1. Bacterial identification
Using API® Kits
by
Noman Farrukh
2015-Mphil-1108
Department of Microbiology
University of Veterinary and Animal Sciences, Lahore
2. Introduction
• API= Analytical profile index
• 23 identification database
• 967 bacterial and yeast species
• 56,277 Strains
• API web 1.3.0
• Because of this, only known bacteria can be identified.
3. Various identification systems
• API® Gram negative Identification
• API 20E – 18-24 hour identification of Enterobacteriacae and other
non-fastidious gram negative bacteria
• API Rapid 20E – 4-hour identification of Enterobacteriaceae
• API 20NE – 24 to 48-hour identification of Gram negative non-
Enterobacteriaceae
• API NH – 2-hour identification of Neisseria Haemophilus and
Branhamella catarrhalis
• API Campy – 24-hour identification of Campylobacter species
4. Various identification systems
• API® Gram positive Identification
• API Staph – Overnight identification of clinical staphylococci
and micrococci
• RAPIDEC® Staph – 2-hour identification of the commonly
occurring staphylococci
• API 20 Strep – 4 or 24-hour identification of streptococci and
enterococci
• API Coryne – 24-hour identification of Corynebacteria and
coryne-like organisms
• API Listeria – 24-hour identification of all Listeria species
5. Various identification systems
• Other API System
• API® Anaerobe Identification
• API 20A®– 24-hour identification of anaerobes
• Rapid ID 32A – 4-hour identification of anaerobes
• API® Yeast Identification
• API 20C AUX – 48 to 72-hour identification of yeasts
• Others
• API ® 50 CH – Performance of carbohydrate
metabolism tests
• API ZYM® – Semi quantitation of enzymatic activities
6. Manufacturer and Supplier
• Biomerieux®
France & USA
http://www.biomerieux.com
• Muhammad Shahid iqbal
Director Sales and Marketing
Global Marketing services
+92-321-5011171
Rawalpindi,Pakistan
www.gms-world.com
7. API 20E
• Aim
• Principle
• API test strips consists of microtubes (cupules)
containing dehydrated substrates to detect the
enzymatic activity
fermentation of sugars
• During incubation, metabolism produces colour
changes.
• When the carbohydrates are fermented, the pH within
the cupule changes and is shown by an indicator.
CupuleTube
8. • The API 20E Strip, For Identification Of The
Enterobacteriaceae
• It consists of 20 microtubules.
• These tests are inoculated with a bacterial
suspension that reconstitute the media.
API 20E
9. API 20E
• Kit contains
• API 20e Strip
• Media
• Reagents
• API strip Trays
• API code Chart
• API score Sheet
• API web/API Catalogue
10. Analytical Profile Index (API)
• Material Needed.
1. API 20 E Strips
2. Incubation boxes
3. Report sheets
4. Disposable Plastic pipettes
5. Disposable plastic
inoculating loop
6. 5 ml sterile distilled water
7. Mineral Oil
8. MacConkey agar plate.
• Reagent needed.
1. TDA reagent
2. James reagent
3. VPI reagent
4. VP2 reagent
11. 1. Preparation of the inoculum
• Open an ampule of API NaCl
0.85 % Medium (5 ml) or
use 5 ml of sterile saline
or sterile distilled water
• Use colony from an isolation plate.
12. 2. Preparation of the tray
• Dispense about 5 ml of
tap water into the
tray
15. Create anaerobiosis in the tests ADH, LDC, ODC,
H2S and URE. by overlaying with mineral oil.
Since the media in |CIT|, |VP|, and |GEL|
compartments require oxygen, completely fill both the
cupule and tube of these compartments.
Fill only the tube (and not the cupule) of the other
tests.
3.Inoculation of strip
16.
17. 4. Incubation of strip
• Inoculate and streak MacConkey purity plate.
• Close the incubation box. Incubate the box
along with the MacConkey agar at 37°C for 18-
24 hours.
18. Results and interpretations
• Evaluation of tests, all reactions will be recorded
on the laboratory report and test reagents will be
added to some compartments.
• The seven digit profile number will be determined
so the unknown organism can be looked up in the
API 20E analytical profile index.
• If the MacConkey purity plate give mix culture,
then repeat the test.
19. Results and interpretations
Reveal the test which require the addition of
reagent as follows:
Well Reagent
TDA One drop of TDA reagent
IND One drop of James reagent
VP One drop of VP1 then one drop of VP2
Also perform an oxidase on the purity plate
20. Results and interpretations
• Read the API strip according to the interpretation
table, and record the result on the report sheet.
21. Results and interpretations
TEST REACTION NEGATIVE POSITIVE
ONPG -galactosidase Colourless Yellow (maybe pale)
ADH Arginine dihydrolase Yellow Orange or red
LDC Lysine decarboxylase Yellow Orange or red
ODC Ornithine decarboxylase Yellow Orange or red
CIT Citrate utilisation Light green Blue-green or blue
H2S H2S production Colourless Black
URE Urea hydrolysis Yellow Pink
TDA Tryptophan deamination Yellow Dark brown
IND Indole production Colourless
reagent
Pink
VP Acetoin production Colourless Pink or red
GEL Gelatin hydrolysis Colourless Black diffuse pigment
GLU Glucose fermentation Blue Yellow
MAN Mannitol Blue Yellow
INO Inositol Blue Yellow
SOR Sorbitol Blue Yellow
RHA Rhamnose Blue Yellow
SAC Sucrose Blue Yellow
MEL Melibiose Blue Yellow
AMY Amygdalin Blue Yellow
ARA Arabinose Blue Yellow
Oxidase Cytochrome oxidase Colourless Purple
22. Results and interpretations
• On the report sheet, the test are separated
into groups of three and number 1 , 2 or 4 is
allocated for each test.
24. Results and interpretations
The 7-digit profile is then compared with the
numerical profile in the API 20 E analytical profile
index book to obtain the organism identification.
28. Advantages and Limitations
• Advantages
• Easy to use.
• Economical.
• Better as compared to traditional biochemical
tests.
• Rapid Identification
• Better identification.
29. Limitations
• Based on traditional biochemical testing
rather than more sensitive molecular
diagnostics.
• Results may vary from person to person
• Identify only those species that are already
present in API database
• Interpretation may become difficult at some
levels.
