Book on the eighth penalty Biological Laboratory
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\01\v01 Page 2 of 4
Technical Manual
Procedure
1. Preparation of Inoculum
a) Only pure cultures of a single organism should be used (heavily inoculated sheep
BAP x 3; incubate for 24 hours at 35
0
C in 5% CO 2
).
b) Using a sterile swab, harvest all the culture from 3 BAP and inoculate into 3 ml.
sterile saline to give a turb idity of at least McFarland #6.
2. Preparation of the Strip
a) An incubation tray is supplied for each strip.
b) Dispense 5 ml of water into the wells of the tray.
3. Inoculation of the Strip
a) Inoculate tests 1 → 11 of the strip (NIT to GEL).
b) Avoid bubbles by tilting the strip slightly forward while placing the pipette tip on
the side of the cupule.
c) Add 3 drops into each cupule for tests NIT to ES.
d) For the URE test fill the tube portion only.
e) For the GEL test, fill both the tube and cupule. Then:
f) For the last nine tests of the strip (O to GLYG transfer the rest of the bacterial
suspension to an ampoule of GP medium . Mix well.
g) Distribute the new suspension into the tubes only of tests O to GLYG
h) Overlay cupules URE and O to GLYG with mineral oil, forming a slight convex
meniscus.
i) Cover with incubation lid and incubate the strip for 24 hours at 35
0
C (non-CO2
).
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\01\v01 Page 3 of 4
Technical Manual
Interpretation
REACTIONS TABLE
TESTS REACTONS NEGATIVE
RESULTS
POSITIVE
RESULTS
NIT NITrate reduction NIT A + NIT B (10 mn)
Colourless Very pale pink
Dark pink
Red
PYZ PYraZinamidase PYZ (10 mn)
Colourless Very pale brown
Very pale orange
Brown
Orange
PyrA Pyrrolidonyl Arylamidase ZYM A +ZYM B
PyrA → B
NAG (10 mn)
Colourless Pale orange
Orange
PAL Alkaline Phosphatase Colourless
Beige-pale purple
Pale orange
Purple
β GUR Beta GlucURonidase Colourless
Pale grey
Pale beige
Blue
β GAL Beta GALactosidase Colourless
Beige-pale purple
Purple
∝ GLU
Alpha GLUcosidase Colourless
Beige-pale purple
Pale green
Purple
BNAG N-Acetyl-B Glucosaminidase Colourless
Beige-pale purple
Pale brown
Pale grey
Brown
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY
Page 16
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\01\v01 Page 4 of 4
Technical Manual
REACTIONS TABLE (Cont'd)
TESTS REACTONS NEGATIVE
RESULTS
POSITIVE
RESULTS
ESC ESCulin ( β Glucosidase)
Colourless
Grey
Black
URE UREase Yellow Orange
Red
Pink
[GEL] GELatine (hydrolysis) No diffusion
of black pigment
Diffusion of
black pigment
O
GLU
RIB
XYL
MAN
MAL
LAC
SAC
GLYG
Control (Fermentation )
GLUcose }
RIBose }
XYLOSE }
MANnitol } Fermentation
MALtose }
LACtose }
Sucrose }
GLYcoGen }
Red
Orange
Yellow
Yellow-orange
CAT CATalase (ESC or GEL test) H 2 O2
3% 1 min
No bubbles Bubbles
References
1. Coyle, Marie B., Benjamin Lipsky. 1990. Cor yneform Bacteria in Infectious Diseases:
Clinical and Laboratory Aspects. Clinical Microbiology Reviews. 3:227-246.
2. Freney, J.M.T. Duperron, C. Couturier, W. Hansen, F. Allard, J.M. Boueufgras, D.
Monget, J. Fleurette. Evaluation of API Coryne in Comparison with Conventional
Methods for Identifying Coryneform Bacteria, Journal of Clinical Microbiology, January
1991, Vol. 29, p. 38-41.
3. Murray P.A., et al. Manual of Clinical Microbiology, 7
th
ed., 1999.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 17
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 1 of 7
Section: Technical Manual Subject Title: API Test Strips - API 20E
Issued by: LABORATORY MANAGER Original Date: July 31, 2000
Approved by: Laboratory Director
Revision Date: February 15, 2002
IDENTIFICATION OF ENTEROBACTERIACEAE (API 20E)
Principle
The API 20E system facilitates the 24-hour identification of Enterobacteriaceae as well as 24 or
48-hour identification of othe r Gram negative bacteria.
The API 20E strip consists of microtubes containing dehydrated substrates for the demonstration
of enzymatic activity and carbohydrate (CHO) fermen tation. The substrates are reconstituted by
adding a bacterial suspension. After incubation, the metabolic end products are detected by
indicator systems or the addition of reagents. CHO fermentation is detected by colour change in
the pH indicator.
Materials
API 20E strips - store at 2-8
0
C
0.85% sterile saline
Nitrate A - store at 2-8
0
C
Nitrate B - store at 2-8
0
C
Mineral oil
Zinc dust
Kovacs Reagent
Voges - Proskauer Reagents
Ferric Chloride Store at 2-8
0
C
H2 O2
Oxidase Reagent
OF Dextrose ID of non-
Motility Medium Enterobacteriaceae
Procedure
1. Preparation of Inoculum
a) Add 5 ml. of 0.85% saline to a sterile test tube.
b) Using a sterile inoculating loop, carefully touch the centre of a well isolated
colony (2-3 mm. Diameter) and thoroughly emulsify in the saline.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
Page 18
TML/MSH Microbiology Department
Policy & Procedure Manual
Policy # MI\TECH\04\02\v01 Page 2 of 7
Technical Manual
2. Preparation of the Strip
a) An incubation tray and lid is supplied for each strip.
b) Dispense 5 ml of water in to the tray.
3. Inoculation of the Strip
a) Remove the cap from the tube containing the bacterial suspension and insert a 5
ml. Pasteur pipette.
b) Tilt the API 20E incubation tray and fill the tube section of the microtubes by
placing the pipette tip against the side of the cupule.
Note: The ADH , LDC , ODC , H2S , AND URE reactions can be interpreted best
if these microtubes are slightly underfilled.
c) Fill both the TUBE and CUPULE section of [CIT ], [VP ] and [GEL ] tubes.
d) After inoculation, completely fill the cupule section of the ADH , LDC , ODC ,
H2S and URE tubes with mineral oil.
e) Using the excess bacterial suspension, i noculate an agar slant or plate (non-selective media such as nutrient agar, blood ag ar or tryptic (tryp ticase) soy agar is
suggested) as a purity check and for oxida se testing, serology, and/or additional
biochemical testing. Incubate the slant or plate for 18-24 hours at 35
0
C.
4. Incubation of the Strip
a) After inoculation, place the plastic lid on the tray and incubate the strip for 18-24
hours at 35
0
C in a non-CO2
incubator.
b) Weekend incubation: The biochemical reactions of the API 20E should be read
after 18-24 hours incubation. If the strips cannot be read after 24 hours
incubation at 35
0
C, the strips should be removed from the incubator and stored at
2-8
0
C (refrigerator) until the reactions can be read.
5. Reading the Strip
a) After 18 hours of incubation and before 24 hours incubation, record all reactions
not requiring the addition of reagents.
b) If the GLU tube is negative (blue or green), do not add reagents. Reincubate a
further 18-24 hours.
PROCEDURE MANUAL
TORONTO MEDICAL LABORATORIES / MOUNT SINAI HOSPITAL MICROBIOLOGY DEPARTMENT
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