Carte Practic Engleza Sem 1. Final23.09.2017 [310507]

“VICTOR BABES”

UNIVERSITY OF MEDICINE AND PHARMACY

TIMISOARA

MICROBIOLOGY DEPARTMENT

GENERAL MICROBIOLOGY

practical work notebook FOR INTERNAL USE

FOR MEDICINE STUDENTS

PROF. [anonimizat] M.D., Ph.D.

PROF. [anonimizat] M.D., Ph.D.

IULIA CRISTINA BAGIU M.D., Ph.D.

DANA BREHAR CIOFLEC M.D., Ph.D.

FLORIN HORHAT M.D., Ph.D.

PILUT CIPRIAN M.D., Ph.D.

2016

TABLE OF CONTENTS

RELEVANT DEFINITIONS

Pathogens = microorganisms or infectious agents (bacteria, fungi, viruses, rickettsiae, chlamydiae, mycoplasmas, parasites and prions) [anonimizat].

Infection = Invasion by and multiplication of a microorganism or infectious agent in a body part or tissue ± tissue injury and progress to disease.

Colonisation = first stage of infection; pathogen situated at possible appropriate entry site (urogenital, digestive, respiratory, conjunctiva) – not necessarily followed by infection.

Infectivity = [anonimizat] a susceptible host.

Pathogenicity = Capacity of pathogen to produce disease in a susceptible host.

Susceptible host = Organism (human, animal) which can be infected by pathogen (defense mechanisms are defeated by pathogen).

The infectious process:

Dynamic: reciprocal influences

Specific: host & tissue specificity (certain pathogens selectively infect certain tissues/cells)

Depends on:

Virulence of pathogen vs. Susceptibility / Resistance of host

Exposure (pathogen meets susceptible = suitable host)

Some factors which influence the occurrence of infection:

Pathogen related:

Survival time of pathogen outside (suitable) host

Infective dose (quantity of pathogen required to produce infection)

Transmission routes (inhalation, ingestion, percutaneous, sexual, etc)

Host related:

Susceptibility (age, immune status)

Preventive means (vaccination, [anonimizat], etc.)

Survival outside host (after Walther B.A., Ewald P.W., 2004):

Variola virus:

– years – [anonimizat], dark

~ 80 days – [anonimizat], dark

Mycobacterium tuberculosis:

– 90 – 300 days – [anonimizat], dark

– 10 – 120 days – in dust (floor/objects/fabric), [anonimizat]: ˂ 1 day – on glass/plastic/fabric, suspension, room T°, dark

– Hepatitis B Virus: at least 7 days

Minimal infective dose

Table no. 1: Minimal infective dose

CFU= colony forming unit

Antiseptic = agent that causes destruction or inhibition of growth of microorganisms (bacteria, viruses, fungi) on living surfaces such as skin & mucous membranes.

Disinfectant = agent that causes destruction or inhibition of growth of microorganisms (bacteria, viruses, fungi) on non living surfaces (instruments, equipments, [anonimizat], etc).

MIC (minimal inhibitory concentration) = the lowest quantity of antibiotic completely inhibiting the multiplication of a bacterial strain.

MBC (minimal bactericidal concentration) = the lowest quantity of antibiotic able to kill 99.9-100% of the germs of a tested bacterial strain.

GENERAL PRINCIPLES IN THE MICROBIOLOGY LABORATORY. Occupational health and BIOsafety rules. [anonimizat] (1-4)

Risk group 1 (no / low individual & community risk)

– human / [anonimizat]

– e.g. E.[anonimizat], Lactobacillus spp.

Risk group 2 (moderate individual & low community risk)

ability to cause human / animal disease

laboratory exposure → possible infection (severe)

– availability of prevention & treatment

– e.g. Corynebacterium diphtheriae, Haemophilus influenzae, E. coli, Orthomyxoviridae (Influenza viruses)

Risk group 3 (high individual & low community risk)

serious human / animal disease

respiratory transmission (aerosolization)

no interpersonal transmission (usually) + exceptions!*

availability of prevention and/or treatment

e.g. E.coli O157:H7, Francisella tularensis type A, Flaviviridae (West Nile Virus), Hepadnaviridae (HBV),

+ M. tuberculosis* (interpersonal transmission)

Risk group 4 (high individual & high community risk)

serious human & animal disease

respiratory transmission

interpersonal transmission possible

NO prevention & treatment

e.g. Filoviridae (Ebola, Marburg), Arenaviridae (Lassa)

Requirements for sample processing

Premises (location, construction)

Equipments

Materials

Staff related requirements (number, qualifications, training)

Procedures

Biosafety Levels (BSL)

Table no. 2: Biosafety Level I (BSL I) (EBSA pre-conference course, Basel, 18 June 2013)

PPE= Personal Protective Equipments

Table no. 3: Biosafety Level II (BSL II) (EBSA pre-conference course, Basel, 18 June 2013)

Figure no. 1 Biosafety cabinets (BSC) www.slideshare.com

Table no. 4: Biosafety Level III (BSL III) (EBSA pre-conference course, Basel, 18 June 2013)

Respiratory protection

Figure no. 2: Respiratory protection www.slideshare.com

Table no. 5: Biosafety Level IV (BSL IV) (EBSA pre-conference course, Basel, 18 June 2013)

Table no. 6. CORRELATION: Risk group – BSL (EBSA pre-conference course, Basel, 18 June 2013)

THE ROLE OF THE CLINICAL MICROBIOLOGY LABORATORY:

The clinical microbiology laboratory deals with the study of:

– biology of infectious agents,

– relationship between the infectious agents and human organism,

-pathogenicity of infectious agents,

-anti-infectious resistance patterns,

–etiological diagnosis of infectious diseases,

-bases of anti-infectious therapy,

-bases of anti-infectious prevention.

The academic microbiology laboratory is also interested in investigating mechanisms of microbial reproduction and genetics, metabolism, etc.

ANTISEPTICS AND DISINFECTANTS

DECONTAMINATION = removal or destruction of microorganisms. Includes:

1. CLEANING: removal of dirt, dust, earth

2. DISINFECTION: reducing the number of germs (bacteria, not spores) to reduce the risk of contamination;

– Apply to: Furniture, swimming pools, rooms, thermometers, endoscope.

– 3 levels: – high level: bacteria, BK, viruses, fungi, spores

– Average level: bacteria, viruses, fungi, not the spores

– Low level: bacteria

– Germicides: Effect: bactericidal, bacteriostatic, sporicidal, mycobactericidal, fungicidal, virucidal.

– Two types: – Antiseptic: on living tissues: skin, mucous membranes.

– Disinfectant: objects, surfaces, toxic, irritant on human tissues.

– Ideal Properties of antiseptics/disinfectants (not all are achievable in one single product):

Microbicidal activity

Non staining & good odor

Active against all pathogens

Active in presence of pus, blood & exudates

Rapid activity

Non irritating to tissues / non corrosive

Non absorbable

Non sensitizing

– Mechanisms of action: disrupt cellular structures and/or processes

Oxidation of bacterial protoplasm: Hydrogen peroxide (H2O2, Halogens, potassium permanganate

Coagulation (denaturation) of proteins : Phenols, chlorhexidine, alcohols, aldehydes

Increasing permeability of bacterial cell membrane : Cetrimide, soaps

3. STERILIZATION: the destruction of all microorganisms, including bacterial spores.

– Risk of infection:

– Low risk: no direct contact with humans: walls, floors

– Medium risk: in contact with injured skin: respiratory equipment, endoscopes, thermometers

– High risk: penetrating normally sterile areas: surgical interventions, vascular catheters.

A. CLEANING

– Manual: cold water; brushing (decontaminate and dry the brush)

– Cleaning floors, surfaces, furniture and sanitary.

– Gloves

B. DISINFECTION

– Thermal and chemical disinfection

– Thermal disinfection: – is preferred, it is not toxic.

Methods:

1 – boiling at 100 degrees Celsius at least 5 minutes

• HBV, HIV, not the spores

2 – temperatures lower than 80 ° C for 5 minutes.

3 – disinfection with hot water in special machines that combine the drying temperature: dishwasher, bedding

Chemical disinfectants: – 1. For biological products: faeces, urine

– 2. thermolabile medical instruments: thermometers, endoscopes

– 3. strips, pipettes – 24 hours.

Factors that influence the action of disinfectant:

– disinfectant concentration,

– germ type;

– the degree of contamination. There are 3 groups: A, B, C. Group A is the most easily destroyed (normal bacteria), group C contains bacterial spores, the most difficult to destroy.

– Cleanliness of surfaces: dirt = protection of germs

– Contact time

– Physical and chemical properties: high water hardness, calcium and magnesium precipitate and neutralize disinfectants.

ALCOHOLS: ethyl, methyl, concentration of 50-80 %

– Advantages: quick effect, no staining, leaves no residue.

– Disadvantages: not sporicidal;

– Stethoscopes, thermometers, skin

HALOGENATED SODIUM HYPOCHLORITE

– Advantages: cheap, very effective in the free chlorine form, fast effect, low toxic and irritant effect,

– Disadvantage: corrosive on metals, not sporicidal;

– Swimming pools, toilets, dialysis equipment, floors, lingerie, walls

Chlorine – potent germicidal effect against most bacteria, viruses, protozoa, and fungi at a concentration of 0.1 ppm, but much higher concentrations are required in the presence of organic matter

Alkaline pH ionizes chlorine and decreases its activity by reducing its penetrability.

Irritant to the skin and mucous membranes

Widely used to disinfect water supplies and inanimate objects (e.g. utensils, bottles, pipelines)

Sodium hypochlorite solutions (bleach) 2–5% can be used as a disinfectant, and a more diluted form (0.5%) can be used for irrigating suppurating wounds, but it dissolves blood clots and delays clotting

Root canal therapy in dentistry

IODOFORS:

Iodine tinctures: 2% iodine + 2.4% sodium iodide (NaI) in 50% ethanol; it is used as a skin disinfectant. Strong iodine tincture contains 7% iodine and 5% potassium iodide (KI) dissolved in 95% ethanol; it is more potent but also more irritating than tincture of iodine.

Iodine solutions: 2% iodine + 2.4% NaI dissolved in aqueous solution; it is used as a nonirritant antiseptic on wounds and abrasions. Strong iodine solution (Lugol solution) contains 5% iodine and 10% KI in aqueous solution.

– Advantages: bacteria, fungi, mycobacteria, viruses, destroyed quickly;

– Disadvantages: corrosive effect on metals, destroy living tissue, synthetic or plastic materials stain, do not destroy spores

– Thermometers, hydrotherapy baths.

PHENOL & DERIVATIVES

Phenol

– Earliest use (19th century), reference standard

– Mechanism: denaturation of bacterial proteins

– 0.5 to 3 %

– Advantages: wide action; disinfection of surfaces/areas contaminated with urine/faeces/pus

– Disadvantages: irritating and skin depigmentation, corrosive, not destroy spores

– Surfaces, furniture, flooring, medical decontamination equipment which does not come in direct contact with the patient (ultrasound, ECG)

Chloroxylenol (Dettol)

Does not coagulate proteins

Non corrosive, Non irritating to skin

Commercial 4.8 % solution used for surgical antisepsis

Skin cream and soap: 0.8%

Mouth wash 1%

Cresol

– Methyl derivative of phenol, less damaging to tissues than phenol

– 3-10 times more active

– Used for disinfection of utensils, excreta & for washing hands

AMMONIUM COMPOUNDS ( NH4)

– 0.1-0.2 %

– Advantages: slightly irritating to the hands on adequate amounts

– Disadvantages: does not destroy spores

– For surfaces, rooms, furniture, not for medical instruments.

Detergents: destroy bacteria, fungi & viruses by altering permeability of cell membrane

– Efficiently remove dirt and grease

– Widely used as antiseptics & disinfectants for surgical instruments, gloves

Soaps: Anionic detergents

– Weak antiseptics with cleansing action

– Washing with soap and warm water – one of the most effective methods of preventing disease transmission

ALDEHYDES:

– GLUTARALDEHYDE 2%:

– Advantages: compatible with optical instrumentation, metal, plastic, rubber.

– Disadvantages: – unstable: two weeks – one month maximum.

