MICLAB-090 Stock Suspensions of Micro Organisms

NOTES:
The microorganisms to be used in Stasis work and Media checks are as follows:

i)       Vegetative organisms of Candida albicans                       ATCC 10231

          Vegetative organisms of Staphylococcus aureus              ATCC 6538

          Vegetative organisms of Escherichia coli                         ATCC 8739

          Vegetative organisms of Pseudomonas aeruginosa          ATCC 9027

          Spores of Bacillus subtilis                                               ATCC 6633

          Spores of Clostridium sporogenes                                   ATCC 19404

          Spores of Bacillus sphaericus                                          ATTC 4525

          Spores of Aspergillus niger                                              ATCC 16404

KWIK-STIK TM Plus Microorganisms are sourced from BIOMERIEUX (brand).

The microorganisms may be used after the noted expiry date providing the purity and growth is of the correct standards.  The correct standards are verified by following steps 1 and 2 of this SOP.

ii) Appropriate strains of microorganisms collected from the manufacturing environment.

Procedure

Stock suspensions of the noted microorganisms are used to perform monthly dilutions, which are used for growth promotion and stasis checks (See sections 6 and 7).  Stock suspensions and dilutions must be placed in the refrigerator immediately after use for preservation.  See MICLAB 035 for the requirements for Media Runs and Soup Tests. Record the bacterial counts from the dilutions.

Method for obtaining microorganisms from KWIK-STIK TM Plus Microorganisms

1.1.         Remove the cylinder for the relevant strain of microorganism from the fridge.

1.2.         Take one pouch from the cylinder and reseal.  Return the cylinder to the fridge.  Allow the pouch to equilibrate to room temperature.

1.3.         Tear open the pouch at the notch and remove the KWIK-STIK^TM.

1.4.         Tear off the pull-tab portion of the label and attach to the primary culture plate.  Use Nutrient Agar (NA) for all organisms except Cl.Sporogenes – use Reinforced Clostridial Agar (RCA) and A.niger – use Saboraud Dextrose Agar (SDA).

1.5.         With the pellet in the down position, crush the reservoir in the cap to release the hydrating fluid.

1.6.         Squeeze the hydrating fluid through the shaft of the swab into the bottom of the unit containing the pellet.

1.7.         Using a pinching action on the bottom portion of the unit, crush and mix the pellet in the fluid until the pellet particles are uniform in size and the solution is homogenous in appearance.

1.8.         Saturate the swab with the hydrated material and transfer the material to the primary culture plate appropriate for the organism.

1.9.         With pressure, rotate the swab, and inoculate approximately an inch area of the medium.  {Hint: Use the sticker label as a guide.}

1.10.      Using a sterile loop, streak through the inoculated area approximately 10 – 20 times to facilitate colony isolation.

1.11.      Discard the remaining hydrated material in the Biohazard Bin according to MICLAB 020.

1.12.      Plates are to be incubated at 32°C (±1.5°C) for 24 – 48 hours.  {Cl.sporogenes is to be incubated anaerobically i.e. in an anaerobe jar with an anaerobe pack. A.niger is to be incubated at 25°C (±1.5°C) for 5-7 days in isolated incubator, wrapped in alfoil.}

1.13.      This will produce microorganisms that are at the 3^rd Passage from the original culture. (KWIK-STIK TM Plus Microorganisms are all at the 2^nd passage).

2. Positive Identification of Reference Culture Micro-organisms

2.1.         Full ID work is to be carried out immediately on all subcultured organisms.

• Gram stain
• Vitek

NOTE: Cl.sporogenes cannot be identified on Vitek, as it is not available on the Database.  A.niger is a mould that cannot be identified using standard procedures. Visual identification is sufficient (see Appendix 2).

2.2.         If a positive ID is not achieved, the culture is to be discarded as per MICLAB 020 and a new culture started (return to section 1).

3. Cryogenic Storage of Reference Culture Micro-organisms

3.1.         The microorganisms are to be stored on porcelain beads treated with a cryogenic solution, i.e. Protect or Microbank^TM.
(A.niger is not stored on porcelain beads, as it cannot be stored cryogenically.)

