Biochemical Tests PDF

Nitrite Reduction Test Other Biochemical Tests Motility Test...

Nitrite Reduction Test Other Biochemical Tests Motility Test To determine the ability of an organism to reduce nitrites to gaseous nitrogen or to other compounds Thursday, 09 January 2025 05:27 containing nitrogen. Determines the motility of bacterium and differentiates between motile and non motile bacteria. Urease Test Principle of Nitrite Reduction Test Detects the alkaline fermentation of urine (urea) with the resultant production of ammonia by Principle of Motility Test microorganisms. > Nitrate reduction by bacteria is mediated by nitrate Media Used in Motility reductase and indicates that the organism can use Flagellum has a very rigid, helical structure, and actual motility results Test NO3– as an electron acceptor during anaerobic from the rotation of the flagellum in a manner similar to that of a boat propeller. SIM Medium respiration and reduce nitrate to nitrite. The fermentation of urea occurs in the presence of the enzyme > Nitrite, on the other hand, may be reduced to a variety ‘urease’, resulting in two molecules of ammonia and carbon Motility by the bacterium is demonstrated in a semi-solid agar medium. Composition per liter: of nitrogen products including NO, N2O, N2, and NH3, dioxide. The medium mainly used for this purpose is SIM medium (Sulphide depending on the enzyme system of the organism and the Indole Motility medium) which is a combination differential medium During the test, the organisms utilize urea as the sole Urease activity is one of the important characteristics for the identification Pancreatic digest of casein 20 g atmosphere in which it is growing. of Proteus species and allows for Proteus to be distinguished from non- that tests three different parameters, Sulfur Reduction, Indole Peptic digest of animal tissue 6.1g source of nitrogen, producing a sufficient amount of lactose-fermenting members of the Enterobacteriaceae. Production, and Motility. Agar 3.5g > Microorganisms capable of reducing nitrite to ammonia to overcome the buffering capacity of the medium. Fe(NH4)2(SO4)2·6H2O 0.2 g This media has a very soft consistency that allows motile bacteria to Na2S2O3·5H2O 0.2g nitrogen do not turn color but the production migrate readily through them causing cloudiness. of gas is visible in the nitrite reduction test. The inoculum is stabbed into the center of a semisolid agar deep using pH 7.3 ± 0.2 at 25°C > N2 gas is usually visible in the Durham tube. Media Used Microorganisms Tested a sterile inoculating needle. > In the absence of gas, the product is assumed to be Some organisms grow throughout the entire medium, whereas others > Brain-heart infusion broth 2 g other than N2 gas. The change in color of the medium as a result of the change in show small areas or nodules that grow out from the line of inoculation. > Pancreatic digest of casein 10 g Directly, this test is performed > The reduction of nitrite is determined by adding pH is indicative of the test result. The non-motile bacteria will only grow in the soft agar tube and only > Peptic digest of animal tissue 5 g as a rapid test on gastric biopsy sulfanilic acid and alpha-naphthylamine. the area where they are inoculated. > Yeast extract 3 g samples to detect the presence > After the addition of the reagents, no red coloration in > NaCl 5 g of H. pylori. the test tube and the presence of gas in the Durham > NaNO2 0.1 g tube indicates a positive test indicating nitrite reduction to nitrogen gas. Procedure Result Interpretation of Motility Test > The test does not require the addition of zinc dust. per 1000 mL Principle of Urease Test pH 6.9 Organisms will spread out into the medium from the site of With a sterile straight needle, touch a colony of a inoculation, diffuse zone young (18 to 24 hour) culture growing on agar Non-motile: organisms remain at the site of inoculation ► Urea medium, whether a broth or agar, contains medium. urea and the phenol red as a pH indicator. Single stab down the center of the tube to about ► Many organisms, especially those that cause half the depth of the medium. Nitrite reduction medium urinary tract infections, produce the urease Incubate at 35°-37°C and examine daily for up to 7 enzyme. days. ► The ammonia combines with the carbon dioxide Beef extract 3.0g and water to form ammonium carbonate, which A positive test is demonstrated by an intense magenta to Gelatin peptone 5.0g turns the medium alkaline, turning the indicator bright pink color in 15 min to 24 hours. Quality Control of Motility Test Potassium nitrite (KNO 2) 1.0g from its original orange-yellow color to bright A negative test shows no color change. Deionized water 1000ml pink. Positive: Escherichia coli (ATCC25922) Negative: Staphylococcus aureus (ATCC25923) Media Used Urease Procedure Both urea agar slants and broth media can be used A loopful of a well-isolated colony is taken with an for the detection of urease production. inoculating loop and inoculated on the agar slants. Procedure of Nitrite Reduction Test The composition of the urea agar base is given below: Results of Nitrite Reduction Test S.N Ingredients Gram/liter The inoculation should be done on just the  Inoculate nitrite broth with organism from a 24-hour broth culture. 