strongly with cytoplasmic components of bacteria, especially the nucleus that is basic in nature. Stains can be either natural (eg: carmine and haematoxylin) or coal-tar derivatives /aniline stains (eg: methylene blue, crystal violet).
Page 111

 Supravital (cells removed from the body) and intravital (cells still a part of the body).

Who invented Gram stain?

Hans Christian Gram invented this stain in 1884. The original formulation was Aniline Gentian violet, Lugols iodine, absolute alcohol and Bismark brown.

Explain the theories/principles of Gram staining?

Cell wall theory: Cell wall of Gram positive bacteria are 40 times thicker than those of Gram negative cells, hence they are thought to help retain the dye-iodine complex. Lipid Content Theory: In gram positive bacteria, the alcohol/acetone mixture causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. In the case of gram-negative bacteria, the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the cell wall and may also damage the cytoplasmic membrane to which the peptidoglycan is attached. The single thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the cell is decolourized. Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more acidic than those of Gram negative ones. Hence the dye is said to bind with more affinity to Gram positive cells.

List out the primary stains that can be used in gram staining.
i. Crystal violet ii. Methyl violet iii. Gentian violet

List out the counterstain that can be used in gram staining.

i. Safranin ii. Dilute Carbol fuchsin

Page 112

State the functions of the following in gram staining.

i. Grams iodine mordant (forms a complex with the dye and fixes into the cell) ii. Acetone decolourizer

Name some gram positive cocci.

Staphylococcus Streptococcus Enterococcus

Name some gram positive bacilli.

Bacillus (sporing) Corynebacterium (non-sporing) Clostridium (sporing)

Name some gram negative cocci.

Neisseria gonorrhoeae Neisseria meningitidis

Name some gram negative bacilli

Escherichia coli Salmonella Shigella and other Enterobacteriaceae Pseudomonas

What objective lens is used to observe the gram stained smear?

100x oil immersion objective.

Page 113

Why do we use immersion oil?

Placing a drop of oil with the same refractive index as glass between the glass slide and objective lens eliminates two refractive surfaces and improves the resolution i.e. the image will be clear.

Which part of the bacteria actually gets stained?

It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as Mycoplasma and L forms are Gram negative.

Which are the bacteria or bacterial component that cant be stained by Gram stain?
Extremely slender bacteria such as Treponema Cells containing waxy substances impermeable to stain such as Mycobacteria Minute intracellular bacteria such as Chlamydia and Rickettsia Cell organelles such as capsule, spore, flagella etc

What are the alternatives used in Gram stain?

Primary stain: Crystal violet, Methyl violet and Gentian violet Mordant: Grams iodine, rarely Lugols iodine Decolorizer: Alcohol, acetone, acetone-alcohol mixture (1:1) Counterstain: Dilute carbol fuchsin, safranin, neutral red, (Sandiford stain for Gonococci)

What are the conditions when Gram positive bacteria can appear Gram negative?
When over-decolourized by either prolonged exposure to decolourizer or using acetone alone. When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin. Old cultures, where cell wall is weakened or action of autolytic enzymes Those bacteria that are phagocytosed, where cell wall is acted upon by lysosomal contents

Page 114

Name the more important step in Gram stain?

Decolourization is the most important step as this step differentiates between Gram positive and Gram negative bacteria. Over-decolourization can result in Gram positive bacteria appearing Gram negative and under-decolourization can result in Gram negative bacteria appearing Gram positive.

What are the applications of Gram staining?

Rapid presumptive diagnosis of diseases such as bacterial meningitis Selection of empirical antibiotics based on Gram stain finding Selection of suitable culture media based on Gram stain finding Screening of quality of clinical specimens, such as sputum that should contain many pus cells and few epithelial cells Counting of bacteria Appreciation of morphology and types of bacteria in a clinical specimen

Name a fungus that is Gram positive?

Candida spp.

What is acid fast staining?

