Ex # 24, p 61-64 API 20E Multiple Test Strip

Ex # 24, p 61-64
API 20E Multiple Test Strip
Clinical Rapid Identification of Enterobacteriaceae and other Gram Negative Rods
Additional references:
1. Textbook
a. Ch
2. Photo Atlas
a. p 45-46
Notes:
1. Prepare a suspension of the bacteria in the saline
tube: Inoculate a large colony (2-3mm diameter) of
the bacterium (pure culture) into the 0.85% NaCl
solution, making sure that the suspension is
homogenous and without clumps of floating bacteria
2. Inoculate the API strip:
a. Holding the strip at a slight angle up from the
table top, inoculate the bacterial suspension
into each well with a sterile pipette
b. Touch the end of the pipette to the side of the cupule, allowing capillary action to
draw the fluid into the well as you slowly squeeze the bulb. This should eliminate
any bubbles forming in the wells. Each well should be filled up to the neck (see
diagram)
c. CIT, VP, and GEL have boxes around their names. These test wells will be
filled all the way up to the top of the well
d. LDC, ODC, ADH, H2S, and URE are filled as described in step B, but they
will then be filled up to the top with sterile mineral oil
3. Incubate the strip in its chamber:
a. The bottom of the incubation chamber has small indented wells: fill it with water
just enough to fill these indentations
b. Place the strip onto the bottom of the chamber. There should not be so much
water that it slops onto the top of the API strip
c. Place the top of the incubation chamber over the bottom, and label it
d. Place the strip at 37o C for 18-24 hours
4. Interpretation:
a. Add the proper reagents to the compartments:
I.
II.
III.
1 drop of Kovac's to the IND (read within a couple of minutes)
1 drop of Barritt's A and B to VP (a + reaction may take up to 10 minutes)
1 drop of FeCl3 to TDA
b. Read all other tests as described (chart below) without reagents
c. Record results on the diagram handed out to you in lab
I.
II.
1, 2, or 4 points for + reaction, 0 points for - reaction
The oxidase test reaction should be negative, and is added as the last test
result
d. Three test reactions are added together to give a 7-digit number, which can then
be looked up in the codebook
READING THE API 20
TESTS
SUBSTRATE
REACTION TESTED
- RESULTS
+ RESULTS
ONPG
ONPG
beta-galactosidase
colorless
yellow
ADH
arginine
arginine dihydrolase
yellow
red/orange
LDC
lysine
lysine decarboxylase
yellow
red/orange
ODC
ornithine
ornithine decarboxylase
yellow
red/orange
CIT
citrate
citrate utilization
pale green/yellow
blue-green/blue
H2S
Na thiosulfate
H2S production
colorless/gray
black deposit
URE
urea
urea hydrolysis
yellow
red/orange
TDA
tryptophan
deaminase
yellow
brown-red
IND
tryptophan
indole production
yellow
red (2 min.)
VP
Na pyruvate
acetoin production
colorless
pink/red (10 min.)
GEL
charcoal gelatin
gelatinase
no diffusion of black
black diffuse
GLU
glucose
fermentation/oxidation
blue/blue-green
yellow
MAN
mannitol
fermentation/oxidation
blue/blue-green
yellow
INO
inositol
fermentation/oxidation
blue/blue-green
yellow
SOR
sorbitol
fermentation/oxidation
blue/blue-green
yellow
RHA
rhamnose
fermentation/oxidation
blue/blue-green
yellow
SAC
sucrose
fermentation/oxidation
blue/blue-green
yellow
MEL
melibiose
fermentation/oxidation
blue/blue-green
yellow
AMY
amygdalin
fermentation/oxidation
blue/blue-green
yellow
ARA
arabinose
fermentation/oxidation
blue/blue-green
yellow
OX
oxidase
oxidase
colorless/yellow
violet
QUESTIONS:
1. What is the purpose of the water in the tray?
2. What is the function of the mineral oil?
3. What are the advantages of this test (compared to regular biochemical tube media)?
4. What are the disadvantages of this test?
Good web sites for ‘extra’ information:
http://www.jlindquist.net/generalmicro/102bactid2.html
www.biology.ed.ac.uk/.../microbes/proteus.htm
http://pages.usherbrooke.ca/biomedias/techniques_microbio.htm
O
AL OCHUT I
GGMI S R S MA A
culture N
V
D D D I 2 R D N E L A N O H A E M R identification
no. P
P
HC C T S E AD L UN OR AC L Y A
G
8101 + – + + – – – – + – – + + – + + + + – +
Escherichia
coli