Growth Promotion Test for Culture Media
This Document describes (Standard Operating procedure) SOP for Growth Promotion Test for Microbiological Culture Media
SOP for Growth Promotion Test for Microbiological Culture Media
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Growth Promotion Test for Microbiological Culture Media.
- This Standard operating procedure shall be applicable for Growth Promotion Test for Microbiological Culture Media used in microbiology Department.
II. Responsibilities:
- All Quality Control and Quality Assurance personnel shall be responsible to follow and implement this SOP.
III : Introduction and Procedural Part :
Preparation of Media
- Prepare and sterilize the media as per procedure respective SOP.
- Growth Promotion Test shall be done for each received lot of dehydrated or ready to use media and each In-house lot of prepared media.
Growth Promotion Test (Dehydrated Media Container) :
- Perform the growth promotion test (GPT) of each received lot of dehydrated media; perform the test on a single container of each batch.
- Prepare and sterilize the media as per the appropriate instructions given by the manufacturer. For solid media, pour the media in petri plates and prepare media plates.
- Use appropriate culture suspensions of test micro-organisms for growth promotion test of media (refer annexure-I).
- For solid media, prepare required no. of plates for GPT. Label them as media name, in-house lot no., name of test organism and date of analysis. Use individual culture suspension containing NMT 100 cfu of test organism to inoculate the media plate. Perform the test by spread plate method.
- For Liquid media, inoculate a portion of the appropriate medium quantity (10 ml to 100 ml) with NMT 100 cfu of specified individual test organisms.
- Use separate plates/tubes/flask for each organism. Always keep an uninoculated media plate/tubes/flask as a negative control.
- Incubate the plates or tubes at respective temperature for specified period of time (refer annexure-I for incubation temperature and time).
- By following the shortest incubation period, growth must be detected in growth promotion test.
- Observe the plate/tube for characteristic growth/turbidity. Record the results in Annexure-II.
- For solid media, record the number of colonies observed as colony forming unit (cfu).The growth promotion test of these media passes, if the number of ‘cfu’ recovered are not differing by more than a factor of 2 from the ‘cfu’ value of the used inoculums.
- In case of Selective solid media (for example: MCA, MSA, CA, XLDA, VRBGA, PAP, PAF, EMB, TSI) desired characteristic growth/ indicative properties and inhibitory properties of the media shall be observed. For growth characteristics refer Annexure-I.
- For the inhibitory test of selective media use atleast 100 cfu of test micro-organism and incubate the plate/tube at specified temperature for longest incubation period. The growth shall not be observed in ‘inhibitory test’ of media.
- For GPT of Liquid media, the clearly visible or luxuriant growth/ turbidity shall be observed in the broth tubes.
- If media passes the GPT test, update the label on media bottles for GPT status. For label details refer Annexure-III.
- If open media container is not finished within 3 months from the date of its opening, then perform the whole GPT analysis (retesting) of that individual container after every 3 months interval (quarterly). Record the results in Annexure-II.
- Affix the ‘Media retest label’ on container mentioning the details of retest number, retest done on and retest due date. Replace the old retest label with new one, after every retest. For label details refer Annexure-IV.
Growth promotion test for In-House lot of media prepared (Positive control/Media fertility test):
- Prepare and sterilize the media as per the appropriate instructions given by the manufacturer. For solid media, pour the media in petri plates and prepare media plates.
- Perform the Media fertility test by using appropriate culture suspension of 10 to 100 cfu of test micro-organism for media fertility test (using only single organism, refer annexure-I).
- For solid media perform the test by spread plate method. For liquid media, use appropriate quantity of medium (10 ml to 100 ml) to inoculate with specified microorganism by direct inoculation method. Consider these as a positive control.
- Keep an uninoculated media plate/tube/flask as a negative control.
- Incubate the plate or tube at respective temperature for specified period of time (refer annexure-I for incubation temperature and time).
- By following the shortest incubation period, growth must be detected in media fertility test.
- Observe the plate/tube for characteristic growth/turbidity. Record the results in Annexure.
- The in-house media lot complies for the Growth Promotion test (GPT), if clear evidence of characteristic growth appears in the inoculated media within the shortest incubation period. No evidence of growth shall be observed in the negative control.