– Chemical burns if in contact with the tissues for a long period of time,

-For: Endoscopes, respiratory equipment, anesthesia equipment.

– FORMALDEHYDE – gaseous or liquid.

OXIDIZING AGENTS

Peroxides

– Short-acting germicidal effect through release of nascent oxygen, which irreversibly alters microbial proteins

– Little or no action on bacterial spores

– Nascent oxygen rendered inactive when it combines with organic matter

e.g. Hydrogen peroxyde solution (3%)

– releases oxygen in contact with catalase on wound surfaces and mucous membranes; effervescent action mechanically helps remove pus and cellular debris from wounds and is valuable for cleaning infected tissue.

– Advantages: sporicidal, ecological.

– Disadvantages: bad for some endoscopes components, soft contact lenses.

Peracetic acid

Broad antimicrobial spectrum (like hydrogen peroxide) + greater lipid solubility

Effective against bacteria, yeasts, fungi, and viruses (0.001–0.003%)

Sporicidal at 0.25–0.5%

– Solutions of 0.2% peracetic acid applied to compresses are effective at reducing microbial populations in severely contaminated wounds

– Advantage: sporicidal at low temperatures

– Disadvantage: corrosive, strong oxidant, unstable.

– Used for meat, milk, alcoholic and non-alcoholic.

BIGUANIDES

Chlorhexidine

Acts by disrupting bacterial cell membrane & denaturation of bacterial proteins

Nonirritant, more active against Gram positive bacteria

Surgical scrub, neonatal bath, mouth wash & general skin antiseptic

Most widely used antiseptic in dentistry 0.12-0.2% oral rinse or 0.5 -1 % tooth paste

ACIDS

Boric acid

Weak antiseptic, bacteriostatic

Used for mouth wash, irrigation eyes, glossitis

Adverse effect: vomiting, abdominal pain on systemic absorption

METALS: METALLIC SALTS

Silver

– Silver ions precipitate proteins + interfere with essential metabolic activities of microbial cells

– 0.1% aqueous silver solution – bactericidal but irritating

– 0.01% solution – bacteriostatic

– 0.5% solution – sometimes applied as a dressing on burns to reduce infection

Colloidal silver compounds – slowly releasing silver ions

– More sustained bacteriostatic effect

– Non-irritant

– Mild antiseptics, also used in ophthalmic preparations

DYES

Gentian violet (Crystal violet)

Topical antiseptic; commonly used for:

Marking the skin for surgery preparation and allergy testing

Effective against Candida albicans and related infections such as thrush, yeast infections, tinea, etc.

In settings with limited resources, gentian violet is used to manage burn wounds, inflammation of the umbilical cord stump (omphalitis) in neonates, oral candidiasis, mouth ulcers

MCQ examples:

Antiseptic solutions are used on:

A. Surfaces

B. Objects

C. Medical instruments

D. Living tissues

E. Skin

Factors that influence the action of disinfectant:

A. Disinfectant concentration

B. Germ type

C. The degree of contamination

D. The sensibility to antibiotics

E. Cleanliness of surfaces

CLEANING, DISINFECTION AND STERILIZATION

PHYSICAL METHODS:

A. Heat

A.1 DRY – HEAT:

Flame sterilization:

– Bunsen burners – small gas burners with an adjustable flame, manipulated at the base by controlling the amount of gas and air admitted

– Bacteriological loops, needles, scalpels

– Buckling: test tubes, flasks, pipettes

Sterilization ovens – dry heat + vacuum (hot air oven): 180 degrees Celsius, one hour.

Figure no. 3: Hot air oven www.romedic.ro

Components:

– Sterilization chamber

– Electrical heating system

– Thermal insulation (asbestos)

– Thermometer + thermostat

– Fan (mechanical air convection)

Advantages:

Usable for items that cannot be sterilized by moist heat or chemical methods e.g. powders, oils, items prone to rust

Usable for equipments that cannot be disassembled

Non-corrosive

– Disadvantages:

Slow and uneven penetration → longer time required

Less effective than hot steam sterilization (autoclave) – see below

– Higher temperatures required → harmful to some materials, special packaging materials

– Control methods:

Physical methods: indicators for reaching the high temperatures: thermometer or temperature reading on the display monitor.

Chemical methods – Filter paper strips impregnated with thermochromic substances – color change indicates the required temperature has been reached

– Biological test consists in the use of Bacillus subtilis vials, which after sterilization will be incubated for 48 hours at 56 degrees Celsius.

A.2 MOIST HEAT:

Autoclave sterilization: – include the effect of pressure and steam, at 121 degrees Celsius for 30 minutes to 1 atmosphere.

Figure no.4. Autoclave www.sfatulmedicului.ro

Components:

metal container, resistant to high pressures, heavy lid, sealed closure (rubber or silicone)

evacuation valve for air (upon start of sterilization cycle) and steam (at the end of the sterilization cycle)

safety valves, pressure gauges, pressure regulator

manometer: pressure correlated to temperature i.e. 2.03 atmospheres – 121°C; 2.65 atmospheres – 130°C

thermometer

Advantages:

More rapid and even penetration

Better bactericidal/virulicidal effect (denaturation and coagulation of proteins)

Disadvantages:

May corrode or cause rusting of some instruments

Control methods:

Chemical tests: – Based on the melting point of certain substances: e.g.120°C for benzoic acid

Biological tests: cotton fibers impregnated with spores: e.g. B.subtilis/B.stearothermophylus subjected to autoclavation and then inoculated in culture media – lack of bacterial growth means the sterilization has been effective

Suitable for:

Biological waste (hospitals, laboratories)

Culture media

Oral/nasal swabs

Items made of autoclavable rubber (caps, tubes)

Cotton items (laboratory gowns, surgical cloth)

There is a flash autoclave: 134 degrees Celsius for 4 minutes; used in operating rooms.

2. Boiling of 100 degrees Celsius, 30 minutes.

– spores are not destroyed,

– linens, patient’s cookware

3. Pasteurization:

– bath water, conservation of food for a small period of time: milk, dairy products, beer, wine, fruit juices (commercial-scale sterilization of food is not common because it adversely affects the taste).

– foods retain vitamins

– not sporicidal activity.

– NOT a” true” sterilization: it does not” kill all vegetative forms of microorganisms and spores” BUT aims to reduce the number of viable microbes so they are unlikely to cause disease

The method: heating followed by swift cooling and packaging

High pasteurization: 85-90°C, several seconds

Medium pasteurization: 70-75°C, 20 minutes

Low pasteurization: 60-65°C, 1 hour

4. Moist heat – Tyndallization

– The method: staged heating (boiling) for 15 minutes/day during 3 days

Day 1: after the 15 minutes of boiling all vegetative forms of bacteria are killed BUT the spores survive

Day 2: some of the surviving spores, in favorable conditions (moisture, warmth) have turned into vegetative forms; the second round of boiling kills them

Day 3: late germinating spores have survived the second stage of boiling as they were still in a sporulated condition but upon the 3rd boiling round they have already generated vegetative bacteria which will be killed by the 3rd boiling round

– Used for items that cannot withstand autoclavation: e.g. plant seeds

5. Ultra high pasteurization: injecting superheated steam under pressure directly into the fluid;

– it is a sterilization based on instant heating at 150 degrees.

B. Filtration: vaccines, special culture media, biological fluids.

– Mechanical retention of microorganisms from fluids by passing them through special membrane filters with adequate pore size

– Used for fluids which would be damaged by heat or other sterilization agents (radiations, chemicals) e.g. heat labile pharmaceutic substances, wine, beer, etc.

– microfiltration, pore size 0.2 µm for bacteria

– nanofiltration, pore size 20-50 nm for viruses

– The smaller the pore size the higher the retaining effectiveness.

C. Radiations: – Non-penetrating: UV – radiation: – air, smooth surfaces (operating rooms, dental offices)

– 2537 Å wavelength

– Kill bacteria, viruses, fungi in the air by altering their nucleic acids

– Spores are not killed

Penetrating – Ionizing radiations

– Gamma irradiation: Co-60 – industrial sterilization of disposable, single use, medical devices e.g. syringes, pipette tips, surgical wires, implants, etc.; causes microbial death as a direct effect of the destruction of a vital molecule or by an indirect chemical reaction.

– Electron beam (e-beam sterilization) – industrial sterilization of disposable medical devices; same mechanism and dose as in gamma irradiation; advanced electronics precisely control the use of electron beams in the sterilization of medical devices; less material degradation than with gamma irradiation.

– X-ray – alternative for sterilizing large packages and pallet loads of medical devices; no toxic residues; no high temperature rises → more suitable for plastics

CHEMICAL METHODS

Ethylene oxide (CH2OCH2)

bacteria killed under the action of highly active oxygen at relatively low temperatures (20-60°C)

Suitable for medical devices that cannot be subjected to high temperatures, moisture or abrasive chemicals e.g. Electronics, optical equipment, paper, non-autoclavable rubber and plastics

Disadvantages: toxic, allergy, irritant, can only be used in strictly controlled conditions.

Formaldehyde: – liquid sterilizing agent

By immersion, provided that the immersion time is sufficiently long e.g. over 24 hours to kill all spores in a clear liquid

By spraying e.g. closed rooms

Highly volatile, and toxic by both skin contact and inhalation

Many vaccines, such as the original Salk polio vaccine, are sterilized with formaldehyde

HANDS WASHING AND DECONTAMINATION

– SCRUBBING: – water and soap for at least 10 seconds, then dry with paper towels

• After using the toilet, before food use, before the patient consultation, when your hands are dirty

Figure no. 5: Scrubbing technique www.who.com

– HYGIENICAL WASHING: – antiseptic soap or alcohol

• Before invasive maneuvers, before and after contact with immunocompromised patients, before and after the contact with infected wounds, catheters, or contact with any product pathological

• Chlorhexidine 4% or 5%

• Povidone -iodine solution – 0.75% iodine

• 10 to 15 seconds, rinse and dry

– SURGICAL WASHING: – surgical procedures;

• sterile nail brushes, alcohol 70%

– Gloves are not considered as a substitute for hand washing.

MCQ examples:

The correct answers are:

A. Autoclaving includes the effect of pressure and water, at 121 degrees Celsius for 30 minutes to 1 atmosphere.

B. Autoclaving includes the effect of pressure and water, at 121 degrees Celsius for 30 minutes to 2 atmospheres.

C. Autoclaving includes the effect of pressure and water, at 124 degrees Celsius for 60 minutes to 2 atmospheres

D. A flash autoclave uses: 134 degrees Celsius for 4 minutes

E. A flash autoclave uses: 134 degrees Celsius for 20 minutes

Which is the correct answer?

A. Bactericidal effect = destroys bacteria

B. Bacteriostatic effect= destroys bacteria

C. Bacteriostatic effect = stops the multiplication of bacteria

D. Bactericidal effect = stops the multiplication of bacteria

E. Dry – heat sterilization involves the use of autoclave ovens

CULTURE MEDIA

Most bacteria and fungi grow on average inert acellular media. Viruses, rickettsia, chlamydia cells grow on culture, embryonated eggs, or animal experience.

– Culture media conditions:

– Sterile;

– Nutrients: water, carbon, nitrogen, vitamins, minerals, amino acids.

– Optimum pH: 7.2-7.4; Vibrio cholerae -PH = 9

– To be clear, to see the development of microbial colonies

– Aerobic or anaerobic conditions.

The composition of the culture medium: – synthetic environments – most.

Content: peptone, meat extract, yeast extract, sodium chloride, mono- and / or polysaccharides, vitamins.

1. Peptone: by enzymatic or acid hydrolysis of origin animal proteins are rich sources of nitrogen, for the preparation of all culture media.

2. Meat extract: 2 elements necessary for: nitrogen and carbon.

Nitrogen source: creatine sources, xanthine, hypoxanthine, uric acid, adenylate acid, glycine, urea. Carbon sources: glycogen, lactic acid.

3. Yeast extract: – controlled cultivation of yeasts, contains many B vitamins

4. Sodium chloride: 0.9%

5. Mono- or polysaccharides, glycerin, mannitol.

Incubation – usually 37 degrees Celsius.

