Biochemical test of bacteria. Informatio
- 1. Biochemical test of bacteria By: Raju Yadav M.Sc. Medical Microbiology
- 2. Introduction • Biochemical tests are laboratory methods used to identify and differentiate microorganisms based on their unique metabolic characteristics, such as enzyme production or utilization of specific substrates. • Biochemical reactions can reveal the vital information necessary for accurately identifying the genera of various bacteria within a sample. By their nature, bacteria produce large volumes of enzymes, and it is these enzymes that allow for their identification via biochemical methods. The type of enzymes produced by a bacterium can usually be used to classify its species given that bacteria have distinct enzymatic profiles. • Each species of bacteria has specific metabolic needs and relies on different enzymes to fuel those unique needs. The presence of catalase, gelatinase, oxidase, urease, for example, can be used to identify the species of bacteria. • Biochemical tests have become common place in the fields of healthcare, where they are relied upon for assisting with disease diagnosis; epidemiology, for the tracking and tracing of disease outbreaks; pharmaceutical, for the analysis of environmental microbes that may have health implications; and forensic science, where microbial investigation can assist in the investigation of bioterrorism threats.
- 3. Continue….. • For the identification of microorganism especially bacteria, there are following types of biochemical tests. S.N Biochemical tests for gram positive bacteria 1 Mannitol Salt Agar (MSA) 2 Catalase Test 3 Blood Agar Plates (BAP) Streak-stab technique 4 Coagulase Test 5 Taxos P (Optochin sensitivity testing) 6 Taxos A (Bacitracin sensitivity testing) 7 CAMP Test 8 Bile Esculin Agar 9 Spirit Blue agar
- 4. Continue…. • Biochemical tests for gram negative bacteria: S.N Biochemical TEST for gram negative bacteria 1 Oxidase Test 2 Sugar (eg glucose) broth with Durham tubes 3 Methyl Red / Voges-Proskauer (MR/VP) 4 Kliger’s Iron Agar (KIA)/Triple sugar Iron agar (TSI) 5 Indole test 6 Citrate test 7 Urease Test 8 MacConkey agar
- 5. Continue…. • Mannitol Salt Agar (MSA): Mannitol Salt Agar (MSA) is a selective and differential growth medium commonly used in microbiology to isolate and identify bacteria, particularly Staphylococcus species. • Composition of MSA: a) Mannitol: A sugar alcohol that serves as a fermentable carbohydrate source. b) Salt (NaCl): High salt concentration (~7.5%) inhibits the growth of most bacteria, making the medium selective for salt-tolerant organisms (e.g., Staphylococcus species). c) Agar: Provides a solid medium for bacterial growth. d) Phenol Red: A pH indicator that changes color based on the acidity or alkalinity of the medium: • Yellow: Indicates acid production (mannitol fermentation). • Red: Neutral pH (no fermentation of mannitol). • Pink: Alkaline pH.
- 6. Continue…. e) Selective Medium: The high salt concentration selectively allows for the growth of halotolerant organisms, primarily Staphylococcus species. f) Differential Medium: Differentiates bacteria based on their ability to ferment mannitol: • Mannitol-fermenting bacteria (e.g., Staphylococcus aureus) produce acid, turning the medium yellow around colonies. • Non-mannitol-fermenting bacteria (e.g., Staphylococcus epidermidis) do not produce acid, so the medium remains red or pink. g) Uses: • Isolation of Staphylococcus species: MSA is commonly used to distinguish Staphylococcus aureus from other Staphylococcus species. • Clinical microbiology: Often used to identify pathogens in clinical samples like wound swabs or nasal swabs. • Environmental studies: Useful for studying halotolerant organisms in high-salt environments.