IV: Annexure:
Annexure I: Media Preparation and Growth Promotion test Chart
Annexure II: Growth Promotion test Report
Annexure III: Microbiological Media status label
Annexure IV: Media Retest Label
Annexure I
Media Preparation and Growth promotion test chart
Page No. : 1 of 5
| Sr. no. | Name of media | Inhouse Code No | pH
(± 0.2) |
Organism for GPT
(For Dehydrated Media container ) |
Organism for GPT
(For In-house Prepared Media) |
Organism for
Inhibitory test (For Dehydrated Media container ) |
Incubation temperature & period | Growth Characteristics |
| 01 | Soybean Casein Digest Agar | SCDA | 7.3 | Bacillus subtilis,
S . aureus Ps . aeruginosa |
Bacillus subtilis |
– |
30 – 35°C for 48 to72 hrs. |
Luxuriant growth |
| C . albicans
A .brasiliensis |
– | – | 20-25ºC for 72 to 120 hrs. | Luxuriant growth | ||||
| 02 | Soybean Casein Digest Medium | SCDM | 7.3 | Bacillus subtilis,
S . aureus Ps . aeruginosa |
Bacillus subtilis |
– | 30 – 35°C for 18 to 24 hrs. |
Luxuriant growth in form of turbidity |
| C . albicans
A .brasiliensis |
20 -25ºC for 72 to 120 hrs. | |||||||
| 03 | Sabouraud Chloramphenicol Agar | SCA | 5.6 | C.albicans | C.albicans | – | 20-25°C for 72 to 120 hrs. |
Luxuriant growth |
| 04 | Antibiotic assay medium11 | AAM11 | 8.3 | S. epidermidis | S. epidermidis | – | 30- 35ºC for 18 to 24 hrs. | Luxuriant growth |
Annexure I (Cont…)
Media Preparation and Growth promotion test chart
Page No. : 2 of 5
| Sr. No | Name of media | Inhouse Code No | pH
(± 0.2) |
Organism for GPT
(For Dehydrated Media container ) |
Organism for GPT
(For In-house Prepared Media) |
Organism for
Inhibitory test (For Dehydrated Media container ) |
Incubation temperature & period | Growth Characteristics |
| 05 | Sabouraud Dextrose Agar | SDA | 5.6 | C.albicans |
C.albicans |
– | 20-25°C for 72 to 120 hrs. |
Luxuriant growth |
| 06 | Cetrimide agar | CA | 7.2 | Ps. aeruginosa |
Ps. aeruginosa |
Escherichia coli |
30-35°C for 18 to 72 hrs. |
Greenish colonies |
| 07 | Fluid Thioglycollate Medium (Sterility) | FTGM | 7.3 | Staphy.aureus
Ps. aeruginosa Cl.sporogenes |
Cl.sporogenes
|
– |
30-35°C for 48 to 72 hrs. |
Luxuriant in form of turbidity |
| 08 | Mac Conkeys Agar | MCA | 7.2 |
Escherichia coli |
Escherichia coli |
Staphy.aureus |
30 – 35°C for 18 to 72 hrs. |
brick red in colour and have a surrounding zone of precipitated bile |
| 09 | R2A Agar | R2A | 7.2 | Bacillus subtilis,
Ps. aeruginosa C.albicans |
Bacillus subtilis |
– | 30-35°C for 48 to 72 hrs. |
Luxuriant growth |
Annexure I (Cont..)
Media Preparation and Growth promotion test chart
Page No. : 3 of 5
| Sr. No. | Name of media | Inhouse Code No | pH
(± 0.2) |
Organism for GPT
(For Dehydrated Media container ) |
Organism for GPT
(For In-house Prepared Media) |
Organism for
Inhibitory test (For Dehydrated Media container ) |
Incubation temperature & period | Growth Characteristics |
| 10 | MacConkeys broth | MCB | 7.2 |
Escherichia coli |
Escherichia coli |
Staphy.aureus |
42 – 44°C for 24 to 48 hrs. |
Yellow colour turbidity observed |
| 11 | Mannitol Salt Agar | MSA | 7.4 | Staphy.aureus | Staphy.aureus | Escherichia coli | 30 – 35°C for 18 to 72 hrs. | Yellow colonies with yellow zones |
| 12 | Rappaport vassiliadis salmonella enrichment broth | RVSEB | 5.2 |
Salmonella abony |
Salmonella abony |
Staphy.aureus |
30 – 35°C for 18 to 24 hrs. | Luxuriant in form of turbidity |
| 13 | EMB Agar, Levine | EMB | 7.1 | Escherichia coli | Escherichia coli | – | 30-35°C for 24 to 48 hrs. | Blue to black with characteristic metallic sheen |
| 14 | Xylose Lysine Deoxycholate Agar | XLDA | 7.4 | Salmonella abony | Salmonella abony | – | 30 – 35°C for 24 to 48 hrs. | Red with or without black centers |
| 15 | Dey engley neutralizing agar | DENA | 7.6 |
Bacillus subtilis, C.albicans |
Bacillus subtilis,
|
– |
30-35°C for 48 to 72 hrs. |
Luxuriant growth |
| 16 | Violet Red Bile Glucose Agar | VRBGA | 7.4 |
Escherichia coli |
Escherichia coli |
Staphy.aureus |
30 – 35°C for 18 to 24 hrs. |
Pink to Red colonies |
Annexure I (Cont..)
