Pathogenic Micro Exam 1: Traditional Culture & Identification

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What are the two phases of media?

1) Liquid (Broth)
2) Solid (Agar) *
* used more frequently

What are the four general categories of media?

1) Enrichment
2) Supportive
3) Selective
4) Differential

Enrichment Media

- Contains specific nutrients for the growth of particular bacterial pathogens that may be present alone or with other spp
- Is used to enhance the growth of a particular bacterial pathogen from a mixture of organisms using bacterial specificity

Supportive Media

Contains nutrients that support growth of most nonfastidious organisms without giveing any particular organism a growth advantage

Selective Media

- Contains one or more agents that are inhibitory to all organisms except those being sought
- Inhibitory agents include dyes, bile salts, alcohols, acids, and antibiotics

Differential Media

Employs some factor (or factors) that allows colonies of one bacterial spp or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria

Blood Agar

Type: Supportive & Differential
Content: Trypticase soy agar and 5-10% sheeps blood
Purpose: Cultivation of fastidious microorganisms and the determination of hemolytic reactions; important for determining Streptococcus beta hemolytic reaction

Chocolate Agar

Type: Enriched
Content: Peptone base, enriched soln of 2% hemoglobin or IsoVitalex (BBL)
- Releases intercellular nutrients
- Brown color comes from the denaturation
Purpose: Cultivation of Haemophilus spp and pathogenic Neisseria spp

MacConkey agar

Type: Selective & Differential
Content: Peptone base with lactose. Gram positive organisms inhibited by crystal violet and bile salts. Neutral red as indicator
Purpose: Isolation and differentian of lactose fermenting and non-lactose fermenting enteric bacilli; lactose positive is bright pink, lactose negative is clear

Hektoen Enterik (HE) agar

Type: Selective & Differential
Content: Bile salts, bromthymol blue, acid fuchsin, Lactose fermentation (orange, salmon), Ferric ammonium citrate (H2S production)
Purpose: Differential, highly selective medium for the isolation and differentiation of Salmonella and Shigella spp. from other gram negative enteric bacilli

Xylose-Lysine-Deoxocholate (XLD) agar

Type: Selective & Differential
Content: Yeast extract with lysine, xylose, lactose, sucrose (yellow colonies) and ferric ammonium citrate. Sodium desoxycholate inhibits gram-postive organims; phenol red is indicator
Purpose: Isolation and differentiation of Salmonella and Shigella spp. from other gram negative enteric bacilli

Thayer-Martin Agar

Type: Enrichment & Selective
Content: Blood agar base enriched with hemoglobin; contaminating organisms inhibited by colistin, vancomycin, nystatin, trimethoprim.
Purpose: Selective for N. gonorrhoeae and N. menigitidis

Phenylethyl alcohol (PEA) agar

Type: Selective
Content: Nutrient agar base. Phenolethyanol inhibits growth of gram-negative organisms
Purpose: Selective isolation of gram-negative bacilli

Brain Heart Infusion

Type: Enrichment
Content: Enriched broth or agar; Infusion from animal tissues, peptone, dextrose
Purpose: Liquid culture media for blood cultures

Thioglycolate broth

Type: Enrichment
Content: Nurtients, reazurine, 0.075% agar. Clear when oxygen present, purple when oxygen present (oxidation-reduction indicator)
Purpose: Supports growth of anaerobes, aerobes, microaerophilic and fastidious microorganisms (broad purpose medium)

Enzyme-Based Tests

Designed to measure the presence of one specific enzyme or a complete metabolic pathway that may contain several different enzymes

Catalase Test

- The enzyme catalase catalyzes the release of water and oxygen from hydrogen peroxide
- Produces bubbles when hydrogen peroxide is added to bacterial growth
- Is key to the identification scheme of many gram-positive organisms
- Staphylococci is positive, streptococci is negative

Oxidase Test

- Cytochrome oxidase participates in electron transport and in the nitrate metabolic pathways of certain bacteria
- Test is initially used for differentiating between groups of gram-negative.
- Enterbacteriaceae is negative, Pseudomonas spp is positive

Indole Test

- Bacteria that produce the enzyme tryptophanase are able to degrade the amino acid tryptophan into pyruvic acid, ammonia and indole
- Is used in numberous identification schemes, especially E.coli, for gram-negative bacillus

Urease Test

- Urease hydrolyzes the substrate urea into ammonia, water and carbon dioxide
- Helps identify certain spp of Enterbacteriaceae, such as Proteus spp.

PYR Test

- The enzyme L-pyrroglutamylaminopeptidase hydrolyzes the substrate PYR to produce beta naphthylamine.
- Helps indentify gram-positive cocci
- Streptococcus p. and Enterococcus spp are positive

Oxidation & fermentation Test

- This test is used to determine wheather an organism utilizes carbohydrates (glucose) by fermentation or oxidation.
- Uses two tubes. One tube is sealed with oil.
- If it oxidizes glucose = open tube turns yellow. If it ferments glucose = oil tube and open tube turn yellow

Amino acid degradation

- Determines the ability of the bacteria to produce enzymes that deanimate, dihydrolyze or decarboxylate certain amino acids
- Moeller decarboxylase base
- Overlay with mineral oil (anaerobic)

What are the four components of bacterial identification schemes?

1) Selection and inoculation of tests
2) Incubation for substrate utilization
3) Detection of metabolic activity
4) Analysis of metabolic profiles

What are the phenotypic characteristics that can be quickly determined?

1) Gram-stain reaction
2) Morphology
3) Growth characteristics
4) Rapid, single-enzyme tests

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