Book on the ninth penalty Biological Laboratory
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 3 of 7
Technical Manual
c) If the GLU is positive (yellow):
i. Perform the oxidase test.
A portion of the growth from the agar slate or plate, inoculated from the
20E bacterial suspension, should be rubbed onto filter paper to which a
drop of oxidase reagent (1% tetramethyl-p-phenylenediamine
dihydrochloride) has been added. Th e area where the growth has been
added will turn dark purple within 10 seconds if the reaction is positive
and will 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 positive
reaction.
(b) The oxidase test should NOT be performed using bacterial
growth from selective media such as MacConkey, EMB,
etc.
Note: (a) Before addition of reagents, observe GLU tube (positive or
negative) for bubbles.
(b) The nitrate reduction and indole tests must be performed
last since these reactions re lease gaseous products which
interfere with the interpretati on of other tests on the strip.
The plastic incubation lid shoul d not be replaced after the
addition of these reagents.
ii. Add the reagents to TDA and VP tubes. If positive, the TDA reactions
will be immediate, whereas the VP reaction may be delayed up to 10
minutes.
iii. The Kovacs' reagent should then be added to the IND tube.
iv. The Nitrate Reduction test should be performed on all oxidase positive
organisms. The reagents should be added to the GLU tube after the
Kovacs Reagent has been added to the IND tube.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 20
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 4 of 7
Technical Manual
Interpretation
a) Use the API 20E analytical profile index. (For 18-24 hour tests, use white pages. For 36-48 hour tests, use blue pages.)
b) The tests are separated into groups of three. The following numerical value is assigned to
each reaction recorded:
1- positive reaction in the fi rst test of the group
2- positive reaction in the second test of the group
4- positive reaction in any test
0- negative reaction in any test
Reference
1. Murray P.A., et al. Manual of Clinical Microbiology, 7
th
ed., 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 21
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 5 of 7
Technical Manual
SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE
TUBE INCUBATION POSITIVE NEGATIVE COMMENTS
ONPG Yellow Colourless (1) Any shade of yellow is a positive
reaction.
(2) VP tube, before the addition of reagents,
can be used a negative control.
ADH 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Orange reactions occurring at 36-48 hours
should be interpreted as negative.
LDC 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Any shade of orange within 18-24 hours is a
positive reaction.
At 36-48 hours, ora nge decarboxylase
reactions should be interpreted as negative.
ODC 18-34 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
Orange reactions occurring at 36-48 hours
should be interpreted as negative.
CIT Turquoise or
Dark Blue
Light Green
Or Yellow
(1) Both the tube and cupule should be filled.
(2) Reaction is read in the aerobic (cupule)
area.
H2
S Black Deposit No Black Deposit (1) H2
S production may range from a heavy
black deposit to a very thin black line
around the tube bottom. Carefully
examine the bottom of the tube before
considering the reaction negative.
(2) A "browning" of the medium is a
negative reaction unless a black deposit
is present. "Browni ng" occurs with TDA
positive organisms.
URE 18-24 hr
36-48 hr
Red or Orange
Red
Yellow
Yellow or Orange
A method of lower sensitivity has been chosen .
Klebsiella, Proteus and Yersinia routinely give
positive reactions.
TDA Add 1 drop 10% Ferric chloride.
Brown-Red
Yellow
(1) Immediate reaction.
(2) Indole positive organisms may produce a
golden orange colour due to indole
production. This is a negative reaction.
IND Add 1 drop Kovacs Reagent
Red Ring
Yellow
(1) The reaction should read within 2
minutes after the addition of the Kovacs
reagents and the results recorded.
(2) After several minutes, the HCl present in
Kovacs reagent may react with the plastic
of the cupule resulting in a change from a
negative (yellow) colour to a brownish-red. This is a negative reaction.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 22
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 6 of 7
Technical Manual
SUMMARY OF RESULTS - 18-24 HOUR PROCEDURE (cont'd)
TUBE INCUBATION POSITIVE NEGATIVE COMMENTS
Add 1 drop of 40% Potassium Hydroxide, then 1 drop of alpha-napthol.
VP
Red Colourless
(1) Wait 10 minutes before considering the
reaction negative.
(2) A pale pink colour which appears
immediately after the addition of reagents
but which turns dark `pink or red after 10
minutes should be interpreted as positive.
Motility may be observed by hanging drop or
wet mount preparation.
GEL Diffusion of the
pigment
No diffusion (1) The solid gelatin particles may spread
throughout the tube after inoculation.
Unless diffusion occurs, the reaction is
negative.
(2) Any degree of diffusion is a positive
reaction.
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
Yellow
Or Gray
Yellow
Blue or
Blue-Green
Blue or
Blue-Green
COMMENTS FOR ALL CARBOHYDRATES
Fermentation
(Enterobacteriaceae, Aeromonas, Vibrio)
(1) Fermentation of the carbohydrates begins
in the most anaerobic portion (bottom) of
the tube. Therefore, these reactions should
be read from the bottom of the tube to the
top.
(2) A yellow colour at the bottom of the tube
only indicates a weak or delayed positive
reaction.
Oxidation (Other Gram-negatives)
(1) Oxidative utilization of the carbohydrates
begins in the most aerobic portion (top) of
the tube. Therefore, these reactions should
be read from the top to the bottom of the
tube.
(2) A yellow colour in the upper portion of the
tube and blue in the bottom of the tube
indicate oxidative utilization of the sugar.
This reaction should be considered
positive only for non- Enterobacteriaceae
gram negative rods. This is a negative
reaction for fermentative organisms such
as Enterobacteriaceae.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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