Department | Micro Laboratory | Document no | MICLAB 160 | ||
Title | Handling of Media Diluents and Reagents in the Microbiology Laboratory | ||||
Prepared by: | Date: | Supersedes: | |||
Checked by: | Date: | Date Issued: | |||
Approved by: | Date: | Review Date: | |||
1. DOCUMENT OWNER
Laboratory/Quality Manager
2. PURPOSE
This document describes the general procedure for the receival, use and storage of all media, diluents and reagents in the Microbiology Laboratory at a GMP site.
3. SCOPE
All Microbiological media, reagents and diluents must comply with specified documentation, preparation, storage and quality control requirements before permitting their use in the laboratory. All batches of agar prepared in the Microbiology Laboratory must be examined for their ability to support the formulation of colonies by organisms that are designed to grow.
4. RESPONSIBILITY \ BUSINESS RULES
All microbiology staff.
5. PROCEDURE
5.1 Receival of Dehydrated Media (Including Supplements)
5.1.1. Upon receival of media check the invoice against the purchase order. Record the date of receival on the container.
5.1.2. Check expiry date and storage conditions.
5.1.3. Store according to manufacturer’s instructions and in a manner which allows oldest media to be used first.
5.2 Preparation of Media
5.2.1. Media preparation should take place in a clean environment relatively free of draughts and moisture.
5.2.2. Use only clean and dry glassware and utensils and prepare the medium in a container approximately twice the final volume to allow adequate mixing. (ie. for 1L of media, prepare in a 2L beaker).
5.2.3. Use distilled or RO water.
5.2.4. Check expiry of dehydrated media prior to weighing out. Discard any media on which expiry has elapsed. Examine dehydrated media before use; if solid or discolored, discard (Media becomes solid with exposure to moisture).
5.2.5. All used and unusable dehydrated media must be autoclaved or incinerated prior to disposal. eg. expired media.
5.2.6. When opening a new container, record date opened on the bottle.
5.2.7. Ensure balance, dispensers and pH meters are verified. Record in pH meter and dispenser instrument log book.
5.2.8. When weighing out powder wear a protective dust mask to avoid inhalation of the powder and safety glasses. All weighing of dehydrated media must be performed under a dust extractor.
5.2.9. Label all vessels containing media during preparation with media type, batch number.
5.2.10. Prepare all media according to procedures in Media Manual.
5.2.11. Media which is sterilized by boiling should be kept covered throughout this process.
5.2.12. Bismuth Sulphite Agar (BSA) and EMB (Eosin Methylene Blue) agars are light sensitive and should be covered immediately after pouring.
5.2.13. Record all details of media preparation in form “Media Quality Control Report” (Appendix 1).
5.2.14. Label all bottles (50 mL and upwards) with media name, laboratory batch number, date prepared and expiry, prior to dispensing. Baskets containing smaller bottles (such as McCartneys) should be labelled with the same information and a small strip of autoclave tape should be applied to the basket.
5.2.15. A MacCartney bottle may be used to test the batch pH. Only set up fertility and sterility QCs on a batch when a new lot number of dehydrated media has been opened.
5.2.16. Fertility is to be set up using positive and negative control cultures, or as required.
5.3 Media Volume Checks
5.3.1. Media dispensed into volumes requiring final volumes of 9.9mL or 9.0mL require volume checks after autoclaving.
5.3.2. After autoclaving randomly select thirteen bottles.
5.3.3. Place an appropriate beaker onto a balance and tare.
5.3.4. Pour media from one bottle into a beaker and record value on “Media Quality Control Report” (Appendix 1).
5.3.5. Repeat steps 5.3.3 – 5.3.4 for the remaining 12 bottles.
5.4 Quality Control of Prepared Media – Sterility
5.4.1. To check sterility of broths, incubate at 30°C±1oC for 72 hours. For agars, melt down, pour plates and incubate for 48 hours at 30°C±1oC.
5.4.2. Plain agar, water, saline, hard water and all diluents do no require a fertility test.
5.5 Quality Control of Prepared Media – Fertility
5.5.1. Fertility of Broth Media
5.5.1.1 To prepare a bacterial culture, inoculate 10 mLs of Tryptone Soya Broth (TSB) with the required organism for the media being tested (table 5.5.1.8). Incubate for 24 hour at 30 ± 1°C.
