Analytical Profile Index (API) & Automated Machines. principle and procedure

1. Analytical Profile Index (API)
& Automated machines
Zari Esa M.Saleh
MSc. Medical Microbiology

2. Analytical Profile Index (API)
Analytical Profile Index or API are commercial
miniaturized biochemical test panels that cover a
significant number of clinically-important groups
of bacteria, as well as food- and water-associated
microorganisms.
API is a classification of bacteria based on
experiments, allowing fast identification (rapid
identification).
The system is developed for quick identification of
clinically relevant bacteria.
Only known bacteria can identified.

3. Analytical Profile Index (API)
API test strips consists of microtubes (cupules)
containing dehydrated substrates to detect the
enzymatic activity or the fermentation of sugars
by the inoculated organisms.
During incubation, metabolism produces colour
changes that are either spontaneous or revealed
by the addition of reagents
When the carbohydrates are fermented, the pH
within the cupule changes and is shown by an
indicator.

4. Analytical Profile Index (API)
API Gram negative Identification
1. API 20E. 18-24 hour identification of
Enterobacteriacae and other non-fastidious gram
negative bacteria.
2. API Rapid 20E. 4-hour identification of
Enterobacteriaceae.
3. API 20NE. 24 to 48-hour identification of Gram
negative non-Enterobacteriaceae.
4. API NH. 2-hour identification of Neisseria
Haemophilus and Branhamella catarrhalis.
5. API Campy. 24-hour identification of
Campylobacter species

5. Analytical Profile Index (API)
API Gram positive Identification
1. API Staph. Overnight identification of clinical
staphylococci and micrococci.
2. RAPIDEC Staph. 2-hour identification of the
commonly occurring staphylococci.
3. API 20 Strep. 4 or 24-hour identification of
streptococci and enterococci.
4. API Coryne. 24-hour identification of
Corynebacteria and coryne-like organisms.
5. API Listeria. 24-hour identification of all Listeria
species

6. Analytical Profile Index (API)
Other API System include:
Anaerobe Identification
1. API 20A. 24-hour identification of anaerobes.
2. Rapid ID 32A. 4-hour identification of anaerobes
API Yeast Identification
1. API 20C AUX. 48 to 72-hour identification of yeasts.
Others
1. API 50 CH. Performance of carbohydrate
metabolism tests.
2. API ZYM. Semiquantitation of enzymatic activities

7. The API 20E Strip, For Identification Of The
Enterobacteriaceae and other Gram negative rods,
it consists of 20 microtubules containing dehydrated
substrates.
These tests are inoculated with a bacterial
suspension that reconstitute the media.
During incubation, the metabolism of the organism
produces color changes that are either spontaneous
or revealed by the addition of reagent.
Analytical Profile Index (API)

8. 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

9. Preparation of the inoculum
Open an ampule of API NaCl 0.85 % Medium (5
ml) or use any tube containing 5 ml of sterile
saline or sterile distilled water, without additives.
Use colony from an isolation plate.
It is recommended to use young cultures (18-24
hours old).
Carefully emulsify to achieve a homogeneous
bacterial suspension.
This suspension must be used immediately after
preparation.

10.  Dispense about 5 ml of tap water into the tray
 Vortex mix the saline suspension to get uniform
dispersal
 Inoculate all the tubes on the test strip with the
pipette by depositing the suspension into the
cupules as you tilt the API tray
Preparation of the inoculum

13.  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.
 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.
Preparation of the inoculum

15. 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.

16. Results and interpretations
 Reveal the test which require the addition of reagent as
follows:
1. Vp test: add one drop of VPI and VP2 reagent and wait for
10 min for. color development. The pale pink color that
occurs immediately has no significance . A positive
reaction is dark pink or red.
2. TDA test: add one drop of TDA reagent. A positive
reaction brown-red will occur immediately. A negative
reaction color is yellow.
3. IND test: add one drop of James reagent. Look for the
positive red ring reaction within 2 minutes. After several
minutes the acid in the reagents reacts with the plastic
cupule to produce a color change from yellow to
brownish-red, which consider negative .

17. Results and interpretations
Read the API strip according to the interpretation
table, and record the result on the report sheet.
On the report sheet, the test are separated into
groups of three and number 1 , 2 or 4 is allocated
for each test.
By adding the numbers corresponding to the
positive reaction within each group, a 7-digit
profile number is obtained for 20 tests of the API
20E strip.

18. 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.

19. 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

20. VITEK 2 system
The VITEK 2 system has everything healthcare
laboratories need for fast, accurate microbial
identification, and antibiotic susceptibility testing.
The VITEK 2 compact system is a fully automated
system that performs bacterial identification by
biochemical analysis using colorimetry.
It focuses on the clinical microbiology laboratory
and provide increased levels of automation and
capacity for higher volume laboratories.

21. VITEK 2 system
• The VITEK 2 compact system offers Advance
Colorimetric technology that allows for the
identification of 98% of clinical isolates.

22. VITEK 2 system
VITEK 2 system can accurate identify of some
bacterial strains in as little as two hours.
In addition to being able to identify bacteria, the
VITEK 2 compact system is able to identify
multiple species of yeast.
They also provide an option of automatic
pipetting and dilution for antimicrobial
susceptibility testing.

23. VITEK 2 system
There are currently four reagent cards available
for the identification of different organism classes
as follows:
1. GN. Gram-negative fermenting and non-
fermenting bacilli.
2. GP. Gram-positive cocci and non-spore-forming
bacilli.
3. YST. Yeasts and yeast-like organisms.
4. BCL. Gram-positive spore-forming bacilli

24. VITEK 2 system
The reagent cards have 64 wells that can each
contain an individual test substrate.
Substrates measure various metabolic activities
such as acidification, alkalinization, enzyme
hydrolysis, and growth in the presence of
inhibitory substances.

25. VITEK 2 system
The reagent cards have 64 wells that can each
contain an individual test substrate.
Substrates measure various metabolic activities
such as acidification, alkalinization, enzyme
hydrolysis, and growth in the presence of
inhibitory substances.

26. Phoenix
Phoenix automated microbiology system is
intended for the in vitro rapid identification (ID)
of Gram Positive bacteria from pure culture
belonging to the genera Staphylococcus,
Enterococcus, and other Gram Positive cocci and
Gram Positive bacilli.
The Phoenix Automated Microbiology System is
also intended for the quantitative determination
of antimicrobial susceptibility by minimal
inhibitory concentration (MIC) of most Gram
Positive bacteria from pure culture belonging to
the genera Staphylococcus and Enterococcus.