3.2.         Bead stocks are to be prepared in duplicate.

3.3.         Using the freshly subcultured plate, which has been positively identified (exception Cl.sporogenes), select isolated colonies using a sterile disposable plastic loop and inoculate a single vial of beads, by rotating the loop.  Ensure the cryogenic solution is visibly turbid.

3.4.         Re-cap and rotate the vial 6 times to coat the beads with the inoculated solution and sit for at least 30 seconds.

3.5.         Repeat with a new loop to inoculate the second vial of beads.

3.6.         Using a sterile pipette remove ALL the cryosolution.  If all the liquid is not removed from the vial it may hinder the recovery of the microorganism later on.
NOTE: Remember to remove the liquid from the lid.

3.7.         Re-cap the vials and label with the:

• Name of the microorganism {or appropriate abbreviation}
• Date of preparation and
• Number the vials for usage purposes, (i.e. Vial #1 and Vial #2).

NOTE: There is no expiry set as the organisms’ viability and purity is checked with each stock set up.

3.8.         Place the vial in a pre-frozen cryoblock and store in the freezer at –20°C (±1°C) {preferably towards the back for the coldest temperature}.
NOTE: The removal of individual beads will be made easier if the vial is placed horizontally in the freezer for the first few minutes.

4. Set Up of Stock Cultures

4.1.         Vegetative Micro-organisms

Candida albicans                 ATCC 10231

Staphylococcus aureus         ATCC 6538

Escherichia coli                   ATCC 8739

Pseudomonas aeruginosa     ATCC 9027

4.1.1.     Remove the vial for the organism required from the freezer and place in a separate pre-frozen cryoblock.

4.1.2.     Transfer using a sterile disposable plastic loop, 2 beads onto 2 separate Tryptone Soy Agar (TSA) slopes.

4.1.3.     Discard the loop.

4.1.4.     Using a fresh sterile plastic disposable loop streak the bead onto the slope surface.

4.1.5.     Remove the bead from the slope by tipping into a Hibitane-containing waste container and pulling out with the loop.

4.1.6.     Repeat for the second slope using a fresh sterile plastic disposable loop.

4.1.7.     Incubate slopes at 32°C (±1.5°C) for 24 – 48 hours.  If sufficient growth has not occurred slopes may be incubated for up to 4 days.  If suitable growth has not been obtained after 4 days incubation, re-streak the organism from the beads onto a fresh set of TSA slopes and incubate as above.
Once substantial growth has occurred, place one slope in the fridge to be kept for future use if required (spare).  Label with the Organism Name, Date of Subculture, and the fact that it is a spare.

4.1.8.     Wash the growth off the remaining slope with 5 – 10mL sterile Peptone Water and transfer aseptically into a sterile 20mL McCartney bottle.  Store the prepared stock culture in the refrigerator at 4°C(±1°C).

NOTE:
Slopes can be kept for a period of 3 months.  Upon expiry a fresh stock must be prepared.  If the stock becomes contaminated prior to expiry, the spare slope can be used to produce another stock suspension.

4.2.         Spore Formers

Bacillus subtilis              ATCC 6633

Clostridium sporogenes        ATTC 19404

Bacillus sphaericus        ATTC 4525

4.2.1.     Remove the vial for the organism required from the freezer and place in a separate pre-frozen cryoblock.

4.2.2.     Transfer using a sterile disposable plastic loop, in the biohazard cabinet, 2 beads onto 2 separate slopes.  (Use RCA slopes for Cl.Sporogenes and TSA or Sporulation Agar (SA) slopes for B.subtilis and B.sphaericus).

4.2.3.     Discard the loop.

4.2.4.     Using a fresh sterile plastic disposable loop streak the bead onto the slope surface.

4.2.5.     Remove the bead from the slope by tipping into Hibitane-containing waste container and pulling out with the loop.

4.2.6.     Repeat for the second slope using a fresh sterile plastic disposable loop.

4.2.7.     Incubate slopes at 32°C (±1.5°C) for 24 – 48 hours (Cl.Sporogenes under anaerobic conditions).  If suitable growth has not been obtained after 4 days incubation, re-streak the organism from the beads onto a fresh set of slopes and incubate as above.  Once substantial growth has occurred, place one slope in the fridge to be kept for further use, if required (spare).  Label with the Organism Name, Date of Subculture, and the fact that it is a spare.

