BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA In the name of God Yasuj University of Medical Sciences Department of Microbiology BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA By: Dr. S. S. Khoramrooz Department of Microbiology, Faculty of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran

SUSCEPTIBILITY TO BACITRACIN The bacitracin susceptibility test is used for the presumptive identification of group A β-hemolytic streptococci. The test is performed on a blood agar medium with a bacitracin differential disk (e.g., Bacitracin Disk, 0.04 unit). Any zone of inhibition around the disk is considered a positive test Although this test is simple, inexpensive, and fairly accurate for presumptive identification of group A streptococci, it is not highly specific. Dr. S. S. Khoramrooz

Groups C and G streptococci are usually susceptible to SXT Over 10% of group C and G streptococcal strains are also susceptible to bacitracin, as are about 5% of group B strains. Consequently, this test is often performed along with the sulfamethoxazoletrimethoprim (SXT) susceptibility test because: Groups C and G streptococci are usually susceptible to SXT Whereas groups A and B streptococci are resistant Dr. S. S. Khoramrooz

However, this method will identify only 50-60% of isolates. Some workers have advocated the use of bacitracin disks directly on primary, nonselective blood agar for rapid detection and identification of group A streptococci in throat cultures. However, this method will identify only 50-60% of isolates. Placement of bacitracin disks on primary plates containing selective media is considerably more sensitive. The laboratory report should reflect the use of a presumptive method: β-hemolytic streptococci, presumptively group A by bacitracin” or "β-hemolytic streptococci, presumptively not group A by bacitracin." Dr. S. S. Khoramrooz

SUSCEPTIBILITY TO SULFAMETHOXAZOLE TRIMETHOPRIM (SXT) The SXT susceptibility test presumptively distinguishes groups A and B streptococci from other β-hemolytic streptococci. When used in conjunction with the bacitracin test, the SXT susceptibility test helps screen out those non- A, non- B S streptococci that may be susceptible to bacitracin because both group A and B strains are SXT- resistant, whereas groups C, F, and G are SXT- susceptible. Dr. S. S. Khoramrooz

Any zone of inhibition indicates susceptibility to SXT The test is performed in the same way as the bacitracin test, except that a commercial disk containing 1.25 µg trimethoprim and 23.75 µg of sulfamethoxazole is used. Any zone of inhibition indicates susceptibility to SXT Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

Bile solubility test The bile (sodium deoxycholate) solubility test distinguishes S. pneumoniae from all other alpha - hemolytic streptococci. S. pneumoniae is bile soluble whereas all other alpha- hemolytic streptococci are bile resistant. Sodium deoxycholate (2% in water) will lyse the Pneumococcal cell wall Dr. S. S. Khoramrooz

A.Preparation of 2% sodium deoxycholate (bile salt) solution 1. Dissolve 2 g of sodium deoxycholate into 100 ml sterile distilled water. Dr. S. S. Khoramrooz

B.Performing the bile solubility test 1. Grow the isolate (s) to be tested for 18-24 hours on a BAP at 35- 37°Cwith ~5% CO2 (or in a candle-jar) 2. Add bacterial growth from the overnight BAP to 1.0 ml of 0.85% saline to achieve turbidity in the range of a 0.5-1.0 McFarland standard. 3. Divide the cell suspension equally into 2 tubes (0.5 ml per tube). 4.Add 0.5 ml of 2% sodium deoxycholate (bile salts) to one tube. Add 0.5 ml of 0.85% saline to the other tube. Mix each tube well. Dr. S. S. Khoramrooz

5. Incubate the tubes at 35-37°C in CO2 6.Vortex the tubes. 7.Observe the tubes for any clearing of turbidity after 10 minutes. Continue to incubate the tubes for up to 2 hours at 35- 37°C in CO2 if negative after 10 minutes. Observe again for clearing. Dr. S. S. Khoramrooz