Classification of culture media:

A. Depending on the state of matter: liquids, semi- solid, solid

B. Complexity: simple, or rich.

C. By order of use: selective, diagnostic, special.

A. Depending on the state of matter:

1. Liquid media: Nutrient Broth

2. Solid media

– Are obtained from liquid media by solidification with agar-agar. Agar is a gelification agent extracted from marine red algae from East Asia. Is colorless, tasteless, non-toxic, and it has no smell. Insoluble in cold water but soluble in boiling water. Solid at the incubation temperatures used in bacteriology (35-37°C). At 80-100 °C is liquid.

Figure no. 6: Culture media

http://www.medicotips.com/2011/07/culture-media-for-bacteria-types-of.html

Figure no. 7: Pouring a plate www.slideshare.com

Figure no. 8: Pouring a plate www.slideshare.com

Figure no. 9: The technique for obtaining isolated colonies www.slideshare.com

B. Depending on complexity

1. Simple media : solid/liquid media- (above).

2. Enriched media: blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious microbes e.g. blood agar, chocolate agar

a. 10% blood agar: – Add 10 ml blood agar melted and cooled to 45 degrees Celsius. Sheep blood is used (Romania, U.S.) Horse (England). In this environment follows hemolytic activity.

– Used to isolate microorganisms and detect hemolytic activity:

– β-hemolysis – lysis and complete digestion of red blood cell contents surrounding colony, e.g. Streptococcus pyogenes

– α-hemolysis – partial lysis – incomplete hemoglobin digestion → green or brown (due to the conversion hemoglobin to methemoglobin), e.g. Streptococcus viridans

– γ-hemolysis (or non-hemolytic) – lack of hemolytic activity

b. Chocolate agar

– variant of blood agar in which red blood cells have been lysed by slow, by gradual heating to 80°C in order to provide additional growth factors contained in red blood cells

– Does not contain chocolate!! The name is suggestive for the brownish color resulted after red blood cell lysis

– Used for growing fastidious respiratory bacteria, e.g. Haemophilus influenzae, Neisseria meningitidis

c. Enriched with sugars glucose bullion

d. Enriched ascites, serum, yeast extract.

Figure no. 10. Blood-agar www.umftgm.ro

Attention!

Enriched media are non-selective – i.e. they contain additional substances aiming to a better growth & multiplication

≠

Enrichment media are selective i.e. content adjusted to favor certain germs and inhibit others

C. Depending on intended use:

– Selective & enrichment media

– Diagnostic media

– Special media

1. Selective & enrichment media:

– Favors the growth and the multiplication of species while suppressing other species.

– Very useful for polymicrobial biological products when attempting to isolate pure cultures

– Used for inoculation of biological products (primary isolation)

– Composition & cultivation conditions (temperature, aero/anaerobiosis, etc.) adjusted according to the known growth characters & requirements of the suspected microbe

– Examples of components or conditions which increase the selectivity of culture media:

Nutrient broth + acid sodium selenite – Salmonella spp

Peptone water – Vibrio cholerae – the alkaline pH (9) inhibits other species

Temperature: +4°C – inhibits the growth of most bacteria EXCEPT Listeria spp

Sodium chloride Staphylococcus -not inhibited. Streptococcus is inhibited.

Antibiotics. E.g. vancomycin.

Example: Gram negative bacteria – resistant to vancomycin, while gram-positive bacteria are destroyed by vancomycin.

Figure no. 11: Blood agar for antibiotic testing www.slideshare.com

2. Diagnostic media: they contain: the main nutrients, sugar, pH indicator that modifies their color.

E.g.: Fermentation of sugars:

-nutrients + sugar + pH indicator – if fermentation occurs the color will change indicating the presence of bacteria which ferment that sugar

Identification relies on performing a number of tests and analyzing the ”profile” which is further matched to known metabolic & growth characters of bacteria

Figure no. 12. Mannitol Salt Agar (CHAPMAN)

www.slideshare.com

-It is used to isolate Staphylococcus aureus and Staphylococcus epidermidis

-Incubation 24 hours in an aerobic atmosphere, 37°C.

– It contains a high concentration (about 7.5%-10%) of salt (NaCl), making it selective for some Gram-positive bacteria (Staphylococcus and Micrococcaceae) since this level of salt is inhibitory to most other bacteria.

– It is also a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the indicator phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci. Staphylococcus aureus produces yellow colonies with yellow zones, whereas other coagulase-negative staphylococci produce small pink or red colonies with no color change to the medium. If an organism can ferment mannitol, an acid byproduct is formed that causes the phenol red in the agar to turn yellow.

– It is used for the selective isolation of presumptive pathogenic (pp) Staphylococcus species.

Figure no. 13. Staphylococcus aureus versus Staphylococcus epidermidis – CHAPMAN medium

www.slideshare.com

Yellow colonies = mannitol positive Staphylococcus aureus with yellowish halo around colonies.

Whitish colonies = mannitol negative colonies of Staphylococcus epidermidis.

S. aureus – mannitol fermentation

S.epidermidis – no mannitol fermentation

Figure. No. 14. MAC CONKEY medium

www.slideshare.com

MacConkey agar is a selective and differential culture medium for bacteria designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and differentiate them based on lactose fermentation.

The crystal violet and bile salts inhibit the growth of gram-positive organisms which allows for the selection and isolation of gram-negative bacteria. Enteric bacteria that have the ability to ferment lactose can be detected using the carbohydrate lactose, and the pH indicator neutral red.

It contains:

– bile salts (to inhibit most Gram-positive bacteria),

– crystal violet dye (which also inhibits certain Gram-positive bacteria),

– neutral red dye (which turns pink if the microbes are fermenting lactose).

Figure no. 15: Mac Conkey agar

www.slideshare.com

Figure no. 16: Lactose fermenting/non-fermenting

www.slideshare.com

3. Special media, for certain bacteria.

Examples: – Lowenstein – Jensen – for Koch bacillus

– Tinsdale – for Corynebacterium diphtheriae bacillus

– Bordet Gengou – for Bordetella pertussis.

Figure no. 17: Lowenstein Jensen medium www.slideshare.com

Sources of error in preparing culture media:

– Choosing the wrong media;

– Error in measuring the amount of water;

– Inappropriate pH;

– Imprecise distribution;

– Failure of proper sterilization;

– Dirty and contaminated glass;

– Interruption of electricity during the night with lower incubator temperature.

– Improper collection of samples.

Companies that produce culture media: Romania (Cantacuzino Institute), U.S.(DIFCO), Germany (Merck), England (Oxoid, Unipath), France (Sanofi, BioMerieux), etc.

MCQ examples:

A culture medium has the following nutrients:

A. Peptone

B. Meat extract

C. Yeast extract

D. Fat from animals

E. Vitamins

2. For the preparation of blood-agar media we use:

A. Horse blood

B. Sheep blood

C. Dog blood

D. Human blood

E. Rabbit blood

STEPS IN BACTERIOLOGICAL DIAGNOSIS

AIMS

To provide accurate information about the presence or absence of microorganisms in a specimen.

Three main categories:

– Identification of microorganisms by isolation and culture,

– Identification of a specific microbial product,

– Detection of specific antibodies to a pathogen.

IDENTIFICATION OF MICROORGANISMS BY ISOLATION AND CULTURE

– growth – in artificial media = bacteria;

– in cell cultures = viruses.

Quantitation is important – e.g.: > 105 bacteria/ml of urine=infection

Once an organism has been isolated in culture, its susceptibility to antimicrobial agents can be determined.

IDENTIFICATION OF A SPECIFIC MICROBIAL PRODUCT

Non-cultural techniques= do not depend on the growth and multiplication of microorganisms;

More rapid results

Include – detection of:

– structural components of the cell ( cell-wall antigens)

– extracellular products ( toxins).

Alternatively: specific gene sequences detected by the application of DNA probes to clinical specimens -amplification of DNA by the polymerase chain reaction (PCR).

Potentially applicable to all microorganisms

Antimicrobial susceptibilities cannot be determined without culture (although the presence of resistance genes may be detected by specific probes).

DETECTION OF SPECIFIC ANTIBODIES TO A PATHOGEN

Method of choice when the pathogen cannot be cultivated in laboratory media (e.g. Treponema pallidum, many viruses) or when to do so would be very hazardous to laboratory staff.

The usual method is by detection of a rise (four-fold or greater) in antibody titer between "paired" sera, collected in the acute phase of an infection and in convalescence. Alternatively, a single test for “ acute phase ” specific antibodies (IgM).

The diagnostic cycle

Laboratory testing – series of events:

Pre-analytical

Analytical = observation & isolation & identification & enumeration

Post-analytical

Performance must be monitored throughout the entire cycle for quality assurance i.e. accurate, reliable results

Pre-analytic phase

SPECIMEN COLLECTION

Crucial for confirming a certain microorganism as cause of the clinically suspected infectious disease

Improper specimen collection may cause:

* Failure to recover the microorganism (no growth on culture medium)

* Incorrect / harmful therapy e.g. directed against a commensal / contaminant microorganism

E.g. Klebsiella pneumoniae:

– recovered from sputum of pneumonia patient;

– recognized causative agent of pneumonia BUT also may colonize the naso-pharynx

– If sputum sample consisted mostly of saliva then isolating K.pneumoniae might not reflect the true cause of the patient‘s pneumonia but saliva contamination of the sputum sample

Specimen collection -performed with care, avoiding harm or unnecessary discomfort to the patient.

When the patient is asked to collect a specimen (e.g. of urine or sputum), clear instructions must be given

Representative specimen – good quality and adequate quantity.

Rules for correct specimen collection:

Source: actual infection site; minimal contamination from adjacent tissues, organs, secretions e.g. throat swabs from peritonsillar fossae and posterior pharyngeal wall, avoiding contact with other oral areas

Optimal moment: depending on the natural history and pathophysiology of the infectious process e.g. Typhoid fever: blood – 1st week; feces and urine – 2nd-3rd week

Sufficient quantity

Appropriate collection devices, containers + transport systems (container ± transport medium): main objective to decrease time between collection and inoculation to prevent lack of recovery of certain bacteria

Sample collection before antibiotics (if possible)

Smears performed to supplement culture (if possible)

Assessment of inflammatory nature of specimen → aid the clinical integration (meaningfulness) of the culture result

Gram smears e.g. Gram negative bacilli + no growth on aerobic culture (wrong atmosphere or wrong media i.e. fastidious microbes e.g. Legionella)

Labeling of specimen containers & Request form:

Legible

Minimum information:

Patient name; identification number (hospital file, practice log book, etc.)

Source of specimen; clinician + contact data

Date and hour of collection

Clinical diagnosis (suspected infection)

Treatments (antibiotics?…)

Specimens for culture

Can be divided into two main types:

– fluid (including exudate and excreta)

– tissue

Swabs – collect samples of fluids (e.g. wound exudate),

– sampling surfaces (e.g. skin)

Sterile containers

Specimens for detection of microbial products

It is important to minimize the risk of contamination by external (enviroment) organisms

Specimens for detection of antibodies

Samples of serum (and sometimes cerebrospinal fluid) are used to detect antibody responses.

'Paired' serum, collected in the acute and convalescent phases of the disease (ideally 10-14 days apart), are tested in parallel.

Serum samples can be stored in the cold (-20°C) for months or years without loss of antibody titer.

SPECIMEN LABELLING AND REQUEST FORMS

All specimens must be properly labelled by the person collecting the specimen.

Correct identification continues during the specimen's passage through the laboratory.

The results of all tests on the specimen should be compiled into a report

Each specimen should be accompanied by the information about the patient, the clinical diagnosis and current antimicrobial therapy.

TRANSPORT OF SPECIMENS

Specimens should be transported to the laboratory as quickly as possible.

In general, most specimens should be processed in the laboratory within 1 to 2 hours after collection.

In practice, a 2-to 4-hour time limit is probably more practical during a normal working day.

A continuous effort must be made in order to ensure proper collection and transportation of clinical specimens.

Refrigeration for short periods may preserve the organisms in a urine specimen, but similar conditions will kill certain fastidious organisms and greatly reduce the chances of isolating small numbers of bacteria, from blood cultures for example.

Transport media

Fluids and tissue specimens should be transported to the laboratory in sterile containers, without the addition of preservative.

