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Bacterial Identification Methods
1. Acetamide Utilization - Purpose : Differentiate organisms based on the ability to use acetamide as the sole carbon source. - Principle : o Bacteria producing acylamidase deaminate acetamide, releasing ammonia. o Ammonia increases pH, changing the medium from green to royal blue. - Expected Results : o Positive : Blue color indicates acetamide utilization. o Negative : No color change; medium remains green. - Limitations : o Heavy inoculum may lead to false negatives. - Quality Control : o Positive : Pseudomonas aeruginosa (growth; blue color). o Negative : Escherichia coli (no growth; green color). 2. Acetate Utilization - Purpose : Differentiate organisms based on the ability to use acetate as the sole carbon source. - Principle : o Utilization of sodium acetate leads to alkaline pH, turning the medium blue. - Expected Results : o Positive : Blue color due to alkaline reaction. o Negative : No color change; medium remains green. - Limitations : o Some E. coli strains may give false negatives. - Quality Control : o Positive : Escherichia coli (growth; blue ). o Negative : Shigella sonnei (little growth; green ). 3. l-Alanine- 7 - amido- 4 - methylcourmarin (Gram-Sure) Test - Purpose : Distinguish aerobic gram-positive rods or coccobacilli that may appear gram-negative. - Principle : o Detects aminopeptidase enzyme activity; gram- negative organisms produce fluorescence under UV light. - Expected Results : o Positive : Blue fluorescence (gram-negative). o Negative : No fluorescence (gram-positive). - Limitations : o May not work with obligate anaerobes. - Quality Control : o Positive : Escherichia coli (fluorescence). o Negative : Staphylococcus aureus (no fluorescence).
4. Bacitracin Susceptibility Test - Purpose : Differentiate β-hemolytic group A streptococci (susceptible) from other streptococci and distinguish staphylococci (resistant) from micrococci (susceptible). - Principle : o Bacitracin inhibits cell wall synthesis; susceptibility indicated by a zone of inhibition. - Method : BAP, Bacitracin Disk - Expected Results : o Positive : Zone of inhibition ≥10 mm (susceptible). o Negative : No zone (resistant). - Limitations : o Disk integrity affects performance. - Quality Control : o Positive : Streptococcus pyogenes , Micrococcus luteus. o Negative : Streptococcus agalactiae , Staphylococcus aureus. 5. Bile Esculin Test - Purpose : Identify enterococci and group D streptococci by their ability to hydrolyze esculin in the presence of bile. - Principle : o Esculin hydrolysis produces esculetin, which reacts with ferric ions to form a dark precipitate. - Method : bile esculin agar slant. - Expected Results : o Positive : Blackening of the medium. o Negative : No color change. - Limitations : o Some organisms may not grow due to nutritional requirements. - Quality Control : o Positive : Enterococcus faecalis (black precipitate). o Negative : Escherichia coli , Streptococcus pyogenes. 6. Bile Solubility Test - Purpose : Differentiate Streptococcus pneumoniae (positive) from other alpha-hemolytic streptococci. - Principle : o Bile salts lyse pneumococcal colonies due to the presence of an autolytic enzyme. - Method : BAP, sodium deoxycholate - Expected Results : o Positive : Colony disintegration. o Negative : Intact colonies. - Limitations : o Old cultures may yield false negatives. - Quality Control : o Positive : Streptococcus pneumoniae (bile soluble). o Negative : Enterococcus faecalis , viridans streptococci.
10. Cetrimide Agar Test - Purpose : Isolate and purify Pseudomonas aeruginosa. - Principle : o Cetrimide inhibits many bacteria; P. aeruginosa resists and grows, often producing pigments. - Method : cetrimide agar slant. - Expected Results : o Positive : Growth with color change (yellow-green to blue-green). o Negative : No growth. - Limitations : o Some enteric bacteria may grow weakly. - Quality Control : o Positive : Pseudomonas aeruginosa. o Negative : Escherichia coli. 11. Citrate Utilization - Purpose : Identify organisms that use sodium citrate as the sole carbon source. - Principle : o Citrate utilization leads to alkaline pH, turning bromothymol blue indicator from green to blue. - Method : Simmons citrate agar slant. - Expected Results : o Positive : Growth with or without blue color. o Negative : No growth; medium remains green. - Limitations : o Some organisms may grow without changing color. - Quality Control : o Positive : Klebsiella aerogenes (growth; blue color). o Negative : Escherichia coli (no growth). 12. Coagulase Test - Purpose : Differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci. - Principle : o Slide Test : bound coagulase (clumping factor). o Tube Test : free coagulase clot formation. - Method : o Slide Test : Coagulate with EDTA, observe clumping o Tube Test : Observe clot formation - Expected Results : o Positive : Clumping (slide); clot formation (tube). o Negative : No clumping or clot. - Limitations : o Some strains may autoagglutinate; confirm negatives with tube test. - Quality Control : o Positive : Staphylococcus aureus. o Negative : Staphylococcus epidermidis.