4.2.8.     On the remaining slope perform a spore stain (see MICLAB 065).  Approximately 80% of the cells must yield spores before continuing further.  If not, re-incubate the slope at 37°C (-0.5 to +1.5°C) for 24 – 48 hours to shock the organism and perform a spore stain once again to determine the level of sporulation.  The slope can be incubated for up to 1 week at 37°C (-0.5 to +1.5°C).  If sufficient sporulation is not achieved after this time, return to 4.2.1.

4.2.9.     Wash the growth off the slope with 5–10mL sterile Peptone Water and transfer aseptically into a sterile 20mL McCartney Bottle.

4.2.10.  Centrifuge at approximately 4000 rpm for 10 minutes or until the suspension has spun down sufficiently.

4.2.11.  Decant and discard the supernatant.

4.2.12.  Add 5 – 10mL sterile Peptone Water to resuspend the centrifuged spores.  Repeat steps 4.2.10 and 4.2.11 so that the spore suspension is washed three times in total.

4.2.13.  After the third wash, decant the supernatant and resuspend the spores in 5–10mL sterile Peptone Water.

Fill an attest incubator with 200mL Distilled Water and heat the water until it reaches 56°C.  Place the sealed spore suspension in the incubator and cover it with foil to trap the heat.  Heat the suspension for 30 minutes to kill the vegetative cells.  Store the heat-treated suspension in the refrigerator at 4°C(±1°C).
NOTE: Slopes can be kept for a period of 6 months.  Upon expiry a fresh stock must be prepared.  If the stock becomes contaminated prior to expiry, the spare slope can be used to produce another stock suspension.

Aspergillus niger            ATTC 16404

4.2.14.  Subculture A.niger from KWIK-STIK ^TM (plus Microorganisms) to produce organisms at the 3^rd passage (see section 1).  Transfer microorganisms to 2 SDA slopes and incubate for 5-7 days at 25°C (±1.5°C).  This step produces organisms at the 4^th passage.  Once substantial growth has occurred, place one slope in the fridge to be kept for further use, if required (spare).  Label with the Organism Name, Date of Subculture, and the fact that it is a spare.

4.2.15.  Wash the growth from the other slope using 5-10mL sterile Peptone Water and transfer to a sterile 20mL McCartney bottle.  Store the suspension in the refrigerator at 4°C(±1°C).

NOTE: Aspergillus niger is not stored on Microbank beads, only in liquid suspension.  A.niger can be stored for a period of 6 months.  Upon expiry a fresh stock must be prepared as per section 4.2.14.  If the stock becomes contaminated prior to expiry, the spare slope can be used to produce another stock suspension.

4.3.         Environmental Isolates

4.3.1.     Streak the factory microorganism onto an NA plate and incubate at 32°C (±1.5°C) for 24–48 hours.

4.3.2.     Identify the organism by gram stain and Vitek (brand).

4.3.3.     Streak the organism onto 2 TSA slopes and incubate at 32°C (±1.5°C) for 24–48 hours.  If sufficient growth is not obtained, incubate for a further 24 hours.  If sufficient growth is not obtained after this time the organism is to be re-streaked onto another 2 TSA slopes and incubated as above.

4.3.4.     Once substantial growth has occurred, place one slope in the fridge to be kept for further use. if required (spare).  Label with the Organism Type, Isolation location, Date of Subculture, and the fact that it is a spare.

4.3.5.     Based on the above identification, if the organism is Vegetative follow the instructions from 4.1.8 onwards.  If the organism is a Spore Former follow the instructions from 4.2.8 onwards.

5. Serial Dilutions

Serial dilutions from prepared stock culture suspensions (section 4) are conducted on a monthly basis.  These dilutions are plated out weekly to quantitatively verify the number of organisms in the dilutions and to check the following:

• That organism numbers do not decrease dramatically from week to week.
• That the purity of the dilutions is maintained throughout the month.

If either of the above requirements is not met then the dilutions are to be re-made from the stock culture.