5.5.1.2. Prepare yeast suspensions in the same manner with TSB substituted for Sabouraud Liquid Medium.
NOTE: Z.rouxii requires 25 + 1°C incubation.
5.5.1.3. Inoculate media using a 1 microlitre plastic disposable loop.
5.5.1.4. Immerse only the loop itself and not the stem.
5.5.1.5. Use a fresh loop after each media inoculation.
5.5.1.6. The incubation time and temperature will depend on the test organism and media tested.
5.5.1.7. Only media which have growth (turbidity) pass the fertility test.
NOTE:Due to the medias turbidity the fertility bottles for Letheen Broth + 2% Lecithin + 4% Tween 80 must be streaked onto a Tryptone Soya Agar Plate and incubated at 30°C±1oC for 24 hours to check growth
5.5.1.8. Control Microorganisms Used For Fertility Testing of Broth Media
MEDIUM | INCUBATION CONDITIONS | CONTROL ORGANISMS POSITIVE | CONTROL ORGANISMS NEGATIVE | ACCEPTABLE RESULTS |
|---|---|---|---|---|
Asparagine Broth | Aerobic 48hrs, 30°C | P. aeruginosa (ATCC 9027) | S. aureus (NCTC 6571) E. coli (NCTC 9001) | Growth Growth Growth |
Bacteriological Peptone Plus 20% Sucrose | Aerobic 48hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
Bacteriological Peptone (Diluent) Plus 0.1% Tween | Aerobic 24-48 hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
Bacteriological Peptone (0.1%) Diluent | Aerobic 24-48 hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
Brain Heart Infusion Broth | Aerobic 24 hrs, 37°C | S. aureus (NCTC 6571) E. coli (NCTC 9001) C. albicans (ATCC 10231) | N/A | Growth Growth Growth |
Buffered Peptone Water | Aerobic 18 hrs, 37°C | S. salford (IMVS 1710) | C. freundii (NCTC 9750) | Growth |
Cooked Meat Medium | Anaerobic 24 hrs, 37°C | Cl. perfringens (NCTC 8237) | S.aureus (NCTC 6571) | Growth |
Double Strength Lauryl Tryptone Broth | Aerobic 48hrs, 37°C | E. coli (NCTC 9001) | Enterobacter aerogenes (NCTC 10006) | Positive Control – Growth and Gas Production Negative Control – Growth and no or slight Gas Production |
EC Broth | Aerobic 24-48 hrs, 44.5 °C | E. coli (NCTC 9001) | E. aerogenes (NCTC 10006) | Positive Control –Growth and Gas Production Negative Control –Growth and no Gas Production |
Lauryl Tryptone Broth | Aerobic 48hrs, 37°C | E. coli (NCTC 9001) | Enterobacter aerogenes (NCTC 10006) | Positive Control – Growth and Gas Production Negative Control – Growth and no or slight Gas Production |
Letheen Broth | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth |
Letheen Broth – DIFCO | Aerobic 24-48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth |
Letheen Broth + 2% Lecithin + 4%Tween80 | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth |
Letheen Broth + 4% Tween 80 | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth |
Lysine Decorboxylase Broth | Aerobic 24 hrs, 37°C | S. salford (IMVS 1710) | C. freundii (NCTC 9750) | Positive Control -Growth Negative Control -Growth Yellow |
Mannitol Selenite Cystine Broth | Aerobic 18 hrs, 37°C | S. salford (IMVS 1710) | C. freundii (NCTC 9750) | Growth Growth |
Nutrient Broth + 3% Tween 80 | Aerobic 48 hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
ONPG Broth | Aerobic 24 hrs, 37°C | C. freundii (NCTC 9750) | S. salford (IMVS 1710) | Positive Control – Growth Yellow Negative Control – Growth Colourless |
Rappaport-Vassiliadis Enrichment Broth | Aerobic 24 hrs, 42°C | S. salford (IMVS 1710) | C. freundii (NCTC 9750) | Growth |
