.
1.0 Equipments Required
LAF
Measuring Cylinder
Sterile Petri Plates
2.0 Material Required
Disinfectant
Sterile Saline solution
Sterile & Molten cooled Soybean casein digest agar
Test
organism – E. coli, Staph. aureus, Ps. aeruginosa, C. albicans, Salmonella.
3.0 Procedure:
3.1 From a recently grown
stock culture of each of the test organisms; subculture on the surface of
respective media slants as given below.
S.No.
|
Organism
|
Type
|
Medium
|
Incubation
temperature and duration
|
1
|
Candida
albicans
|
MTCC 227
|
Sabouraud
Dextrose Agar
|
20-25°C for
48 hours
|
2
|
E.coli
|
MTCC4313
|
Soybean Casein
Digest Agar
|
30-35°C for 24
hours
|
3
|
Ps. aeruginosa
|
MTCC1688
|
Soybean Casein
Digest Agar
|
30-35°C for 24
hours
|
4
|
S. aureus
|
MTCC737
|
Soya bean
Casein
Digest Agar
|
30-35°C for 24
hours
|
5
|
Salmonella
|
MTCC 3858
|
Soya bean
Casein
Digest Agar
|
30-35°C for 24
hours
|
3.2 Using sterile solution about 10 ml of sterile
normal saline with peptone (0.1 % w/v), harvest the cultures and dilute
suitably with sterile saline solution to bring the count to about 1 X 108 per
ml
3.3 Determine the number of cfu per ml in each
suspension by a ten-fold serial dilution .
3.4 Transfer 1.0 ml of culture suspension to a
tube containing 9.0 ml of sterile saline solution to obtain 10-1 dilution.
3.5 Transfer 1.0 ml of the 10-1 dilution
to a tube containing 9.0 ml of sterile saline solution to obtain test dilution
10-2.
3.6 Similarly serially dilute the culture
suspension to obtain dilution of 10-3, 10-4, 10-5, 10-6,
10-7 and 10-8.
3.7 Plate 1.0 ml from test dilution 10-6,
10-7 and 10-8 in duplicate into 90 mm sterile
petri plates.
3.8 Pour approximately 20 to 25 ml of sterile
molten cooled soybean casein digest agar in each plate.
3.9 Carry out the same dilution method for all
the cultures and label accordingly with dilution number and test organism.
3.10 Allow to solidify the plates at room
temperature, invert and incubate individually as specified in above table.
3.11 Count the number of colonies and find out the
concentration of culture suspension and tabulates the results in specified
format.
3.12 Apply the above known culture cells (approx
108 cfu) individually to a surface of 25 cm2.
3.13 Allow to air dry the applied cultures at room
temperature.
3.14 Prepare the disinfectant solution to be
tested as per SOP for Preparation of Disinfectant Solution and apply on the
surface of artificially contaminated area.
3.15 Take a swab immediately at 0 min, after 5
min, 10 min, 20 min and after 30 minutes, and then proceed the swab testing as
per SOP for Swab Testing.
3.16 Incubate
the plates as specified in above table and tabulate the result in specified
format.
3.17 The
tested sample is effective at specified concentration when there is no growth
in swab tested after 20 and 30 minutes for each culture.
4.0 Precaution
4.1 Artificially contaminated area should be
cleaned immediately after completion of work.
4.2 Use hand gloves and nose mask during testing.
4.3 All the glassware used for dilution and
testing, immediately clean and sterilize as per SOP.
Related: Microbial Resistance against Disinfectants
Related: Microbial Resistance against Disinfectants
5.0 Frequencies
For each consignment6.0 Abbreviation
SOP : Standard Operating Procedure
QA : Quality Assurance
QC : Quality Control
NA : Not Applicable
WFI : Water for Injection
what volume of culture suspension and disinfectant do you apply on the surface?
ReplyDeleteWhat volume of culture suspension and disinfectant do you apply on the surface?
ReplyDelete