C. Reading the bile solubility test results A clearing of the turbidity in the bile tube but not in the saline control tube indicates a positive test. D.Troubleshooting Partial clearing (partial solubility) is not considered positive for pneumococcal identification. Partially soluble strains that have optochin zones of inhibition of less than 14 mm are not considered pneumococci. Dr. S. S. Khoramrooz

S. pneumoniae strain ATCC 49619 can be used as a positive control Q uality control Each new lot of sodium deoxycholate should be tested with positive and negative QC strains. S. pneumoniae strain ATCC 49619 can be used as a positive control S. mitis strain ATCC 49456 can be used as a negative control. Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

Optochin test S. pneumoniae strains are sensitive to the chemical optochin (ethylhydrocupreine hydrochloride). Optochin sensitivity allows for the presumptive identification of alpha-hemolytic streptococci as S. pneumoniae, although some pneumococcal strains are optochin-resistant. Other alpha-hemolytic streptococcal species are optochin- resistant. Dr. S. S. Khoramrooz

Performing the optochin test Optochin (P) disks (6 mm, 5 μg) can be obtained from a commercial vendor. Optochin disks are often called “P disks” and many commercial versions are labeled with a capital “P”. If a commercial source of P disks is not available, a 1:4000 solution of ethylhydrocupreine hydrochloride can be applied to sterile 6 mm filter paper disks. Dr. S. S. Khoramrooz

1. Grow the strain(s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO2 (or in a candle-jar). 2. Use a disposable loop to remove an isolated colony from the overnight culture on the BAP and streak onto one half of a BAP. Dr. S. S. Khoramrooz

Two different isolates can be tested on the same plate, but care must be taken to ensure that the cultures do not overlap. 3. Place a P disk within the streaked area of the plate and incubate the BAP overnight at 35-37°C with ~5% CO2 (or in a candle-jar). 4. Observe the growth on the BAP near the P disk and measure the zone of inhibition, if applicable. Dr. S. S. Khoramrooz

B. Reading the optochin test results Using a 6 mm, 5 μg disk, a zone of inhibition of 14 mm or greater indicates sensitivity and allows for presumptive identification of pneumococci. Zones of inhibition should be measured from the top surface of the plate with the top removed. Use either calipers or a ruler with a handle attached for these measurements. Measure the diameter of the zone holding the ruler over the center of the surface of the disk when measuring the zone of inhibition. In the case of an isolate completely resistant to optochin, the diameter of the disk (6 mm) should be recorded. Dr. S. S. Khoramrooz

C. Troubleshooting A smaller zone of inhibition (< 14 mm) or no zone of inhibition indicates that the bile solubility test is required. It is important to remember that pneumococci are sometimes optochin-resistant. D. Quality control Each new lot of optochin disks should be tested with positive and negative controls. The growth of S. pneumoniae strain ATCC 49619 is inhibited by optochin and growth of S. mitis strain ATCC 49456 is not inhibited by optochin. Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

Susceptibility to Novobiocin for Identification of Staphylococcus saprophyticus NOVOBIOCIN DISK TEST I. Principle Coagulase-negative staphylococci can be divided into novobiocin-susceptible and novobiocin-resistant species. Among the novobiocin-resistant species, S. saprophyticus is the one commonly recovered from humans as a cause of urinary tract infections. II. Reagents I. Novobiocin disks. 5 µg (NB 5, BD Microbiology Systems, Franklin Lakes, NJ) 2. Sheep blood agar plate III. Quality Control A known S. saprophyticus strain and a S. epidermidis strain should be tested with each new lot of novobiocin disks or on a weekly basis. Dr. S. S. Khoramrooz

IV. Procedure 1. Prepare a suspension of the organism to be identified in sterile distilled water or broth. 2. The suspension should be equivalent in turbidity to a 0.5 McFarland standard. 3. With a sterile swab, spread some of the suspension over half of a blood agar plate. 4. Aseptically place a novobiocin disk on the inoculated area. Susceptibility to furazolidone may be assessed on the same plate by placing the disks about 4 cm apart on the inoculated area. Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

