Utf-8''Principle Diagnostic Microbiology

8/3/2019 Utf-8''Principle Diagnostic Microbiology

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Principle Diagnostic

Microbiology


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Concerned with the etiologic

diagnosis of infection.

Laboratory procedures used in the diagnosis ofinfectious disease in humans include thefollowing: (1) Morphologic identification of the agent in stains of

specimens or sections of tissues (light and electron

microscopy). (2) Culture isolation and identification of the agent.

(3) Detection of antigen from the agent byimmunologic assay (latex agglutination, EIA, etc) orby fluorescein-labeled (or peroxidase-labeled)

antibody stains. (4) DNA-DNA or DNA-RNA hybridization to detectpathogen-specific genes in patients' specimens.

(5) Detection and amplification of organism nucleicacid in patients' specimens.

(6) Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.


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In the field of infectious diseases,

laboratory test results depend largely on:

The quality of the specimen,

The timing and the care with which it iscollected, and

The technical proficiency andexperience of laboratory personnel.


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Diagnostic microbiology encompasses the

characterization of thousands of agents that

cause or are associated with infectiousdiseases.

The techniques used to characterize infectious

agents vary greatly depending upon: the clinical syndrome and

the type of agent being considered, be it virus,

bacterium, fungus, or other parasite.


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Communication between

Physician & Laboratory no single test will permit isolation or

characterization of all potential pathogens

clinical information is much more important

for diagnostic microbiology than it is for

clinical chemistry or hematology.


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the physician should inform the laboratory

staff of the tentative diagnosis

e.g. type of infection or infectious agentsuspected.

Infected part to be sampled


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Treatment of samples:---

Proper labeling of specimens including:

clinical data the patient's identifying data (at least two

methods of definitive identification)

the requesting physician's name and pertinent

contact information.


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Many pathogenic microorganisms grow slowly,and days or even weeks may elapse before they

are isolated and identified. the physician should begin treatment with drugs

aimed at the organism thought to be responsiblefor the patient's illness.

As the laboratory staff begins to obtain results,they inform the physician, who can thenreevaluate the diagnosis and clinical course ofthe patient and perhaps make changes in thetherapeutic program.

The "feedback" information from the laboratoryconsists ofpreliminary reports of the results ofindividual steps in the isolation and identificationof the causative agent.


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Diagnosis of Bacterial & Fungal

Infections Specimens


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A few general rules apply to all

specimens: (1) The quantity of material must be adequate. (2) The sample should be representative of the infectious

process (eg, sputum, not saliva; pus from the underlyinglesion, not from its sinus tract; a swab from the depth of

the wound, not from its surface). (3) Contamination of the specimen must be avoided byusing only sterile equipment and aseptic precautions.

(4) The specimen must be taken to the laboratory andexamined promptly. Special transport media may behelpful.

(5) Meaningful specimens to diagnose bacterial andfungal infections must be secured before antimicrobialdrugs are administered. If antimicrobial drugs are givenbefore specimens are taken for microbiologic study, drugtherapy may have to be stopped and repeat specimens

obtained several days later.


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Microscopy & Stains

relatively simple and inexpensive much less sensitive method than culture for

detection of small numbers of bacteria.

A specimen must contain at least 105 organisms

per milliliter. Specimens containing 102103 organisms per

milliliter produce growth on solid media, andthose containing ten or fewer bacteria permilliliter may produce growth in liquid media.

Gram staining is a very useful procedure indiagnostic microbiology.

All specimens submitted when bacterial infectionis suspected should be smeared on glass slides,G

ram-stained, and examined microscopically.


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Gram staining

Gram reaction (purple-blue indicates gram-positive organisms; red, gram-negative) and

morphology (shape: cocci, rods, fusiform, orother) of bacteria should be noted.

The appearance of bacteria on Gram-stainedsmears does not permit identification of species.

Reports of gram-positive cocci in chains are

suggestive of, but not definitive for, streptococcalspecies; gram-positive cocci in clusters suggesta staphylococcal species.

Gram-negative rods can be large, small, or even

coccobacillary. Some nonviable gram-positive bacteria can staingram-negatively. Typically, bacterial morphologyhas been defined using organisms grown onagar. However, bacteria in body fluids or tissue

can have highly variable morphology.


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Specimens submitted for examination for

mycobacteria should be stained for acid-fast

organisms, using eitherZiehl-Neelsen stain or

Kinyoun stain. An alternative fluorescent stain for mycobacteria,

auramine-rhodamine stain, is more sensitive

than other stains for acid-fast organisms but

requires fluorescence microscopy and, if results

are positive, confirmation of morphology with an

acid-fast stain


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Table 471. Gram and Acid-Fast Staining Methods.

Gram stain

(1) Fix smear by heat.

(2) Cover with crystal violet.

(3) Wash with water. Do not blot.

(4) Cover with Gram's iodine.


(6) Decolorize for 1030 seconds with gentle agitation in acetone (30 mL) and alcohol (70 mL).


(8) Cover for 1030 seconds with safranin (2.5% solution in 95% alcohol).

(9) Wash with water and let dry.

Ziehl-Neelsen acid-fast stain

(1) Fix smear by heat.

(2) Cover with carbolfuchsin, steam gently for5 minutes over direct flame (or for20 minutes over a water

bath).

(3) Wash with water.

(4) Decolorize in acid-alcohol until only a faint pink color remains.

(5) Wash with water.

(6) Counterstain for 1030 seconds with Loeffler's methylene blue.

(7) Wash with water and let dry.

Kinyoun carbolfuchsin acid-fast stain

(1) Formula: 4 g basic fuchsin, 8 g phenol, 20 mL 95% alcohol, 100 mL distilled water.

(2) Stain fixed smear for3 minutes (no heat necessary) and continue as with Ziehl-Neelsen stain.


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Utf-8''Principle Diagnostic Microbiology

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