- 7. Continue.. • Table showing the Staphylococcus species fermenting mannitol: Test S. aureus S. epidermidis S. saprophyticus Mannitol* + - +
- 8. Continue…. • Catalase Test: The catalase test tests for the presence of catalase, an enzyme that breaks down the harmful substance hydrogen peroxide into water and oxygen. If an organism can produce catalase, it will produce bubbles of oxygen when hydrogen peroxide is added to it. • The catalase test helps differentiate between bacterial species based on their ability to produce catalase. It is commonly used to distinguish between Staphylococci (catalase-positive) and Streptococci (catalase-negative). • Principle: • When a catalase-positive bacterium is exposed to hydrogen peroxide, the enzyme catalase breaks it down into water and oxygen. The release of oxygen forms visible bubbles. • Reaction: • 2H2O2→2H2O+O2↑
- 9. Continue…. • Procedure: A. Materials Needed: • Hydrogen peroxide solution (usually 3%) • Glass slide or petri dish • Sterile wooden stick or loop • Bacterial culture (fresh or 24-hour-old) B. Steps: • Place a drop of hydrogen peroxide on a clean glass slide. • Using a sterile loop or stick, transfer a small amount of bacterial colony to the hydrogen peroxide. • Observe for immediate bubble formation.
- 10. Continue… • Interpretation of Results: C. Positive Test: • Rapid and vigorous bubbling (oxygen release). • Indicates the presence of catalase. • Example: Staphylococcus species, Bacillus species. D. Negative Test: • No bubbles or very few bubbles. • Indicates the absence of catalase. • Example: Streptococcus species, Enterococcus species.
- 11. Continue…. • Oxidase Test: The oxidase test is a biochemical reaction that assays for the presence of cytochrome oxidase, an enzyme sometimes called indophenol oxidase. In the presence of an organism that contains the cytochrome oxidase enzyme, the reduced colorless reagent becomes an oxidized colored product. • The final stage of bacterial respiration involves a series of membrane embedded components collectively known as the electron transport chain. The final step in the chain may involve the use of the enzyme cytochrome oxidase, which catalyzes the oxidation of cytochrome c while reducing oxygen to form water. • The oxidase test often uses a reagent, tetra-methyl-p-phenylenediamine dihydrochloride, as an artificial electron donor for cytochrome c. When the reagent is oxidized by cytochrome c, it changes from colorless to a dark blue or purple compound, indophenol blue.
- 12. Continue… • Reagent required: A. Kovács oxidase reagent 1% tetra-methyl-p-phenylenediamine dihydrochloride, in water Store refrigerated in a dark bottle no longer than 1 week. B. Gordon and McLeod reagent. C. Gaby and Hadley oxidase test • 1% α-naphthol in 95% ethanol • 1% p-aminodimethylaniline oxalate. Store refrigerated in dark bottles no longer than 1 week. • Oxidase test may performed by different methods such as Filter Paper Test Method, Filter Paper Spot Method, Direct Plate Method, and Test Tube Method.
- 13. Continue…. Procedure: • Filter Paper Test Method: 1. Soak a small piece of filter paper in 1% Kovács oxidase reagent and let dry. 2. Use a loop and pick a well-isolated colony from a fresh (18- to 24hour culture) bacterial plate and rub onto treated filter paper. Observe for color changes. 3. Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds or sometimes it may give purple color within 60 to 90 seconds (delayed positive). Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes.
- 14. Continue… • Direct Plate Method:- 1. Grow a fresh culture (18 to 24 hours) of bacteria on nutrient agar using the streak plate method so that well-isolated colonies are present. 2. Place 1 or 2 drops of 1% Kovács oxidase reagent or 1% Gordon and McLeod reagent on the organisms. Do not invert or flood plate. Observe for color changes. 3. When using Kovács oxidase reagent, microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds or sometimes it may give purple color within 60 to 90 seconds (delayed positive). Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes. 4. When using Gordon and McLeod reagent, microorganisms are oxidase positive when the color changes to red within 10 to 30 minutes or to black within 60 minutes. Microorganisms are oxidase negative if the color does not change.
