CLINICAL BACTERIOLOGY LABORATORY IDENTIFICATION OF GRAM POSITIVE COCCI OUTLINE • Introduction o Gram Positive Cocci • Tests for Identification of Gram Positive Cocci o Bacitracin Test o Beta Lactamase Test o Bile Esculin Test o Bile Solubility Test o CAMP Test o Catalase Test o Coagulase Test o Hippurate Hydrolysis Test o Deoxyribonuclease (DNase) Test o Leucine Amino Peptidase (LAP) Test o Microdase Test o Novobiocin Susceptibility Test o Optochin Susceptibility Test o PYR Test o Salt Tolerance Test INTRODUCTION • Health professionals need to understand the important difference between gram-positive and gram-negative bacteria. • Gram-positive bacteria are bacteria classified by the color they turn in the staining method. • Hans Christian Gram developed the staining method in 1884. The staining method uses crystal violet dye, which is retained by the thick peptidoglycan cell wall found in gram-positive organisms. This reaction gives gram-positive organisms a blue color when viewed under a microscope. Although gram-negative organisms classically have an outer membrane, they have a thinner peptidoglycan layer, which does not hold the blue dye used in the initial dying process. Other information used to differentiate bacteria is the shape. Gram-positive bacteria comprise of cocci, bacilli, or branching filaments. • Staph - cluster • Strep - chains GRAM POSITIVE COCCI • Staphylococcus aureus o Gram-positive, catalase-positive, coagulase-positive cocci in clusters. o Can cause inflammatory diseases, including skin infections, pneumonia, endocarditis, septic arthritis, osteomyelitis, and abscesses o Can also cause toxic shock syndrome (TSST-1), scalded skin syndrome (exfoliative toxin, & food poisoning (enterotoxin; cause food px with B. cereus ). • Staphylococcus epidermidis o Cram-positive, catalase-positive, coagulase-negative cocci in clusters and is novobiocin sensitive. o Commonly infects prosthetic devices and IV catheters producing biofilms. o Novobiocin resistant and is a normal flora of the genital tract and perineum. o Accounts for the second most common cause of uncomplicated urinary tract infection (UTI). • Streptococcus pneumoniae o Gram-positive, encapsulated, lancet-shaped diplococci, most commonly causing otitis media, pneumonia, sinusitis, and meningitis. o Consist of Strep. mutans and Strep mitis found in the normal flora of the oropharynx commonly that cause dental carries and subacute bacterial endocarditis ( Strep. sanguinis ). • Streptococcus pyogenes o Most pathogenically important Strep o Gram-positive group A cocci which can cause pyogenic infections (pharyngitis, cellulitis, impetigo, erysipelas), toxigenic infections (scarlet fever, necrotizing fasciitis), and immunologic infections (glomerulonephritis and rheumatic fever). o ASO titer detects S. pyogenes infections. • Streptococcus agalactiae o Gram-positive group B cocci that colonize the vagina and is found mainly in babies. o Pregnant women need screening for Group-B Strep (GBS) at 35 to 37 weeks of gestation. • Enterococci o Gram-positive group D cocci found mainly in the colonic flora and can cause biliary tract infections and UTIs. Usually bacteria in lab surfaces o Vancomycin-resistant enterococci (VRE) are an important cause of nosocomial infections/hospital acquired infections (HAIs). TESTS FOR IDENTIFICATION OF GRAM POSITIVE COCCI BACITRACIN TEST • Identification of Streptococcus pyogenes and Enterococcus • Bacitracin is a polypeptide antibiotic produced by organisms of the licheniformis group of Bacillus subtilis var Tracy. It is used to determine its effect of a small amount (0.04 IU or 0.05 IU not higher) on different microorganisms. The presumptive identification of beta hemolytic Group A streptococci from beta hemolytic non-Group A Streptococci is usually done by testing for sensitivity to bacitracin. Many laboratories use this test as the sole test for diagnosing GAS infections due to difficulty in performing sero-grouping and high cost of antisera.
