Bacterial Identification General

Function of Clinical Microbiology Lab Participation in management of patients with infectious diseases by processing of clinical specimens: 1- Microscopy. 2- Cultivation & isolation of pathogens. 3- Identification of isolates: macroscopy, microscopy, biochemical tests, serology. 4- Antimicrobial susceptibility testing (AST) of isolates.

 Early preliminary indicators of identity of suspected bacterial pathogen involved in the infection : Direct microscopy of clinical specimen’s smear. Culture media & incubation conditions supporting its growth.  Indicators of clinical significance of bacterial isolate : Source (body site) of clinical specimen. Direct smear results. Virulence vs Predominance (Relative quantities of each colony type).

Bacterial growth on solid media Colony = The resulting visible mass of sufficiently large bacterial numbers (population) that is considered to be originating from a single mother bacterial cell. All bacterial cells within a single colony are a single clone (belong to the same genus & species, having identical genetic & phenotypic characteristics). Bacterial cultures derived from a single individual colony or clone are considered pure.

Bacterial culture Pure culture Growth containing one type of bacteria only. Mixed culture Growth containing > one type of bacteria.

Bacterial colonies Description Size (diameter): usually measured in millimeters or described in relative terms as tiny pinpoint (Punctiform), small, medium, large. Morphology (Shape): includes: 1- Form (basic morphology) of whole colony. 2- Elevation (side view). 3- Margin/border (edge). Surface e.g. smooth, rough, wrinkled, glistening, dull, dry, powdery. Opacity e.g. transparent (clear), opaque, or translucent (like looking through frosted glass). Colour (pigmentation) e.g. white, buff, yellow, red, purple, etc. Odor: e.g. Proteus, Pseudomonas etc. Changes in agar media resulting from bacterial growth (e.g. hemolytic pattern on blood agar, changes in color of pH indicators, pitting of agar surface).

Colony Shape GLISTEN or FLAT

Colony color & size

Pigments & agar changes

Bacterial growth in liquid media Patterns

Principles of Identification Identification scheme = set & order-in-use of laboratory tests that provide the characteristic profiles of bacterial isolate leading to its definite identification. There are two identification schemes of bacteria: Genotypic. Phenotypic.

Genotypic Criteria involve characterization of some portion of bacterium’s genome using molecular techniques for DNA or RNA analysis. highly specific & very sensitive. presence of specific gene or particular nucleic acid sequence → definitive identification.

Phenotypic Criteria based on observable physical or metabolic characteristics of bacteria. provides preliminary identification & information necessary for determining what other identification procedures should follow to confirm the final identification. most commonly used include: Microscopic cellular morphology & staining characteristics. Macroscopic (colony) morphology. Environmental requirements for growth. Resistance or susceptibility to antimicrobial agents. Nutritional requirements & metabolic capabilities.

1- Microscopic Cellular Morphology & Staining Characteristics Gram stain of bacterial growth from isolated colonies on various media is usually the first step in any identification scheme. Most pathogenic bacteria can be classified into four distinct groups: 1- Gram-positive cocci. 2- Gram-positive bacilli. 3- Gram-negative bacilli. 4- Gram-negative cocci.

Some bacterial species are morphologically indistinct & are described as “Gram-negative coccobacilli,” “Gram-variable bacilli,” or pleomorphic (i.e. various shapes). Other morphologies include curved and/or rods and spirals. Even without staining, examination of wet preparation of bacterial colonies under oil immersion (1000× magnification) can provide clues to possible identity.

2- Macroscopic (Colony) Morphology Evaluation includes considering colony size, shape, color (pigment), surface appearance, & any changes that colony growth produces in the surrounding agar medium (e.g. hemolysis of blood in blood agar plates).

3- Environmental Requirements for Growth Organisms growing only in bottom of tube containing thioglycollate broth are NOT likely to be strictly aerobic bacteria. Organisms growing on blood agar plates incubated in ambient (room) atmosphere are NOT likely to be anaerobic bacteria. Organism’s requirement, or preference, for increased CO 2 concentrations e.g. Strep pneumoniae, Haemophilus influenzae, & Neisseria gonorrhoeae. Organism’s ability for survival or growth in temperatures that exceed or are well below normal body temperature of 37°C e.g. survival of Yersinia enterocolitica at 0°C & growth of Campylobacter jejuni at 42°C.

4- Resistance or Susceptibility to Antimicrobial Agents Organism’s ability to grow in presence of certain antimicrobial agents or specific toxic substances is widely used to establish preliminary identification information. This is accomplished by using agar media supplemented with inhibitory substances or antibiotics. Or by directly measuring organism’s resistance to antimicrobial agents that may be used to treat infections.

Nature of media on which organism is growing provides the first clue for identification of isolated colony: Gram-negative bacteria grow well on MacConkey agar. Columbia-CNA agar (with colistin & naladixic acid), will support growth of Gram-positive organisms by inhibiting growth of most Gram-negative bacilli. Chocolate agar will support growth of all Neisseria spp., but antibiotic-supplemented Thayer-Martin formulation will almost exclusively support growth of pathogenic spp. N. meningitidis & N. gonorrhoeae.

Organism’s uncertain Gram stain “status”: Susceptibility to vancomycin: truly Gram- negative bacteria are resistant & many Gram-positive bacteria are susceptible. Susceptibility to colistin or polymyxin: most Gram-negative bacteria are susceptible & Gram-positive bacteria are frequently resistant.

5- Nutritional Requirements & Metabolic Capabilities the most common approach used for determining the genus & species of an organism. In general, all methods use combination of tests to establish:  Enzymatic capabilities of a given bacterial isolate.  Isolate’s ability to grow or survive in presence of certain inhibitors (e.g. salts, surfactants, toxins, & antibiotics.

A- Establishing Enzymatic Capabilities Enzymatic content of an organism is a direct reflection of the organism’s genetic makeup, which, in turn, is specific for individual bacterial species. In diagnostic bacteriology, enzyme-based tests are designed to measure:  Either Presence of one specific enzyme(Single Enzyme Tests):  Although most single enzyme tests do not yield sufficient information to provide species identification, they are used extensively to determine which subsequent identification steps should be followed

 Complete metabolic pathway that may contain several different enzymes.  Assays for metabolic pathways can be classified into three general categories: A. Carbohydrate oxidation & fermentation. B. Amino acid degradation. C. Single substrate utilizations.

Bacterial Enzymes Intracellular Catalse. Oxidase. IMViC. Urease. Carbohydrate fermentation. Nitrate reduction. Extracellular Blood hydrolysis (Streptolysin). Starch hydrolysis (Amylase). Casein hydrolysis (protease). Lipid hydrolysis (Lipase). Gelatin hydrolysis (Gelatinase).

B- Establishing Inhibitor Profiles Ability of bacterial isolate to grow in presence of one or more inhibitory substances can provide valuable identification information. Examples: Growth in presence of various NaCl concentrations (identification of enterococci & Vibrio spp.). Susceptibility to optochin & solubility in bile (identification of Strep pneumoniae). Ability to hydrolyze esculin in presence of bile (identification of enterococci).