1. Dextrose 1.0 slant, and the butt shouldn’t be stabbed.  Incubate for 48 hours at 35°-37°C.  Examine 48-hour nitrite broth cultures for nitrogen gas in the inverted Durham 2. Peptic digest of animal tissue 1.5 The tubes are then incubated with loosened caps at 35 tube and add 5 drops each of the nitrate reagents A and B to 3. Sodium chloride 5.0 to 37°C. determine whether nitrite is still present in the medium. 4. Monopotassium phosphate 2.0 For non-fermenters, the tubes are incubated at 30°C. 5. Phenol red 0.012 6. Agar 15.0 The tubes are observed for the development of pink color for as long as 7 days. Limitations of Nitrite Reduction Test If no growth is seen on the slant, further inoculation Final pH at 25°C: 6.8 ±0.2 with heavy inoculum should be done. If the broth does not become red and no gas production is observed, zinc dust is added to determine if the nitrite has not been oxidized to nitrate (thus invalidating the test). Control organisms Limitations of Urease Test If oxidation has occurred, the mixture turns red after the addition of zinc. Positive test: Proteus mirabilis. When performing overnight tests from a medium that contains peptone, Negative test: Escherichia coli. the alkaline reaction may be due not to urease but to hydrolysis of peptone. Urea is light sensitive and might undergo autohydrolysis. The medium Quality Control thus must be stored at 2 to 8°C in the dark. Positive test: No color change to red 2 minutes after the addition of the reagents; gas production in the Positive: Proteus mirabilis (ATCC12453): colorless, gas production The test is less sensitive if the medium is not buffered. Durham tube. Negative: Acinetobacter baumannii (ATCC19606): red coloration, no gas production Negative test: The broth becomes red after the addition of the reagents; no gas production. Principle of Catalase Test Lysine Iron Agar Test Catalase Test The presence of the catalase enzyme can be demonstrated by The metabolic activity of aerobic and facultative To differentiate gram-negative bacilli based on decarboxylation or deamination of lysine and the adding hydrogen peroxide to the bacterial inoculum, which anaerobic microorganisms produce toxic by- results in the rapid liberation of oxygen bubbles. The lack of formation of hydrogen sulfide (H2S). Biochemical test for aerobic organisms that detects the production of catalase enzyme in the organism. products like hydrogen peroxide and superoxide enzyme is demonstrated by the absence of such bubbles. radical (O2—). These products are toxic to the organisms and might even result in cell lysis if not broken down. Principle of Lysine Iron Agar Test Lysine decarboxylation is Lysine deamination is Catalase enzyme is a common enzyme that is found in all In the case of pathogenic organisms, different an anaerobic process that occurs in an aerobic process that occurs living beings that survive in oxygen and catalyzes the mechanisms are found that break down these the butt of the media. on the slant of the media. decomposition of hydrogen peroxide, releasing water and products to non-toxic substances.  The medium has an aerobic slant and an oxygen. The production of catalase thus protects the anaerobic butt. Catalase is an essential enzyme in pathogenic organisms organism against the lethal effect of hydrogen Cadaverine neutralizes the The enzyme neutralizes the bactericidal  The indicator in LIA is bromcresol purple. organic acids formed by If the decarboxylase is not produced, as it protects the organism from oxidative damage from effects of hydrogen peroxide, and its peroxide accumulated at the end of the aerobic the butt remains acidic (yellow).  Bromocresol purple, the pH indicator, is yellow glucose fermentation, and the reactive oxygen species. concentration in bacteria has been metabolism. the butt of the medium correlated with the pathogenicity of the at or below pH 5.2 and purple at or above pH 6.8. reverts to the alkaline state organism.  When glucose is fermented, the butt of the (purple). Under the aerobic condition, 3% H2O2 is used, whereas Procedure of Catalase Test 15% H2O2 is used under anaerobic conditions. medium becomes acidic (yellow). Reagents and Supplies Used  If the organism produces lysine decarboxylase, There are more than one method or procedure variations for the cadaverine is formed. Media Used for Lysine Iron Agar Test Hydrogen peroxide reagent catalase test.  