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Microbiology Principles of Diagnostic Microbiology

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19 DIAGNOSTIC MICROBIOLOGY - Encompasses the characterization of thousands of agents that cause or are associated with disease - Concerned with the etiologic diagnosis of infection SERVICES OF A MICROBIOLOGY LABORATORY: - Provide relevant information - Establish guidelines for proper specimen collection (MOST IMPORTANT: good quality specimen) - Timely specimen transport - Identification of pathogens - Susceptibility testing (to know what drug to prescribe) - Rapid reporting test result RESPONSIBILITY OF MICROBIOLOGISTS TO PATIENT AND CLINICIAN - Identify and control nosocomial pathogens - Track resistant organisms - Coordinate the antimicrobial agents tested with the Therapeutics Committee o Therapeutics Committee decide what drugs should be available in the pharmacy - Prepare and update manuals regarding appropriate specimen collection and handling - Save patients’ money by providing accurate diagnosis in a timely manner using the most cost-effective techniques FUNCTIONS OF A MICROBIOLOGY LABORATORY: 1. Etiologic diagnosis of an infectious disease Subject: Microbiology Topic: Principles of Diagnostic Microbiology & Antibiotic Susceptibility Immunoassays Lecturer: Dr. Teresa Barzaga Date of Lecture:06/23/2011 Transcriptionist: Group 5 SY 2011- 2012
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- Encompasses the characterization of thousands of agents that cause or are associated with disease

- Concerned with the etiologic diagnosis of infection


- Provide relevant information - Establish guidelines for proper

specimen collection (MOST IMPORTANT: good quality specimen)

- Timely specimen transport- Identification of pathogens - Susceptibility testing (to know what

drug to prescribe)- Rapid reporting test result


- Identify and control nosocomial pathogens

- Track resistant organisms- Coordinate the antimicrobial agents

tested with the Therapeutics Committeeo Therapeutics Committee decide

what drugs should be available in the pharmacy

- Prepare and update manuals regarding appropriate specimen collection and handling

- Save patients’ money by providing accurate diagnosis in a timely manner using the most cost-effective techniques


1. Etiologic diagnosis of an infectious disease

a. Isolation and identification of an infectious agent

b. Demonstration of an immunologic response

2. Set guidelines for rational use of antimicrobials


1. COLLECTION- From active site of infection

o If suspected CNS infection, collect CSF not sputum

- Timingo During acute stage of disease,

before giving antibioticso Antibiotics can kill the organisms,

giving false negative results- Amount

o Adequate for culture

Subject: MicrobiologyTopic: Principles of Diagnostic Microbiology & Antibiotic Susceptibility ImmunoassaysLecturer: Dr. Teresa BarzagaDate of Lecture:06/23/2011Transcriptionist: Group 5Pages: 19

SY 2




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o Sputum: 5ml or 1 teaspoon- Proper tools and containers

o Urine, stool, CSF: Use sterile containers without transport or preservative- Preservatives can kill the

microorganismso Tissue: Use sterile, dry, wide-

mouth, screwcapped bottle or plastic container without water or saline

o Blood:Sterile tubes containing broth or anticoagulant

STERILE SWABS- Most convenient and most

commonly used tool for specimen collection

- Provides the poorest conditions for survival and can only absorb a small volume of inflammatory exudates


2. DELIVERY, TRANSPORT, PROCESSING- Immediately - Viability may be lost if specimen

is delayed- Transport media stabilize

conditions and prevent drying Minimizes the effects of delay

between specimen collection and laboratory processing

- Example: Stuart, Amies and Cary Blair transport media

3. STORAGE- Appropriate conditions- All specimens can be kept at room

temperature EXCEPT:o 4°C

- To prevent overgrowth of organism present in the specimen Urine Stool Viral specimen Sputum Swabs Foreign devices Gastric biopsies Rectal swabs Stool for culture

o 37°C CSF

o -20°C Serum serologic studies

(stored up to one week)o -70°C

Tissues or specimens for long-term storage

o Use a low temperature or deep freezer

o Not all freezers have the same temperature

o Ordinary household freezer is about -20°C

o Ultra low freezer is about -70° C

o Liquid nitrogen freezer is about -196°Cfor very long storage purposes.

