C-1 Catalase I. Principle Catalase is an enzyme that decomposes hydrogen peroxide (H 2 O 2 ) into water and oxygen. Chemically, cat- alase is a hemoprotein, similar in structure to hemoglobin, except that the four iron atoms in the molecule are in the oxidized (Fe 3+ ), rather than the reduced (Fe 2+ ), state. Excluding the streptococci, enterococci, and a few other genera, 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. Catalase converts hydrogen peroxide into oxygen and water as shown by the following reaction: 2H 2 O 2 → 2H 2 O + O 2 (gas bubbles) The catalase test is most commonly used to differentiate members of the Micrococcaceae, which are posi- tive, from members of the Streptococcaceae. 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 IV. Procedure 1. 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. Because some bacteria possess enzymes other than catalase that can decompose hydrogen peroxide, a few tiny bubbles forming after 20–30 seconds is not considered a positive test. In addition, catalase is present in red blood cells; so care must be taken to avoid carryover of red blood cells with the colony material. Bile-Solubility Test I. Principle Bile salts, specifically sodium deoxycholate and sodium taurocholate, have the capability to lyse Streptococ- cus pneumoniae selectively when added to actively growing bacteria in agar or broth media. S. pneumoniae produces autolytic enzymes (autolysins) that account for the central depression or umbilication character- istic of older pneumococcal colonies on agar media. The addition of bile salts activates the autolysins and accelerates the natural lytic reaction observed with cultures or pneumococci. CHART 1-2 CHART 1-1 Charts