- 15. Continue….. • Test Tube Method:- 1. Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth. 2. Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% paminodimethylaniline oxalate (Gaby and Hadley reagents). Observe for color changes. 3. Microorganisms are oxidase positive when the color changes to blue within 15 to 30 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 2 to 3 minutes. Microorganisms are oxidase negative if the color does not change.
- 16. Note….. • The reagents used in the oxidase test have been shown to autooxidize, so it is very important to use fresh reagents, no older than 1 week. Steel found that the autooxidation can be slowed by the addition of 1% ascorbic acid. • Nickel, steel, and other wire loops may give false-positive results, so it is important to use only platinum or inert transfer loops, such as sterile wood sticks commonly used in teaching laboratories. Other acceptable examples include sterile plastic loops, sterile toothpicks, and sterile swabs. • Bacteria grown on media containing dyes may give aberrant results. The test reagents will effectively kill the microorganisms, so sub culturing should be done prior to adding any reagent to an active culture. • Older cultures are less metabolically active and are thus unreliable for this test in a clinical setting. In the classroom, if by necessity older cultures must be used, expect longer reaction times.
- 17. Coagulase test: • A coagulase test is a biochemical test that detects the presence of coagulase, an enzyme that causes blood plasma to clot. It’s used to identify Staphylococcus aureus (S. aureus) and used to differentiate coagulase-positive staphylococci with coagulase negative Staphylococci species. • Most strains of S.aureus produce two types of coagulase, free coagulase and bound coagulase. While free coagulase is an enzyme that is secreted extracellulary while bound coagulase is a cell wall associated protein. The bound coagulase is also known as clumping factor. • It cross-links α and β chain of fibrinogen in plasma to form fibrin clot that deposits on the cell wall. As a result, individual coccus stick to each other and clumping is observed. • The free coagulase secreted by S. aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma. Free coagulase is heat labile while bound coagulase is heat stable. • The coagulase test can be performed using two different procedures. The slide test is simple, giving results within 10 seconds, but it can give false negatives.
- 18. Continue… • The tube test is the definitive test, however, it can take up to 24 hours to complete. For both tests, clumping or clots of any size indicate a positive response. Both forms of the coagulase test require fresh bacterial cultures (24 hours or younger), grown in non-inhibitory media. Acceptable non inhibitory media include blood agar, trypticase soy agar, and chocolate agar. • Phenylethylalcohol and mannitol salt agars are considered inhibitory media. Agar media with antibiotics are also considered inhibitory. Coagulase plasma is used for this test which is obtained by treating rabbit blood (or some time human blood can also be used) with lyophilized citrated or EDTA and rehydrated with sterile distilled water immediately prior to use. • Procedure: The procedure for both slide and tube tests are mention below a) Slide test: The slide test is done to detect the bound coagulase. Dense suspensions of Staphylococci from culture are made on two ends of clean glass slide. One should be labeled as “test” and the other as “control”. The control suspension serves to rule out false positivity due to auto agglutination. The test suspension is treated with a drop of coagulase plasma and mixed well. Agglutination or clumping of cocci within 5-10 seconds is taken as positive. Some strains of S.aureus may not produce bound coagulase, and such strains must be identified by tube coagulase test.
- 19. Continue…. b) Tube test: The tube test is done to detect free coagulase. Three test tubes are taken and labeled “test”, “negative control” and “positive control”. Each tube is filled with 0.5 ml of 1 in 10 diluted rabbit plasma. To the tube labeled test, 0.1 ml of overnight broth culture of test bacteria is added. To the tube labeled positive control, 0.1 ml of overnight broth culture of known S. aureus is added and to the tube labeled negative control, 0.1 ml of sterile broth is added. All the tubes are incubated at 37oC and observed up to four hours. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube. If the test remains negative until four hours at 37oC, the tube is kept at room temperature for overnight incubation.