Quality of the specimen is crucial

Types of specimens


- Blood- Urine- CSF

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- Secretion- Aspirate- Cavity fluids- From cystic cavities




- Corneal- Skin


- Tissues- Hair and nails- Swabs- Stool


- IUD (intrauterine device)- I.V. catheters

o Common source of organism because when we do intravenous administration, we puncture the skin, and if it is not properly cleaned, then you introduce organism on the skin into the blood stream and cause infection.

o We recommend time for change of IV catheter in 48 hrs / whenever there is a sign of infection.

- Pins- Prosthatic valves

The Specimen

A. DIRECT TISSUE OR FLUID SAMPLES- Collected from normally sterile

tissues (lung, liver) and body fluids (CSF, blood)

Methods by which it can be obtained are:1. Needle aspiration2. Surgical biopsy (very sterile)*Specimen collected contains only the pathogens - Positive findings: diagnostic- Negative findings: can exclude- Infection at suspected site



- Pathogen is localized in sterile and must pass through a site containing normal flora in order to be collected


- SPECIMENS OF INFLAMMATORY EXUDATES (expectorated sputum, voided urine) that have passed through sites colonized with normal flora.

- For example, urine is sterile when it’s in the urinary bladder but when patient voids, it passes through urinary tract and passes through urethral orifice and causes contamination.

- Sputum is sterile when it is in the lungs, but when it is expectorated to the upper respiratory tract to the

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mouth, it gets contaminated because the mouth contains millions of organism.

*Degree of contamination is related to the skill with which the normal flora site was “bypassed"in specimen collection

- Bypassing normal flora requires extra efforto In obtaining sterile urine, there

issuprapubic aspiration: Don’t allow patient to void so don’t allow the specimen to pass through the urethral meatus but puncture directly through suprapubic area on the skin with syringe and aspirate but that’s very invasive unless in an emergency, avoid doing that.

o For sputum, some doctors use broncho-alveolar lavage or endotracheal aspiration; but still, this is very invasive

- Result require interpretive evaluation of contamination


- Primary site of infection: in an area known to be colonized with many organisms (pharynx and large intestines)o The large intestine contains

millions of gram negative organisms.

o E.coli, which are called resident flora, don’t cause any disease in the large intestine but once the large intestine is ruptured;

bacteria spills out into sterile peritoneal cavity and cause the disease peritonitis


*Both are collected and the non-pathogens are either inhibited by use of selective culture methods or discounted in interpretation of culture results.


- Easier: essential little normal viral to confuse interpretation

- Lack of normal viral flora simplifies interpretation Allows selection guided by the

knowledge of which sites are most likely to yield the suspected etiologic agent

MUST KNOW (*LIKELY TO BE ASKED IN EXAM)SAMPLING SITES AND NORMAL FLORANORMALLY STERILES SITES (no flora)- Blood and bone marrow- Peritoneal and pulmonary cavity- Posterior urethra- Urinary bladder- Accessory nasal sinuses- Trachea, bronchi, alveoli- CSF- Kidney, prostate- Uterus- Larynx- Middle and inner ear


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- Mouth- Nose- Skin- Female genital tract- External genitalia- External ear canal- Throat- Oropharynx- Gastrointestinal tract- Urethra- Eye


1. ANTISEPSISa. 1-2minutes contact time with a

disinfectant (70% alcohol or betadine) especially for blood culture

2. By-pass areas with normal flora (invasive)a. Transtracheal aspirationb. Open biopsyc. Percutaneous needle biopsyd. Suprapubic aspiration of urine

3. Culture specifically for organisms known to be etiologica. Agents (e.g. Group A streptococci

in throat)4. Quantities culture results (e.g.

quantitative urine cultures)5. Decontamination for mycobacterial

culturesa. 4-10% NaOH or hypochlorite

solutionb. e.g. We use this for TB

mycobacterial because sputum is contaminated in mouth

6. Selective mediaa. Salmonella-Shigella agar

b. TCBS – thiosulfate-citrate bile salt solution (for Vibrio cholera)


1. Patient information on requisition slipa. Erroneous entry, incomplete

data- PhilHealth nowadays has become very strict about the patients information (age, date of birth etc.)