Organisms that deaminate lysine, form L-Lysine Hydrochloride 10.0 gm 30% H2O2 for Neisseria alpha – ketocarboxylic acid, which 15% H2O2 for anaerobes  These methods include the slide or drop catalase test, the Peptone 5.0 gm reacts with iron salt near the surface 3% H2O2 for other bacteria tube method, the heat-stable catalase used for the Yeast Extract 3.0 gm (purchase or dilute 30% 1:10 differentiation of Mycobacterium species, the of the medium under the influence of in deionized water prior to semiquantitative catalase for the identification oxygen to form a reddish-brown Dextrose 1.0 gm use) of Mycobacterium tuberculosis, and the capillary tube and compound. Ferric Ammonium Citrate 0.5 gm coverslip method.  If deamination does not occur, the LIA Sodium Thiosulfate 0.04 gm slant remains purple. Bromcresol Purple 0.02 gm The most popular method of catalase test in clinical bacteriology is the slide or drop catalase method as it requires a small number Agar 15.0 gm of organisms and works on a relatively uncomplicated technique. Final pH 6.7 +/- 0.2 at 25ºC. Procedure of Lysine Iron Agar Test Sodium thiosulfate is incorporated into this medium as the source of hydrogen sulfide and acts as the substrate for enzymatic reduction and the resultant colorless hydrogen sulfide Limitations of Catalase Test 1. With a straight inoculating needle, gas reacts with ferric ammonium citrate to produce ferrous sulfide, an insoluble black precipitate that blackens the medium. inoculate LIA by stabbing through the The test should not be tested from Mueller-Hinton agar. center of the medium to the bottom of the Collecting colonies with metal bacteriological loop materials might yield false-positive tube and then streaking the slant while results; however, platinum loops do not yield false-positive results. Result Interpretation of Lysine Iron Agar Test Because the enzyme is present in viable cells only, colonies that are older than 24 withdrawing needle. hours should not be used. Older cultures may give false-negative results. 2. Incubate tubes aerobically with loose caps Reversing the order of adding the reagent to the colony might result in false-negative at 35-37ºC for 18-48 hours and examine results. The reagent and the colony should not be mixed. reaction of medium. Some strains of S. aureus may appear catalase-negative by drop method so the test should be repeated with the tube method. 30% H2O2 is extremely caustic to the skin. If contact occurs, Limitations wash immediately with 70% ethyl alcohol, not water. Caps must be loosened during this test or erroneous results will occur. Quality Control LIA is not as sensitive in detecting hydrogen sulfide in comparison to other iron containing Staphylococcus aureus: Catalase positive. mediums, such as Sulfide Indole Motility (SIM) Lysine Decarboxylation (detected in Lysine Deamination (detected on a slant): Streptococcus pyogenes: Catalase-negative. Medium and TSIA. H2S production may not be the butt): seen with organisms that do not produce lysine Positive Test: Red slant Bacillus is catalase-positive, and Clostridium spp. are catalase-negative. decarboxylase, such as Proteus spp., since acid in Positive Test: Purple slant/purple Negative Test: Slant remains purple Neisseria gonorrhoeae produces an enhanced elaboration of bubbles butt (alkaline), the butt reaction may the butt may suppress H2S formation. not seen with other members of the genus due to superoxol. be masked by H2S production Certain species or strains may give delayed Negative Test: Purple slant/yellow H2S Production: reactions or completely fail to ferment the butt (acid), fermentation of glucose carbohydrate in the stated manner. However, in only Positive Test: Black precipitate Negative Test: No black color most cases if the organism fails to ferment development dextrose within 48 hours, and growth is definitely Gas production: demonstrated by the present, the organism is most likely not in the presence of bubbles or cracks in the Enterobacteriaceae family. medium Coagulase Test Slide Test Coagulase Positive organisms:Staphylococcus aureus and A positive test is the demonstration of the agglutination other animal host bacteria like S. pseudintermedius, S. Differentiates the species of the genus Staphylococci into two groups; Coagulase positive Staphylococci and Coagulase Negative of the bacterial cells after the plasma is added. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae, S. Staphylococci. A negative test is demonstrated by the lack of hyicus agglutination. Coagulase Negative organisms:Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc. Principle of Coagulase Test Test Tube A positive test meets one of the following criteria: ▫ The coagulase enzyme acts as a virulence factor in some organisms as it interacts with the fibrinogen present on the host’s cell surface. 1. Complete clot formation or any degree of clot formation ▫ Coagulase is of two types; free coagulase and bound coagulase, each of which is detected by different methods. before 24 hours. Limitations of Coagulase Test Study Guide Page 1  Staphylococci. A negative test is demonstrated by the lack of hyicus agglutination. Coagulase Negative organisms:Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc. Principle of Coagulase Test Test Tube A positive test meets one of the following criteria: ▫ The coagulase enzyme acts as a virulence factor in some organisms as it interacts with the fibrinogen present on the host’s cell surface. 1. Complete clot formation or any degree of clot formation ▫ Coagulase is of two types; free coagulase and bound coagulase, each of which is detected by different methods. before 24 hours. Limitations of Coagulase Test ▫ The bound coagulase is called the clumping factor and is detected rapidly by a slide test. 2. No clot formation after the addition of 1 or 2 drops of ▫ The free coagulase, in turn, is detected in the test tube as a result of the formation of a clot. 5% CaCl2 to a tube without a clot at 24 hours. Coagulase testing cannot be performed from A negative test meets one of the following criteria: growth on mannitol salt agar. 3. A lack of clot formation at 24 h at 25°C. Coagulase is an enzymatic protein that is a thermostable thrombin-like substance, which converts 4. No clot after 24 hours at 35°C, but after the addition of fibrinogen into fibrin resulting in clotting or clumping. 1 or 2 drops of 5% CaCl2 to the tube, a clot forms. In S. aureus, two different forms of coagulase are found; free coagulase and bound coagulase. Result Interpretation of Coagulase Test Free coagulase Bound coagulase Free coagulase is different from bound The clumping factor, termed bound coagulase, can be detected rapidly in the slide coagulase in that the clotting mechanism of test, but this test requires several colonies and lacks sensitivity. free coagulase requires the activation of a S. aureus produces another substance in its cell wall, protein A, which binds to Reagent Supplies plasma coagulase-reacting factor (CRF), which the FC moiety of human immunoglobulin G (IgG) and thus acts as a coagulase- is a modified or derived thrombin molecule, to Frozen plasma (preferably rabbit Loops or sterile sticks reacting factor. form a coagulase-CRF complex. plasma) with EDTA. Glass or plastic tubes When latex or erythrocyte particles are coated with IgG and with human The plasma added to the tube is preferably Human plasma is commonly not used Glass slides fibrinogen, a staphylococcus will agglutinate if either clumping factor or protein A rabbit plasma which acts as a binding factor. is present in the bacterial cell wall. for the test, as it is less sensitive and The complex then reacts with fibrinogen to The presence of the clumping factor is demonstrated by the ability of the potentially infectious with human Lower A- Slide coagulase test for clumping factor. The left side is positive; the right side is negative. form the fibrin clot in a test tube. organism to act directly on the fibrinogen in the plasma to clump it in a slide assay. pathogenic viruses. The test for the clumping factor is rapid but requires several colonies, and also 5% CaCl2 (optional) Lower B- Tube coagulase test for free coagulase. the factor might not be present in all S. aureus organisms. The tube on the left is positive, exhibiting a clot. The tube on the right is negative. Procedure of Coagulase Test Above: Negative coagulase test in Staphylococcus epidermidis (left) and positive coagulase tests in Staphylococcus aureus (center and right). Slide Test Tube Test About 10 µl of deionized water or The plasma is diluted with physiological saline. (Add 0.2 ml physiological saline is added to a slide. plasma in 1.8 ml saline) TSIA Test Several colonies from a fresh culture are 5 ml of the diluted plasma is then added to a test tube. About collected with an inoculating loop and are 5 drops of the test organism culture are added to the test emulsified into the water to obtain a tube. smooth milk-colored suspension. The test tube is mixed and incubated at 37°C for an hour. Biochemical test used to differentiate bacteria based on their ability to ferment these three sugars and release acid and hydrogen A drop of a rabbit or human plasma is The tube is finally observed for clot formation. sulfide gas. added to the slide, and the clumping is If no clotting is observed, the tube should be examined at 30 observed immediately, not to exceed 10 minutes intervals of up to 6 hours. seconds. TSIA test is similar to Kligler’s Iron Agar (KIA) test except that the TSIA medium contains ‘1% sucrose’ as extra sugar in addition to glucose and lactose in the KIA medium.  