• Uses o The test is used for presumptive identification and differentiation of beta-hemolytic group A streptococci ( Streptococcus pyogenes – susceptible) from other beta-hemolytic streptococci . o It is also used to distinguish staphylococci species (resistant) from micrococci (susceptible). • Principle o The antibiotic bacitracin inhibits the synthesis of bacterial cell walls by interfering the peptidoglycan synthesis of bacteria. o A disk impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate, allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms. After incubation, the inoculated plates are examined for zones of inhibition surrounding the disks. o If the organism grows up to the edge of the disk, it is resistant to the antimicrobial compound infusing the disk. If there is a zone around the edge of the disk where the organism has not grown, the organism is susceptible to the antimicrobial in the disk. • Medium Used o Mostly, blood agar plate is used (trypticase soy agar + 5% sheep blood). • Expected Results o Positive : Any zone of inhibition greater than 10 mm; susceptible. o Negative : No zone of inhibition; resistant LAB EXERCISE: BACITRACIN TEST • Materials o PPE o 6.5% NaCl o Prepared Blood agar plate or TSA o Alcohol lamp o Sheep’s blood o Bacterial cultures o Inoculating loop o slides o Bacitracin disk/Taxo A • Procedure 1. Select a beta-hemolytic colony and heavily inoculate a quadrant of a 5% sheep blood agar plate. 2. Drop an “A” disk in the heaviest zone of inoculation. 3. Tap disk lightly to ensure that it adheres to the agar. 4. Incubate the plate at CO2 incubator at 35OC for 24 hrs. • Reading o Any zone of inhibition is considered a positive test. BETA LACTAMASE TEST • Different bacteria produce an important class of enzymes called beta-lactamases, which may be mediated by genes on plasmids or chromosomes. • These enzymes confer resistance to a number of penicillin antibiotics by cleaving the beta-lactam ring of penicillins and cephalosporin antibiotics, resulting in inactivation of these drugs. • They are capable of inactivating “penicillinase-labile-penicillins”, such as amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin. • β-lactamases thus play a key role in bacterial resistance to beta-lactam agents, and detection of their presence can provide useful information. • Various assays are available to detect β-lactamases, such as the iodometric method, the acidometric method, and by the use of chromogenic substrates. • Objective o To detect the enzyme beta-lactamase, which confers penicillin resistance to various bacterial organisms. • Principle o One of the most useful tests in clinical laboratories for β- lactamase detection is the chromogenic cephalosporinase test. The test disk employed consists of a chromogenic cephalosporin which is used as the substrate. Organisms possessing β-lactamases when applied to the disk, exert their effect by opening the β-lactam ring of the substrate. This process results in a colored product which is conspicuous and hence allows detection. On hydrolysis by the bacterial inoculum, a deep pink color is produced. Lack of color production indicates the absence of β-lactamase. • Test Disk o The Cefinase disk is an example of a commercially available chromogenic test (BD Microbiology Systems, Cockeysville, Maryland). o The disk incorporates nitrocefin as the substrate. o It exhibits a very rapid colour change from yellow to red as the amide bond in the beta lactam ring is hydrolyzed by a beta-lactamase. o When a bacterium produces this enzyme in significant quantities, the yellow-colored disc turns red in the area where the isolate is smeared. • Procedure 1. Using a single disk dispenser, dispense the disk from the cartridge into an empty petri dish or onto a microscope slide. 2. Moisten disc with 1 drop of sterile distilled water. 3. With a sterilized loop or applicator stick remove several well-isolated similar colonies and smear onto a disk surface. 4. Observe disk for color change. • Expected Results o Positive : Yellow to pink-red color change on the area where the culture is applied. o Negative : No color change.
• Uses o Useful applications include detection of: ▪ N. gonorrhoeae resistance to penicillin ▪ H. influenzae resistance to ampicillin ▪ Staphylococcal resistance to penicillin BILE ESCULIN TEST • Esculin is a glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin). The two moieties of the molecule (glucose and 7-hydroxycoumarin) are linked together by an ester bond through oxygen. • Many bacteria can hydrolyze esculin, but few can do so in the presence of bile. • Thus the bile esculin test is based on the ability of certain bacteria, notably the group D streptococci and Enterococcus species, to hydrolyze esculin in the presence of bile (4% bile salts or 40% bile). • Bacteria that are bile-esculin positive are able to grow in the presence of bile salts and the hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin. • Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate) to form a black diffusible complex. • Principle o Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe3+ and forms a dark brown to black precipitate. Thus the tolerance to the presence of bile and the hydrolysis of esculin provide the means to presumptively identify organisms. If an organism can hydrolyze esculin, the media will turn dark brown or black. o Esculin + Acid → β-D-glucose + Esculetin o Esculetin + Fe3+ → Dark Brown Color • Procedure 1. Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant. 2. Incubate at 35°-37°C in ambient air for 48 hours. 3. Observe for growth and blackening of the medium. • Expected Results o Positive : Growth and blackening of the agar slant o Negative : Growth & no blackening of medium ; No growth • Uses o This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group. o The test differentiates enterococci and group D streptococci from non –group D viridans streptococci. BILE SOLUBILITY TEST • Bile Solubility Test is the test which differentiate Streptococcus pneumoniae (positive- soluble) from alpha- hemolytic streptococci (negative- insoluble). Streptococcus pneumoniae is bile soluble whereas all other alpha- hemolytic streptococci are bile resistant. • Principle o S. pneumoniae has an autolytic enzyme which can be demonstrated by allowing a broth culture to age in the incubator; at 24 hours the broth is turbid; after a few days the medium will become clear. o Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process. Bile salts activate the autolytic enzyme which induces clearing of culture. • Reagents o 2% sodium deoxycholate (bile salt) solution ▪ Dissolve 2 gram of sodium deoxycholate into 100 ml sterile distilled water. o 10% sodium deoxycholate (bile salt) solution ▪ Dissolve 10 gram of sodium deoxycholate into 100 ml sterile distilled water. • Tube Method Procedure 1. Prepare a heavy suspension of a pure culture in 2 ml of 0.85% saline. 2. Divide the organism suspension into two tubes. 3. Adjust the turbidity to that of 0.5-1 McFarland standard. 4. To one tube (test tube), add 2 drops of 2% sodium desoxycholate and mix. 5. To the other tube (control tube), add 2 drops of sterile water distilled water and mix. 6. Leave both tubes for 10-15 minutes at 35-37°C. 7. Observe for a clearing of turbidity in the tube containing 2% sodium deoxycholate. 8. If negative, continue to incubate up to 3 hours. Observe again for clearing. • Plate Method Procedure 1. Incubate the sample on 5% sheep blood agar for 12 to 24 hours. 2. Place one to two drops of 10% sodium desoxycholate to the side of a freshly isolated colony (18 -24 hours) on 5% sheep blood agar. 3. Gently wash the solution over the colony with dislodging the colony from the medium. 4. Incubate the culture plate at 35-37°C for 30 minutes. 5. Examine for lysis of colony (Disappearance of colony). • Modified Bile Solubility Test (Rapid Test) o The rapid identification of microorganisms recovered in blood can provide information about the source of the infectious process and guide the specific therapeutic management of the septic patient. 1. Blood (10%, vol/vol) was inoculated into one bottle each of tryptic soy broth and Thiol broth under vacuum with C02 and 0.025% sodium polyanetholsulfonate. 2. On one-half of the slide, 1 drop of blood was mixed with 1 drop of 2% sodium deoxycholate. 3. On the other half of the slide, 1 drop of blood was mixed with 1 drop of water, which served as the control.