- Sometimes, there are patients who have exactly the same names! Thus, the samples collected might be interchanged.

2. Containera. Improperb. Leaking

- Improper and leaking containers are immediately rejected.

3. Specimena. Improperly collected

- If this occurs, the personnel in charge (medtechs) are required to collect a new sample. This applies especially on tissues placed in fixatives (stabilizing or preserving agents).

b. Dried up/dried swabc. Placed in a fixatived. Pooled specimen (24 hour)

- A pooled specimen is a sputum sample.

- In the past, pooling of sputum samples is advised because it is

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believed that the more pool samples that you have, the greater the chance of isolating or obtaining a positive sample. (e.g. Mycobacterium tuberculosis)

- Nowadays, pooling samples is not advised because it was realized that the more positive results that you obtain, there is also a greater chance of contamination.

- Three early morning sputum samples are the best samples that can be collected (e.g. for TB cultures).

e. Insufficient quantity- Quantity is a very important criterion for collection of specimen.

4. Transporta. Improper temperatureb. Inappropriate transport

containerc. Delay of collection and arrival in

the laboratory- It is a daily routine to stamp the specimen. This is done to avoid confusion and to confirm the actual time when the specimen was obtained.

5. Processinga. Produce information of

questionable medical value


I. Direct ExaminationII. Growth and Cultivation

- Agar media

III. Analysis of Cultivated Organisms


1. Microscopy- Most commonly used method for direct examination

Bright field microscope

- Visible light passed thru specimen – series of lenses which reflects light in a manner that result in magnification of organisms present.

- Total magnification is a product of the lenses used (objective lens + eyepiece).

Phase-contrast microscopy – translates differences in phase within the specimen into differences in light intensities resulting in contrast among objects within the specimen.

- Allows observation of viable organisms

- Identification of important fungi grown in the culture.

Fluorescent microscopy – when dye molecules (fluorochromes) which have been raised to a higher energy level

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after absorbing UV light returns to the lower energy state, excess energy is released in the form of viscible (fluorescent) light.

- In the example mentioned, the acid fast bacilli stained yellow against a black background.

Darkfield Microscopy

- Condenser doesn’t allow light to pass directly through the specimen but directs the light to hit the specimen at an oblique angle.

- Used to detect spirochetes (T. pallidum)

Electron microscope – this uses electrons instead of photons (light). The electrons are focused by electromagnetic field and form an image on a fluorescent screen.

Have 2 types:

- Scanning- Transmission

2. Staining techniquesa. Smear preparation

- Apply sufficient material to slide.

Wet Mounts

- Direct fecal smear, KOH mount, NSS mount, hanging drop.

- It is used to demonstrate: blood cells and microbes in

fluid specimens, cysts, eggs and parasites, fungi in skin, protozoa in blood and tissues.

Stained smears

(Bacteria must be stained!!)

i. Simple stains – uses single stain and all cells and structure stain same color.

*Positive – stain is attracted to cells which take on the color.

*Negative – stain is repelled by the cells and background takes on color.

ii. Differential stains - specific types of organisms or structures exhibit different staining reactions that can be distinguished by different colors.

Gram stain – principle: numerous teichoic acid cross linkages contribute to the ability of gram (+) organisms to resist alcohol decolorization.

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1. Heat fixation allows the bacteria to stick into the slide to avoid from being washed away.

2. Crystal violet as the primary stain will stain all of the organisms blue or violet gram(+) or gram(-), INITIALLY.

3. Iodine is a mordant (an element which aids the chemical reaction that takes place between the dye and the fiber so that the dye is absorbed).

