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Subject Title: API Test Strips - API 20NE Original Date: August 3, 2003 Revision Date:
IDENTIFICATION OF NON-ENTERIC GRAM-NEGATIVE RODS (API 20NE) Principle The API 20NE system facilitates theidentification of non-fastidious Gram-negative rods not belonging to the Enterobacteriaceae within 48 hours. The API 20NE strip consists of microtubes containing dehydrated media and substrates. The media microtubes containing conventional tests are inoculated with a bacterial suspension which reconstitutes the media. After incubation, the metabolic end products are detected by indicator systems or theaddition of reagents. The substrate microtubes contain assimilation tests and are inoculated with a minimal medium. If the bacteria are capable of utilizing the corresponding substrate, then they will grow. Materials API 20NE strips - store at 2-80C 0.85% sterile saline Mineral oil Zinc dust AUX Medium } James Reagent } } Nitrate 1 - store at 2-80C Nitrate 2 - store at 2-80C } Oxidase ReagentProcedure 1. Preparation of Inoculum a) b) Add 2 ml. of 0.85% saline to a sterile test tube. Using a sterile inoculating loop, carefully touch the centre of a well isolated colony (2-3 mm. Diameter) and thoroughly emulsify in the saline. The suspension turbidity should be equal to a 0.5 McFarland standard.
Store at 2-80C
Preparation of the Strip a) b) An incubation tray and lid are suppliedfor each strip. Dispense 5 ml of distilled water in to the tray.
PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page 24
TML/MSH Microbiology Department Policy & Procedure Manual Technical Manual 3. Inoculation of the Strip a) b) c) d) e) f)
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Remove the cap from the tube containing the bacterialsuspension and insert a sterile pipette. Tilt the API 20NE incubation tray and fill the TUBE section of the NO3 to PNPG microtubes by placing the pipette tip against the side of the cupule. Open an ampule of AUX Medium and add 200 uL of the bacterial suspension to the ampule. Mix well with a pipette while avoiding the formation of air bubbles. Using the AUX Medium bacterial suspension, fill both theTUBE and CUPULE section of [GLU] to [PAC]. Do not overfill the cupules. Fill to a flat or slightly convex meniscus. After inoculation, completely fill the CUPULE section of the 3 underlined tests, GLU, ADH and URE tubes with mineral oil. Using the excess bacterial suspension, inoculate an agar slant or plate (nonselective media such as nutrient agar, blood agar or tryptic (trypticase) soy agar issuggested) as a purity check and for oxidase testing, and/or additional biochemical testing. Incubate the slant or plate with the API 20NE strip.
Incubation of the Strip a) After inoculation, place the plastic lid on the tray and incubate the strip for 24 hours at 300C in a non-CO2 incubator.
Reading the Strip a) b) After 24 hours incubation, record all reactions not requiring theaddition of reagents. Perform the oxidase test. A portion of the growth from the agar slate or plate, inoculated from the 20NE bacterial suspension, should be rubbed onto filter paper to which a drop of oxidase reagent (1% tetramethyl-p-phenylenediamine dihydrochloride) has been added. The area where the growth has been added will turn dark purple within 10 seconds if the reaction is positive andwill be colourless or light purple if negative. Note: (a) Nichrome wire loops should NOT be used in performing the oxidase test. Nichrome wire can cause a false positivereaction. (b) The oxidase test should NOT be performed using bacterial growth from selective media such as MacConkey, EMB, etc.
PROCEDURE MANUAL TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT Page...