V. Results A. Interpretation Gently tamp the disk(s) with sterile forceps to assure contact with the agar surface. 5. Incubate the plate aerobically for 18 to 24 hours at 35°C. V. Results A. Interpretation 1. S. saprophyticus are novobiocin-resistant and will show zones of inhibition of 6 mm (no zone) to 12 mm. 2. Other coagulase-negative staphylococci and S. aureus are novobiocin-susceptible and will show zones of 16 mm or larger. Dr. S. S. Khoramrooz

Slide Coagulase Test Most strains of S. aureus have a bound coagulase or "clumping factor" on the surface of the cell wall. This factor reacts directly with fibrinogen in plasma, causing rapid cell agglutination. The test can be performed with growth from blood agar, CNA agar, or other nonselective nutrient medium, but should not be performed from media with a high salt content (e.g., mannitol salts agar) since the high salt content causes some strains of S. aureus to autoagglutinate. Dr. S. S. Khoramrooz

Any strain that is negative on the slide coagulase test must be confirmed with a tube coagulase test, because strains deficient in clumping factor will usually produce free coagulase. Some strains of the human coagulase-negative species S. lugdunensis and S. schleiferi subsp schleiferi also produce clumping factor and may be positive with the slide test Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

II. Media and Reagents Rabbit plasma with EDTA (commercially available in lyophilized form). Reconstituted plasma should be refrigerated. III. Quality Control Coagulability of plasma may be tested by adding one drop of 5% calcium chloride to 0.5 mL of the reconstituted plasma. A clot should form within 10 to 15 seconds. Dr. S. S. Khoramrooz

A known Staphylococcus aureus strain and a Staphylococcus epidermidis strain serve as positive and negative controls, respectively. Each reconstituted vial of rabbit plasma with EDTA should be tested with 18- to 24-hour cultures of the control strains. Dr. S. S. Khoramrooz

IV. Procedure 1- Slide test (bound coagulase): Place two drops of sterile water or saline in two circles drawn on a glass slide with a wax pencil. Gently emulsify colony material from the organism to be identified in liquid in each of the circles. Place a drop of coagulase plasma in the suspension in one of the circles and mix with a wooden applicator stick. Place another drop of water or saline in the other circle as a control. Rock the slide back and forth, observing for agglutination of the test suspension. Dr. S. S. Khoramrooz

2. Tube test (free coagulase): Emulsify a small amount of the colony growth of the organism in a tube containing 0.5 mL of coagulase plasma. Incubate the tube at 35°C for 4 hours and observe for clot formation by gently tilting the tube. If no clot is observed at that time, reincubate the tube at room temperature and read again after 18 hours. Dr. S. S. Khoramrooz

V. Results A. Interpretation 1. Slide test: A positive reaction will be detected within 10 to 15 seconds of mixing the plasma with the suspension by the formation of a white precipitate and agglutination of the organisms in the suspension. The test is considered negative if no agglutination is observed after 2 minutes. The saline control should remain smooth and milky. Dr. S. S. Khoramrooz

If the control suspension agglutinates as well, the test is uninterpretable. All strains that are coagulase-positive can be reported as coagulase-positive Staphylococcus or, less precisely, Staphylococcus aureus. All strains producing negative slide tests must be tested with the tube coagulase test. Dr. S. S. Khoramrooz

Tube Coagulase Test The coagulase detected by this method is secreted extracellularly and reacts with a substance in the plasma called coagulase-reacting factor (CRF) to form a complex, which, in turn, reacts with fibrinogen to form fibrin (clot formation) Tests that are negative after 4 hours of incubation at 35°C should be held at room temperature and read again after 18 to 24 hours, Because some strains will produce fibrinolysin on prolonged incubation at 35°C, causing dissolution of the clot during the incubation period. Rare S. aureus strains may be coagulase-negative, and some animal isolates (S. intermedius, S. hyicus, S. delphini, and S. schleiferi subsp. Coagulans) may be tube coagulase_positive. Dr. S. S. Khoramrooz