If anaerobic bacteria are suspected and a pus sample is obtainable – aspire into a syringe – the needle removed – the syringe tip capped and the specimen sent without delay to the laboratory to minimize exposure to air.

The addition of antibiotics to viral transport media helps to reduce bacterial contamination

Main transport related objectives:

Sample related: Transport media

Maintain the sample as similar to its original state as possible

Human & environment related: Packaging and transport systems and regulations

Prevent contamination/infection of healthcare staff & environment & general population (biosafety)

”Triple” Packaging of biological samples:

Outer box (usually cardboard, rigid, secure closure system, adequately labeled to state content)

Inner container (waterproof, resistant to pressure, usually plastic, securely closed by lid, contains additional materials to absorb shocks e.g. bubbled plastic bags and leakages e.g. absorbent material

Sample containers (tubes, plates) inserted in the inner plastic container, wrapped in above mentioned shock- and fluid absorbent materials

+ request form and other documents inserted in sealed plastic bags, inserted in outer box or sticked to its exterior

Analytical phase= observation & isolation & identification & enumeration

SPECIMEN PROCESSING

Examples of acceptance/rejection criteria (checklist):

Request form & labels contain all info required (check for consistency !!!)

Improperly packaged, leaking / broken containers

Time from collection to receipt – too long to allow recovery

Improper / lack of transport media

Insufficient quantity e.g. single swab for multiple requests

Overgrown / dried out culture plates

Each lab must have such a list and share it with collaborators!

Rejecting samples must be avoided as much as possible!

Collection & transport requirements must be shared with clinicians!!!

Specially designed area / room for receiving and recording samples

Rules for manipulating samples and accompanying documents (UNIVERSAL PRECAUTIONS):

Samples: biological safety cabinet (BSC), personal protective equipment (PPE): lab coat, gloves, eye&respiratory protection

Documents – handled by different person / at different stage e.g. either before or after preliminary examination/processing of sample (after removal of gloves & hand washing) – purpose: avoid cross contamination of objects (log record book, computer, pens, etc.)

Preliminary actions upon receipt of specimens

Data entry into lab log book/computer database

(Unpacking and) visual examination – check for acceptance criteria

– Microscopic examination of wett mounts/stained smears → presumptive diagnosis

– Sample(s) taken to area/room where the analytical phase begins

Specimens intended for the cultivation of microorganisms can be divided into two types:

– from sites that are normally sterile

– from sites which usually have a commensal flora

Specimens from sterile sites, (urine, sputum from the lower respiratory tract), are usually collected after passage through orifices that have a normal flora, which may contaminate the specimens. This needs to be considered when interpreting culture results of these specimens.

TEHNIQUES FOR DIAGNOSIS

Microscopical examination

= first step in the examination of all specimens

Types of microscopes

Optical – Magnification objectives

10x; 40x; 100x for bacteria

Phase contrast

Dark field (dark ground)

Fluorescence – UV light

Electron

LIGHT MICROSCOPY

A.Bright field microscopy

Wet preparations – blood cells and microbes in fluid specimens (urine, faeces or cerebrospinal fluid),

– cysts, eggs and parasites in faeces,

– fungi in skin

– protozoa in blood and tissues. Living organisms can be examined to detect motility

Dyes are used to stain cells so that they can be seen more easily.

Stains are usually applied to dried material that has been fixed (by heat or alcohol) onto the microscope slide.

The most important differential staining technique in bacteriology is the 'Gram' stain =>

Gram – positive (stain purple)

Gram-negative (stain pink)

Figure no. 18:Gram positive and Gram negative bacteria

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The Ziehl-Neelsen stain

– is a differential staining procedure

– utilizes heat to drive the fuchsine stain into the cells;

– mycobacteria stained with fuchsine withstand decolorizing with acid and alcohol and hence they are known as 'acid-' and 'alcohol-fast' (other bacteria lose the stain after acid and alcohol treatment).

B. Dark ground (dark field) microscopy

The light microscope – adapted – the object appears brightly lit against a dark background.

Organisms examined in the living state

Also used for visualizing very thin cells such as spirochetes because the light reflected from the surface of the cells makes them appear larger and thus more easily visible than they are under bright field.

C. Phase contrast microscopy

This technique enhances the very small differences in refractive index and density between living cells and the fluid in which they are suspended.

A higher degree of contrast than that achieved by bright field microscopy.

Rarely used in the diagnostic laboratory.

D. Fluorescence microscopy

Some biological substances are naturally fluorescent; others can be stained with fluorescent dyes and viewed in a microscope with an ultraviolet light source instead of white light.

It is widely used in microbiology and immunology

Developed to detect microbial antigens in specimens by 'staining' with specific antibodies tagged with fluorescent dyes (immunofluorescence). Direct or indirect staining methods can be used.

ELECTRON MICROSCOPY (EM)

A beam of electrons instead of light and magnets are used to focus the beam.

The whole system is operated under a high vacuum conditions.

Electrons pass through the section -> an image on a fluorescent screen, images are photographed and enlarged so that the original specimen is magnified many thousand fold

Used to identify virus particles (e.g. rotaviruses, hepatitis A virus).

Fluid for examination is dried onto a copper grid and examined.

The sensitivity can be increased by treating the fluid with antiviral antibodies so that clumps of virus particles are visible. This is known as immunoelectron microscopy, a technique analogous to immunofluorescence in light microcopy.

Detection of microbial antigens in specimens

The methods include those which detect antigens by their interaction with specific antibodies.

For example, the common causative agents of bacterial meningitis (Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis types A and C) can be detected in cerebrospinal fluid (CSF) by mixing the specimen with specific antibody coated onto latex particles

CULTIVATION (CULTURE) OF MICROORGANISMS

Bacteria and fungi grow on the surface of solid nutrient media – produce colonies composed of thousands of cells derived from a single cell inoculated on the agar surface

It takes 12-48 hours for colonies of most species to become macroscopically visible,

Parasites such as Leishmania, Trypanosoma and Trichomonas can be cultivated in liquid media to allow small numbers present in the original specimen (e.g. blood or vaginal secretions) to multiply and thus become easier to detect by microscopically examination.

Parasites do not form colonies on solid media in the same way as bacteria and fungi.

Viruses, chlamydia and rickettsia must be grown in cell or tissue cultures because they are incapable of independent growth.

IDENTIFICATION OF MICROORGANISMS (CULTURES)

Identification of bacteria:

• microscopic characteristics: cell morphology (e.g. rod or coccus) and arrangement (e.g. pairs or chains);

• cultural characteristics: ability to grow under aerobic or anaerobic conditions; growth requirements (simple or fastidious).

• Further identification is made on the basis of biochemical properties such as:

• ability to produce enzymes that can be detected by simple tests: e.g. coagulase, catalase, oxidase, lecithinase;

• ability to metabolize sugars by oxidation or fermentation

• ability to utilize a range of substrates for growth: e.g. glucose, lactose, sucrose etc.

Antibiotic susceptibility tests

– Pure cultures;

– A lawn of the test organism inoculated on an agar plate on whichdiscs impregnated with antibiotics are placed.

– During overnight incubation the organisms grow and multiply and the antibiotics diffuse out from the discs and inhibit growth around the disc.

In vitro testing for the sensitivity of microbes to various antibiotics; expressed as:

MIC (minimal inhibitory concentration) – the lowest quantity of antibiotic completely inhibiting the multiplication of a bacterial strain

MBC (minimal bactericidal concentration) – the lowest quantity of antibiotic able to kill 99.9-100% of the germs of a tested bacterial strain

In vivo – concentration of antibiotic at the infection site.

MCQ examples:

Which of the following statements are false:
A . Collect the specimen before the administration of antimicrobial agents

B. The prevention of contamination of the specimen with externally present organisms or normal flora of the body is very important

C. Collect the specimen after the administration of antimicrobial agents

D. The prevention of contamination of the specimen with enviromental organisms or normal flora of the body is not important

E. All specimens must be properly labelled by the person collecting the specimen.

2. The true statesments are:

A. In general, most specimens should be processed in the laboratory within 1 to 2 hours after collection

B. In practice, a 2-to 4-hour time limit is probably more practical during a normal working day

C. In practice, a 1-to 2-hour time limit is probably more practical during a normal working day

D. In general, most specimens should be processed in the laboratory within 5 to 6 hours after collection
E. Refrigeration for short periods may preserve the organisms.

COLECTION OF SPECIMENS

COLLECTION AND TRANSPORTATION OF CLINICAL SPECIMENS

The laboratory diagnosis of an infectious disease begins with the collection of a clinical specimen for examination or processing in the laboratory (the right one, collected at the right time, transported in the right way to the right laboratory).

The first step in obtaining an accurate laboratory diagnosis of an infectious disease is proper collection of an appropriate clinical specimen

The guidelines must emphasize two important aspects:

– Collection of the specimen before the administration of antimicrobial agents.

– Prevention of contamination of the specimen with environmental organisms or normal flora of the body.

CRITERIA AND CONDITIONS

– Apply strict aseptic techniques throughout the procedure.

– Wash hands before and after the collection.

– Collect the specimen at the appropriate phase of disease.

– Make certain that the specimen is representative of the infectious process (e.g. sputum/ saliva) and is adequate in quantity for the desired tests to be performed.

– Collect or place the specimen aseptically in a sterile and/or appropriate container.

– Ensure that the outside of the specimen container is clean and uncontaminated.

– Close the container tightly so that its contents do not leak during transportation.

– Label and date the container appropriately and complete the collection form/request form.

– Arrange for immediate transportation of the specimen to the laboratory.

CRITERIA FOR REJECTION OF SPECIMENS

Examples:

– Missing or inadequate identification

– Insufficient quantity

– Specimen collected in an inappropriate container

– Contamination suspected

– Inappropriate transport or storage

– Unknown time delay

– Haemolysed blood sample

COLLECTION OF SPECIMENS

The clinical state of the patient will not necessarily be reflected by the result of laboratory investigation despite correct laboratory performance unless the specimen is in optimal condition required for the analysis.

BLOOD

Whole blood is required for bacteriological examination.

– Serum separated from blood – serological techniques.

– Skin antisepsis is extremely important at the time of collection of the sample.

Ideal agents: – tincture of iodine (1-2%),

– povidone iodine (10%)

– chlorhexidine (0.5% in 70% alcohol)

While collecting blood cultures, the following points must be remembered:

– Collect blood during the early stages of disease (the number of bacteria in blood is higher in the acute and early stages of disease).

– Collect blood during paroxysm of fever (higher at high temperatures in patients with fever).

– In the absence of antibiotic administration, 99% culture positivity can be seen with three blood cultures

– Small children usually have higher number of bacteria in their blood as compared to adults and hence less quantity of blood needs to be collected from them (Table no. 7).

Table no. 7: Volume of blood to be collected at different ages

CEREBROSPINAL FLUID (CSF)

Examination of CSF – meningeal irritation or affected cerebrum.

Almost 3-10 ml of CSF is collected and part of it is used for:

– biochemical,

– immunological

– microscopical examination

A.D.A.M.INC

Figure no. 19: Collection technique of cerebrospinal fluid

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A.D.A.M.INC

Figure no. 20: Collection technique of cerebrospinal fluid

IMPORTANT PRECAUTIONS:

– Collect CSF before antimicrobial therapy is started.

– Collect CSF in a screw – capped sterile container and not in an injection vial with cotton plug

– Do not delay transport and laboratory investigations

– Transport in a transport medium if delay in processing is unavoidable

– CSF is a precious specimen, handle it carefully and economically. It may not be possible to get a repeat specimen.

– Perform physical inspection immediately after collection and indicate findings on laboratory request form.

– Store at 37°C, if delay in processing is inevitable.

Figure no. 21: Cerebrospinal fluid: diagnostic steps www.slideshare.com

NORMAL VALUES

Pressure: 70 – 180 mm H20

Appearance: clear, colorless

CSF total protein: 15 – 60 mg/100 mL

Gamma globulin: 3 – 12% of the total protein

CSF glucose: 50 – 80 mg/100 mL (or greater than 2/3 of blood sugar level)

CSF cell count: 0 – 5 white blood cells (all mononuclear), and no red blood cells

Chloride: 110 – 125 mEq/L

Table no. 8: The characteristics of the appearance of cerebrospinal fluid

GLUCOSE

– Increased CSF glucose = high blood sugar

– Decreased CSF glucose = hypoglycemia, bacterial or fungal infection (meningitis), tuberculosis, or certain other types of meningitis.