Sabouraud Liquid Medium | Aerobic 48 hrs, 25°C | C. albicans (ATCC 10231) Z. rouxii (NCYC 381) | N/A | Growth Growth |
Synthetic Broth | Aerobic 24 hrs, 37°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) S. aureus (NCTC 6571) | N/A | Growth Growth Growth |
Thioglycollate Media + 0.5% Tween 80 | Aerobic 24-48 hrs, 30°C | Cl. perfringens (NCTC 8237) | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | Growth Growth Growth |
Tryptone Soya Broth | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) S. aureus (NCTC 6571) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
Tryptone Soya Broth + 4% Tween 80 | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) S. aureus (NCTC 6571) E. coli (NCTC 9001) | N/A | Growth Growth Growth |
Tryptone Water | Aerobic 48 hrs, 37°C | E. coli (NCTC 9001) | E.aerogenes (NCTC 10006) | Growth Growth |
5.5.2. Fertility of Agar Media – Ecometric Quality Control Evaluation
5.5.2.1 Materials Required List
– Sterile Petri Dishes
– Ecometric Quality Control Evaluation stamp and black stamp pad
– Test cultures
– 1 microlitre plastic disposable loop
– Vortex
– Tryptone Soya Broth (TSB) – 10mL in MacCartney bottles
– Biohazard safety cabinet
5.5.2.2 Work Instruction
This procedure involves the inoculation of standardised cultures on agar plates with a carefully standardised sequential streaking technique.
Based on the principal of an ever decreasing number of colony forming units per surface area, this is a quantitative evaluation of the ability of media to support the growth of certain micro-organisms.
5.5.2.3 Culture Preparation
Determine cultures required to perform fertility testing by referring to Table 5.5.2.4.
Follow steps 5.5.1.1 – 5.5.1.2 for culture preparation (follow table 5.5.2.4 for agars).
5.5.2.4 Control Microorganisms for Ecometric Quality Control Evaluation of Media
MEDIUM | INCUBATION CONDITIONS | CONTROL ORGANISMS POSITIVE | CONTROL ORGANISMS NEGATIVE | ACCEPTABLE RESULTS |
|---|---|---|---|---|
Baird Parker Agar | Aerobic 24 hrs, 37°C | S. aureus (NCTC 6571) | S.epidermidis (NCTC 6513) | Growth index ≥ 3 |
Bismuth Sulphite Agar | Aerobic 48 hrs, 37°C | S. salford (IMVS 1710) | C. freundii (NCTC 9750) | Growth index ≥ 3 |
Brain Heart Infusion Agar | Aerobic 24 hrs, 37°C | S. aureus (NCTC 6571) E. coli (NCTC 9001) C. albicans (ATCC 10231) | N/A | Growth ≥ 3 |
Cetrimide B Agar | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) | E. coli (NCTC 9001) | Growth index ≥ 3 |
CLED | Aerobic 24 hrs, 37°C | S. salford (IMVS 1710) | P. vulgaris (NCTC 4635) E. coli (NCTC 9001) | Growth index ≥ 3 |
DNase Agar | Aerobic 18 hrs, 37°C | S. aureus (NCTC 6571) | S.epidermidis (NCTC 6513) | Growth index ≥ 3 |
Eosin Methylene Blue Agar | Aerobic 48 hrs, 37°C | E. coli (NCTC 9001) | E. aerogenes (NCTC 10006) |
Growth index ≥ 3 |
Letheen Agar | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) S. aureus (NCTC 6571) | N/A | Growth index ≥ 3 |
MacConkey Agar | Aerobic 48 hrs, 37°C | E. coli (NCTC 9001) | P. aeruginosa (ATCC 9027) | Growth index ≥ 3 |
Malt Extract Agar | Aerobic 5 days, 25°C | C. albicans (ATCC 10231) | N/A | Growth index ≥ 3 |
Malt Extract Agar + 35% Sucrose + 10% Glucose | Aerobic 5 days, 25°C | Z. rouxii (NCYC 381) | N/A | Growth index ≥ 1 |
Membrane Lauryl Sulphate Agar | Aerobic 24 hrs, 37°C | E. coli (NCTC 9001) | P. aeruginosa (ATCC 9027) | Growth index ≥ 3 |