- 20. Taxos P (Optochin sensitivity testing) • Taxos P is a biochemical test that determines whether an organism is sensitive to the antibiotic optochin. This test is used to differentiate between Streptococcus pneumoniae, which is sensitive to optochin, and other alpha-hemolytic streptococci, which are resistant. • The enzyme responsible for the optochin sensitivity of Streptococcus pneumoniae is H+-ATPase (proton pump). Optochin (ethylhydrocupreine hydrochloride) disrupts the activity of H+-ATPase, an essential enzyme found in the cell membrane of S. pneumoniae. • H+-ATPase plays a critical role in maintaining proton gradients and energy metabolism within the bacterial cell. Optochin inhibits this enzyme, leading to the collapse of the proton motive force, impaired ATP production, and ultimately cell death. • This sensitivity is unique to S. pneumoniae, as other alpha-hemolytic streptococci, such as the Viridans group, have variations in their membrane structures or enzyme activities that confer resistance to optochin.
- 21. Methodology • Culture Medium: Blood agar is commonly used. • Inoculation: A colony of the suspected alpha-hemolytic streptococci is streaked on the agar plate. • Optochin Disc Placement: A Taxos P disc (impregnated with optochin) is placed on the inoculated agar. • Incubation: The plate is incubated at 35-37°C in a 5-10% CO₂-enriched environment for 18-24 hours. Interpretation of Results: • Sensitive: A clear zone of inhibition (≥14 mm with a 6-mm disc or ≥16 mm with a 10-mm disc) forms around the Taxos P disc. This indicates S. pneumoniae. • Resistant: No significant zone of inhibition indicates non-pneumococcal streptococci (such as the Viridans group).
- 22. Limitations • The test must be performed on isolated alpha-hemolytic colonies. • False positives or negatives can occur if the test is not conducted under appropriate conditions (e.g., improper CO₂ levels). • Other confirmatory tests, such as bile solubility, may be needed to definitively identify S. pneumoniae.
- 23. Taxos A (Bacitracin sensitivity testing) • Taxos A (bacitracin sensitivity testing) is a differential test that determines if an organism is sensitive to the antibiotic bacitracin. It’s used in clinical laboratories to identify and differentiate between different types of bacteria. • Bacitracin is a peptide antibiotic that disrupts cell membranes and inhibits cell wall synthesis. It’s produced by the bacteria Bacillus subtilis, Bacillus licheniformis • The bacitracin susceptibility test is used to distinguish Group A streptococci, from other streptococci. This test is used to determine the effect of small amount of bacitracin (0.04U) on an organism. Sterptococcus pyogenes is inhibited by the small amount of bacitracin in the disk; other beta-hemolytic streptococci usually or not. Procedure: • With a sterile inoculating loop, select 2-3 well isolated colonies of suspected beta-hemolytic organism from an 18-24 hours old culture. • Streak the colonies onto a blood agar plate.
- 24. Continue… • Using heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Tap the disk with forceps to ensure adequate adherence with the agar surface. • Incubate the plate in ambient air at 35°C-37°C for 18-24 hours. • After incubation, observe the zone of inhibition around the bacitracin disk.
- 25. CAMP Test (Christie-Atkins-Munch-Petersen test) • It is a biochemical test that identifies the presence of group B streptococci (Streptococcus agalactiae) and Listeria monocytogenes. The test is based on the production of a extracellular diffusible protein called CAMP factor, which enhances the hemolysis produced by Staphylococcus aureus (beta-lysin producing strain, NCTC 1803 or ATCC 25923). • The synergistic reaction results in an enhanced and very visible zone of hemolysis in the region between the two cultures. The synergistic zone is not observed in group A, C, and G Streptococcus • This test is performed by streaking a known beta-lytic Staphylococcus aureus across a 10% blood agar plate (usually sheep blood) and then inoculating the test organism at right angles to it. The test organism must not touch the staphylococcal inoculum. • An Enterococcus species is also inoculated as a negative control. The test organism is presumed to be S.agalactiae. After overnight incubation at 35-370 C, there is an arrow-head shaped area of hemolysis where the staphylococcal organism meets the test organism.