BLOOD CELLS

– Increased white blood cells = meningitis, acute infection, beginning of a chronic illness, tumor, abscess, stroke or demyelinating disease (multiple sclerosis)

– Red blood cells = bleeding into the spinal fluid or the result of a traumatic lumbar puncture.

CSF PRESSURE

– Increased CSF pressure – increased intracranial pressure

– Decreased CSF pressure – spinal cord tumor, shock, fainting, or diabetic coma

GAMMA GLOBULIN

– Increased CSF gamma globulin levels may be due to diseases such as multiple sclerosis, neurosyphilis or Guillain-Barre syndrome

3. SPUTUM

Sputum is processed in the laboratory for etiological investigation of bacterial and fungal infections of the lower respiratory tract.

It is the most important in the diagnosis of pulmonary tuberculosis.

Figure no. 22: Sputum sample collection technique

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4. URINE

– Under normal circumstances urine is sterile.

– The lower part of the urethra and the genitalia are normally colonized by bacteria, many of which may also cause urinary tract infection.

– Since urine is a good growth medium for all sorts of bacteria, proper and aseptic collection assumes greater importance for this specimen.

– For microbiological examination urine must be collected as a "clean catch-mid-stream" specimen.

– Urine specimens should be transported to the laboratory within one hour for bacteriological examination, because of the continuous growth of bacteria in vitro thus altering the actual concentration of organisms.

5. STOOL

– Faecal specimens – etiological diagnosis (acute infectious diarrheas);

– Collected – early stage of illness and prior to treatment with antimicrobials.

– A stool specimen rather than a rectal swab is preferred.

– The faeces specimen should not be contaminated with urine.

– Do not collect the specimen from bedpan.

– 1 to 2 gr quantity is sufficient.

– lf possible, submit more than one specimen on different days.

-The fresh stool specimen must be received within 1-2 hours of passage.

-Store at 2-8°C.

-Modified Cary and Blair medium = good transport medium. It is a very stable medium. It is a semi-solid transport medium

-Use screw – capped containers

-At least two swabs should be inoculated

-Most pathogens will survive for up to 48 hours at room temperature.

-Specimens are unacceptable if the medium is held for more than one week or if there is detectable drying of the specimen.

6. THROAT SWAB

– Depress the tongue with a tongue blade.

– Swab the inflamed area of the throat, pharynx or tonsils with a sterile swab taking care to collect the pus or piece of membrane.

– Transport in sterile transport tube.

Figure no. 23: Throat swab collection technique

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7.NASAL SWAB

Figure no. 24: Nasal swab collection technique

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8. RECTAL SWAB

Insert swab at least 2.5 cm beyond the anal sphincter so that it enters the rectum.

Rotate it once before withdrawing.

Transport in Cary and Blair or other transport medium.

TRANSPORTATION OF SPECIMENS

In general, most specimens should be processed in the laboratory within 1 to 2 hours after collection.

In practice, a 2-to 4-hour time limit is probably more practical during a normal working day.

A continuous effort must be made in order to ensure proper collection and transportation of clinical specimens.

MCQ examples

Which is the right amount of blood collected from a child under 2 years?

A. 1ml

B. 2m

C. 6ml

D. 10 ml

E. 20ml

2. Which is the right quantity of cerebrospinal fluid collected?

A. Almost 3-10 ml

B. Almost 20 ml

C. Almost 5 ml

D. Almost 30 ml

E. Almost 40 ml

8. MICROSCOPIC AND MACROSCOPIC EXAMINATION IN BACTERIOLOGICAL DIAGNOSIS

MACROSCOPIC EXAMINATION

THE CEREBROSPINAL FLUID

– color

– turbidity

– deposits

– clot before being subjected to centrifugation.

Normally CSF = transparent

– no color

– clear like water

If we have: fever, neck stiffness, photophobia, and a clear CSF => viral meningitis or tuberculosis .

A turbid CSF = indicates a bacterial infection

– pneumococcus

– Haemophilus influenzae

PUS

Is important to follow:

– Color

– Texture

– Smell

E.g.: * Staphylococcus aureus

– The most common bacterium involved

– Produces a creamy yellow pus

* Pseudomonas (Pseudomonas aeruginosa)

– Secrets a diffusible pigment

– The pus is bluish green with aromatic odor.

Figure no. 25: Pseudomonas aeruginosa culture

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URINE

It is important to notice:

– Color

– Degree of turbidity

– The presence and nature of the sediment (if it exists)

– The presence of filaments, blood (hematuria), pus (pyuria)

If the urine is turbid this is due to the presence of leukocytes or salts.

A mild heating flame, will lead to the disappearance of turbidity due to salts.

Normal aspect Hematuria Pyuria

Figure no. 26: Urine sample appearance

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SPUTUM

It is important to notice:

– Color

– Adhesion

– The presence of pus fragments

– Consistency

In pneumococcal pneumonia = sputum has a rusty pathognomonic aspect.

Bright red sputum = tuberculosis (hemoptysis)

Yellowish – white sputum = presence of white blood cells (infection)

Figure no. 27: Types of sputum samples

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FAECES (THE STOOL)

Look for the presence of:

– Pus

– Blood

– Excess mucus

– Color

– Consistency

In dysentery mucopurulent and bloody stools are pathognomonic.

MICROSCOPICAL EXAMINATION

BRIGHTFIELD MICROSCOPY

Typically used 3 objectives

– A lens that magnifies

– 10x is used to obtain an overview of the preparation examined

– A 40x objective – higher – for example fungi, parasites

– Immersion lens that enhances the image 90 to 100x

MICROSCOPE WITH A DARK BACKGROUND

Allows only light to enter from the periphery of the lens.

Examined native preparations (wet mounts) between slide and cover slip

– Made ​​directly from a product: bacteria < 0.1-0.2 micrometers – Treponema pallidum in syphilitic chancre

– In a liquid medium

– Culture

PHASE CONTRAST MICROSCOPY

– wet mounts

– It is equipped with filters that allow observity of the phase difference of light as it passes through the items of different density

– Three-dimensional micro-organisms and cells plus and additional morphological details.

U.V. LIGHT MICROSCOPE

– It is used to examine the preparations stained with fluorescent substances

– Light source: ultraviolet light instead of white light

– UV light wavelength = 180 – 400 nm

– White light wavelength = 400 – 700 nm

– allows visibility of smaller microorganisms (smaller wavelength → smaller resolution power)

– allows observation of substances absorbed by microorganisms (become fluorescent under UV light)

– UV radiations – not visible → images impressed on photographic film (image converter tube) / captured by phototube and projected on screen

By optical microscopy are examined:

> Wet mounts in direct light, dark field or phase contrast;

> Smears fixed and stained.

> Preparations stained with fluorochromes (fluorescein isothiocyanate, auramine) UV light microscopy.

Wet mounts in direct light is used to highlight:

– Cells (neutrophils, erythrocytes)

– Mobile germs in liquid samples such as urine, CSF

– Fluid cysts, protozoa in urine, faeces etc.

DARK FIELD MICROSCOPY

– Is used to detect Treponema pallidum in conjunction with clinical data

Direct smear provides data:

– Presence of inflammatory cells (neutrophils, macrophages)

– The morphology of bacteria (cocci, bacillus, Vibrio, spirils)

– The arrangement (pairs, diplo, Tetrades, chains, uppercase letters or Chinese letters)

– Their relationship with existing cells in pathological products (intra and extracellular)

– It also provides triage of evidence, if evidence came too late in the laboratory, microbes already dead.

Stained smears

– Cleaning and degreased slides;

– A bacteriological loop

– Pasteur pipettes

– Dyes

A fluid pathological product:

– We can make smears directly from the product itself, with a Pasteur pipette

– We put one drop of product on the slide, and spread the smear product, taking care to let free the edge of the slide

– After use, the pipette is inserted into a container with disinfectant.

– Smear was allowed to dry at room temperature.

When germ density is low germs in a biological fluid (CSF, urine)

– We have to centrifuge to 3000 rotations/ min for 10 minutes

– The smear it is performed from the sediment

-With a sterile loop put one drop of the sediment in the middle.

– After use, sterilize the loop into the flame until it becomes red.

FIXATION – is used

– to stick the product on the slide,

– to ease the dye absorption on the surface of bacteria.

Fix before staining by passing through flame 3-4 times, warming the smear on the opposite side.

CLASIFICATION OF STAINS

Routine stains – Simple stain: methylene blue

Differential stains (counter staining): – Gram and Ziehl-Neelsen.

– Del Vecchio, May-Grunwald Giemsa.

SIMPLE STAINING: METHYLENE BLUE

Flame fixed smear is covered with methylene blue

-> leave for 1-2 minutes -> rinse with water -> dry it at room temperature

The background is light blue and bacteria appear dark blue.

Figure no. 28: Methylene blue staining method

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COMBINED STAINING

GRAM

A colony is dried on a slide and treated as follows:

Step 1. Staining with crystal violet.

Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.

Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram negative) and not others (Gram positive).

Step 4. Counterstaining with safranin I Fuxin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink.

Figure no. 29: Gram staining technique

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– The background appears red.

Figure no. 30. Gram staining method- Gram positive bacteria

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ZIEHL- NEELSEN STAIN

Is used to identify Mycobacterium tuberculosis.

We need to use heat to color the bacteria, and the process is difficult.

Technique:

– Fix the smear with flame -> cover with fuchsine -> heat up occur vapor (repeat 3 times within 10 minutes)

For heating use a metal rod which has at one end an alcohol swab

– Fade with diluted nitric acid or sulfuric acid.

– Wash with water -> Recolor methylene blue 1-2 minutes. Wash with water -> dry and examine.

– Alcohol – resistant acid-fast bacilli (BK), appear in red on a blue background.

Figure no. 31: Ziehl- Neelsen staining method https://it.wikipedia.org/wiki/Mycobacterium_tuberculosis

GIEMSA STAIN

– Preparations of blood, vaginal, urethral secretions, exudates

Technique:

– Methyl alcohol or ethylic-alcohol in equal parts for 2-3 minutes -> cover with diluted Giemsa solution for 20-23 minute -> wash with water -> dry and examine under a microscope.

Figure no. 32: Giemsa staining method

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MICROSCOPIC EXAMINATION OF CEREBROSPINAL FLUID

– Centrifuge at 3000 rotations / minute for 5 minutes.

From the sediment three smears are performed: Gram stain, Ziehl- Neelsen and a reserve smear…

MICROSCOPIC EXAMINATION OF THE PUS

– Gram

– PMN cells, and then bacteria.

MICROSCOPIC EXAMINATION OF SPUTUM

Wash with saline solution ->Fix into the flame and stain with Gram, Ziehl- Neelsen, or Giemsa coloration.

MICROSCOPIC EXAMINATION OF URINE

Centrifuged urine

From the sediment we obtain smears, and then we use Gram coloration and Ziehl- Neelsen coloration (renal TB).

Bacteria and PMN cells.

MICROSCOPIC EXAMINATION OF THE PRODUCTS OBTAINED FROM BIOPSY OR AUTOPSY

Tap the fragment from the biopsy (or autopsy) to obtain a pres imprint or touch imprint smear. Smear is fixed with flame and stained Gram and Ziehl- Neelsen coloration.

MCQ examples:

The correct affirmation are:

A. Staphylococcus – Arrangement in clusters

B. Streptococcus – Arrangement in chains

C. Streptococcus pneumoniae – Arrangement in diplo like two coffee beans

D. Neisseria gonorrhoeae – Arrangement in diplo like two candle lights

E. Streptococcus pneumoniae – Arrangement in chains

2. Cocci morphology – correct affirmation:

A. Round – Staphylococcus

B. Oval – Streptococcus

C. Lanceolate – Streptococcus pneumoniae

D. Reni form – Neisseria (gonococci, meningococci)

E. Oval – Staphylococcus

9. BACTERIAL CULTURES. IDENTIFICATION OF BACTERIA BASED ON MORPHOLOGICAL CHARACTERS

Isolation of bacterial colonies – essential to work with pure cultures. Why?

