1.0 Objective:
The purpose of this SOP is to provide guidelines for the
procedure of Growth
Promotion Test to be performed for different media.
2.0 Scope:
This SOP covers the media use in microbiological work
3.0 Responsibility:
Microbiologist:
Responsible for preparation the media, handling the culture and maintain the necessary record.
QA Officer/QA Manager: Review the records and governing the document.
4.0 Procedure:
4.1 Procedure for agar medium use for total
aerobic count test and maintenance of micro- organisms:
4.1.1 Prepare the media as per preparation for mibiological media
4.1.2 Transfer the media to the LAF bench.
4.1.3 Prepare a dilution of the organism shown in
the table using Sterile Saline to obtain a count of 10 to 100 cfu/ml as
follows:
4.1.3.1 Sterilize an inoculation loop by burning over
the flame till red hot and allow it to cool.
4.1.3.2 Using the above loop, transfer a loop full of
the required bacterial culture to 10 ml of Soyabean Casein Medium and fungal
& yeast culture to 10 ml Sabouraud Dextrose Broth.
4.1.3.3 Incubate the medium for 18 to 24 hours at 30°
to 35° C for bacteria and at 20° to 25° C for yeast and mold.
4.1.3.4 Make serial dilution using Sterile Saline up to
106 and further if required.
4.1.3.5 Using
pre-sterilized pipette, transfer 1 ml of the prepared dilutions to 2
pre-sterilized petri-plates each and pour 15 to 20 ml of the sterile liquified Soyabean
Casien Digest
agar
at a temperature of NMT 45° C for
bacteria.
4.1.3.6 Using pre-sterilized pipette, transfer 1 ml of
the prepared dilutions to 2 pre-sterilized petri-plates each and pour 15 to 20 ml of the
sterile liquified Sabouraud Dextrose Agar at a temperature of NMT 45° C for yeast
and mold.
4.1.3.7 Incubate the plates for 48 to 72 hours.
4.1.3.8 Store the dilutions in the refrigerator for the
growth promotion test and use within 48 to 72 hours.
4.1.3.9 Record the total number of colonies in the
plates and identify the dilution giving approximately between 10 to
100 cfu/ml and use this dilution for growth promotion test in next batch of
media.
4.1.4 Using pre-sterilized pipette transfer 1 ml
of the identified dilution to 2 pre-sterilized petri-plates each and pour 15
to 20 ml of the Sterile Liquified Media at a temperature of NMT 45° C with reference to
Table - 1. Similarly stores pre-sterilized media at room temperature and
inoculate with the identified dilution at the end of 1 week, 15 days and 30 days.
4.1.5 Mix the plates by rotating on the LAF bench
such that the media does not touch the lid.
4.1.6 Allow the agar to set at room temperature.
4.1.7 Incubate at the conditions specified in
Table - 1 in inverted condition.
4.1.8 The media under test satisfies the test if a
clearly visible early growth of the micro- organism occurs.
4.2. Procedure for media used in test for
specified micro-organisms:
4.2.1 Lactose broth:
Inoculate
loopful of culture of E coli or Salmonella abony in 10 ml of
broth and incubator for 18 - 24 hours
at 30° - 35° C and observe the growth. Record the observations.
4.2.2 Nutrient broth:
Inoculate loopful of culture of E coli or
Salmonella abony in 10 ml of broth and incubator
for 18
- 24 hours at 30° - 35° C and observe the growth. Record the observations.
4.2.3 Maconkey broth, Maconkey agar, EMB agar, EE
broth, VRA agar :
4.2.3.1 Inoculate loopful of E. coli in 10 ml
lactose broth, incubate at 30° - 35° C for 18 - 24 hours and observe the growth.
4.2.3.2 From 24 hours culture of E coli loopful
of culture inoculate in broth medium and streak on agar surface and incubate
at 35° - 37° C for 18 - 48 hours and
observe the growth.
Record the observations.
4.2.4 Tetrathionate broth, Selenite broth,
Brillian green agar, Deoxycholate citrate agar, Bismuth sulphite agar, TSI agar,
Urea broth :
4.2.4.1 Inoculate loopful of Salmonella abony in
10 ml lactose broth or TSB and incubate at
30° - 35° C for 18 - 24 hours and
observe the growth.
4.2.4.2 From 24 hours old culture of Salmonella a transfer
loopful culture in Tetrathionate broth and Selenite broth and Incubate for 18 -
24 hours at 35° - 37° C.
4.2.4.3 From TT broth or selenite broth transfer
loopful of culture and streaked on said agar and incubate at 35°- 37° C
for 18 - 48 hours and observe the growth. Record the observations.
4.2.4.4 Loopful of culture of Salmonella abony
inoculate in urea broth and incubate and check for colour change.
4.2.4.5 Loopful of culture of Salmonella abnoy streak
on surface and stab with inoculation needle in TSI agar slant
and incubate at 35° - 37° C for 18 - 48 hours and observe the growth. Record the
observations.
4.2.5 Cetremide agar, Pseudomonas agar for
pyocyanin, Pseudomonas agar for fluorescein :
4.2.5.1 Inoculate loopful of P-aeruginosa in 10
ml TSB and incubate at 30° - 35° C for 18 - 24 hours and observe the growth.
4.2.5.2
From 24 hours old culture of P-aeruginosa
streak loopful on cetrimide agar incubate at
35°
- 37° C for 18 - 48 hours and observe
the growth
4.2.5.3 From cetremide agar transfer isolated colony
and streak on Pseudomonas agar for fluorescein and
Pseudomonas agar for pyocyanin and incubates at 35° - 37° C for 18 - 48 hours and observes the
growth. Record the observations.
4.2.6 Vogel Johnson
agar, Manitol salt agar :
4.2.6.1 Inoculate loopful of S.aureus in 10 ml
TSB and incubate at 30° - 35° C for 18 - 24 hours and observe the growth.
4.2.6.2 Streak
loopful of 24 hours old culture of S.aureus on VJA and Manitol salt agar
and incubate at 35° - 37° C
for 18 - 24 hours and observe the growth.
4.3 B12 assay agar, B12 culture agar :
4.3.1 Prepare suspension of E. coli
(mutant) culture in normal saline by adding one loopful of culture into 25 ml of normal saline.
4.3.2 Transfer 1 ml of suspension into two
Petri-plates and pour 15 ml to 18 ml of B12 assay agar and B12 culture
agar. Mix by rotating the plates. Allow it to solidify.
4.3.3 Incubate inverted plates at 30° - 35° C for
18 - 24 hours. Observe for growth exhibition.
4.4 Record
the relevant information’s in the Growth promotion test log book in log book
4.5 Affix approved labels on the media lot
containers passing Growth promotion test bearing the following details:
|
APPROVED
|
|
A.R.No :
Date of Received :
Date of Opening :
Opened By :
|
|
|
5.0 Precautions:
5.1 Handle the tubes with the culture
carefully to avoid direct contact.
5.2 Ensure use of hand gloves and nose mask
during the experiment.
5.3 In case of accidental spillage of the
culture on the skin or clothing wipe the area with 70 % IPA and wash
immediately with 2 % Dettol / savlon, rinse the clothing in soap solution immediately.
6.0 Frequency:
Growth
promotion test shall be done for each lot or batch of dehydrated media received
in the lab.