Stock Culture 10⁰                           10^-3                  10^-4                     10^-5               10^-6

5.1.         Take 0.1mL of the Stock Culture (100), (prepared in section 4) and transfer to 99.9mL of Peptone Water.  This serves as the 10-3dilution.  Invert the bottle 15 to 20 times to mix.  Label the bottle with the appropriate organism name and date.

5.2.         To dilute by a factor of ten transfer 10mL from bottle to bottle ensuring that at each transfer the contents are mixed by inverting the bottle 15 to 20 times.

5.3.         If one dilution level is skipped, i.e. from 10^-3 to 10^-5, then transfer 1mL from the 10^-3 dilution to 99mL of Peptone Water.  This then serves as the 10^-5 dilution.

5.4.         If two dilution levels are to be skipped, i.e. from 10^-3 to 10^-6, then transfer 0.1mL from the 10^-3 dilution bottle to 99.9mL of Peptone Water. This then serves as the 10^-6 dilution.

Therefore in summary:

Number of Jumps	Transfer Volume	Diluent Volume
1 jump	10mL	90mL
2 jumps	1mL	99mL
3 jumps	0.1mL	99.9mL

5.5.         Dilute all prepared stock suspensions to 10^-7, (except E.coli, which should be diluted to 10^-8).

5.6.         Perform spread plates on NA using the last 3 dilutions in the series.  This involves taking 100µL of the dilution and using a sterile spreader to distribute the sample over the surface of the plate.  Plates are then incubated at 32°C (±1.5°C) for 48hrs.

The exceptions to this are:

• Cl.sporogenes: Use the pour plate technique with RCA and incubate anaerobically at 32°C (±1.5°C) for 48hrs.
• A.niger: Use SDA and wrap plates in foil prior to incubating at 25°C (±1.5°C) for 5 days in a designated incubator.

5.7.         Count all plates and record the number of cfu/0.1mL in the Reference Culture File and stasis spreadsheet.

5.8.         The following week, plate out 2 or 3 dilutions based on the number of cfu/0.1mL obtained from the dilutions plated from the previous week.  Based on the counts from the previous week, plate out the dilutions, which will most likely give rise to 10-100 cfu/0.1mL.  Incubate according to the conditions listed in 5.6.  Record results as in 5.7.

NOTE: Dilutions can be stored for a period of 1 month from the preparation date.  Upon expiry a fresh dilution series must be made from stock suspensions.  If the dilutions become contaminated prior to expiry, the dilutions can be re-made from the stock.

6. Positive Control (Stasis) Details

All media and Peptone Waters both manufactured in the laboratory and sourced from external suppliers must be challenged for growth promotion according to the procedures outlined in the following section.

6.1.         Liquid Growth Media

Four bottles from every batch of liquid media serving as positive controls must be inoculated with a fixed number of organisms to determine if the media is capable of supporting growth.  Because of the nature of our injection solutions it is likely that any microbial contamination level will be low and we must therefore ensure the media is sufficiently sensitive to pick up these low levels.  A suitable level of inoculum is 10‑20 organisms nominally, but not more than 100 viable organisms.  2 bottles from each media type are to be inoculated as per table outlined below:

Organisms to be used for Liquid Media Stasis

Medium	Challenge Organism	Incubation Temp.
TSB x 2	Candida albicans	25°C (±1.5°C)
TSB x 2	Bacillus subtilis	25°C (±1.5°C)
FTM x 2	Clostridium sporogenes	32°C (±1.5°C)
FTM x 2	Staphylococcus aureus	32°C (±1.5°C)
LB x 2	Escherichia coli	32°C (±1.5°C)
LB x 2	Staphylococcus aureus	32°C (±1.5°C)
NB x 2	Escherichia coli	32°C (±1.5°C)
NB x 2	Staphylococcus aureus	32°C (±1.5°C)
RCM x 2	Escherichia coli	32°C (±1.5°C)
RCM x 2	Clostridium sporogenes	32°C (±1.5°C)
Lac B x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)
Lac B x 2	Pseudomonas aeruginosa	37°C (-0.5°C to +1.5°C)
Mac B x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)
EEB x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)

6.1.1.     Check the reference culture file for the most recent counts from dilutions for the specific organisms required for the media being challenged, e.g. For TSB check the counts for C.albicans and B.subtilis.