4. The slide was air-dried at ambient temperature. 5. Gram staining was done and examined for cocci. 6. The numbers of organisms present in the blood treated with sodium deoxycholate and in the untreated blood were compared. • Result Interpretation o Tube Method ▪ Positive Result: Suspension clears in tube labelled test and remains turbid in control tube. ▪ Negative Result: Suspension remains turbid. ▪ Note: Partial clearing (partial solubility) is not considered positive for S. pneumoniae identification. o Plate Method ▪ Positive Result : Colony disintegration or flattening of the colony within 30 minutes, leaving an alpha-hemolytic where colony may be located. ▪ Negative Result : No change; colonies remain intact. ▪ Figure: Bile solubility (desoxycholate) test. A, Colony lysed (Positive). B, Intact colony (Negative). o Modified Bile Solubility Test ▪ Positive Result : No cocci are seen in the test smear and control smear shows intact bacteria. ▪ Negative Result : Cocci are seen in both test smear and control smear. • Quality Control o Positive Control : Streptococcus pneumoniae NCTC 12977 o Negative Control : Streptococcus mitis NCTC 10712 CAMP TEST • CAMP test is used to distinguish the species Streptococcus agalactiae from other species of beta- hemolytic Streptococcus. S. agalactiae , a member of the Lancefield Group B streptococci, is one of the causative agents of mastitis in cows. CAMP is an acronym for the authors of this test (Christie, Atkinson, Munch, and Peterson) which was identified in 1944. • Principle o Certain organisms (including group B streptococci) produce a diffusible extracellular hemolytic heat-stable protein (CAMP factor) that acts synergistically with the beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells. The group B streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar. A positive reaction appears as an arrowhead zone of hemolysis adjacent to the place where the two streak lines come into proximity. o The hemolytic activity of the beta-hemolysin produced by most strains of Staphylococcus aureus is enhanced by extracellular protein produced by group B streptococci. Interaction of the beta-hemolysin with this factor causes “synergistic hemolysis,” which is easily observed on a blood agar plate. This phenomenon is seen with both hemolytic and non-hemolytic isolates of group B streptococci. • Uses o It is used to distinguish the species Streptococcus agalactiae from other species of beta-hemolytic Streptococcus. o It is used to identify Listeria monocytogenes which also produces a positive CAMP reaction. • Result Interpretation o Positive : Enhanced hemolysis is indicated by an arrow head-shaped zone of beta-hemolysis at the junction of the two organisms. o Negative : No enhancement of hemolysis. • Quality Control for CAMP test o Positive : Streptococcus agalactiae (ATCC13813) — enhanced arrowhead hemolysis. o Negative : Streptococcus pyogenes (ATCC19615) — beta-hemolysis w/o enhanced arrowhead formation. LAB EXERCISE: CAMP TEST • Materials o PPE o Culture of gram positive cocci o Test tubes o 3% H2O2 o Inoculating loop o Incubator o Applicator stick Alcohol o Lamp
• Slide Method 1. Use a clean and dry glass slide, place a drop of 3% H2O2 on to the slide. 2. Pick a small amount of bacterial colony using a loop or sterile applicator stick. Gently mix. 3. Presence of bubbles or effervescence within 5-10 seconds indicates a positive result. 4. Absence of bubbles or effervescence indicates a negative result. 5. Record the result and dispense in an infectious waste container/bag. • Tube Method 1. Pour 1-2 ml of hydrogen peroxide solution into a test tube. 2. Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution. 3. Observe for immediate bubbling. CATALASE TEST • Principle o The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The lack of catalase is evident by a lack of or weak bubble production. The culture should not be more than 24 hours old. o Bacteria thereby protect themselves from the lethal effect of Hydrogen peroxide which is accumulated as an end product of aerobic carbohydrate metabolism. • Results and Observations • Quality Control of Catalase Test o Positive : Staphylococcus aureus – ATCC 33592 o Negative : Enterococcus faecalis – ATCC 29212 • Precautions of Catalase Test o The test organisms should not be taken from blood agar culture. Red Blood cells contain catalase and their presence will give a false positive test. o Culture should be 18 to 24 hours old. o Hydrogen peroxide must be fresh as it is very unstable. o Iron wire loop should not be used. o Some bacteria produce a peroxidase that catalyzes a breakdown of H2O2 causing the reaction to be weakly positive; (a few bubbles elaborated slowly). This should not be confused with a truly positive reaction. o Do not add organism to reagent, particularly if iron-containing inoculating loops are used. Iron containing loops will cause false positive test results if exposed to hydrogen peroxide. LAB EXERCISE FOR THE CATALASE TEST • Materials o PPE o Culture of gram positive cocci o Test tubes o 3% H2O2 o Inoculating loop o Incubator o Applicator stick Alcohol o Lamp • Slide Method 1. Use a clean and dry glass slide, place a drop of 3% H2O2 on to the slide. 