-to determine

-colonial characteristics,

– biochemical properties, and

– other details

Selection of primary media depends on the suspected causative bacteria from clinical sample.

Primary inoculation – loops / swab

Bacterial streaking to obtain isolated colonies

Bacterial culture streaking

Purpose: to identify and isolate pure bacterial colonies from a mixed population i.e. biological specimen

spread specimen across agar plate

incubate at a certain temperature for a period of time

Colonial Characters on Solid Media

Size

Shape

Margins

Transparency

Relief

Type S / R

Further description

Color

Consistency

Adherence to medium

Changes induced in the medium

Colonial characters: Colony Size

Large: Staphylococcus

Small: Shigella, Salmonella

Pinpoint (”powder grains”): Streptococcus

Very small: Neisseria

Figure no. 33: Colonial characters – Colony Size

Left: Staphylococcus (large colonies);
Right: Streptococcus (small, powder-like colonies)

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Colonial characters: Colony shape

Round, convex, smooth (S type of colonies) (Staphylococcus, Streptococcus, Neisseria)

Figure no. 34: Colonial characters – Colony Shape

”Daisy flower”, rough (R type of colonies) – Corynebacterium

Figure no. 35:Colonial Characters – Colony Shape

Klebsiella pneumoniae: Mucous colonies

Color of colonies (bacterial pigment)

White / colorless: Staphylococcus epidermidis

Golden: Staphylococcus aureus

Green: Pseudomonas aeruginosa

Figure no. 36: Colonial Characters – Colony Color

Golden: Staphylococcus aureus

Figure no. 37: Colonial Characters – Colony color

Green: Pseudomonas aeruginosa

Changes induced in medium components

Hemolysis:

β-hemolysis – complete digestion of red blood cell contents surrounding colony e.g. Streptococcus pyogenes

α-hemolysis – partial lysis – incomplete hemoglobin digestion → green or brown (conversion of hemoglobin to methemoglobin) e.g. Streptococcus viridans

γ-hemolysis (or non-hemolytic) – lack of hemolytic activity

Figure no. 38: Colonial Characters – Changes induced in medium components

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MICROSCOPY

This involves putting the specimen on a slide.

Without any preparation (urine, CSF)

Staining steps is required (methylene blue stain, Gram stain, Ziehl-Nielsen stain).
Staining of a specimen – make it easier to observe microorganisms and also the color of the bacterial cells after the staining

– May provide some preliminary information

Figure no. 39: Bacteria morphology

http://www.medicalbacteriology.com/understanding-bacteria-morphology-using-gram-stain-technique.html

Cocci

Morphology:

– Round – Staphylococcus

– Oval – Streptococcus

– Lanceolate – Streptococcus pneumoniae

– Reniform – Neisseria (gonococci, meningococci)

Arrangement:

In clusters – staphylococcus

In chains – streptococcus

In diplo like two coffee beans (Neisseria gonorrhoeae)

In diplo like two candle lights (Streptococcus pneumoniae)

Figure no. 40: Bacteria arrangement

http://www.threesology.org/bio-physiological-3s-5.php

Figure no. 41: Bacterial shape and arrangements

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Figure no. 42. Staphylococcus – Optical Microscopy Gram stain

https://mgsii.files.wordpress.com/2014/09/coloratia-gram.pdf

Figure no. 43. Bacteria arrangement – Gram positive Cocci, disposed in cluster

Figure no. 44: Bacteria arrangement – Gram positive Cocci

Figure no. 45: Staphylococcus – Electron microscopy

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Figure no. 46: Bacteria arrangement Streptococcus – Optical Microscopy Gram stain

http://www.microbiologybook.org/Infectious%20Disease/s%20pneumoniae.jpg

Figure no. 47: Streptococcus – Electron microscopy aspect

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Figure no. 48: Neisseria – Electron microscopicy aspect

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Bacilli

Rod shape bacteria with their ends:

round (Enterobacteriaceae)

straight cut and disposed in chains (Bacillus anthracis)

resembling Chinese letters (Corynebacterium diphtheria)

fusiform (Fusobacterium)

Figure no. 49: Morphology of bacterial cell

http://www.generalmicroscience.com/bacteriology/microbiology-morphology-of-bacterial-cell/

Arrangenment:

Isolated.

Diplobacilli: If two bacilli cells are attach to each other it is called as diplobacilli.

Streptobacilli: It bacilli cells are arranged in the form of long chain it is called as streptobacilli.

Figure no. 50: Gram stain – gram positive bacilli

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Figure no. 51: Electron Microscopy aspect of bacilli

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Encurved bacteria

Vibrio

Spirochetes (Treponema pallidum)

Leptospira (Leptospira interrogans)

Spirillum (Borrelia burgdorferi)

Figure no. 52: Vibrio – Gram stain

http://www.farmamed.ro/enciclopedie_medicala/afectiuni_digestive/index-10.html

Figure no. 53: Electron Microscopy aspect

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Figure no. 54 : Spirochetes – Treponema pallidum, Leptospira Borrelia

https://en.wikipedia.org/wiki/Spirochaete

Figure no. 55: Leptospira interrogans

http://tolweb.org/onlinecontributors/app;jsessionid=4188D559D54B894F8534B730AB716146?page=ViewImageData&service=external&sp=6428

Figure no. 56: Spirillum bacteria

http://www.allposters.com/-sp/Spirillum-Bacteria-LM-X600-Posters_i9013548_.htm

Figure no. 57: Electron Microscopy aspect

http://www.wisegeek.org/what-is-spirillum.htm

Coccobacilli

Haemophilus

Bordetella

Yersinia

Figure no. 58: Haemophilus Influenzae – Electron Microscopy aspect www.pinterest.com

Figure no. 59: Haemophilus influenzae – Gram stain

Schneierson`s Atlas of Diagnostic Microbiology – Seventh Edition, Edward J. Bottone 1979

Branched forms

Actinomyces

Nocardia

Figure no. 60: Actinomyces – Gram coloration aspect

www.studyblue.com

Figure no. 60: Actinomyces – Electron microscopy aspect

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MCQ examples:

1. CHAPMAN medium – Which of the statements are true:

A. Yellow, mannitol positive Staphylococcus aureus with yellowish halo around colonies

B. Whitish, mannitol negative colonies of Staphylococcus epidermidis

C. It contains salt and lactose

D. It contains salt and mannitol

E. CHAPMAN mediums is a simple medium

2. Colonial characters: Colony Size- True affirmations:

A. Large: Staphylococcus

B. Small: Shigella, Salmonella

C. Pinpoint (”powder grains”): Streptococcus

D. Very small: Neisseria

E. Small: Staphylococcus

10. IDENTIFICATION OF BACTERIA IN PATHOLOGICAL SAMPLES, BASED ON MICROSCOPICAL, CULTURAL, BIOCHEMICAL CHARACTERS AND PATHOGENICITY TESTS

Samples collected can be examined by microscopy, culture and various antigen – antibody tests. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on culture plates and isolated colonies of bacteria (which are visible to the naked eye) appear after incubation for one – several days. Each colony consists of millions of bacterial cells. Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacterial identification. Whether the organism requires oxygen for growth is another important differentiating characteristic.

1.Microscopy

This involves putting the specimen on a slide. It may be possible to look at the specimen without any preparation (urine, CSF). Often a series of staining steps is required (methylene blue stain, Gram stain, Ziehl-Nielsen stain). Staining of a specimen with dyes may make microorganisms easier to observe and also the color of the bacterial cells after the staining process may provide some preliminary information as to the kind of bacteria present.

THE GRAM STAIN

A colony is dried on a slide and treated as follows:

Step 1. Staining with crystal violet.

Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple or blue.

Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram negative) and not others (Gram positive).

Step 4. Counterstaining with safranin I Fuxin. Gram positive bacteria are already stained with crystal violet and remain purple. Gram negative bacteria are stained pink.

Culture

This involves placing some of the specimen in conditions where the organism, or organisms, of interest can grow and multiply. The specimen may be put (inoculated) into a liquid or onto a solid surface. A liquid used to culture cells is generally referred to as a "broth" or a "liquid culture medium".A solid surface used to grow cells is a "solid culture medium". Most solid culture media are prepared by adding a substance called agar to a hot liquid culture medium.

Then while the liquid is still hot it is poured into a series of shallow plates (Petri dishes). When the liquid cools the agar solidifies giving a smooth surface on which the specimen can be spread. A culture medium (liquid or solid) must contain nutrients necessary for bacterial growth. When culturing for virus and for some special kinds of bacteria the specimen must be placed in a vial containing living cells (tissue culture). In this case the living cells will need a liquid culture medium to provide the cells with nutrients. In addition to nutrients laboratory culture of microorganisms requires that the medium be placed at a suitable temperature and in a suitable gas mixture. For most organisms of interest in clinical microbiology the temperature used
is 35 to 37°C. Air is a suitable gas mixture for many micro-organisms but some require an atmosphere free of oxygen (anaerobic organisms). Another may require a concentration of oxygen less than that normally present in air (micro-aerophilic organisms).

Media use for laboratory culture of microorganisms must 'have one other property. It must be sterile, that is all organisms in the media must be killed before it is used. If this was not done one could not know if organisms growing on the medium are from the specimen or were things that got into the media during preparation.

Identification

If the lab has detected/isolated a microorganism by culture the next question is to determine what kind or organism has been detected. This is the process of identification. We need to identify only the isolated bacteria, which it is of practical importance in making clinical or infection control decisions. For example antibacterial sensitivity and a rough guess at identification is often all that is needed for an organism cultured from the urine of an otherwise healthy person with a urinary tract infection. In the case of a blood stream infection in a person in an Intensive Care Unit (ICU) one would normally want to know exactly what the organism is.

Labs usually begin by looking at the colony on the plate. With experience it is possible to make a very good judgment about what one is dealing with from the size, shape and color of the colony and the effect the colony has on the culture media around it. This may be all the identification performed in some cases.

The next step is often a Gram-stain. Cells are attached to a glass slide and exposed to particular colored dyes. Afterwards, on looking down the microscope it is possible to observe the color, size and shape of the bacterial cells. Pink = Gram negative; Purple = Gram-positive. Round cells are cocci and rods shaped cells are bacilli.

At this point one has a general idea of what kind of bacteria one is dealing with and which additional tests, if any, are required. Additional tests may include biochemical tests:

– enzyme producing: coagulase, catalase, oxidase

– fermentation of different sugars

– H2S producing

3. Biochemical tests

Purpose: to assess the biochemical / metabolic activity of bacteria → identification

Methods: re-inoculation of primary isolate

onto a series of differential test media

into solutions

Proceeded by:

Preliminary observation

Direct (rapid) biochemical tests

Assessment of Biochemical Characters

3 steps:

Preliminary observation

Direct rapid tests

Testing for enzyme systems

Preliminary observation of biochemical / metabolic characters

may offer identification to a clinically useful level e.g.

