D-1016

Microbial Identification via Biolog Microstation

Section D — Laboratory Operations and Specifications 22 pages

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1.0 Purpose

The Biolog MicroStation System is an optical reading instrument for identifying
microorganisms. BioLog’s patented technology uses each microbe's ability to use particular

carbon sources or chemical sensitivity assays to produce a unique pattern or "Phenotypic

Fingerprint" for that microbe and compares that fingerprint to their identification databases. This
procedure details processes used to identify microbial organisms with the Biolog MicroStation.

2.0 Scope

Any samples collected for quality or environmental testing may be used on this analyzer to

identify gram-negative and gram-positive aerobes, anaerobes, and yeasts.

3.0 Responsibility

3.1 It is the responsibility of QC Laboratory Analysts to follow this procedure.

3.2 It is the responsibility of QC Laboratory Management to implement this procedure and to
ensure it is being followed.

3.3. It is the responsibility of QC Laboratory Management to keep this procedure aligned with

current practices.

4.0 Definitions

4.1 Microplate -GENIII plates purchased from Biolog with 95 carbon source utilization tests
(one in each well), with Al well as control; can be used for aerobes and anaerobes; yeast

microplate has 94 tests and two control wells (Al and D1)

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4.2 Pattern — a unique arrangements of reactions on a microplate, which occur from the
metabolism of different reagents in each well; the Microstation system reads these

patterns with a lamp and optical filters, and compares them to a data library of microbes

4.3 GEN III — microplate used for the identification of aerobic gram negative and gram

positive microbes; also refers to the process of preparing plates, and to criteria used on

the Biolog analyzer

4.4 AN — used for ID of anaerobic microbes; also refers to the process of preparing plated

and to the criteria used on the Biolog analyzer

4.5 YT — type of microplate used for the identification of yeasts, also refers to the process of

preparing plates, and to the criteria used on the Biolog analyzer

4.6 Culture — growth on media, used to obtain isolate

4.7 Culture Media — agar used to grow and isolate microorganisms. In most cases BUG
(Biolog Universal Growth), BUG+B, TSA, or CHOC will be used.

4.8 Isolate — pure microbiological culture, not mixed with any other species

4.9 Inoculum — liquid suspension of isolate and appropriate IF mixed to a specific %T

4.10 IF-A, IF-B, IF-C, AN-IF — Inoculation Fluid; media used to grow isolates in
microplates. The letters A,B,C,& AN refer to the protocol followed for positive

identification on the MicroStation.

4.11 %T — Percent Transmittance; the cell density measured with the turbidometer, used to
make the IF at the correct concentration for cellular growth on the microplates

4.12 Turbidometer — instrument used to measure %T of IF before the second incubation

4.13 Protocol — refers to a setting in the Biolog analyzer software

4.14 MicroLog Secure 6.3 — software used to operate the Biolog MicroStation

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4.15 Audit trail viewer — software used to document all activity with MicroLog Secure 6.3 as
required by 21 CFR part 11

4.16 Aseptic technique — standard laboratory procedures used to prevent contamination

4.17 SIM — an abbreviation for a similarity index value used to assess how well a sample is

identified: a value of 1 indicates a perfect match; a value of 0 indicates no match; a larger
value is a better match; a value of 0.5 is the threshold for GEN III

4.18 DIST — indicates the approximate number of mismatches between MicroPlate results and

the database pattern aggregate for that species; a smaller value is a better match

4.19 PQV — Process Quality Verification

5.0 References

5.1 Biolog Microstation Identification Validation Protocol

3.2 MicroLog Secure 6.2 User Guide

a3 Biolog Microstation User Guide

5.4 D-113, SOP, Microbiological Media Validation

+ D-1016-F1, Form, Microbial Identification Log

5.6 A-106, SOP, Documentation Guidelines for cGMP Records

5.7 C-502, SOP, Record Storage, Retention, and Destruction

6.0 Limitations

6.1 With the exception of yeast, the instructions provided in this procedure are not for any
mold or fungi.

6.2 The product is not for human in vitro diagnostic use.

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6.3. The Biolog analyzer is not able to quantify or count any colonies. It is solely for
identification.