2. Pick a small amount of bacterial colony using a loop or sterile applicator stick. Gently mix. 3. Presence of bubbles or effervescence within 5-10 seconds indicates a positive result. 4. Absence of bubbles or effervescence indicates a negative result. 5. Record the result and dispense in an infectious waste container/bag. • Tube Method 1. Pour 1-2 ml of hydrogen peroxide solution into a test tube. 2. Using a sterile wooden stick or a glass rod, take several colonies of the 18 to 24 hours test organism and immerse in the hydrogen peroxide solution. 3. Observe for immediate bubbling. COAGULASE TEST • Purpose o The test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (negative). • Principle o S. aureus produces two forms of coagulase, bound and free. Bound coagulase, or “clumping factor,” is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in precipitation of fibrinogen on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. o The presence of bound coagulase correlates with free coagulase, an extracellular protein enzyme that causes the formation of a clot when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a plasma coagulase-reacting factor (CRF), which is a modified or derived thrombin molecule, to form a coagulase-CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.
• Result Interpretation o Slide Test ▪ Positive : Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop. ▪ Negative : No clumping in either drop. o Tube Test ▪ Positive : Clot of any size. ▪ Negative : No clot. • Quality Control o Positive : Staphylococcus aureus (ATCC25923) o Negative : Staphylococcus epidermidis (ATC12228) LAB EXERCISE FOR COAGULASE TEST • Materials o PPE o Culture of gram positive cocci o Slides o Rabbit’s plasma o Inoculating loop o Incubator o Applicator stick o Alcohol Lamp • Procedure: Slide Method 1. Place a drop of coagulase plasma (preferably rabbit plasma with ethylenediaminetetraacetic acid [EDTA]) on 2. a clean, dry, glass slide. 3. Place a drop of distilled water or saline next to the drop of plasma as a control. 4. With a loop, straight wire, or wooden stick, emulsify a portion of the isolated colony being tested in each drop, inoculating the water or saline first. Try to create a smooth suspension. 5. Mix well with a wooden applicator stick. 6. Rock the slide gently for 5 to 10 seconds. • Procedure: Tube Method 1. Emulsify several colonies in 0.5 mL of rabbit plasma (with EDTA) to give a milky suspension. 2. Incubate tube at 35°-37°C in ambient air for 4 hours. 3. Check for clot formation. HIPPURATE HYDROLYSIS TEST • Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyse substrate hippurate into glycine and benzoic acid by action of hippuricase enzyme present in bacteria. Hippuricase is a constitutive enzyme that hydrolyzes the substrate hippurate to produce the amino acid glycine. Glycine is detected by oxidation with Ninhydrin reagent, which results in the production of a deep purple color. Hippurate hydrolysis test is used in the presumptive identification of Gardnerella vaginalis, Campylobacter jejun i, Listeria monocytogenes and group B streptococci by detecting the ability of the organism to hydrolyze hippurate. • Objective o To detect the production of the enzyme hippuricase for the presumptive identification of a variety of microorganisms. • Principle o Hippurate is the glycine conjugate of benzoic acid. When hippurate is hydrolysed by an organism glycine and benzoic acid are formed. Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during the process. The end products of the ninhydrin oxidation react to form a purple-colored product. • Procedure 1. Add 0.1 mL of sterile water to a 12 ×75 mm plastic test tube. 2. Make a heavy suspension of the organism to be tested. 3. Using heated forceps, place a rapid hippurate disk in the mixture. 4. Cap and incubate the tube for 2 hours at 35°C; use of a water bath is preferred. 5. Add 0.2 mL ninhydrin reagent and reincubate for an additional 15 to 30 minutes. 6. Observe the solution for the development of a deep purple color. • Expected Results o Positive : A positive test is indicated by the appearance of a deep blue/violet color in 30 minutes. o Negative : Colorless or slightly yellow pink color • Uses o The hippurate test is most frequently used in the identification of Gardnerella vaginalis, Streptococcus agalactiae, Campylobacter jejuni , and Listeria monocytogenes. o Used in differentiation of β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci. o Aids in the separation of Campylobacter jejuni and Campylobacter coli strains.