Media Preparation and Growth promotion test chart
Page No. : 4 of 5
| Sr. no. | Name of media | Inhouse Code No | pH
(± 0.2) |
Organism for GPT
(For Dehydrated Media container ) |
Organism for GPT
(For In-house Prepared Media) |
Organism for
Inhibitory test (For Dehydrated Media container ) |
Incubation temperature & period | Growth Characteristics |
| 17 | Triple Sugar Iron Agar | TSI | 7.4 | Salmonella abony | Salmonella abony | – | 30 – 35°C for 24 to 48 hrs. | Red(alkaline)slant and Acid(yellow)butt with gas(H2S) production |
| 18 | Reinforced Clostridial medium | RMC | 6.8 | Cl.sporogenes
|
Cl.sporogenes
|
– | 30-35°C for 24 to 48 hrs. (Anaerobically) | Luxuriant growth in form of turbidity |
| 19 | Pseudomonas Agar (For Fluorescein) | PAF | 7.0 | Pse. aeruginosa | Pse. aeruginosa | – | 30-35°C for 48 to 72 hrs. | Yellowish colonies under U.V
lamp |
| 20 | Pseudomonas Agar (For Pyocyanin) | PAP | 7.0 | Pse. aeruginosa | Pse. aeruginosa | – | 30-35°C for 48 to 72 hrs. | Greenish colonies under U.V lamp |
|
21 |
Soybean Casein Digest Medium with Tween 80and Lecithin |
SCDMTL | 7.3 | Bacillus subtilis,
S . aureus Ps . aeruginosa |
Bacillus subtilis |
– |
30 – 35°C for 18 to 24 hrs. |
Luxuriant growth in form of turbidity |
| C . albicans
A .brasiliensis |
20 -25ºC for 72 to 120 hrs. |
Annexure I (Cont..)
Media Preparation and Growth promotion test chart
Page No. : 5 of 5
| Sr. no. | Name of media | Inhouse Code No | pH
(± 0.2) |
Organism for GPT
(For Dehydrated Media container ) |
Organism for GPT
(For In-house Prepared Media) |
Organism for
Inhibitory test (For Dehydrated Media container ) |
Incubation temperature & period | Growth Characteristics |
| 22 | Sabouraud Dextrose broth | SDB | 5.6 | C.albicans | C.albicans | – | 30-35°C for 48 to 72 hrs. | Luxuriant growth in form of turbidity |
| 23 | Enterobacteria enrichment broth | EEB | 7.2 | Escherichia coli | Escherichia coli | Staphy.aureus | 30-35°C for 24 to 48 hrs. | Luxuriant growth in form of turbidity |
| 24 | Columbia agar | CLA | 7.3 | Cl.sporogenes
|
Cl.sporogenes
|
– | 30-35°C for 48 to 72 hrs. (Anaerobically). | Luxuriant growth |
Annexure II
Growth Promotion Test Report
Page No.: 1 of 1
Name of the media: ——————————————- Code no : ———————–
Batch no : ——————————— Container no: ———————
Date of receipt : ————————— In house lot Number:——————–
Date of Testing/Retesting: ———————– Date of Release: ———————–
Retest due on: —————————–
| Culture used for GPT | Incubation Temp./Time | Incubator
I.D |
Details of inoculums
( cfu/ml) |
Observations |
Observed by |
Checked by |
||
| Growth
promoting |
indicative | inhibitory | ||||||
Negative Control :_______________
Remark : Growth Promoting Test of Given media Complies/Does not Comply as per acceptance criteria.
Annexure III
Microbiological Media Status Label
Page No.: 1 of 1
|
Microbiological Media Status Label Name of Media : ___________________________________ Date of Received: __________________ Container Number : __________ Valid Up to : _____________ Opened on/ By : ______________ GPT Status : _______________ Retest due on : _______________
|
Annexure IV
Media Retest Label
|
Media Retest Label
Retest No. :_______________
Retest done on / by :______________
Next Retest due on :_______________
|