6.1.2.     Given that 1 organism will give rise to 1 cfu, take the dilution of the particular organism with a concentration of 10-20 organisms/0.1mL, (i.e. if the result was
20 cfu/0.1mL, then the concentration of that dilution is 20 organisms/0.1mL).

6.1.3.     Inoculate the liquid medium with 0.1mL of this dilution and invert to mix.  Repeat for the second bottle of medium.  Note that the suitable level of inoculum is 10-20 organisms, but not more than 100 viable organisms.  If the concentration of organisms per 0.1mL in the dilution does not fall between 10-20 organisms the volume may be adjusted so that the number of organisms inoculated falls into this range.

6.1.4.     Label on the bottle the organism inoculated, the number of organisms inoculated and the inoculation date.  Incubate according to the conditions in the table above.

In all cases growth is to be apparent within 48 hours.  If growth is not apparent repeat the test using the original samples.  If again the media fails consult the Microbiology Quality Manager or a Level 4 Technician.  Record details in the ‘Comments’ section of the media report in MicroTrack.

6.2.         Peptone Water

For each batch of Peptone Water, a positive control must be run to determine that it is not inhibitory to the growth of low numbers of microorganisms.  An inoculum of microorganisms as indicated in the table below is added to the Peptone Water.  A suitable level of inoculum is 10-20 nominally but definitely not more than 100 viable organisms.

Initial medium	Challenge organism	Media	Incubation Temp.
Peptone Water 1	Candida albicans or Bacillus subtilis	TSB x 2	25°C (±1.5°C)
Peptone Water 2	Clostridium sporogenes or Staphylococcus aureus	FTM x 2	32°C (±1.5°C)

6.2.1.     Follow sections 6.1.1 – 6.1.3 to inoculate two bottles of Peptone Water.  Inoculate one bottle with Candida albicans or Bacillus subtilis and the other with Clostridium sporogenes or Staphylococcus aureus according to the table above.

6.2.2.     Filter each bottle of inoculated Peptone Water through a separate sterile 0.45M membrane filter and aseptically divide (cut) the filter into 2 parts.

6.2.3.     Transfer one part to each of 2 bottles of the appropriate medium for the organism inoculated.

6.2.4.     Label on the media bottles the Peptone Water expiry date, the organism inoculated, the number of organisms inoculated and the inoculation date.  Incubate according to the conditions in the table above.

Growth must be apparent in the bottles of media after 48 hours incubation.  If growth is not apparent repeat the test using samples from the same batch.  If again the media fails consult the Microbiology Quality Manager or a Level 4 Technician.  Record details in the ‘Comments’ section of the media report in MicroTrack.

6.3.         Solid Growth Media

For each batch of solid media prepared a positive control must be run to analyse the selective and productive properties of that media.  This is to ensure that the intrinsic, extrinsic and implicit factors are correct in order for that media to sustain growth.  For stasis procedures pertaining to media sourced from external suppliers please see section 6.4.

6.3.1.     Pour four plates of the prepared media and allow to dry overnight at 32C (±1.5°C).

6.3.2.     From the Stock Culture, inoculate a loop full of the appropriate organisms (according to the table below) into either TSB or FTM and incubate at 32C (±1.5°C) to obtain an 18-hour culture (log phase).  Use 20mL of FTM for Clostridium sporogenes and use 10mL of TSB for all other organisms.

An exception is Candida albicans where you need to use 50mL to inoculate into the TSB and incubate for 3 days at 32C (±1.5°C).

Organisms to be used for solid media stasis

Medium	Challenge Organism	Incubation Temperature
LA x 2	Escherichia coli	32C (±1.5°C)
LA x 2	Staphylococcus aureus	32C (±1.5°C)
NA x 2	Escherichia coli	32C (±1.5°C)
NA x 2	Staphylococcus aureus	32C (±1.5°C)
PSA x 2	Pseudomonas aeruginosa	37C (-0.5°C to +1.5°C)
PSA x 2	Staphylococcus aureus	37C (-0.5°C to +1.5°C)
RCA x 2	Clostridium sporogenes	32C (±1.5°C) anaerobically
RCA x 2	Escherichia coli	32C (±1.5°C) anaerobically
SA x 2	Clostridium sporogenes	32C (±1.5°C) anaerobically
SA x 2	Escherichia coli	32C (±1.5°C) anaerobically
SDA x 2	Candida albicans	25C (±1.5°C)
SDA x 2	Escherichia coli	25C (±1.5°C)
TSA x 2	Staphylococcus aureus	25C (±1.5°C)
TSA x 2	Escherichia coli	25C (±1.5°C)
R2A x 2	Escherichia coli	32oC (±1.5°C)
R2A x 2	Staphylococcus aureus	32oC (±1.5°C)
MSA x 2	Staphylococcus aureus	32°C (±1.5°C)
BPA x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)
BPA x 2	Staphylococcus aureus	37°C (-0.5°C to +1.5°C)
Mac A x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)
VRBGA x 2	Escherichia coli	37°C (-0.5°C to +1.5°C)

6.3.3.     Dilute the 18-hour culture adding 5mL culture to 100mL of Peptone Water.  The exception to this is Clostridium sporogenes, which is diluted 10mL into 100mL of Peptone Water.

6.3.4.     Divide each plate into 4 quadrants and streak 2 plates with each organism according to the diagram below.  Each quadrant should contain five streak lines overlapping once only at the edge of each quadrant, as well as an additional line bisecting all quadrants.

Diagram for streaking solid media

6.3.5.     Incubate non-selective media for 48hr and selective media for 4 days at the appropriate temperature.

6.3.6.     Score the rate of growth, awarding a score of 0.2 for each streak line with growth.  The maximum score for growth in all quadrants plus the additional line bisecting all quadrants is 4.2.

NOTE: A minimum combined score of 2 must be evident for the agar to be considered acceptable for use in the Laboratory.  If the media does not meet the acceptance criteria, then repeat the test using new samples of media.  If again the media fails, consult the Microbiology Manager or a senior Technician.  Record details in the log book.

6.3.7.     The average of the scores for the replicate plates is to be determined and this information is to be recorded in log book.

6.4.         Verification of pre-poured media purchased from External suppliers

Verification of growth promotion of plated media supplied from an external supplier is to take place for every new batch for each media type purchased from the external supplier.  That is, verification is to be conducted on each new lot number of media for each media type delivered to the laboratory.  Stasis testing and analysis of results is to be conducted as per Section 6.3 of this SOP.  Growth results are recorded in the media preparation field for each lot number verified at which time the batch may be signed off for use.

6.5.         Recording Stasis Results

It is the responsibility of the person on Stasis testing to enter the results from media growth promotion and verification tests into appropriate log book.

7. Media Check after Sterility Testing (Stasis Test)

7.1.         Membrane Filtration and Direct Inoculation

Once every 12 months media containers from a product tested by Membrane Filtration and, if possible those from a product tested by Direct Inoculation, must be subjected to a challenge of a low number of microorganisms after the sterility test has been completed.  This is to ensure that any inhibitory effects present in the products have been successfully inactivated, either by the dilution effect or the washing procedure, as appropriate to the Test Method.  This challenge is to demonstrate the effectiveness of the sterility test method, ensuring that the testing procedures are allowing for the potential growth of any microorganisms within the product.

It is important that on an annual basis, the range of sterile products are subjected to a Stasis Test.  A Stasis Check on Sterility Tests results must be recorded on an annual basis. It should include the product type and the date that the Stasis test was conducted.

An inoculum of microorganisms as indicated in the table below is added to the test canisters or bottles after the sterility test.  A suitable level of inoculum is 10-20 nominally but definitely not more than 100 viable organisms.

Medium	Challenge organism	Incubation Temperature
FTM	Staphylococcus aureus ATCC 6538 or Clostridium sporogenes ATCC 19404	32ºC (±1.5ºC)
TSB	Candida albicans ATCC 10231 or Bacillus subtilis ATCC 6633 or Aspergillus niger ATCC 16404*	23ºC (±1.5ºC)

Periodically (approx. every 12 months) the strains of bacteria referred to above, should be substituted with appropriate strains of microorganisms collected from the manufacturing environment.