4. Decolorization is done by adding alcohol.Gram (+) will maintain the blue/violet color and gram (-) will be colorless.

5. Applying safranin on a gram(+)stain will retain its blue color while applying it on a gram (–) one will produce a red color.

* Gram positive bacteria retain purple iodine dye complexes.

* Gram negative bacteria do not retain complexes when decolorized.

* Properly decolorized background should be red.

* Gram reaction plus morphology guide clinical decisions e.g. giving antibiotics.

there are some antibiotics which are selective only on g + or g – bacteria.

Acid fast stain – Principle: bacteria whose cell wall contain mycolic acids render cells resistant to decolorization even with acid alcohol decolorizers.

- Weak initial staining and strong retention is related to the HIGH LIPID CONTENT of the mycobacterial cell wall.

*This is just the same as gram staining EXCEPT that it uses carbolfuchsin red as the primary stain, the counter stain is methylene blue and it doesn’t use iodine (mordant in gram staining).

Endospore stain – permits differentiation of endospore from vegetative cell.

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*Herpes viruses under the electron microscope

*Summary of the different microscopic methods

II. GROWTH AND CULTIVATION: required for definitive identification and characterization; most sensitive and specific way of diagnosis

1. Grow and isolate all bacteria in an infection

2. Etiological agents are identified3. Allow identification and

characterization4. Assess susceptibility of the pathogen to

the antibiotics5. Direct observation will just provide a

presumptive diagnosis, like when doing gram staining


A. Nutritional requirements

o Phases of growth media Liquid (broth) – bacterial

growth is indicated by change in appearance from clear to turbid Large numbers of bacteria

in the broth produce turbidity

Solid (agar)

Agar is a convenient gelling agent for solid media

o Media classification and function Enrichment media – enhance

growth of a particular bacterial pathogen from a mixture of organisms using nutrient specificity (e.g. buffer charcoal yeast extract (BCYE) for Legionella pneumophilia; upon discretion of clinician)

Supportive media – contain nutrients that support the growth of most non-fastidious organisms (e.g. xylose lysine deoxycholate agar: for the growth of Shigella only)

Selective media – contain one or more agents inhibiting all organisms except what is being searched for. (e.g. Phenyl ethyl alcohol agar, which allows only the enterococci to grow; Deoxycholate-citrate agar: inhibits E. coli and allows salmonella,shigella.

Differential media – use factors that allow colonies of one bacterial species to exhibit metabolic or cultural characteristics that will distinguish them from other bacteria on the same plate. (e.g. MacConkey agar and eosin methylene blue (EMB) agar: used to grow gram (-) organisms

Special media - only for specific organisms Sabouraud dextrose agar:

fungi; the low pH

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encourages the growth of most fungi

Lowenstin Jensen (LJ) media: favor growth of mycobacteria; contain glycerol and malachite green (inhibitory for all other bacteria except mycobacteria)

Bordet-Gengou agar: for Bordetella pertussis

Fletcher medium: for Leptospira

B. Environmental requirements

o Oxygen and CO2 availability Aerobic: thrive withO2

Facultative anaerobes: thrive with or without O2

Anaerobes: do not thrive with O2

o Temp: Optimum 35°C-37°Co pH 6.5-7.5o Moisture of medium and atmosphere

is needed

Process of bacterial cultivation – expose media

A. Establishing a pure culturea. Sterilization

i. Exposure to elevate temperatureii. Toxic

iii. Radiationiv. Filtration 0.20-0.45v. Dry heat

vi. Autoclaving: resulting in a particular smell in the laboratories

b. Aseptic techniquesi. Contact of the pure culture,

sterile medium, and sterile

surfaces of the growth with contaminated organisms

c. Isolationi. Streak plate – a loopful of

bacterial cells is streaked across the surface of a sterile agar plate that contains media