DEOXYRIBONUCLEASE (DNase) TEST • Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is way too large to enter the cell membrane. In order to utilize external DNA, bacteria cells secrete exoenzymes (DNases) outside of the cell that hydrolyze DNA into nucleotides. The nucleotides can then move across the cell membrane via transport proteins to be utilized. The cell can use nucleotides to make nucleic acids and to use as a source of nitrogen, phosphate and carbon. Extracellular DNases have been reported in a relatively small subset of prokaryotes, including a number of human pathogens. • DNA hydrolysis is tested by growing an organism on a DNase Test Agar plate (providing nutrients and DNA) and then checking the plate for hydrolysis. DNA Hydrolysis test or Deoxyribonuclease (DNase) test is thus used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth. It helps in the detection of deoxyribonuclease activity of bacteria and fungi, and especially for identification of pathogenic Staphylococci. • Objective o To differentiate organisms based on the production of deoxyribonuclease. • Principle o The test is used to determine the ability of an organism to hydrolyze DNA. DNase agar is a differential medium that tests the ability of an organism to produce an exo-enzyme, called deoxyribonuclease. DNase are extracellular endonucleases that cleave DNA and release free nucleotides and phosphate. DNase agar contains nutrients for the bacteria, DNA, and mostly methyl green as an indicator. Methyl green is a cation which binds to the negatively-charged DNA. o Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around the areas where the DNase-producing organism has grown. o In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing of the agar after addition of HCL (oligonucleotides dissolves in acid but DNA salts are insoluble). The acid precipitates unhydrolyzed DNA making the medium opaque. Therefore, DNase producing colonies hydrolyze DNA and produce a clear zone around the growth. • Procedure 1. Using a sterile loop, inoculate the DNase agar with the organism to be tested on the test area. 2. Incubate the plate at 35-37°C for 24 hours. 3. After incubation observe the color change in DNase with methyl green. o In DNase agar without indicators: 4. Flood the surface of agar with 1N HCL solution. Tip off the excess acid. 5. Allow the reagent to absorb into the plate. 6. Observe for clear zone around the colonies within 5 minutes. • Expected Results o Positive : Medium is colorless around test organism. o Negative : If no degradation of DNA occurs, the medium remains green. • Uses o Used to determine the ability of an organism to hydrolyze deoxyribonucleic acid. o Used to differentiate Staphylococcus aureus which produces the enzyme deoxyribonuclease from other Staphylococci which do not produce DNase. o Particularly useful if plasma is not available to perform coagulase test or when the result of coagulase tests are difficult to interpret. o It is also used to distinguish Serratia (positive) from Enterobacter sp. o Moraxella catarrhalis (positive) from Neisseria LEUCINE AMINO PEPTIDASE (LAP) TEST • Leucine amino peptidase (LAP) test is a rapid test for detection of enzyme leucine aminopeptidase. Leucine- β- napthalamide impregnated disk serves as a substrate for the detection of leucine aminopeptidase. This test is usually used, in conjunction with other tests, for the identification of streptococci and other catalase negative gram positive cocci. • Objective o To perform the preliminary characterization of catalase negative, gram-positive cocci. • Principle o LAP disk is a rapid test for the detection of enzyme leucine amino peptidase. Disks are impregnated with leucine-ß-naphthylamide, which is hydrolyzed by the enzyme leucine aminopeptidase, produced by LAP positive organisms. This enzymatic activity results in the release of ß-naphthylamine, which couples with p-dimethylaminocinnamaldehyde reagent, when it is added, to form a highly visible red Schiff base. • Procedure 1. Aseptically place a LAP disk in a sterile petri dish, and allow disk to warm to room temperature 2. Slightly dampen the LAP disk with reagent grade water or with a small amount of sterile distilled water. 3. Using a wooden applicator stick, rub a small amount of several colonies of an 18 to 24 hour pure culture onto a small area of the LAP disk. 4. Incubate at room temperature for 5 minutes. 5. After the incubation period, add 1 drop of cinnamaldehyde reagent and read within one minute.
