Principles of Laboratory Diagnosis of Infectious Diseases Bacteriology Lab -2- Principles of Laboratory Diagnosis of Infectious Diseases

The general approaches to laboratory diagnosis vary with different microorganisms and infectious diseases. However, the types of methods are usually some combination of direct microscopic examinations, culture, antigen detection, and antibody detection (serology). Nucleic acid amplification assays that allow direct detection of genomic components of pathogens are now numerous, but few are practical for routine use.

A. Direct samples Direct specimens are collected from normally sterile tissues (lung, liver) and body fluids (cerebrospinal fluid, blood). Direct samples give highest quality and risk.

B. Indirect sample: Indirect samples are specimens of inflammatory(expectorated sputum, voided urine) that have passed through sites known to be colonized with normal flora. Results require interpretive evaluation of contamination.

C. Samples from Normal Flora Sites: The pathogen and nonpathogenic flora are mixed at the site of infection. Both are collected and the non-pathogen is either inhibited by the use of selective culture methods or discounted in interpretation of culture results. Examples are throat and stool.

Specimen Collection Proper specimen collection, container labeling, and culture requests are the responsibility of the ordering physician. The volume is important because infecting organisms that are present in small numbers may not be detected in a small sample.

Specimen Transport Specimens should be transported to the laboratory as soon after collection as possible because some microorganisms survive only briefly outside the body. Transport media stabilize conditions and prevent drying. In general, they are buffered fluid or semisolid media containing minimal nutrients and are designed to prevent drying, maintain a neutral pH, and minimize growth of bacterial contaminants.

Direct Examination light (bright-field) microscopy is particularly useful for detection of bacteria, fungi, and parasites. Even the smallest bacteria (1–2 m wide) can be visualized. Viruses are visible only by electron microscopy.

Staining of Bacteria Bacteria cells are almost colorless. A staining techniques is used to determine the shape and size of bacterial cells under microscope.

Types of staining techniques Simple staining differential staining (use of a single stain) (use of two stains separated by a decolorizing agents) Gram staining Acid fast staining Spore staining Capsular staining

Principle of staining Bacteria are slightly negatively charged and the most commonly used dyes are salts that are positively charged (cationic). Dyes are classified as: Basic dyes: e.g.: methylene blue a pronounced attraction produces between these cationic dye and the bacteria. Acidic dyes: Those dyes that have negatively charged are called acidic dyes, e.g.: eosin.

Staining techniques 1. Smear preparation: Preparation and fixation of bacteria for staining. Prepared bacterial smear is one of that withstands washings during staining without lose of organisms. In a smear preparation, cells from a liquid medium or solid media are spread in a thin film over a small area of a microscope slide, dried, and then fixed to the slide by heating.

Staining techniques 2. Staining a) Simple Staining: The use of a single stain to color a bacterial organism is referred to as simple staining. The most common dyes used for simple staining are: Methylene blue Basic fuchsin Crystal violet Simple staining used to show the morphological shapes, size and arrangement of bacterial cells.

Morphology of bacterial cells

2. Staining b) Gram Staining in 1884 by the Danish physician Hans Christian Gram developed a staining technique that separate bacteria into two groups: gram positive and gram negative because it is the most important differential stain that used in bacteriological Laboratory.

Reagents used in Gram Stain Crystal Violet Gram Iodine Decolorizing agent Safranin

Reagents used in Gram Stain 1. Crystal Violet: Primary stain. Causes both gram positive and gram negative bacteria to become purple. 2. Gram´s Iodine: Mordant. Forms Crystal Violet Iodine complexes. The color of gram positive and gram negative bacteria remains the same (purple).

3. Decolorizing agent: 95% ethanol. Gram negative bacteria become colorless. Gram positive bacteria remain purple. 4. Safranin: Counter stain. Decolorized gram negative bacteria become red but Gram positive bacteria remain purple.

Gram staining procedure

2. Staining c) Acid-Fast Staining Acid fastness is a property of the mycobacteria (eg, Mycobacterium tuberculosis) and related organisms. Acid-fast bacteria take stains poorly, once stained, they retain it strongly. These is related to the high lipid content of the mycobacterial cell wall.

Acid-Fast Staining Procedure The slide is flooded with carbol-fuchsin (red) steam over boiling water for 5 minutes. After slide has cooled, decolorize with hydrochloric acid in alcohol for 15-20 seconds then rise with water counterstained with methylene blue for 30 seconds thin rinse with water. Blot dry and examine under oil immersion. acid-fast organisms appear red against a blue background .

3. Isolation and Identification of Bacteria (Culture): Growth and identification of the infecting agent in vitro is usually the most sensitive and specific means of diagnosis and is thus the method most commonly used.