13. Decarboxylase Tests (Moeller’s Method) - Purpose : Differentiate decarboxylase-producing Enterobacterales from other gram-negative rods. - Principle : o Detects enzymatic decarboxylation of amino acids (lysine, ornithine, arginine) to amines, causing alkaline pH and a color change from yellow/orange to purple. - Method : decarboxylase broths (arginine, lysine, ornithine, and a control broth with no amino acid) - Expected Results : o Positive : Alkaline (purple) color change compared to control. o Negative : Acidic (yellow) or no color change in test and control tubes. - Limitations : o False negatives may occur if growth is insufficient. - Quality Control : o Positive : Klebsiella pneumoniae (lysine-positive), Pseudomonas aeruginosa (arginine-positive). o Negative : Proteus vulgaris (ornithine-negative). 14. Deoxyribonucleic Acid Hydrolysis (DNase Test Agar) - Purpose : Differentiate organisms based on DNA hydrolysis, distinguishing species like Serratia spp. , Staphylococcus aureus , and Moraxella catarrhalis from others. - Principle : o DNA hydrolysis clears the green DNA–methyl green complex, creating a colorless zone. - Method : DNase agar - Expected Results : o Positive : Colorless zone around growth. o Negative : Green medium remains unchanged. - Limitations : o Ensure proper inoculation; inadequate growth may yield false results. - Quality Control : o Positive : Staphylococcus aureus. o Negative : Escherichia coli. 15. Esculin Hydrolysis - Purpose : Identify Enterobacterales based on the ability to hydrolyze esculin. - Principle : o Hydrolysis of esculin produces esculetin, which reacts with ferric ions to form a black precipitate. - Method : esculin medium with a drop of a 24-hour broth culture - Expected Results : o Positive : Blackened medium or loss of fluorescence under UV light. o Negative : No blackening or fluorescence loss. - Limitations : o Nonselective medium may require follow-up testing. - Quality Control : o Positive : Enterococcus faecalis. o Negative : Escherichia coli.
19. Growth at 42°C - Purpose : Differentiate Pseudomonas aeruginosa from other Pseudomonas species. - Principle : o Growth at elevated temperature indicates thermotolerance. - Method : TSA slants - Expected Results : o Positive : Growth at both temperatures. o Negative : No growth at 42°C. - Quality Control : o Positive : Pseudomonas aeruginosa. o Negative : Pseudomonas fluorescens. 20. Hippurate Hydrolysis - Purpose : Identify organisms producing hippuricase ( Streptococcus agalactiae , Listeria monocytogenes ). - Principle : o Hydrolysis of hippuric acid yields glycine and benzoic acid. Glycine reacts with ninhydrin to form a purple color. - Method : hippurate disk; ninhydrin reagent - Expected Results : o Positive : Purple color. o Negative : No color or slight yellow. - Limitations : o False positives may occur with over-incubation. - Quality Control : o Positive : Streptococcus agalactiae. o Negative : Streptococcus pyogenes. 21. Indole Production - Purpose : Identify organisms that produce the enzyme tryptophanase, which hydrolyzes tryptophan to indole. - Principle : o Tryptophanase hydrolyzes tryptophan into pyruvate, ammonia, and indole. o Indole reacts with Kovac’s or Ehrlich’s reagent, producing a pink or red color. - Method : o Kovac’s Method : tryptophan broth; Kovac’s reagent o Ehrlich’s Method : xylene, Ehrlich’s reagent - Expected Results : o Positive : Pink-to-wine-colored o Negative : No color change. - Limitations : o Ehrlich’s method is more sensitive and used for anaerobic organisms. - Quality Control : o Kovac’s Method : E. coli (positive), Klebsiella pneumoniae (negative). o Ehrlich’s Method : Haemophilus influenzae (positive), Haemophilus parainfluenzae (negative).
22. Leucine Aminopeptidase (LAP) Test - Purpose : Identify catalase-negative, gram-positive cocci, differentiating enterococci and streptococci. - Principle : o LAP hydrolyzes leucine-beta-naphthylamide, producing beta-naphthylamine, which reacts with cinnamaldehyde reagent to form a red color. - Method : LAP disk, cinnamaldehyde reagent - Expected Results : o Positive : Bright red color. o Negative : No color change or slight yellow. - Limitations : o Disk integrity affects reliability. - Quality Control : o Positive : Enterococcus faecalis. o Negative : Aerococcus viridans. 23. Litmus Milk Medium - Purpose : Differentiate microorganisms based on metabolic reactions in milk, including fermentation, reduction, clot formation, and peptonization. - Principle : o Lactose fermentation turns litmus pink (acidic). o Reduction causes the medium to lose its color. o Protein hydrolysis results in peptonization (clearing of the medium). - Method : litmus milk - Expected Results : o Fermentation : Pink color. o Reduction : Colorless medium. o Peptonization : Clearing of milk. o Clot Formation : Coagulated milk. - Limitations : o Reactions are nonspecific and require additional confirmatory tests. - Quality Control : o Positive (acid) : Lactobacillus acidophilus. o Negative (peptonization) : Pseudomonas aeruginosa. 24. Lysine Iron Agar (LIA) - Purpose : Differentiate gram-negative bacilli based on lysine decarboxylation or deamination and hydrogen sulfide (H₂S) production. - Principle : o Lysine decarboxylation produces an alkaline reaction (purple butt). o Lysine deamination forms a red slant. o H₂S production results in a black precipitate. - Expected Results : o K/K : Lysine decarboxylation (alkaline slant and butt). o K/A : Glucose fermentation only. o R/A : Lysine deamination (red slant, acidic butt). o H₂S Production : Black precipitate in butt. - Limitations : o Proteus spp. may not blacken the medium despite H₂S production. - Quality Control : o K/K, H₂S+ : Salmonella enterica. o R/A : Proteus mirabilis.
28. Lactobacillus MRS Broth - Purpose : Identify gas production during glucose fermentation, primarily in Lactobacillus spp. and Leuconostoc spp. - Principle : o Medium contains carbon, nitrogen, and vitamins to support lactobacilli growth. o Gas production is observed in a Durham tube. - Method : MRS broth - Expected Results : o Positive : ▪ Leuconostoc spp. : Growth and gas production. ▪ Lactobacillus spp. : Growth without gas. o Negative : No growth. - Quality Control : o Positive : Lactobacillus lactis. o Negative : Escherichia coli. 29. 4 - Methylumbelliferyl-β-d-Glucuronide (MUG) Test - Purpose : Presumptive identification of Escherichia coli and other Enterobacterales. - Principle : o Detects the enzyme β-d-glucuronidase, which hydrolyzes the substrate to produce a fluorescent product under UV light. - Expected Results : o Positive : Bright blue fluorescence. o Negative : No fluorescence. - Limitations : False negatives may occur with media containing dyes or oxidase-negative organisms with natural fluorescence. - Quality Control : o Positive : Escherichia coli. o Negative : Klebsiella pneumoniae. 30. Nitrate Reduction Test - Purpose : Determine an organism's ability to reduce nitrate to nitrite or other nitrogenous compounds. - Principle : o Nitrate reductase converts nitrate to nitrite. o Nitrite reacts with sulfanilic acid and alpha- naphthylamine, producing a red azo dye. o If no red color appears, zinc powder is added to detect unreduced nitrate. - Method : nitrate broth, nitrate reagents, zinc powder (in the absence of color) - Expected Results : o Positive : Red color after reagents A and B (nitrate reduced to nitrite). No color after zinc addition (nitrate reduced to other products). o Negative : Red color after zinc addition (nitrate not reduced). - Limitations : Long incubation may lead to false negatives - Quality Control : o Positive : Escherichia coli (red after A and B). o Negative : Acinetobacter baumannii (red after zinc).
31. Nitrite Reduction Test - Purpose : Determine whether an organism reduces nitrite to gaseous nitrogen or other nitrogen compounds. - Principle : o Organisms capable of reducing nitrite produce no color and may form gas in the Durham tube. - Method : nitrite broth, Durham tube for gas production observation, nitrate reagents for residual nitrite. - Expected Results : o Positive : No red color after reagents; gas in Durham tube. o Negative : Red color after reagents; no gas. - Limitations : Zinc addition confirms unreduced nitrite if results are unclear. - Quality Control : o Positive : Proteus mirabilis (no color, gas production). o Negative : Acinetobacter baumannii (red color, no gas). 32. o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test - Purpose : Distinguish late lactose fermenters from non-lactose fermenters among Enterobacterales. - Principle : o ONPG is hydrolyzed by β-galactosidase to form a yellow product (o-nitrophenol). - Method : ONPG disk turning yellow. - Expected Results : o Positive : Yellow color indicates β-galactosidase activity. o Negative : Colorless. - Quality Control : o Positive : Shigella sonnei. o Negative : Salmonella Typhimurium. 33. Optochin (P Disk) Susceptibility Test - Purpose : Differentiate Streptococcus pneumoniae (positive) from alpha-hemolytic streptococci (negative). - Principle : o Optochin interferes with ATPase and ATP production in susceptible organisms, creating a zone of inhibition around the disk. - Method : BAP, optochin disk - Expected Results : o Positive : Zone of inhibition ≥14 mm. o Negative : No zone or <14 mm. - Limitations : o Small zones require confirmation with bile solubility. - Quality Control : o Positive : Streptococcus pneumoniae. o Negative : Streptococcus pyogenes.