• Expected results o Positive : Development of a red/pink color o Negative : No change or a slight yellow color • Uses o The LAP test is often used in conjunction with PYR and other biochemical tests to help differentiate between catalase, gram-positive cocci. o In general, Streptococcus pneumoniae and Streptococcus pyogenes , Pediococcus, Lactococcus, and Enterococcus species are all LAP positive, while other beta-hemolytic Streptococci, Aerococcus and Leuconostoc species are LAP negative which help in their identification. o Aminopeptidase Test has been used to perform a Gram analysis of bacteria isolated from Scleroderma citrinum mycorrhizae, the mycorrhizosphere and bulk soil. MICRODASE TEST • Microdase Disk is a reagent-impregnated disk recommended for use in qualitative procedures to aid in the differentiation of Staphylococcus from Micrococcus by the detection of the oxidase enzyme. • The oxidase method was originally described by Kovacs in 1956 as a method of differentiating gram-negative bacilli. However, in 1981, Faller and Schleifer modified Kovacs’ oxidase reagent by utilizing a tetramethyl-pphenylenediamine (TMPD) in dimethyl sulfoxide (DMSO).This is referred to as the modified oxidase test. • Objective o To differentiate gram-positive, catalase-positive cocci • Principle o The microdase test is a rapid method to differentiate Staphylococcus from Micrococcus spp. by detection of the enzyme oxidase. For the detection of oxidase enzyme a filter paper circular disks impregnated with tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide (DMSO) are used. The modified oxidase reagent is prepared as 1% (w/v) tetramethyl-p-phenylenediamine in certified grade dimethyl sulfoxide. DMSO provides solubility and stability against auto-oxidation and also aids in the permeability of cells to the reagent. In the presence of atmospheric oxygen, the oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the colored compound, indophenol indicated as blue or purplish blue coloration on the disc after the introduction of bacterial colony on the disc. o All micrococci possess cytochrome c, whereas most staphylococci, with few exceptions, lack this type of cytochrome. The oxidase reagent substantiates the presence of type c cytochrome. • Procedure o Testing should be performed on aerobic, catalase-positive, gram-positive cocci. 1. Using forceps, place the disk in an empty petri dish or on a clean glass slide. 2. Using a wooden applicator stick, rub a small amount of several colonies of an 18- to 24-hour pure culture grown on blood agar onto a small area of the microdase disk. o Note: Do not rehydrate the disk before use. 3. Incubate at room temperature for 2 minutes. 4. Examine for a blue color development. • Expected results o Positive : Development of blue to purple-blue color within 2 minutes time o Negative : No color change (white to gray color remains). • Uses o This test is used to differentiate Micrococci from Staphylococci. (Micrococci should yield a positive result. Staphylococci should yield a negative result, with the exception of Staphylococcus sciuri, S. lentus and S. vitulinus . NOVOBIOCIN SUSCEPTIBILITY TEST • Novobiocin is an aminocoumarin antibiotic, produced by the actinomycete Streptomyces nivens , with antibacterial property. In 1975, Kloos and Schleifer reported a simplified scheme for differentiating coagulase-negative Staphylococcus spp. which included a novobiocin disk test. Staphylococcus saprophyticus , a gram positive coagulase negative Staphylococci, is an uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in young, middle aged female patients. Unlike most other CONS, saprophyticus is rarely resistant to most antibiotics active against gram-positive organisms. S. saprophyticus is innately resistant to the antibiotic novobiocin. Therefore, screening coagulase-negative staphylococci from urine cultures for novobiocin resistance is a reliable presumptive identification of saprophyticus. • Objective o To determine the susceptibility pattern of bacterium to antibiotic novobiocin. • Principle o Novobiocin is an antibiotic interfering with the unpackaging and repackaging of DNA during DNA replication and the bacterial cell cycle. Novobiocin binds to DNA gyrase, and blocks adenosine triphosphatase (ATPase) activity. Susceptibility to novobiocin is determined by placing a novobiocin-impregnated paper disk on a agar plate seeded with the organism under investigation. As the organism multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the antibiotic diffusing into the agar from the paper disk.
o If the bacteria are susceptible to novobiocin, there will be a formation of visible zone of inhibition around the disk, representing an area where the antibiotic concentration has prevented bacterial growth. No zone of inhibition around the disk represents that organism is resistant to the anitibiotic. o Hence, in the laboratory, a Mueller-Hinton agar plate is heavily seeded with the test organism to produce a confluent growth on the agar surface. After the seeding, a novobiocin antibiotic disc is applied to the agar surface. Following incubation, the sensitivity of an organism to the antibiotic is determined by the Kirby-Bauer method. • Procedure 1. The test isolate should be 18-72 hours and in pure culture. Prepare a suspension of test isolate in tryptic soy broth equal to a McFarland 0.5 standard or equiv. 2. Immerse a sterile swab into the suspension and rotate it against the side of the tube above the fluid level to remove excess inoculum. 3. Using the expressed swab, inoculate a blood agar or Mueller Hinton agar plate by streaking the swab over the entire agar surface and repeat in 2 planes. 