7.1.1.     Fill out the batch number and product details of the product tested in the sterility test in log book.

7.1.2.     Follow sections 6.1.1 – 6.1.3 to inoculate one test canister filled with FTM and one filled with TSB with the appropriate organisms according to the above table.  For products tested by Direct Inoculation, subject approx. half the number of bottles of medium involved in the test to a challenge by one of the recommended organisms, and the other half to a challenge by the other recommended organism.

7.1.3.     Label on the canister or bottle inoculated, the number of organisms inoculated, the number of organisms inoculated and the inoculation date.  Incubate according to the conditions in the table above.

In all inoculated canisters or bottles conspicuous growth of each of the added microorganisms must occur in less than 48 hours or a period of time appropriate to the added microorganism, i.e. 5 days for Aspergillus niger.  If conspicuous growth is not apparent within 3 days for bacteria and 5 days for fungi, the test is considered to be invalid.

8. Appendix 1: Typical morphological & microscopic appearance of reference culture microorganisms

8.1.         Candida albicans ATCC 10231

Morphological (after 24-48 hrs on a Nutrient Agar plate at 32°C (±1.5°C))
1mm circular convex dull White with yeasty odour.
Microscopic (Gram stain)
Gram positive oval yeasts ‑ may be budding.

8.2.         Staphylococcus aureus ATCC 6538

Morphological (after 24-48 hrs on a Nutrient Agar plate at 32°C (±1.5°C)),
circular, entire, convex, glistening, cell pigmentation can range from a Yellowish tint through to Orange.

Microscopic (Gram stain)
Gram positive cocci (0.5 -1mm in diameter) Cells occur singly and in pairs.  Division may be in more than one plane, giving rise to irregular clusters.

8.3.         Escherichia coli ATCC 8739

Morphological (after 24-48 hrs on a Nutrient Agar plate at 32°C (±1.5°C))
Smooth, shiny, translucent Beige.
Microscopic (Gram stain)
Gram negative, straight rods, (1.1 – 1.5 mm x 2.0 – 6.0mm), occurring singly, or in pairs.

8.4.         Pseudomonas aeruginosa ATCC 9027

Morphological (after 24-48 hrs on a Nutrient Agar plate at 32°C (±1.5°C))
Smooth, translucent irregular.
Microscopic (Gram stain)
Gram negative, straight or slightly curved rods (not helical), 0.5 – 0.7 mm x 1.5 – 3.0 mm.

8.5.         Bacillus subtilis ATCC 6633

Morphological (after 48 hrs on a Nutrient Agar plate at 32°C (±1.5°C))
3‑4 mm irregular flat, dull, rough, Cream, becomes opaque.
Microscopic (Gram stain)
Gram positive rods, (approx. 4 x 1 mm) ‑ may have formed oval central to sub‑terminal spores with slight swelling.

8.6.         Clostridium sporogenes ATCC 19404

Morphological (after 24-48 hrs on a Reinforced Clostridial Agar plate grown anaerobically at 32°C (±1.5°C))
Spherical, woolly, semi-translucent colonies.  Colonies in agar are spherical, with an opaque centre, and a woolly semi-transparent periphery.
Microscopic (Gram stain)
Gram positive straight rods, occur singly, 0.3 – 1.4 mm x 1.3 – 16 mm.  Spores are oval, sub-terminal and distend the cell.

8.7.         Bacillus sphaericus ATCC 4525

Morphological (after 48 hrs on a Nutrient Agar plate at 32°C (±1.5°C))
3‑4 mm irregular flat, dull, rough, Cream, becomes opaque.
Microscopic (Gram stain)
Gram positive rods, (approx. 4 x 1mm) ‑ may have formed oval central to sub‑terminal spores with slight swelling.

8.8.         Aspergillus niger ATCC 16404

Morphological, (after 5-7 days on Sabouraud Dextrose Agar plate at 25°C (±1.5°C)).

Compact White or Yellow basal felt covered by a dense layer of Dark Brown to Black conidial heads (spores).

Microscopic

Large conidial heads (up to 3mm x 15-20mm in diameter), Dark Brown and globose.

9. Appendix 2: Kwik-Stik instruction diagram

10. Summary of Changes

Version #	Revision History
MICLAB 090	New