37. Pyrrolidonyl Arylamidase (PYR) Test - Purpose : Identify group A streptococci ( Streptococcus pyogenes ) and enterococci. - Principle : o PYR hydrolyzes pyrrolidonyl-β-naphthylamide to produce β-naphthylamine, which reacts with cinnamaldehyde reagent to produce a red color. - Method : PYR, cinnamaldehyde reagent (red color in 1min) - Expected Results : o Positive : Bright red color. o Negative : No color change or orange. - Quality Control : o Positive : Streptococcus pyogenes , Enterococcus faecalis. o Negative : Streptococcus agalactiae. 38. Triple Sugar Iron (TSI) Agar - Purpose : Differentiate gram-negative rods based on glucose, lactose, and sucrose fermentation and hydrogen sulfide (H₂S) production. - Principle : o Fermentation of sugars produces acid (yellow). o Oxidation of peptones produces alkaline reactions (red). o H₂S reacts with iron salts to form black precipitate. - Method : TSI slant - Expected Results : o K/A : Glucose fermentation only (red slant, yellow butt). o A/A : Glucose, lactose, and/or sucrose fermentation (yellow slant and butt). o K/K : Non-fermenter (red slant and butt). o H₂S Production : Black precipitate in the butt. - Limitations : Reactions must be read within 24 hours. - Quality Control : o A/A, gas : Escherichia coli. o K/A, H₂S : Salmonella enterica. 39. Pyruvate Broth - Purpose: Differentiate Enterococcus faecalis (positive) from Enterococcus faecium (negative) based on their ability to utilize pyruvate. - Principle: o Organisms capable of metabolizing pyruvate produce metabolic acids, which lower the pH and change the color of bromothymol blue indicator from green to yellow. - Method: Pyruvate broth, sheep blood agar. - Expected Results: o Positive : Indicator changes from green to yellow. o Negative : No color change; yellow-green indicates a weak reaction but should be regarded as negative. - Quality Control: o Positive: Enterococcus faecalis. o Negative: Streptococcus gallolyticus.
40. Salt Tolerance Test
- Purpose: Differentiate enterococci (positive) from non- enterococci (negative) based on their ability to grow in high salt concentrations (6.5% NaCl).
- Principle: o Enterococci can tolerate high salt concentrations and produce visible turbidity and/or acid production (color change). o Bromocresol purple serves as the pH indicator, changing from purple to yellow in the presence of acid.
- Method: NaCl broth
- Expected Results: o Positive: Visible turbidity in the broth with or without color change to yellow. o Negative: No turbidity or color change.
- Quality Control: o Positive: Enterococcus faecalis (turbidity and color change to yellow). o Negative: Streptococcus gallolyticus (no growth or color change). 41. Spot Indole Test - Purpose: Rapidly determine the presence of tryptophanase, which hydrolyzes tryptophan to release indole. - Principle: o Tryptophanase activity is indicated by the reaction of indole with paradimethylamino-cinnamaldehyde reagent, producing a blue-green color. - Method: paradimethylamino-cinnamaldehyde reagent, rapid development of color within 20 seconds. - Expected Results: o Positive: Blue-green color develops. o Negative: No color or slightly pink color. - Limitations: o Do not select colonies from MacConkey agar as the medium’s color can interfere with interpretation. - Quality Control: o Positive: Escherichia coli. o Negative: Klebsiella pneumoniae. 42. Urease Test - Purpose : Identify organisms capable of hydrolyzing urea to ammonia and CO₂, raising the pH. - Principle : o Urease hydrolyzes urea, producing ammonia and CO₂, causing a color change in phenol red from light orange to magenta. - Method : urea agar slant - Expected Results : o Positive : Pink color change. o Negative : No color change. - Quality Control : o Positive : Proteus vulgaris. o Negative : Escherichia coli.