This error can be avoided by always performing a catalase test first. As mentioned above, the recommended medium for both the slide and the tube coagulase procedures is rabbit plasma with EDTA. Citrated plasma should not be used, because organisms that are able to use citrate (e.g., Enterococcus species) will yield positive results if they are inadvertently mistaken for staphylococci. This error can be avoided by always performing a catalase test first. Human plasma (e.g.,outdated material from blood banks) contains variable amounts of CRF and antistaphylococcal antibodies and should not be used to perform coagulase tests. Dr. S. S. Khoramrooz

2. Tube test: The tube coagulase test is considered positive if any degree of clotting is noted. The tube should be gently tilted and not agitated, because this may disrupt partially formed clotted material. Fibrinolysins produced by the organism may also dissolve the clot soon after formation. Tube tests that are negative after 4 hours should be incubated at room temperature overnight and read after 18 hours. Dr. S. S. Khoramrooz

Dr. S. S. Khoramrooz

CYTOCHROME OXIDASE TEST I. Principle The cytochromes are iron-containing hemoproteins that act as the last link in the chain of aerobic respiration by transferring electrons (hydrogen) to oxygen, with the formation of water. The cytochrome system is found in aerobic, or microaerophilic, and facultatively anaerobic organisms, so the oxidase test is important in identifying organisms that either lack the enzyme or are obligate anaerobes. The test is most helpful in screening colonies suspected of being one of the Enterobacteriaceae (all negative) and in identifying colonies suspected of belonging to other genera such as Aeromonas, Pseudomonas, Neisseria, Campylobacter, and Pasteurella (positive). Dr. S. S. Khoramrooz

The cytochrome oxidase test uses certain reagent dyes, such as p-phenylenediamine dihydrochloride, that substitute for oxygen as artificial electron acceptors. In the reduced state, the dye is colorless; however, in the presence of cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized, forming indophenol blue. Dr. S. S. Khoramrooz

II. Media and Reagents A. Tetramethyl-p-phenylenediamine dihydrochloride, 1% (Kovac's reagent) B. Dimethyl-p-phenylenediamine dihydrochloride, 1% (Gordon and McLeod's reagent) III. Quality Control Bacterial species showing positive and negative reactions should be run as controls at frequent intervals. The following are suggested: A. Positive control: Pseudomonas aeruginosa B. Negative control: Escherichia coli Dr. S. S. Khoramrooz

IV. Procedure The test is commonly performed by one of two methods: I) the direct plate technique, in which two to three drops of reagent are added directly to isolated bacterial colonies growing on plate medium; 2) the indirect paper strip procedure, in which either a few drops of the reagent are added to a filter paper strip or commercial disks or strips impregnated with dried reagent are used. The tetramethyl derivative of p-phenylenediamine is recommended because the reagent is more stable in storage and is more sensitive to the detection of cytochrome oxidase and is less toxic than the dimethyl derivative. In either method, a loopful of suspected colony is smeared into the reagent zone of the filter paper. Dr. S. S. Khoramrooz

V. Results A. Interpretation Bacterial colonies having cytochrome oxidase activity develop a deep blue color at the inoculation site within 10 seconds. Any organism producing a blue color in the 10- to 60-second period must be further tested because it probably does not belong to the Enterobacteriaceae. Stainless steel or Nichrome inoculating loops or wires should not be used for this test because surface oxidation products formed when flame-sterilizing may result in false- positive reactions. Dr. S. S. Khoramrooz

PYR TEST I. Principle Rapid test for the presumptive identification of both group A β-hemolytic streptococci and enterococci. Whereas the original test was described as a 16- to 20-hour agar test, subsequent PYR test formats included a 4-hour broth assay and several rapid (10- to 15-minute) tests in which the PYR reagent is impregnated in filter paper disks or strips that are inoculated with the organism to be tested. This chart describes the 4-hour broth PYR test. Disks impregnated with the PYR substrate can also be used for an even more rapid test. Dr. S. S. Khoramrooz