Differentiates Gram-negative enteric bacilli.  Tests the bacteria’s ability to utilize glucose, lactose, and/or sucrose and produce H2S gas.  Differentiates lactose fermenters from non-lactose fermenters. Culture Media Oxidase Test Peptone – 20.00 grams  Differentiation of Enterobacteriaceae from other Principle of TSIA Test HM Extract (Meat Extract) – 3.00 grams Used to assay the ability of bacteria to synthesize cytochrome c oxidase enzymes. Gram-negative bacilli. Yeast Extract – 3.00 grams  Differentiation of Pseudomonas Dextrose (Glucose) – 1.00 grams aeruginosa from Enterobacteriaceae. Lactose – 10.00 grams Principle of Oxidase Test  TSIA test is based on the distinct metabolic pattern of the different bacterial genera to metabolize glucose, lactose, sucrose, Sucrose – 10.00 grams and sodium thiosulfate (a sulfur compound). Sodium Chloride – 5.00 grams  Fermentation of these sugars will produce acid decreasing the pH of the medium and turning it to red Ferric Citrate – 0.300 grams → Cytochrome c oxidase is a large transmembrane protein acting as the terminal enzyme in the electron transport chain of aerobic bacterial and color. Similarly, the metabolism of sodium thiosulfate is indicated by turning the medium black. Sodium Thiosulfate – 0.300 grams mitochondrial respiration system that catalyzes the final electron transfer from cytochrome c to oxygen molecule.  Glucose will initially be fermented by bacteria capable of doing so, producing metabolic acids. This resulting acid will lower the Phenol Red – 0.024 grams → Some bacteria contain this enzyme and have the ability to transfer a terminal electron to molecular oxygen; however, some bacteria lack this pH of the medium, turning the slant and butt regions yellow. But because there isn’t much glucose left, it will quickly run out. Agar – 12.00 grams enzyme and fail to transfer a terminal electron to molecular oxygen or may use different cytochrome to do the job.  If the bacteria are lactose and or sucrose non-fermenter, they won’t be able to use lactose and sucrose as a carbon source. → In the presence of molecular oxygen, the cytochrome c oxidase enzyme of bacteria oxidizes phenylenediamine in the Therefore, once the glucose is exhausted, the oxidative metabolism of peptone in the slant will be triggered. This oxidative Final pH – 7.4±0.2 at 25°C colorless reagent to a deep purple to blue-colored compound, indophenol blue. Hence, if the bacteria possess the metabolism will increase the pH of the medium to an alkaline level and turns the yellow slant to red. However, because cytochrome c oxidase, there will be the development of deep purple/blue color, but if the bacteria lack that enzyme, there won’t be any to very little oxygen in the butt, peptone won’t undergo oxidative degradation there, there won’t be any color change. leaving the butt’s color unchanged. Thus the bacteria that do not ferment lactose and/or sucrose but ferment glucose Procedure of TSIA Test will develop a red slant and yellow butt (written as Red/Yellow or K/A).  In contrast, if the bacteria are lactose and/or sucrose fermenters, they will begin to ferment these sugars and release acid. The 1. Touch a well-isolated colony from a fresh culture of the test Composition of Nutrient Broth per 1000 mL released acid will drop the pH of the medium and the indicator will turn the medium yellow in both the slant and butt area. bacterium that is 18 to 24 hours old using a sterile inoculating Culture Media  As there is abundant carbohydrate (sucrose and lactose), the oxidative metabolism of peptone won’t be triggered. Thus wire. Peptone- 5.00 grams bacteria that ferment the lactose and/or sucrose (or all three sugars) will develop a yellow slant and 2. Stab the butt up to 3 to 5 mm above the base of the test tube While performing the oxidase test following either the disc HM Peptone B (Beef Extract)- 1.50 grams yellow butt (written as Yellow/Yellow or A/A). using the inoculating wire and while withdrawing, streak the method or filter paper method or swab method or direct plate Yeast Extract- 1.50 grams  If the bacteria are not able to ferment any of the sugars, the medium will remain red (written as Red/Red or K/K). slant. method, there is no need for culture media. Sodium Chloride- 5.00 grams  Bacteria that produce H2S can break down the sodium thiosulfate found in culture media and cause it to decrease, generating 3. Incubate the tube aerobically (with a loose cap) at 35±22°C for  Bacteria grown in any selective medium (or pure colonies H2S gas. about 24 hours. from any media) can be used for the test. Final pH 7.4 ±0.2 at 25°C  The resulting H2S gas subsequently interacts with the ferric ions to produce water-insoluble ferrous sulfide, which is black in 4. Examine for color change of the slant and butt and report the  However, for the tube method (Gaby and Hadley method) color. The presence of H2S is indicated by this insoluble black-colored molecule turning the culture media black. color within 24 hours of incubation. (If you want to read the H2S of the oxidase test, nutrient broth medium (or any  The creation of gas bubbles above the medium or breaking or displacement of the medium are signs that gas is being produced production, incubate it for another 24 to 48 hours, but read standard broth medium with low glucose content) is Quality Control during the fermentation of sugar. sugar fermentation and color change within the first 24 hours of required. (Here, we will use nutrient broth.) Positive control: Pseudomonas aeruginosa ATCC 2783 rapidly inoculation and incubation.) produce deep blue or purple color within 10 to 30 seconds. Negative Control: E. coli ATCC 25922 doesn’t result in the formation of deep blue or purple color within 60 seconds. Reagents Result and Interpretation of TSIA Test Kovacs’ Oxidase Reagent (for the disc, filter paper, Gordon and McLeod Oxidase Reagent (for the disc, filter Gaby-Hadley Reagents (for tube method) swab, or direct plate method) paper, swab, or direct plate method) Reagent A (1% α-naphthol) 1. The red slant and yellow butt (Red/Yellow or Alkaline (K)/Acidic (A)) indicate only glucose is 1% N, N, N, N-tetramethyl-p-phenylenediamine 1% dimethyl-p-phenylenediamine dihydrochloride Add 1.0 grams of α-naphthol in 100 mL of 98% ethanol. fermented. dihydrochloride 2. Yellow slant and yellow butt (Yellow/Yellow or Acidic (A)/Acidic (A)) indicate lactose and/or Preparation of Gordon and McLeod Reagent Reagent B (1% p-amino dimethylaniline oxalate) sucrose fermentation or fermentation of all three sugars. Add 1.0 grams of p-amino dimethylaniline oxalate in 100 Preparation of Kovacs’ oxidase reagent: Dissolve 1.0 grams of dimethyl-p-phenylenediamine mL of distilled water. 3. Red slant and red butt (Red/Red or Alkaline (K)/Alkaline (K)) indicate none of the three Dissolve 1.0 grams of this reagent in 100 mL of sterile dihydrochloride in 100 mL of sterile distilled water and mix distilled water and mix well. well. sugars are fermented. 4. Blackening of the media or formation of the black-colored spots indicates H2S production. 5. Cracking of the media, gas bubbles of the media, or forming a gap in the media indicates gas Procedure of Oxidase Test production. Test Bacteria (Sample bacteria (well- isolated colonies)) Kovacs’ oxidase reagent has higher sensitivity than Gordon and McLeod reagent and comparatively gives faster Positive control: Pseudomonas aeruginosa ATCC 2783 and clearer results. Negative control: E. coli ATCC 25922 Therefore, Kovacs’ oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) is mainly used for the oxidase test. TSIA Test Results of Some Common Enteric Pathogens In this procedure, we use Kovacs’ oxidase reagent, but if you have Gordon-McLeod oxidase reagent, you can Filter Paper Method use it.) Name of Bacteria Color of Slant/Butt (pH of Slant/Butt) H2S Gas Production Production 1. In a sterile petri plate, place a strip/disc of Whatman no. 1 filter paper. Soak the filter paper E. coli Yellow/Yellow (Acidic/Acidic) – ve + ve with 1% Kovacs’ oxidase reagent and let it dry. Direct Plate Method Impregnated Disc/Strip (Oxidase Disc) K. pneumoniae Yellow/Yellow (Acidic/Acidic) – ve + ve 2. Using a sterile inoculating loop, pick up a well- Method isolated colony of test bacteria from a fresh (18 to 24 hours old) culture and make a smear on the 1. Over well-isolated (pure culture) colonies of test bacteria 1. Place the impregnated oxidase disc or strip over a clean petri K. oxytoca Yellow/Yellow (Acidic/Acidic) – ve + ve reagent-soaked filter paper piece. from fresh culture, add a few drops of Kovacs’ oxidase plate (or glass slide) and moisten it with sterile deionized water. 3. Observe for color change and note the time reagent. Shigella spp. Red/Yellow (Alkaline/Acidic) – ve – ve 2. Tilt the plate and shake it gently so that the colonies get (Some discs may not need to be moistened. Look for the required for change in color for up to 60 manufacturer’s instructions.) Serratia marcescens Red/Yellow (Alkaline/Acidic) – ve Variable seconds. exposed to oxygen. 2. Using a sterile inoculating loop, pick up a well-isolated colony of 3. Observe for the formation of purple (deep blue) color over test bacteria from fresh culture and make a smear on the oxidase Salmonella Typhi