4. Allow the agar surface to dry no more than 15 minutes before applying a Novobiocin Disk. With a sterile swab, prepare a lawn of growth over the entire plate by swabbing over the entire plate in 3 directions and around the edge of the plate. 5. Using alcohol-dipped and flamed forceps, aseptically apply a novobiocin antibiotic disc to the surface of each inoculated plate. Gently press the discs down with sterile forceps to ensure that they adhere to the agar surface. 6. Incubate plate aerobically for 18-24 hours at 35-37°C. 7. Measure the diameter of the zone of inhibition using sliding calipers or a metric ruler. • Expected Results o Positive – A zone of inhibition greater than 16mm indicates that the organism is sensitive to antibiotic. o Negative – A zone of inhibition less than or equal to 16mm is indicative of novobiocin resistance. • Uses o The CoNS have been subdivided into two groups based on their novobiocin susceptibility pattern. CoNS group that demonstrates novobiocin susceptibility includes epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. hominis, S. lugdunensis, S. saccharolyticus, S. warneri , and other species. o Novobiocin resistant group consists of such species as cohnii, S. kloosii, S. saprophyticus , and S. xylosus . o It is useful for presumptively distinguishing Staphylococcus saprophyticus from other coagulase- negative staphylococci in clinical specimens. OPTOCHIN SUSCEPTIBILITY TEST • Differentiation of Streptococcus pneumoniae from other viridans streptococci depends on demonstrating optochin susceptibility, bile solubility, reaction with a specific DNA probe, or detection of species-specific capsular polysaccharides. Most clinical microbiology laboratories today depend on the optochin susceptibility test. • Although optochin susceptibility was first described for differentiating pneumococci from other α-hemolytic streptococci in 1915, the test was virtually unused by laboratories until the mid-1950s. • Optochin is a chemical, ethylhydrocupreine hydrochloride and is completely soluble in water. It is useful in the identification of Streptococcus pneumoniae , the alpha- hemolytic Streptococcus most commonly susceptible to this chemical. Other alpha-hemolytic streptococcal species are optochin-resistant. This test determines whether the bacterium is either sensitive (susceptible) to optochin or resistant to the chemical. • The optochin test is widely used in the form of filter paper discs, impregnated with ethylhydrocupreine hydrochloride, which are applied directly to inoculated plates before incubation. The optochin test is less time-consuming than the bile solubility test. • Principle o A positive presumptive identification of S. pneumoniae is made when a well defined zone of inhibition results around the impregnated disk. Other alpha-hemolytic streptococci do not display this clear zone of inhibition when in the presence of optochin. The chemical tests the fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes in surface tension. • Result Interpretation o A presumptive identification for S. pneumoniae can be made if the alpha-hemolytic colony produces a zone of inhibition of 14mm or greater around the disk. Organisms producing smaller zone sizes should be tested for bile solubility. Organisms with questionable zone sizes (6-14mm) around the disk should be presumptively identified as a pneumococci only if it is bile soluble. o Positive Test : Zone of inhibition is 14 mm or greater in diameter with 6 mm disk. o Negative Test : No zone of inhibition or a zone of inhibition of <14mm diameter. • Quality Control o Positive : Streptococcus pneumoniae (ATCC ® 6305), S. pneumoniae (ATCC 49619). o Negative : Streptococcus pyogenes (ATCC ® 19615), S. mitis (ATCC 49456).
LAB EXERCISE: OPTOCHIN TEST • Materials o PPE o CO2 incubator or anaerobic jar o Prepared Blood agar plate o Alcohol lamp o TSA/Columbia Agar Base o Bacterial cultures o Sheep’s blood o slides o Inoculating loop o caliper o Optochin disk or Taxo P • Procedures 1. Using an inoculating loop, select three to four well- isolated colonies of the alpha-hemolytic organism to be tested. An 18-24 hour culture of isolated organism can also be used for testing. 2. Streak the isolate onto one-half of a TSA-5% sheep blood agar plate so as to obtain confluent growth. o Important : Use of media other than TSA-5% sheep blood agar is not recommended, as false identification may result. 3. Using sterile forceps, place an optochin disk onto the inoculated surface of the agar. 4. Press disk gently with the sterile forceps or loop so that the disk adheres firmly to the agar surface. 5. Incubate the plate at 35 +/- 2.0 degrees C. for 18-24 hours in 5-10% CO2 enriched environment. 6. If zone of inhibition is present, measure the diameter with a millimeter ruler or caliper. • Reading and interpretation o If a 6 mm disk is used, a zone of inhibition of at least 14 mm in diameter is considered positive for identification of pneumococci. A zone of inhibition between 6 and 14 mm in diameter is considered questionable for identification of pneumococci and a bile solubility test should be performed. Bile soluble strains with optochin zones of inhibition between 6 and 14 mm are considered pneumococci, those strains that are not bile soluble with the same zone sizes are not considered pneumococci. PYR TEST • PYR (Pyrrolidonyl Aminopeptidase) Test is used for the detection of pyrolidonyl arylamidase (also called pyrrolidonyl aminopeptidase) activity in Streptococcus pyogenes (group A strep), Enterococcus spp., some coagulase-negative staphylococci, and some Enterobacteriaceae. It is also known as