Lactose-utilizing properties on MacConkey agar – red pigmentation of colonies (E.coli)

H2S production on Hektoen agar – colonies with black centers (Salmonella)

Figure no. 61: Lactose- fermenting colonies on MacConkey agar (lactose)

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Figure no. 62: H2S production on Hektoen agar (Salmonella colonies)

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Direct rapid tests

Performed directly on isolated colonies recovered on primary culture plates (e.g.):

The Catalase test

The Bile solubility test

The Coagulase test (slide / tube)

The Catalase test

Principle: the enzyme catalase decomposes hydrogen peroxide (H2O2) into water and oxygen: 2H2O2 →2H2O + O2 (gas bubbles)

2-3 drops of hydrogen peroxide placed directly on a colony

POSITIVE TEST: rapid effervescence

differentiates Staphylococcus (+) / Streptococcus (-)

The Bile solubility test

Principle: Bile salts selectively lyse Streptococcus pneumoniae when added onto cultures on agar / broth

POSITIVE TEST on agar: colony ”disappears” upon addition of bile salts

POSITIVE TEST in broth: turbidity of broth (reflecting bacterial growth) clarifies upon addition of bile salts

Negative test: Streptococcus viridans (bile-insoluble)

Figure no. 63: Bile solubility test – Streptococcus mitis versus Streptococcus pneumoniae

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Figure no. 64: Bile solubility test – Streptococcus pneumoniae

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The Slide Coagulase test

Principle: the coagulase of Staphylococcus aureus (aka ”clumping factor”) converts fibrinogen into fibrin →clot

Procedure:

-2 drops of saline solution in 2 circles drawn on glass slide

-Emulsify colony in each of the 2 circles

-1 drop of plasma (rabbit plasma with EDTA) in one circle

-1 drop of water in the other circle (control)

-Rock slide back and forth & observe agglutination

– POSITIVE TEST: white precipitate & agglutination in 10-15 sec (control should remain smooth)

Figure no. 65: Positive Coagulase test white precipitation and agglutination www.slideshare.com

Figure no. 66: Positive Coagulase test – white precipitation

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Testing for enzyme systems

Final characterization of unknown bacterial isolate by testing for characteristic enzyme systems

Method: re-inoculation of isolated colony (primary culture) into a series of culture media containing specific substrates and chemical indicators

Principle: detection of :

-pH changes produced by utilization of substrates /

-color changes produced by specific by-products,

Challenge: Selection of appropriate sets of characteristics to allow bacterial group identification

Capacity to utilize sugars by oxidation / fermentation

Utilization of certain substrates as carbon source (citrate, malonate)

Production of H2S in the presence of heavy metals

TSI (triple sugar iron) agar – assessment of bacterial capacity to:

a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S

TSI= sucrose, lactose, glucose + mehyl red + ferrous sulfate

only glucose fermented →acid production in the butt of tube → yellow, but insufficient acid to affect the methyl red in the slant

either sucrose or lactose fermented → sufficient fermentation products → both the butt and the slant yellow

gas during fermentation → gas bubbles/cracking of agar

no fermentation → slant and butt remain red

If bacterium forms H2S, this chemical will react with the iron to form ferrous sulfide = black precipitate in the butt (black butt)

R = red = no fermentation (obligate aerobe)

Y = yellow = some fermentation (facultative anaerobe)

YG = fermentation + gas

”+” = Black = H2S

Figure no. 67: TSI (triple sugar iron) agar

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Miniaturization of biochemical systems: multiple characteristics can be examined simultaneously (BioMerieux: API galleries or Vitek cards)

Probabilistic models for identification based on multiple characteristics

Results compared to known ”biochemical patterns” of each bacterial group (handbooks or dedicated computer software for the analysis of bacterial biochemical pattern against database)

MCQ examples:

Direct (rapid) biochemical tests- correct affirmation:

The Catalase test

The Bile solubility test

The Coagulase test (slide / tube)

TSI-agar

Tests for enzyme systems

TSI (triple sugar iron) agar –true affirmation:

metabolize lactose and/or sucrose

conduct fermentation to produce acid

produce gas during fermentation

generate H2S

TSI= sucrose, lactose, glucose + methyl red + ferrous sulfate

11. ANTIMICROBIAL TESTING METHODS. TECHNIQUE AND INTERPRETATION

ANTIBIOTIC SUSCEPTIBILITY TESTS

In vitro tests that measure the growth of an isolated microbe in the presence of particular drug or drugs (antibiotics) in order to predict the in vivo success or failure of antibiotic therapy.

The results – guide the choice of antibiotics (+ clinical information and experience)

Bacterial Resistance to Antimicrobial Agents:

Natural: – genetically determined for all members of a bacterial species

Acquired: – Present only in certain strains of a naturally sensitive species

Natural spectrum = list of microbial species naturally sensitive to an antibiotic

1. DISK DIFFUSION TESTING PRINCIPLE (Kirby Bauer)

– Seeding with a swab the bacteria on the surface of a solid medium and applying antibiotic (disks) on the surface of the environment.

– They are held for 18 hours in the incubator

– Then interpret the germ sensitivity depending on the diameter of the zone of inhibition that occurs around the tablet.

MATERIALS:

– Mueller Hinton culture medium, blood agar;

– Germ researched – pure cultures

– Antibiotic disks, the standardized diameter of 5 mm.

– Petri dishes, bacteriological loop.

Figure no. 68: Muller Hinton Agar medium for antibiotic testing

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TECHNIQUE:

– Pour the culture medium in Petri dishes in a layer thickness of 4 mm (25 ml liquid to a plate of10 cm).

– Place inoculum (only pure cultures)

– Dry 5-15 minutes at room temperature, then place disks (minimum 7-8).

– Read the transparent halo surrounding antibiotic disks (ruler). As the diameter is increased then the germ antibiotic sensitivity is higher.

Figure no. 69: Disk diffusion testing principle (Kirby Bauer) – technique

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DISK STORAGE

For long-term storage – at -14° C or below in a non-frost-free freezer.

A working supply of disks can be stored in a refrigerator at 2° to 8° C for at least 1 week.

Be stored in a tightly sealed container. The container should be allowed to warm to room temperature before it is opened to prevent condensation from forming on the disks

INTERPRETATION

Depending on the zone of inhibition, bacteria are:

large zone = susceptible

small / no zone = resistant

zone between the above = intermediate

Disk diffusion antimicrobial susceptibility testing

Antibiotics sets (e.g.):

Current use for Gram positive bacteria: Penicillin G (P), Oxacillin (O), Cefoxitine (Fox), Erythromycin (E), Clindamycin (Da), Gentamycin (Gn), Vancomycin (Va), etc.

Current use for Gram negative bacteria: Ampicillin (A), Gentamycin (Gn), Ciprofloxacin (Cip), Ceftazidime (Caz), Cefepime (Fep), Colistin (Co), etc.

Additional set for digestive infections: Furazolidone (Fu), Neomycin (Ne), Polymyxin B (Po)

Additional set for urinary infections: Nalidixic Acid (Nx), Colistin (Co), Fosfomycine (Fos), Nitrofurantoin (Nf).

In vitro testing for the sensitivity of microbes to various antibiotics; expressed as:

– MIC (minimal inhibitory concentration) – the lowest quantity of antibiotic completely inhibiting the multiplication of a bacterial strain

– MBC (minimal bactericidal concentration) – the lowest quantity of antibiotic able to kill 99.9-100% of the germs of a tested bacterial strain

DETERMINATION OF MIC

Measurement methods:

Broth dilution

E-test (Epsilometer test)

Broth dilution:

Bacteria inoculated in culture broth + antibiotic (various concentrations) and incubated

MIC = the lowest concentration of antibiotic which inhibited bacterial growth

Figure no. 70: Broth dilution method

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– Used to determine:

– Serious infections which threaten the life of the patient (septicemia, endocarditis, meningitis)

– Testing of slow growing bacteria (BK)

– Preliminary testing detection of minimum bactericidal concentration (MBC)

– Technique: Incubate for 18 hours at 37 °C.

E-test:

The principle of establishing an antimicrobial density gradient in an agar plate as a means of determining antimicrobial susceptibility.

Very thin plastic test strips that are impregnated with an antimicrobial concentration gradient and are marked on the upper surface with a concentration index or scale.

MATERIALS:

plastic strips impregnated with decreasing antibiotic concentrations (log scale)

strips applied on agar microbial culture and incubated

elliptic inhibition zone intersects the MIC value scale (µg/ml) at the level of MIC

shape resembles Greek letter ”Epsilon” (ε)

Figure no. 71: E-test

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Figure no. 72: E- test

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TECHNIQUE

The strips are placed in a radial fashion on the surface of an agar plate.

After overnight incubation the test results are read by viewing the plates from the top side with the lids removed.

The antimicrobial gradient that forms in the agar around the E test strips gives rise to elliptic inhibitory areas with each strip.

The MIC is determined where the growth ellipse intersects the E test strip (Figure above).

DETERMINATION OF MBC

Indications:

– severe infections in immunocompromised patients,

– infections in anatomic sites hard to reach with antibiotics e.g. endocarditis, osteomyelitis

Technique:

After reading MIC, pass a fixed amount from the tubes (in which the organism did not grow), on a solid culture medium lacking antibiotic.

– Incubate at 37 degrees Celsius, for18 hours, and note the lowest concentration that the microorganisms did not grown (they were irreparably destroyed). This is MBC.

– Less used than MIC; more time consuming

MCQ examples:

1. Determination of MIC – Measurement methods – true affirmations:

A. Broth dilution

B. E-test (Epsilometer test)

C. Culture mediums

D. Immunofluorescence

E. ELISA method

2. The true affirmations are:

A. MIC (minimal inhibitory concentration) – the lowest quantity of antibiotic completely inhibiting the multiplication of a bacterial strain

B. MBC (minimal bactericidal concentration) – the lowest quantity of antibiotic able to kill 99.9-100% of the germs of a tested bacterial strain

C. MIC (minimal inhibitory concentration) – the lowest quantity of antibiotic able to kill 99.9-100% of the germs of a tested bacterial strain

D. MBC (minimal bactericidal concentration) – the lowest quantity of antibiotic completely inhibiting the multiplication of a bacterial strain

E. Measurement methods used for determination of MIC are: Broth dilution

and E-test (Epsilometer test)

12. IMMUNOLOGICAL REACTIONS IN MICROBIOLOGICAL DIAGNOSIS: AGGLUTINATION AND PRECIPITATION REACTIONS

Definition of terms

Antigen = foreign substance that, when introduced into the body, is capable of stimulating an immune reaction e.g. foreign molecules in bacteria, viruses, protozoa, serum components, etc.

Antibody (immunoglobulin) = large protein produced by plasmocytes which identifies and neutralizes antigens

Immune reaction = reversible binding of antigen to homologous antibody (high specificity)

Immune reaction: Antigen-Antibody reaction (Ag-Ab)

High specificity: Antigen-binding site of the antibody molecule perfectly matches the antigen (” key in hole”)

Figure no. 73: Antigen-antibody reaction

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Ag-Ab Reactions: applications in laboratory diagnosis of infections

Same basic principle: – specific detection and binding of antigen by antibody → Ag-Ab complex

Differences: – the methods used to reveal the Ag-Ab complex

Examples

Agglutination

Immunofluorescence

Enzyme-linked Immunosorbent Assay (ELISA)

Immunoblotting, Western blot

Important factors

1. Affinity – The higher the affinity of the antibody for the antigen, the more stable will be the interaction.

2. Avidity – Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.

3. Ag: Ab ratio – influences the detection of Ag/Ab complexes because the size of the complexes formed is related to the concentration of the antigen and antibody.

4. Physical form of the antigen

– If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody.

– If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble Ag/Ab complexes

Figure no. 74: Antigen- antibody reaction

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Agglutination Tests

1. Agglutination/Hemagglutination

– When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen.

– When the antigen is an erythrocyte the term hemagglutination is used.

Qualitative agglutination test

– the presence of an antigen or an antibody

– The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen.

– Example: a patient's red blood cells can be mixed with antibody to a blood group antigen to determine a person's blood type.

Quantitative agglutination test

– In this test one makes serial dilutions of a sample to be tested for antibody and then add a fixed number of red blood cells or bacteria or other such particulate antigen and determines the maximum dilution which gives agglutination (TITER).