Nutrient Agar | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) S. aureus (NCTC 6571) | N/A | Growth index ≥ 3 |
Oxidative – Fementative (OF) Medium | Aerobic 24 hrs, 37°C | P. aeruginosa (ATCC 9027) | E.coli (NCTC 9001) | Oxidative – P. aeruginosa Fermentative – E.coli |
Perfringens Agar | Anaerobic 24 hrs, 37°C | C. perfringens (NCTC8237) | N/A | Growth index ≥ 3 |
Perfringens Agar Base Plus Tryptose Sulphite Cyclocserine (TSC) Supplement | Anaerobic 24 hrs, 37°C | C. perfringens (NCTC8237 | N/A | Growth index ≥ 3 |
Potato Dextrose Agar | Aerobic 5 days, 25°C | C. albicans (ATCC 10231) | N/A | Growth index ≥ 3 |
Pseudomonas Agar + C-F-C Supp. | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) | N/A | Growth index ≥ 3 |
Pseudomonas Agar Base | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) | N/A | Growth index ≥ 3 |
R2A | Aerobic 48 hrs, 30°C | P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) S. aureus (NCTC 6571) | N/A | Growth index ≥ 3 |
Reinforced Clostridial Agar | Anaerobic 48 hrs, 30°C | C. perfringens (NCTC 8237) C. sphenoides (ATCC 19403) | N/A | Growth index ≥ 3 |
Sabouraud Dextrose Agar | Aerobic 5 days, 25°C | C. albicans (ATCC 10231) | N/A | Growth index ≥ 3 |
Simmons Citrate Agar | Aerobic 24-48 hrs, 37°C | K.edwardsii var altantae (NCTC 10896) | E. coli (NCTC 9001) | Positive Control Growth index ≥ 3 Negative Control – No Growth |
Sporulation Agar | Aerobic 24 hrs, 37°C | B. subtilis (A.TCC 11714) | N/A | Growth index ≥ 3 |
Tryptone Soya Agar | Aerobic 48 hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth index ≥ 3 |
Tryptone Soya Agar + 4% Tween | Aerobic 48 hrs, 30°C | S. aureus (NCTC 6571) P. aeruginosa (ATCC 9027) E. coli (NCTC 9001) | N/A | Growth index ≥ 3 |
Tryptose Sulphite Cycloserine Agar | Anaerobic 24 hrs, 37°C | C. perfringens (NCTC 8237) | E. coli (NCTC 9001) | Growth index ≥ 3 |
Violet Red Bile Glucose Agar | Aerobic 24 hrs, 37°C | E. coli (NCTC 9001) | S. aureus (NCTC 6571) | Positive Control -Growth index ≥ 3 Negative Control – No Growth |
Violet Red Bile Lactose Agar | Aerobic 24 hrs, 37°C | E. coli (NCTC 9001) | S. aureus (NCTC 6571) | Positive Control -Growth index ≥ 3 Negative Control – No Growth |
X L D | Aerobic 48 hrs, 37°C | S. salford (IMVS 1710) | C. Freundii (NCTC 9750) | Growth index ≥ 3 |
5.5.2.5 Plate Preparation
Pour sterile petri dishes with agar retained for the purpose of Ecometric Quality Control Evaluation (EQCE).
When set, stamp the base of agar plates with the EQCE stamp.
5.5.2.6 Inoculation/Streaking Technique
5.5.2.6.1 Turn the UV lamp on in the Biohazard Safety Cabinet for at least 15 minutes prior to use.
5.5.2.6.2 Open cabinet and decontaminate work bench with Viraclean prior to use.
5.5.2.6.3 Immerse only the loop itself and not the stem in the test culture. Begin each plate inoculation with a fresh loop.
During inoculation the loop must be held at approximately 300, flat against the surface of the medium. Avoid lifting, turning or tilting of the loop. The original position of the hand should be retained during the whole operation, using the fingers only to transmit movement.
Homogenise the cultures by vortexing for 2 seconds immediately before each inoculation.
Inoculate four quadrants in succession with series of five parallel lines as shown below.
The incubation time and temperature will depend on test organism and media tested. After the required time of incubation has passed, assess the growth intensity from 1 to 5.