Certain bacteria or their structures have the ability to retain the primary dye (strong carbol fuchsin) and resist decolourization by weak mineral acids such as H2SO4, HCl. Such bacteria or their structure are termed acid fast and this property is termed acid fastness.

What are the types of acid fast staining?

There are two types of acid fast staining, the hot method and the cold method. The hot method (Ziehl-Neelsen) involves heating the slide while the cold methods such as Kinyouns and Gabbetts do not involve heating the slide.

Who introduced Acid fast staining?

Ehrlich in 1882 discovered acid fastness. The original method involved staining with aniline-gentian violet and decolourization with strong nitric acid. It was later improved by Ziehl and Neelsen.

Page 115

Why are Mycobacteria acid fast?

The cell walls of Mycobacteria are made up of waxy substance, Mycolic acid that is relatively impermeable to ordinary staining techniques. But, by application of heat and a mordant (phenol), the cell can be stained. The purpose of heating is to soften the waxy material of the cell wall and allow the stain to enter the cell. Once stained, acid fast bacteria are able to retain stains that are more easily removed in other bacteria using acidic ethanol/dilute acid. This property gives them their designation as "acid fast."

What are the components of Ziehl-Neelsen stain?

Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and Phenol) Decolourizer: 20% sulphuric acid or acid alcohol. Counterstain: Loefflers Methylene blue or 1% Malachite green, Picric acid for colour-blind workers

What is acid-alcohol decolourizer?

3% HCl in 95% alcohol (methylated spirit). This is useful in differentiating saprophytic Mycobacteria from pathogenic Mycobacteria. Pathogenic Mycobacteria are both acid and alcohol fast but saprophytic Mycobacteria are only acid-fast. Saprophytic Mycobacteria can get decolourized by alcohol. 95% alcohol can be used as a secondary decolorizer after decolourizing with acid. Especially used in staining smears prepared from urine that may contain Mycobacterium smegmatis.

Which are the modifications of Ziehl-Neelsens method?

Decolourizer for Mycobacterium leprae is 5% H2SO4 Tissue sections containing Actinomyctes, Nocardia can be decolourized by 1% H2SO4 Cultures of Nocardia are decolourized by 0.5% H2SO4 Bacterial spores are decolourized by 0.25-0.5% H2SO4 Oocysts of Cryptosporidium are decolourized by 10% H2SO4

What are cold methods of acid fast staining?

The two methods namely Kinyouns and Gabbetts dont involve heating of slides, hence called cold methods. Heating is substituted by increased concentration of phenol and prolonging the duration of staining. Gabbetts method has decolourizer and counterstain in one solution.
Page 116

How to interpret the smear?

At least 300 oil immersion fields must be viewed before declaring the smear as negative. The sensitivity of smear is low because it requires the presence of 104 bacilli/ml to be smear positive. If the number of bacilli is less than this, the chances of detecting them are less. In such a case, the sample should be subjected to concentration techniques such as Petroffs method. If the smear is positive for AFB, it should be counted/graded. Failure to detect any AFB does not rule tuberculosis. Grading of smears has prognostic value.

How is the smear graded and reported?

The rule for reporting acid-fast smears for mycobacteria requires scanning the smear for a minimum of 15 minutes (at least 300 oil immersion fields) before calling the slide negative for acid-fast bacilli or "No AFB seen." The following are recommended interpretations and ways to report smear results:

A request for another specimen or a doubtful report is the result of seeing AFB of 1-2/300 fields for the Ziehl-Neelsen (Z-N) staining. A "1+" report for AFB seen = 1-9/100 fields A "2+" report for AFB seen = 1-9/10 fields A "3+" report for AFB seen = 1-9/field A "4+" report for AFB seen = more than 9/field

What other methods are available for staining Mycobacteria?

Sputum smears for Mycobacteria can be stained by fluorescent dyes such as Auramine and Rhodamine as they have affinity for mycolic acid in their cell walls. The fluorescent microscopy is useful in screening large number of specimens. Large area of smear can be quickly observed that too under high power dry objective.

Page 117