*Last quarter (upper left) of the plate wherein you see individual bacterial colonies. In primary streak, they’re all together, hard to identify. From the last streaking, there are individual colonies.

ii. Spread plate – a drop of bacterial suspension is placed on the center of an agar plate and spread over the surface

iii. Pour plate – bacterial suspensions are added to tubes containing cooled melted agar (42°C-45°C); mixed and poured onto sterile petri dishes

d. Maintaining and preserving pure culturesi. Subculture periodically

1. Especially for control strains: organisms to which the bacterial growth is compareda. American type culture

collectionb. Controls are subjected

to culture sensitivity: do biochemical and sensitivity tests to know expected reaction

2. At least once a week or every 3 days

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ii. Refrigeration at 0° C- 5° Ciii. Freezing in liquid nitrogen (-196°

C)iv. Lyophilization (freeze-drying)v. ATCC (American type culture



Principles of identification

1. Phenotypic identification of bacteria – based on observable physical and metabolic characteristics of the bacteria

A. Macroscopic morphology

a) Colony size: measured in mm or described as pinpoint, small, medium, or large

b) Colorc) Elevation, margins, shaped) Colony appearance: dull,

glistening, opaque, or transparente) Changes in agar media resulting

from bacterial growth: whether there is presence of hemolysis on blood agar plates

f) Change in pHg) Odor

i. Proteus species have a particular odor

B. Environmental requirements for growth

C. Resistance or susceptibility

a) Accomplished by the use of agar media supplemented by inhibitory substances or antibiotics, measuring directly the organism’s resistance to antimicrobial agents that may be used to treat the infection

D. Microscopic morphology and staining characteristics

a) Gram staining: usually the first step in the identification schemei. Divide bacteria to four:

1. Gram (+) cocci2. Gram (–) cocci3. Gram (+) bacilli4. Gram (–) bacilli

E. Nutritional requirementsF. Enzymatic capability

Types of enzyme-based tests; she said that this needs to be memorized)A. Single enzyme test

i. Catalase test: for gram (+) identification1. Positive test: rapid production

of bubbles when the bacterial growth is mixed with H2O2 solution

ii. Oxidase test: gram (–) identification 1. Positive test: development of

dark purple color when reagent substrate is inoculated with bacterial growth

iii. Indole test: used to presumptively identify E. coli1. Positive test: development of a

blue green color when a filter paper disk impregnated with test substrate(indole) is inoculated with bacterial growth

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iv. Urease test: helps to identify Proteus species, Helicobacter pylori1. Positive: development of a

bright pink or red color when test substrate(urease) is inoculated with bacterial growth

Test for Presence of Metabolic Pathways

- May involve several interactive enzymes

- End product from the interactions are measureda. Carbohydrate oxidation and

fermentationb. Amino acid degradationc. Single substrate utilizations


A. Cell and Organ Culture- Living cell cultures that can

support replication are the primary means of isolating pathogenic viruses


- Cells are derived from a tissue fragment (explant) or by dispersal with proteolytic enzymes such as trypsin

B. Detection of Viral Growth 1. CPE (cytopathic effect)

*VIRAL CPE IS DUE TO MORPHOLOGIC CHANGES OR CELL DEATH- As viruses replicate in cells, there is production of alterations in cellular morphology (or cell death) which are seen by light microscopy

- Can be seen as cell rounding or cell fusion- eg. From spindle-shape to being round --- that is positive CPE

2. Production of hemagglutinins

*May be present on the infected cell membranes, in culture media as a result of release of free, hemaggluntinatingvirions from the cells

*Addition of erythrocytes results in adherence to cell surfaces (HEMADSORPTION)

*Allantoic and amniotic fluid is used to test the presence of hemagglutinins

*Presence of hemagglutininsis indicative of presence of virus in the sample.

C. In Vivo Isolation Methods

1. Embryonated hen’s egg

- Inoculation of specimen on egg membrane --- incubation

2. Animal inoculation

- Uses suckling mice: viral replication is seen as development of illness

D. Viral Identification

1. Neutralization and serologic identification

*Neutralize infectivity by mixing isolate with specific antibody to known viruses before inoculation into viruses before inoculation into

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cultures --- inhibition of expected viral effects on the cell culture such as CPE or hemagglutination is evidence for the virus

2. Cytology and histology

*Intranuclear inclusions; cytoplasmic inclusions; cell fusion with multinucleated giant cells

3. EM


1. Inability to grow certain fastidious organisms

2. Inability to maintain viability of certain pathogens during transport

3. Extensive delay in cultivation and identification of slowly growing pathogens

4. Lack of reliable methods to identify certain organisms grown in vitro

5. Use of considerable time and resources in establishing the presence and identity of pathogens


- Techniques for the detection and quantitation of antigens/antibodies

- Could either be labeled or non-labeled reagent assays

- Have limitations in sensitivity because they have to form large antigen-antibody complexes for detection

- For rapid detection of bacteria, fungi, parasites, viruses

- It is a non-cultural method which only needs serum

Advantage: yields result faster


A. Precipitin immunoassays- Reaction that forms insoluble

lattice when large complexes of antigen antibody are combined

- Can provide qualitative/semi-quantitative analysis


- Relative proportion of reactants

*On the pattern of precipitin formation, it can be noted that there is a point of maximum precipitation

- Temperature- pH 6.0 to 7.5- Ionic strength of the medium


1. Single immunodiffusion- Antigen diffuses from

overlay into agar containing antibody forming precipitin line at point of equivalence

- Can be carried out in test tubes, petri dishes, slides

- In this method, antibodies are measured

2. Ouchteriony double diffusion

- Patient specimen containing antigen (in a well)

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+antibody directed against the antigen (in an adjacent well) --- Ag and Ab will diffuse toward each other in 18-24hrs --- visible precipitin band at the meeting point (zone of equivalence)

3. Electroimmunodiffusion

- Variation of double immunodiffusion using an electric current to augment the diffusion(rapid completion of assay)

- Use of electric current makes the reaction faster

4. Immunoelectrophoresis

- Couples electrophoretic separation with the 2 dimensional immunodiffusion

5. Radial Immunodiffusion

- Quantitative technique - Same principle as in single

immunodiffusion but uses antigen standards of known amount

6. Flocculation test

- The precipitin end product forms macroscopically or microscopically visible clumps

a. VDRL (Venereal Disease Research Laboratory)

o Reagin (Ab-like protein produced by patients infected with T. pallidum)

binds to the test antigen (cardiolipin-lecithin-coated cholesterol particles), causing the particles to flocculate

o Not highly specific thus replaced by RPR

b. RPR (rapid plasma regain)

o Most commonly used nowadays

o Similar to VDRL but more specific as a screening test for treponemapallidum/syphillis

o Test consists of positive and negative controls, the reaction card, prepared antigen suspension

o Antigen also contains charcoal particles to allow macroscopically visible flocculation

o Sera tested without heating

o Not recommended for testing CSF

B. Particle Agglutination

- Detects antigen via agglutination (clumping) of an artificial carrier particle such as a latex bead with antibody bound to its surface

1. Latex agglutination

o Antigen is present in a specimen being tested bonds to the combining sites of the antibody exposed on the surfaces of the latex

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beads --- forms crosslinked aggregates of latex beads and antigens

2. Hemagglutination (indirect or passive)

o Uses treated animal red blood cells as carriers of antigen for agglutination tests

a. Microagglutination test for antibody to Treponemapallidum (MHA-TP)

b. Hemagglutinationtreponemal test for syphilis (HATTS)

c. Rubella indirect hemagglutination tests

d. Passive hemagglutination tests for antibody to extracellular antigens of streptococci

3. Coagglutination

C. Enzyme immunoassays

- Basic test consists of antibodies bonded to enzymes which remain able to catalyze a reaction yielding a visually discernible end product while attached to the antibodies (change in color might be observed)

- Antibody-binding sites remain free to react with their specific antigen

1. Competitive assays (analyte assays)

2. Noncompetitive/immunometric sandwich assays (reactant excess)

o Includes 2-site immunometric sandwich assays and indirect assays to measure antibodies

o Enzyme reaction product concentration directly proportional to concentration of standard/analyte antigen


1. Reagents are relatively cheap and have long shelf life

2. Sensitive assays can be developed by amplification effect of enzymes

3. Formation of a colored end product allows direct observation of the reaction or automated spectrophotometric reading

4. No radiation hazards5. Equipment is inexpensive and is widely



1. Measurement of enzyme activity can be more complex

2. Enzyme activity may be affected by plasma constituents

Enzymes used in EIA tests:

1. Horseradish peroxidase2. Alkaline phosphatase3. Avidin-biotin

D. Immunofluorescent assays- Uses fluorescent dyes that can be

covalently attached to antibody molecules and made visible by ultraviolet light in the fluorescent microscope

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- Used for detecting antibodies- Antigens are immobilized and fixed

onto glass slides with formalin, ethanol or acetone --- addition of conjugated to fluorescent dyes --- use fluorescent microscope


1. Direct (DFA) FITC is conjugated directly to the

specific antibody Faster than IFA

2. Indirect (IFA) Antigen-specific antibody is

unlabeled and a second antibody (usually raised against the animal species from which the antigen-specific antibody was harvested) is conjugated to the FITC

More sensitive than DFA

E. Chemiluminescent assays

- Uses chemiluminescence-generating molecules as labels (luminal derivatives, acridinium esters, nitrophenyl oxalate derivatives)

- Assay principle and method does not differ from that of heterogenous immunoassays, radioimmunoassays, and fluorescent immunoassays


- Characterization of some portions of the genome using molecular techniques for DNA or RNA analysis

- The presence of a specific gene or a particular nucleic acid sequence is

interpreted as a definitive identification of the organism


A. Agarose gel electrophoresis- Separates DNA fragments or

plasmids - (ubiquitousextrachromosomal

elements composed of double-stranded DNA that is typically circular) based on size*Speed of migration depends on size: small molecules move faster and appear at bottom of gel

B. Restriction endonuclease digestion

- Refines electrophoretic analysis of DNA- Restriction endonuclease: enzymes that

recognize specific nucleotide sequences on DNA molecules and digest (cut) them at all sites at which the sequence appears

C. DNA hybridization

- Allows DNA from different sources to combine

- Opened DNA helix leaves single-stranded DNA exposing nucleotide bases which become available to interact with other single-stranded nucleic acid molecules

D. Polymerase Chain Reaction

- Amplification technique that allows the detection and selective replication of a targeted portion of the genome

- Faster way used in the laboratory

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Methods of Nucleic Acid Analysis

Polymerase chain reaction (PCR)

- Amplification technique that allows detection and selective replication of a targeted portion of a genome

- Faster way to do identification- Allows study of organisms that

cannot be cultured

Application of Nucleic Acid Methods to Infectious Diseases:

When do we use Nucleic Acid methods for diagnosis?

1.Bacterial or Viral genomic sizing

- Used in research, not usually done in the laboratory

- Number and size of plasmids differentiates strains

- Endonuclease digestion of plasmids refines their comparison

2.DNA Probes

- Probe: a fragment of DNA that has been cloned or recovered from a genomic or plasmid source

- Can detect DNA of pathogens directly in clinical specimens

3. Polymerase Chain Reaction

- Allows study of organisms that cannot be cultured

*See Textbook for Route from Patient to Microbial Diagnosis

Antibiotic Susceptibility Assay

General Indications:

1. Isolation of organisms with unpredictable susceptibility to antimicrobial agents (e.g. Staphylococci, Enterobacteria, Pseudomonas)

2. Isolation of organisms of clinical significance (e.g. isolates from normally sterile sources, wounds and abscesses, and urine if present in significant numbers)Importance:

a) When an antibiotic has a narrow therapeutic index a. The concentration required

for successful treatment and the concentration that is toxic to the patient are not very different

b) When the normal route of excretion of antibiotic is impaireda. Patients with renal failure

c) When the absorption of the antibiotic is uncertain a. Particularly after oral

administrationd) In neonates with serious

infectionse) To ascertain concentrations in

sites of infection into which penetration of antibiotics is irregular or unknowna. In cerebrospinal fluid

f) In patients receiving prolonged therapy for serious infections a. Some patients who have

been on antibiotics for a long time -> do testing to see if patient is sensitive/ resistant to treatment

b. See if organism infecting patient is still sensitive to antimicrobial you are prescribing

g) In patients who fail to respond to apparently appropriate therapy

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a. Patient doesn’t seem to be getting well, patient may be resistant, do testing

h) To check on patient compliancea. If patient is really taking

prescription given

Antibiotic Susceptibility Assay

- Not necessary when the organism is known to be susceptible to the primary drug of choice

o E.g., In the province, there’s no lab that can do testing, just give medication

- Usually performed with organisms that grow rapidly and well on artificial media

Susceptibility test results aid in:

1. Adjustment of initial dosage given [add more, etc.]

2. Modification of existing therapy [ex. If not responding]

Selection of agents to be tested

- Begins with coordination between Pharmacy and Therapeutics Committee of Hospital

- In accordance with the National Committee for Clinical Laboratory Standards guidelines [gram (+) = use gram (+) antimicrobial]

2 Categories

1. Diffusion Tests easier

- Isolate is seeded over the entire surface of an agar plate (streak) and filter paper disks

containing the antibiotics are applied, followed by overnight incubation then zones of inhibition around each antibiotic disk are observed

*** Zone diameters are interpreted as signifying susceptibility, intermediate susceptibility, or resistance to each antimicrobial agent tested

2. Dilution Tests preferred by clinicians due to MIC and MBC

- Provides a quantitative estimate of susceptibility to an a antibiotic

- Serial dilutions of the test antibiotics that are prepared in broth or agar medium, then a suspension of test organism is inoculated then incubated overnight

2 Categories of Dilution Tests

Minimum Inhibitory Concentration [MIC]

- Lowest or minimal concentration of the antimicrobial agent that is required to inhibit the growth of a microorganism

- Recorded as the highest dilution in which there is no macroscopic growth

- Expressed in micrograms/milliliter

Minimum Bactericidal Concentration or Minimum Lethal Concentration [MBC]

- Lowest or minimal concentration of antimicrobial agent that kills at least 99.9% of the inoculum used for the test

- Used in the evaluation of a new antimicrobial agent

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Factors affecting tests:

1. Composition of medium- Use Mueller-Hinton medium for testing aerobic and facultative bacteria

a. Demonstrates good batch to batch reproducibility

b. Low in sulfonamide, Trimethoprim and tetracycline inhibitors

c. Supports the growth of most non-fastidious pathogens

2. Medium-related factorsa. Agar depth should be about

4mmi. Too deep: false-resistant results

ii. <4mm: falsely susceptibleb. Media containing excessive

amounts of thymidinei. Can reverse the inhibitory

effects of sulfonamides and trimethoprim: less distinct zones of inhibition

3. Inoculuma. Inoculum size approx. 5x105

CFU/ml in broth dilution testsb. Method of preparation: should be

prepared directly from overnight growth on an agar medium

McFarland’s Standard

Tube that contains barium sulfate (BaSO4)

Turbid liquid used to compare turbidity of organism that you will put into agar plate

Should be approximately the same, compared visually

Signal to start planting into agar plate, putting filter paper

4. Incubationa. General recommendations: 16-18

hours in ambient air at 35oCb. Exceptions

i. S. pneumoniae, H. influenza, N. gonorrhea

ii. 5-7% CO2: 20-24 hoursiii. Staphylococci and Enterococci:

24 hoursiv. Anaerobes and yeasts: 48 hours