5.5.2.7 Interpretation of Results / Acceptance Criteria
SECTIONS CONTAINING GROWTH | RESULT |
Section 1 only | + |
Sections 1 & 2 | ++ |
Sections 1,2, & 3 | +++ |
Sections 1 – 4 inclusive | ++++ |
Sections 1 – 5 inclusive | +++++ |
If growth was obtained only in section 1 or in sections 1 and 2 only, the batch of media must be rejected and cause of the problem investigated.
Only media which have growth index of 3 and more will be accepted for use.
Note: Malt Extract Agar + 35% Sucrose + 10% Glucose only requires a growth index of 1 or more to be accepted for use.
5.5.2.8 Record all results of QC tests in “Media Quality Control Report” (Appendix 1).
5.5.2.9. Media undergoing QC assessment must be kept separately and not used until the QC tests are completed and passed.
5.6 Storage – General
5.6.1. Store reagents and dehydrated media in cool, dry, dark conditions.
5.6.2. Reagents that require refrigeration are to be kept in a designated refrigerator.
5.6.3. Pay particular attention to agars and broths with special storage requirements and short shelf lives.
5.6.4. Regular stock checks should be made of dehydrated and laboratory prepared media regarding quantity and expiry date. Discard any expired media as per 5.2.5.
5.7 Storage of Bottled Agars and Broths
5.7.1. All prepared bottled agars, diluents and broths must be tightly sealed and stored at room temperature in a dry cupboard.
5.7.2. Label all stored bottles as in 5.2.14.
5.7.3. Store Mannitol Selenite Cysteine broth (MSC) and Rappaport – Vassiliadis Enrichment Broth (RV) in capped test tubes at 2 – 8°C.
5.8 Storage of Poured Plates
5.8.1. Pour plates under laminar flow to ensure an aseptic environment.
5.8.2. Label plates on the underside with name of media, date poured, batch number of bottled agar, expiry date and store inverted in a designated refrigerator with the newest plates placed towards the back.
5.8.3. Ready-made plates should be checked for contamination before refrigeration.
5,8.4. Bismuth Sulphite Agar (BSA) and Eosin Methylene Blue (EMB) agars should be wrapped in aluminium foil when storing in the fridge.
5.9 Determination of expiry date for microbiological media
5.9.1. If a new supplier or formulation is used, quality control of microbiological culture media and determination of expiry date needs to be performed.
5.9.2. Follow steps 5.2.1 – 5.2.16 for media preparation.
5.9.3. In required intervals perform quality control of prepared media (eg. fertility and Ecometric Quality Control Evaluation) following steps 5.5.1 – 5.5.2.7.
5.9.4. Once the expiry date is determined update the relevant media procedure to include the expiry date.
6. DEFINITIONS / ACRONYMS
EQCE – Ecometric Quality Control Evaluation
8. SUMMARY OF CHANGES
Version # | Revision History |
MicLab-160 | New |
Appendix 1: MEDIA QUALITY CONTROL REPORT
See the master document stored with the Document Control Officer for authorisation of this form
Date Prepared:
Prepared By:
Media | OI Used | Laboratory Batch Number | Manufacturer Batch No/ Expiry Date | Grams Weighed | Total Volume Prepared | Volume Dispensed | PH prior to adjustment | Adjusted pH | PH after Sterilisation | Load Number | Sterilisation time/ temperature | Volume Checks Required | QC performed on batch# |
For required volume adjustments for media prior to sterilisation
Media Released By:
Date:
Media Quality Control
Critical Volume Checks (9.0mL and 9.9mL). Acceptance Criteria for volume checks +0.2mL
9.0mL volume checks | 9.9mL volume checks | |||||||
1 | 8 | 1 | 8 | |||||
2 | 9 | 2 | 9 | |||||
3 | 10 | 3 | 10 | |||||
4 | 11 | 4 | 11 | |||||
5 | 12 | 5 | 12 | |||||
6 | 13 | 6 | 13 | |||||
7 | 7 | |||||||
STERILITY | FERTILITY | |||||||
Media | Lab Batch Number | Number of Units Tested | Number of Units Showing Growth | Positive Control | Negative Control | Accept/ Reject | ||
Growth Index | Units showing growth | Growth Index | Units showing growth | |||||
Media QC Recorded By: Date: Media QC Checked By: Date: