Catalase and Coagulase Tests

• Biochemical and antimicrobial sensitivity tests help identify microorganisms based on their enzymes and metabolic products.

Catalase Test

• Used to identify microorganisms based on enzyme activity
• Determines the presence of the catalase enzyme, which catalyzes the release of oxygen from hydrogen peroxide.
• Catalase breaks down hydrogen peroxide (H₂O₂) into water and oxygen.
• Oxygen bubbles are released if the bacteria is catalase-positive.
• Procedure: Mix a small amount of culture from an agar medium with 2-3mls of 2H₂ O₂ in a tube, or one drop on a slide
• Do not use iron wire or test organisms from blood agar, as they produce false positives.
• Examine for gas bubbles when bacteria is mixed with 3% hydrogen peroxide reagent.
• Differentiates Staphylococci (catalase-positive) from Streptococci (catalase-negative).

Coagulase Test

• Differentiates Staphylococcus aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus based on coagulase enzyme production
• Coagulase converts fibrinogen to fibrin, causing plasma to clot; S. aureus produce two types of coagulase.
• Free coagulase converts fibrinogen to fibrin via a coagulase reacting factor present in plasma and is detected by a fibrin clot in the tube test.
• Bound coagulase (clumping factor) directly converts fibrinogen without the reacting factor and is detected by clumping in a rapid slide test.
• A tube test is advised for negative slide tests and if the slide test is negative or unclear but there serious staph infection

Coagulase Test: Slide Method

• Determines bound coagulase
• Place a drop of saline on each end of a slide
• Emulsify colonies of the test organism in each drop; blood and nutrient agar are best for this test, whereas mannitol agar is not recommended.
• Add a drop of plasma to one suspension and mix, checking for clumping within 10 seconds.
• A saline drop without plasma is used as a control to differentiate granular appearance from true coagulase clumping.
• Clumping within 10 seconds indicates S. aureus, while no clumping indicates no bound coagulase production
• Staphylococcus aureus serves as a positive control, and Escherichia coli or Staphylococcus epidermidis serves as a negative control.

Coagulase Test: Tube Method

• Dilute plasma at a ratio of 1:10 in physiological saline
• Label three test tubes: T (test organism), Positive Control, and Negative Control
• Introduce 0.5ml of diluted plasma into each of the tubes
• Add 5 drops of the test organism culture to the T test tube, 5 drops of S. aureus broth to the positive control, and 5 drops of sterile broth to the negative control.
• Mix and incubate at 35-37°C
• Examine after 1 hour and then every 30 minutes for up to 6 hours, for clotting
• Fibrin clot formation indicates S. aureus, while no fibrin clot indicates no free coagulase production.

IMViC Test

• Differentiates microorganisms of the Enterobacteriaceae family, found in the intestinal tract of humans and other mammals.
• The family members include Salmonella, Shigella, Proteus, Klebsiella, Escherichia, and Enterobacter.
• The name "IMViC" represents four reactions: Indole test, Methyl red test, Voges-Proskauer test, and Citrate utilization test.

Indole Test

• Detects indole production from tryptophan breakdown using Kovac’s or Ehrlich’s reagent that contains (p)-dimethyl aminobenzaldehyde.
• The reagent reacts with indole to produce a red-colored compound.
• E. coli, P. vulgaris, P. rettgeri, M. morganii, and Providencia species can break down tryptophan and release indole.
• Add Kovac's reagent to 18-24 hour cultures to detect Indole production.

Indole Test: Requirements

• Peptone water/Tryptophane broth and Kovac’s reagent are needed.
• E. coli is the positive control, and Klebsiella is the negative control.
• PROCEDURE: Inoculate culture organism in 10mls of sterile tryptophane or peptone broth and then incubate at 37°C for 18-24 hours.
• Add 0.2 mL Kovac’s reagent, shake, and let stand for a few minutes before reading the results.
• A cherry red ring in the test tube indicates a positive result.

Citrate Utilization Test

• Determines an organism's ability to use citrate as a sole carbon and energy source.
• Bacteria are inoculated in a medium containing sodium citrate and the pH indicator bromothymol blue.
• The medium also contains inorganic ammonium salt as a nitrogen source.
• Citritase enzymes break down citrate into oxaloacetate and subsequently acetate.
• Oxaloacetate is broken down to pyruvate and CO₂.
• Production of Na₂ CO₃ and NH₃ from sodium citrate and ammonium salt results in alkaline pH.
• Alkaline pH changes the medium color from green to blue.

Citrate Utilization Test: Interpretation

• A positive reaction is indicated by a slant with a prussian blue color.
• A negative reaction indicated by no growth and remains green
• Positive bacteria include: Klebsiella pneumoniae, Citrobacter diversus, Enterobacter cloacae, Serratia marcescens, Providencia alcalifaciens, and Vibrio vulnificus.
• Negative bacteria include: Escherichia coli, Salmonella typhi, Salmonella paratyphi A, Shigella species, Yersinia enterocolitica, and Edwardsiella tarda.

Triple Sugar Iron (TSI) Agar

• The three sugars in TSI agar are glucose, lactose, and sucrose
• It is a differential medium to distinguish gram-negative enteric bacteria based on their lactose/sucrose metabolization, fermentation, gas production, and H₂S generation abilities.
• The medium includes 1.0% sucrose, 1.0% lactose, and 0.1% glucose.
• Following glucose fermentation, acid produced in the butt turns it yellow, but there isn't enough to make methyl red in the slant show up.
• If either sucrose or lactose is fermented, the butt and the slant turn yellow due to sufficient fermentation products.
• Gas formed during fermentation can form bubbles or agar cracking.
• No fermentation will result in both the slant and butt remaining red.
• If it generates H₂S, forms ferrous sulfide which appears as a black precipitate in the butt

Reactions of Selected Enterobacteriaceae in TSI Agar

• Escherichia coli: Yellow slant (Y), Yellow butt (YG), commonly found in the gastrointestinal tract.
• Klebsiella pneumoniae: Yellow slant (Y), Red or Yellow-Green butt (R or YG), associated with nosocomial infections.
• Salmonella typhi: Red slant (R), Yellow butt (Y), produces H₂S (+), and causes typhoid fever.
• Salmonella typhimurium: Red slant (R), Yellow-Green butt (YG), produces H₂S (+), and causes food poisoning.
• Shigella dysenteriae: Red slant (R), Yellow butt (Y), linked to food infection and dysentery.

Antibacterial Sensitivity Testing using Kirby-Bauer Method

• A disk-diffusion method, to determine the appropriate antibiotic treatment for an infection.
• It relies on inhibiting bacterial growth measured under standard conditions.
• Mueller-Hinton agar is uniformly inoculated with the test organism, and filter paper discs with specific antibiotic concentrations are placed on the medium
• The organism grows on the agar, while the antibiotic inhibits growth.
• A zone of inhibition around the disc indicates susceptibility to the antibiotic
• The measurement is compared to the criteria by the NCCLS to classify organisms as Resistant (R), Intermediate (I), or Susceptible (S).

Antimicrobial Sensitivity Test (AST): Principle

• A standard suspension of rapidly growing bacteria is inoculated on a Muller Hinton agar plate.
• Filter paper discs containing a specific concentration of a microbial agent are pressed onto the surface.
• Incubate at 35-37°C overnight (18-24 hours).
• The zone of inhibition around each disc is measured to determine susceptibility.

AST Procedure

• Prepare Muller Hinton agar medium and immerse a sterile cotton swab in it to create bacteria suspension.
• Inoculate the surface of the sensitivity agar plate with the swab; repeat the procedure three times, turning the plate.

McFarland Standards

• McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions and standardize microbial testing, e.g. antibiotic susceptibility testing
• If a suspension is used in a test with too high or low bacteria, erroneous results can occur by indicating false resistance or false susceptibility.
• McFarland standards involve mixing specified amounts of barium chloride and sulfuric acid to form a barium sulfate precipitate.
• A 0.5 McFarland standard is prepared by mixing 0.05mL of 1% barium chloride dihydrate (BaCl₂·2H₂O) with 9.95 mL of 1% sulfuric acid (H₂SO₄).

McFarland Standards Chart

• McFarland Standard 0.5: 0.05 mL 1% Barium Chloride, 9.95 mL 1% Sulfuric Acid, 1.5 Approx. cell density (1X10^8 CFU/mL)
• McFarland Standard 1: 0.1 mL 1% Barium Chloride, 9.9 mL 1% Sulfuric Acid, 3.0 Approx. cell density (1X10^8 CFU/mL)
• McFarland Standard 2: 0.2 mL 1% Barium Chloride, 9.8 mL 1% Sulfuric Acid, 6.0 Approx. cell density (1X10^8 CFU/mL)
• McFarland Standard 3: 0.3 mL 1% Barium Chloride, 9.7 mL 1% Sulfuric Acid, 9.0 Approx. cell density (1X10^8 CFU/mL)
• McFarland Standard 4: 0.4 mL 1% Barium Chloride, 9.6 mL 1% Sulfuric Acid, 12.0 Approx. cell density (1X10^8 CFU/mL)
• The 0.5 McFarland standard is commonly used

AST: Continuation

• Arrange antimicrobial agent discs on the inoculated plate for adherence, ensuring a minimum of 20mm spacing between each disc.
• Incubate inoculated plate with disc within 15 minutes from inoculation at 35 -37°C for 18 - 24 hrs and read the results.
• The diameter of each zone should be measured and recorded in mm
• Measurements are compared to a zone-size interpretive chart to characterize each organism as Resistant (R), Intermediate (I), or Susceptible (S).

Notes on Antibiotic Sensitivity

• R-Resistant: Indicates that clinical efficacy has not been reliable in treatment studies.
• I-Intermediate: Implies clinical applicability because the drug is physiologically concentrated in the body or if a high dosage can be used.
• S-Susceptible: Implies that an infection due to an organism may be treated with the concentration of antimicrobial agent used unless otherwise contraindicated.

Zone-Size Interpretive Chart

• Lists antimicrobial agents and their corresponding disc codes
• Provides zone diameter breakpoints to determine resistance (R), intermediate susceptibility (I), and susceptibility (S).
• Examples of zone diameter (mm) breakpoints include: Amikacin (AK30): R ≤14, I 15-16, S ≥17

Factors Affecting AST

• Depth of agar
• Content of the media
• pH
• Storage of prepared media
• Inoculum size
• Disc storage
• Length of Incubation
• Temperature of incubation