PYR (L-pyrrolidonyl- β-naphthylamide) which serve as a substrate for the detection of pyrrolidonyl peptidase. • Facklam, Thacker, Fox and Eriquez reported that 98% of group A streptococci and 96% of group D enterococci hydrolyze PYR. Although Aerococcus species are rarely isolated in the clinical laboratory, these organisms are also expected to hydrolyze PYR. • Facklam et al. further reported that 98% of group B streptococci, 100% of non-group A, B and D streptococci, 100% of group D non-enterococci and 82% of viridans streptococci yield negative PYR test results. • Principle o PYR is a rapid method for presumptive identification of bacteria based on the pyrrolidonyl arylamidase enzyme. The enzyme L-pyrrolidonyl arylamidase hydrolyzes the L-pyrrolidonyl- β-naphthylamide substrate to produce a β-naphthylamine. The β- naphthylamine can be detected in the presence of N,N-methylaminocinnamaldehyde reagent by the production of a bright red precipitate. o Following hydrolysis of the substrate by the peptidase, the resulting b-naphthylamide produces a red color upon the addition of 0.01% cinnamaldehyde reagent. When a visible inoculum of microorganism is rubbed onto a small area of a disk impregnated with the substrate, the hydrolysis occurs within 2 min, at which time the cinnamaldehyde reagent is added to detect the reaction by a color change to purple. • Uses o It is used for the presumptive identification of group A streptococci ( Streptococcus pyogenes ). o It is used for the rapid differentiation of enterococci from group D ß-hemolytic streptococci. o It also differentiate some Staphylococci (positive haemolyticus from negative S. auricularis ). o It is used in the identification of E. coli , separating it from other indole positive, lactose positive, gram-negative rods. • Broth Method Procedure 1. Inoculate PYR broth with 3-5 colonies from 18-24 hours pure culture. 2. Incubate the tube aerobically at 35-37°C for 4 hours. 3. Add 2-3 drop of PYR reagent and observe for color change. 4. Observe for the red color development within 1-2 minutes. • Disk Method (Rapid) Procedure 1. Wet the PYR test disc on the strip with 10 µl sterile distilled water or deionized water. o Note: Do not flood the disk. 2. Put 5-10 colonies of the tested strain from 18-24 hours culture on the surface of the disc with a loop and smear them lightly on it. 3. Incubate the disc for 1-2 minutes at room temperature. 4. After incubation, add 1 drop of N, N- dimethylaminocinnamaldehyde. 5. Observe for red color development within 1-2 minutes. • Result Interpretation o Positive : Bright pink or cherry-red color within 1-2 minutes. ▪ Examples: Group A Streptococci ( Streptococcus pyogenes ), Group D Enterococci ( Enterococcus faecalis and Enterococcus faecium ), Coagulase negative Staphylococcus species such as S. hemolyticus, S. lugdunensis, S. schleiferi ., Enterobacter , Citrobacter , Klebsiella , Yersinia and Serratia , Aerococcus , Gamella , Lactococcus , most Corynebacterium ( Arcanobacterium ) hemolyticum . o Negative : No color change or a blue color due to a positive indole reaction. ▪ Examples: Group B Streptococci ( Streptococcus agalactiae ), Streptococcus mitis , S. bovis, S. equinus, S. milleri.
o Note: A pale pink reaction (weak) is considered negative. • Quality Control o Positive Control : Enterococcus faecalis (ATCC29212), Streptococcus pyogenes (ATCC19615) o Negative Control : Streptococcus agalactiae (ATCC10386) SALT TOLERANCE TEST • The ability of the bacteria to grow in the presence of variable amount of Sodium Chloride (NaCl) has been used to characterize several bacteria. It takes into account the organism’s ability to tolerate various osmotic concentrations. E. faecalis, E. zymogenes, E. liquifaciens , and E. durans are among the Enterococcus species that are salt tolerant. • Objective o To determine the ability of an organism to grow in high concentrations of salt. • Principle o Salt acts as a selective agent for bacteria and interferes with membrane permeability and osmotic equilibrium. A high salt concentration thus inhibits a range of bacteria but allows salt-tolerant organisms such as enterococci to grow. To demonstrate the ability of such organisms in the laboratory, broth containing 6.5% NaCl is used as the test medium. The broth includes the fermentable carbohydrate, dextrose, and the color indicator, bromcresol purple. Organisms capable of growing in the high salinity medium, utilize sugar and release acid as a byproduct of their metabolism. The pH drop results in indicator, bromcresol purple, to change from purple to yellow. • Procedure 1. Inoculate one or two colonies from an 18- to 24-hour culture into 6.5% NaCl broth. 2. Incubate the tube at 35-37°C in ambient air for 48 hrs. 3. Examine tubes for turbidity after 24 hours and if negative again at 48 and 72 hours. • Expected Results o Positive : Visible turbidity in the broth, with or without a color change from purple to yellow. Note: Turbidity alone is indicative of a positive test o Negative : No turbidity and no color change after 72 hours of incubation. o Salt tolerance (6.5% NaCl) test. A, Positive. B, Negative • Uses o It is used to differentiate enterococci (positive) from non-enterococci (negative). o It is used to differentiate non-beta-hemolytic strains of catalase- negative, gram-positive cocci (i.e. Enterococcus and Aerococcus) based on their ability to grow in a 6.5% sodium chloride broth. o Aerococcus species such as A. viridans and A. urinae can also grow in 6.5% NaCl, therefore salt tolerance broth can also be used to differentiate Aerococcus species from other similar organisms such as Stomatococcus and Helcococcus. •