Agglutination: on slide/in tubes

– clumping together by antibodies of microscopic foreign particles:

red blood cells

bacteria

inert particles (latex)

agglutinated particles are visible with the naked eye (pellet-like agglutination product)

Figure no. 75: Agglutination reaction

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Agglutination: applications in microbiology

Serological diagnosis: detection (and quantification) of unknown antibodies by use of known antigens

Principle:

serum from patient is put in contact with serial dilutions of Ag;

if Ab present in serum →agglutination

Titer of Ab = dilution of the tube with agglutination

Bacteriological diagnosis: identification of unknown antigens (bacteria) by use of known antibodies

Principle:

Serum containing known Ab are put in contact with bacterial suspension

Identification of bacteria – indicated by the type of serum which agglutinates the bacterial suspension

Figure no. 76: Antigen: isolated bacteria e.g. Salmonella
Antibody: kit with antibodies (antisera) against Salmonella types

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Passive agglutination (inerte particles)

Ag on latex (latex-agglutination)/RBC (hemagglutination) – If Ab are present in sample →agglutination of latex/RBC

Ab on inert particles: bacterial identification kits e.g. Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, E.coli

MCQ examples:

Ag-Ab Reactions – examples – correct affirmation:

Agglutination

Immunofluorescence

Enzyme-linked Immunosorbent Assay (ELISA)

Immunoblotting, Western blot

TSI-agar test

The most important factors in the agglutination reaction are:

Affinity – The higher the affinity of the antibody for the antigen, the more stable will be the interaction

Avidity – Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect

Ag: Ab ratio – influences the detection of Ag/Ab complexes because the size of the complexes formed is related to the concentration of the antigen and antibody

Physical form of the antigen

Physical form of the antibody

13. IMMUNOLOGICAL REACTIONS IN MICROBIOLOGICAL DIAGNOSIS: COMPLEMENT FIXATION REACTIONS, LABELED ANTIBODY REACTIONS: IMMUNOFLUORESCENCE, RIA, ELISA, WESTERN -BLOT.

IMMUNOFLUORESCENCE

– Bacterial culture (Ag) incubated with specific antibody coupled with fluorescent dye

– If Ab matches Ag (bacterial culture) →Ag-Ab complex + fluorescent dye

– Under UV light bacteria covered with antibodies coupled with fluorescent dye will produce fluorescence

Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, and may be used to analyze the distribution of proteins, glycans, and small biological and non-biological molecules. Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining.

Several microscope designs can be used for analysis of immunofluorescence samples; the simplest is the epifluorescence microscope, and the confocal microscope is also widely used. Various super-resolution microscope designs that are capable of much higher resolution can also be used

Figure no. 77: Treponema denticola – wet mount, dark field microscopy + fluorescent dye staining

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Types of immunofluorescence:

primary (or direct)

secondary (or indirect).

Primary (direct) immunofluorescence

Primary, or direct, immunofluorescence uses a single, primary antibody, chemically linked to a fluorophore. The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy.

Secondary (or indirect) immunofluorescence

Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen.

RIA( radioimmunoassay)

Radioimmunoassay (RIA) =in vitro assay technique used to measure concentrations of antigens (for example, hormone levels in the blood) by use of antibodies.

Although the RIA technique is extremely sensitive and extremely specific, requiring specialized equipment, it remains among the least expensive methods to perform such measurements. It requires special precautions and licensing, since radioactive substances are used.

The RAST test (radioallergosorbent test) is an example of radioimmunoassay. It is used to detect the causative allergen for an allergy.

Method

To perform a radioimmunoassay, a known quantity of an antigen is made radioactive, frequently by labeling it with gamma-radioactive isotopes of iodine, such as 125-I, attached to tyrosine. This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two specifically bind to one another. Then, a sample of serum from a patient containing an unknown quantity of that same antigen is added. This causes the unlabeled (or "cold") antigen from the serum to compete with the radiolabeled antigen ("hot") for antibody binding sites.

As the concentration of "cold" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigen remaining in the supernatant is measured using a gamma counter.

Using known standards, a standard curve can then be generated which allows the amount of antigen in the patient's serum to be derived (often sigmoidal curve fit). Once the terms for the equation are derived, the radioactivity is fed into the equation to obtain concentrations of the unknowns. This function is often integrated into the radioactivity counter, along with functions indicating coefficient of variation and properties of the binding of tracer to antibody. A typical RIA will have somewhere around 50% of tracer bound to antibody in a sample with no analyte. Samples are often run in duplicate to ensure a correct reading.

WESTERN BLOT

It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein. Gel electrophoresis step is included in Western blot analysis to resolve the issue of the cross-reactivity of antibodies. An improved immunoblot method, Western Blot analysis, is able to address this issue without the electrophoresis step, thus significantly improves the efficiency of protein analysis.

There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins.

Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

Other related techniques include dot blot analysis, Western Blot analysis, immunohistochemistry where antibodies are used to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

ELISA (The enzyme-linked immunosorbent assay)

It is a test that uses antibodies and color change to identify a substance. It uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, or an antibody an antigen, in a liquid sample or wet sample.

Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.

Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

Figure no. 78: Solid support for ELISA:
96 microwell plastic plate

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Direct ELISA

Add serum on solid support (plastic microwell plate); adherence to solid support by charge interactions

Add CONJUGATE: Ab conjugated to enzyme

Add SUBSTRATE of enzyme

COLOUR = Ag present in serum

Figure no. 79: Direct ELISA method

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Indirect ELISA

Solid support pre-coated with Ag

Add Patient serum

If Ab in serum→formation of Ag-Ab complex

Add CONJUGATE: Ab anti-human Ab+Enzyme

Formation of Complex: Ag-Ab-Ab anti-human Ab+Enzyme

Add substrate of enzyme → COLOUR develops as a result of enzyme-substrate reaction → presence of Ab in patient serum (” primary antibody”)

Figure no. 80: Indirect ELISA method www.slideshare.com

” Sandwich” ELISA

Solid support pre-coated with ”capture” Ab (speciffic for the Ag tested for)

Add patient serum; if Ag is present, the Ab on plate capture it

Add ”detection” Ab which will bind Ag (Ag bound between 2 Antibodies: sandwich)

Add CONJUGATE: Ab anti-”detection” Ab conjugated to Enzyme

Add SUBSTRATE of enzyme

Color develops

Figure no. 81: Sandwich ELISA method

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THE COMPLEMENT FIXATION TEST

Can be used to detect the presence of either specific antibody or specific antigen in a patient's serum. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as ELISA and by DNA-based methods of pathogen detection, particularly PCR.

The complement system is a system of serum proteins that react with antigen-antibody complexes. If this reaction occurs on a cell surface, it will result in the formation of trans-membrane pores and therefore destruction of the cell. The basic steps of a complement fixation test are as follows:

1. Serum is collected from the patient.

2. The serum is heated in such a way that all of the complement proteins—but none of the antibodies—within it are destroyed. (This is possible because complement proteins are much more susceptible to destruction by heat than antibodies.)

3. A known amount of standard complement proteins are added to the serum. (These proteins are frequently obtained from guinea pig serum.)

4.The antigen of interest is added to the serum.

5. Sheep red blood cells (sRBCs) which have been pre-bound to anti-sRBC antibodies are added to the serum. The test is considered negative if the solution turns pink at this point and positive otherwise.

If the patient's serum contains antibodies against the antigen of interest, they will bind to the antigen in step 3 to form antigen-antibody complexes. The complement proteins will react with these complexes and be depleted. Thus when the sRBC-antibody complexes are added in step 4, there will be no complement left in the serum. However, if no antibodies against the antigen of interest are present, the complement will not be depleted and it will react with the sRBC-antibody complexes added in step 4, lysing the sRBCs and spilling their contents into the solution, thereby turning the solution pink (hemolysis of sRBCs).

MCQ examples:

The correct affirmation are:

There are 2 immunofluorescence methods: Primary (direct) Immunofluorescence and Secondary (indirect) Immunofluorescence

Primary, or direct, immunofluorescence uses a single, primary antibody chemically linked to a metal.

Primary, or direct, immunofluorescence uses a single, primary antibody chemically linked to a fluorophore.

The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the epitope

The primary antibody recognizes the target molecule (antigen) and binds to a specific region called the paratope

Secondary (or indirect) immunofluorescence – true affirmation:

Secondary, or indirect, immunofluorescence uses two antibodies

Secondary, or indirect, immunofluorescence uses one antibody

The unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody and binds to it

Just one secondary antibody can bind a single primary antibody

Multiple secondary antibodies can bind a single primary antibody

14. DIAGNOSTIC METHODS BASED ON NUCLEIC ACID IDENTIFICATION

Nucleic acids

long, linear macromolecules = polymers which carry genetic information

composed of linked nucleotides = monomers

Each nucleotide has 3 components:

5 carbon sugar = pentose:

deoxyribose in DNA or

ribose in RNA

a phosphate group

a nitrogenous base (nucleobase)

Nucleobases (nitrogenous bases)

Nitrogen containing biological compounds found in the structure of nucleotides

Primary nucleobases:

Cytosine (C) (in DNA and RNA)

Guanine (G) (idem)

Adenine (A) (idem)

Thymine (T) (only in DNA)

Uracil (U) (only in RNA)

Base pairing

Base pairs – formed between specific nucleobases due to complementarity i.e.

A with T

C with G

ensures the DNA double helix → folded structure of both DNA and RNA

DNA structure of each species depends on nucleotide sequence = succession on DNA strand (basis of the genetic code)

Figure no. 82:DNA and RNA structure

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Polymerases

DNA-, RNA-polymerase, reverse-transcriptase = enzymes that catalyze the formation of DNA or RNA using an existing strand of DNA or RNA as a template

Semiconservative DNA replication

DNA strands separated

New complimentary DNA strands synthesized by base pairing

RESULT:

2 identical copies (all biological information from ”parental” DNA)

” daughter” DNA molecules are "Half old" and "Half new “= Half of parental DNA is saved (conserved) in each daughter DNA = semi-conservative replication

Figure no. 83: DNA replication

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Primer – strand of nucleic acid that serves as a starting point for DNA synthesis under the action of a polymerase

Polymerase chain reaction (PCR)

Based upon semiconservative DNA replication

Purpose in microbiological diagnosis:

to obtain a huge number of copies of nucleic acid of a certain microorganism (amplification) e.g. bacteria, viruses

to detect and identify the amplified product

PCR – preparatory steps

Extract nucleic acid (NA) from biological product e.g. nasopharyngeal exudate – bacterial / viral NA:

cell lysis

elution

membrane filtration

Prepare ”reaction mix”:

Specific primers (sequence depends on NA to be detected = target NA)

Polymerase

Other components to favor future steps

Add extracted NA to ”reaction mix”

PCR – the cycling reactions(30-40 cycles)

Performed in thermal cyclers (PCR machines) = instruments that employ precise temperature control and rapid temperature changes

Thermal block where PCR tubes are placed in

Thermal prophile is defined:

number of cycles

temperature and duration for each cycle

Figure no. 84: Strip with 8 PCR tubes containing reaction mix

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Figure no. 85: PCR thermal cycler

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Denaturation (around 94 C):

DNA double strand opens → single stranded DNA

Annealing (around 54 – 64 C):

Primers in the reaction mix find complementary nucleobase sequences on each DNA strand and bind in the respective positions (A with T; C with G)

Extension (around 72 C):

Polymerase in the reaction mix catalyzes the synthesis of the 2 new DNA strands

Figure no. 86: PCR – exponential amplification

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PCR: detection and identification of amplified product

Conventional end-point PCR:

gel electrophoresis of amplified products

Visualize sample migration under UV light

Compare bands of samples with bands of positive control

Figure no. 87: PCR method- detection and amplification of product www.slideshare.com

Real time PCR

Figure no. 88: Fluorescence-based detection; compare cycle threshold (Ct) of sample with Ct of positive control

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MCQ examples:

PCR – the cycling reactions includes:

Denaturation

Annealing

Extension

Amplification

Base pairing

Primary nucleobases are:

Cytosine (C)

Guanine (G)

Adenine (A)

Mannitol

Lactose

ANNEXE

CORRECT ANSWERS:

Chapter 3

Question no. 1 : A,D,E

Question no. 2: A,C,E

Chapter 4

Question no. 1 : D

Question no. 2: A,B,C

Chapter 5

Question no. 1 : A,B,C,E

Question no. 2: A,B

Chapter 6

Question no. 1 : C,D,E

Question no. 2: A,B,E

Chapter 7

Question no. 1 : A,B

Question no. 2: A

Chapter 8

Question no. 1 : A,B

Question no. 2: A,B,C,D

Chapter 9

Question no. 1 : A,B,D

Question no. 2: A,B,C,D

Chapter 10

Question no. 1 : A,B,C

Question no. 2: A,B,C,D,E

Chapter 11

Question no. 1 : A,B

Question no. 2: A,B,E

Chapter 12

Question no. 1 : A,B,C,D

Question no. 2: A,B,C,D

Chapter 13

Question no. 1 : A,C,D

Question no. 2: A,C,E

Chapter 14

Question no. 1 : A,B,C

Question no. 2: A,B,C

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