The substrate used for the PYR test is L-naphthylamide- β-naphthylamide. This compound is hydrolyzed by a specific bacterial aminopeptidase enzyme. Hydrolysis of the substrate by this enzyme releases free β- naphthylamide,which is detected by the addition of N,N-dimethylaminocinnamaldehyde. This detection reagent couples with the naphthylamide to form a red Schiff base. Dr. S. S. Khoramrooz

2. PYR reagent (0.01% p-dimethylaminocinnamaldehyde) II. Media and Reagents PYR broth (Todd-Hewitt broth with 0.01% L- pyrrolidonyl-ρ-naphthylamide) dispensed into sterile tubes in 0.20-mL volumes. 2. PYR reagent (0.01% p-dimethylaminocinnamaldehyde) III. Quality Control A. Positive control: Enterococcus faecalis or Streptococcus pyogenes B. Negative control: Streptococcus agalactiae Dr. S. S. Khoramrooz

IV. Procedure I. With a sterile bacteriologic loop, pick up the growth of two to three morphologically similar colonies and emulsify them in the small volume of PYR broth. 2. Incubate the tube at 35°C for 4 hours. 3. Add one drop of the PYR reagent and observe for color change. The reaction should be read and recorded 1 minute after the addition of reagent. Dr. S. S. Khoramrooz

V. Results A. Interpretation I. Positive: the development of a deep cherry red color within a minute of addition of the reagent 2. Negative: a yellow or orange color Dr. S. S. Khoramrooz

VI. Procedure Notes It is essential that testing be performed before the PYR test to determine that the organism is a streptococcus (i.e., gram-positive cocci, catalase- negative). Other organisms (e.g., some aerococci, staphylococci, nutritionally variant streptococci, Arcanobacterium haemolyticum) may also be PYR-positive. Dr. S. S. Khoramrooz

CATALASE I. Principle- Catalase is an enzyme that decomposes hydrogen peroxide (H202) into water and oxygen. Chemically, catalase is a hemoprotein, similar in structure to hemoglobin, except that the four iron atoms in the molecule are in the oxidized (Fe3+), rather than the reduced (Fe2+), state. Excluding the streptococci, most aerobic and facultative bacteria possess catalase activity. Hydrogen peroxide forms as one of the oxidative end products of aerobic carbohydrate metabolism. If allowed to accumulate, it is lethal to bacterial cells. Dr. S. S. Khoramrooz

Catalase converts hydrogen peroxide into oxygen and water as shown by the following reaction: The catalase test is most commonly used to differentiate members of the Micrococcaceae from members of the Streptococcaceae. Dr. S. S. Khoramrooz

A. Hydrogen peroxide 3% stored in a brown bottle under refrigeration II. Reagents A. Hydrogen peroxide 3% stored in a brown bottle under refrigeration B. An 18- to 24-hour culture of the organism to be tested III. Quality Control The hydrogen peroxide reagent must be tested with positive and negative control organisms each day or immediately before unknown bacteria are tested. A. Positive control: Staphylococcus aureus B. Negative control: Streptococcus species Dr. S. S. Khoramrooz

IV. Procedure I. With an inoculating needle or a wooden applicator stick, transfer growth from the center of a colony to the surface of a glass slide. 2. Add one drop of 3% hydrogen peroxide and observe for bubble formation. V. Results A. Interpretation The rapid and sustained appearance of bubbles or effervescence constitutes a positive test. Dr. S. S. Khoramrooz

Because some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide, a few tiny bubbles forming after 20 to 30 seconds is not considered a positive test. In addition, catalase is present in red cells, so care must be taken to avoid carryover of red cells with the colony material. Dr. S. S. Khoramrooz

THE END Dr. S. S. Khoramrooz