6.4 Only use media purchased from Biolog or that has been validated as per SOP D-113
Microbiological Media Validation.

6.5. The microbe to be identified must be from a pure culture that is no more than 24 hours
old.

6.6 The test panels will only identify members of the species in the current databases (GEN

Ill, AN, and YT). Other species and atypical strains will usually yield a “no
identification” result.

7.0 Procedure

Tal These steps are to be followed in the order as written. Keep in mind, this is a two day

process. Each sample must be incubated twice: once for a pure culture, second for the

microplate reactions to take place. Only use fresh growth (22-24 hours for most bacteria)
of isolated colonies to make the inoculum.

7.2 First Culture and Incubation - Materials Needed

7.2.1 Sample for analysis

7.2.2 Appropriate growth media

7.2.3 Sterile swabs

7.2.4 Incubator with appropriate environment for growth

73 First Culture and Incubation — Procedure

7.3.1. See section 11.0 “Media selection and Process Chart” to select the appropriate
media for optimal growth. Label all plates prior to streaking.

7.3.2 Using aseptic technique, streak plates for isolation.

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7.3.3 Incubate for at least 22 hours in the environment appropriate for the organism to
grow. See charts for optimal growth environment. Record the date, time, and tech

initials on form D-1016-F1 Microbial Identification Log.

7.3.4 Use the red biohazard bags for disposal of all contaminated waste generated in

this process.

7.4 Visual Plate Examination - Materials Needed

7.4.1. Colony magnifier lamp

7.4.2 Gram stain kit, if anaerobes have been cultured

7.4.3 Microscope

7.5 Visual Plate Examination — Procedure

7.5.1 After at least 22 hours of incubation, examine areas of confluent growth. If the
lawn is not uniform in texture and color, this indicates that the culture is not pure.

Preparing an inoculum directly from a mixed-growth plate will not ID any

growth on the analyzer.

7.5.2 Conversely, sometimes a culture may be pure, but gives the appearance of

heterogeneity. This is due to a common phenomenon whereby microorganisms
produce more than one colony type. To be certain of its identity, purify and test

each colony type individually. Be aware that different colony types of the same

strain may ID but give different phenotype profiles.

7.5.3. In instances where growth is indistinguishable between yeast and fungi, a wet

prep may be performed to differentiate genus. If the wet prep determines the
growth to be a fungi, it cannot be identified on the analyzer at this time, and

must be sent to a third party laboratory for analysis.

7.5.4 In circumstances where there is not sufficient growth for suspected anaerobic

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microbes, a second sub culture is necessary. Additionally, if the suspected
organism is a historically weak grower, you may swab 2 plates for the first

culture, and take isolates from both plates, so long as the growth on both plate
does not exhibit pleomorphism between the two.

Tadd After identifying a pure culture on the plate, follow the instructions in the next

sections to prepare the IF and Microplates.

7.6 MicroPlate Inoculation and Incubation — Materials Needed

7.6.1 Pure microbial culture

7.6.2 Inoculation Fluid

7.6.3 Sterile swabs or streakerz

7.6.4 Turbidimeter

7.6.5 8 channel pipette

7.6.6 Sterile pipette tips

7.6.7 Sterile multichannel pipette fluid reservoir

7.6.8 MicroPlate ( GEN III or YT)

169 Incubator with appropriate environment for microbial growth

77 MicroPlate Inoculation and Incubation — Procedure

7.7.1 Calibrate the turbidimeter.

7.7.1.1. Before powering on, make sure the dial is turned all the way left, and the

display is at 0%T. If the hand in the display is not at 0, turn the screw on
the front of the turbidimeter to adjust the hand to 0.

7.7.1.2 Turn on the turbidimeter and wait 15 minutes for warm up.

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7.7.1.3 Put a blank IF vial into the tube well on the top of the turbidimeter and
use the screw on the front to adjust the display to read 100%T. The

turbidimeter is now ready for use.

7.7.2 Liquid Inoculum Preparation

ye | Prior to starting, make sure the correct IF is selected and pre-label all IF
tubes.

Ldndad Place the IF tube into the top of the turbidimeter and remove the cap.

Make sure the display is at 100%T.

7.7.2.3 Using a sterile swab, gently select a very small amount of an isolate

colony from the agar plate.

7.7.2.4 Place the swab into the tube and disperse the growth into the fluid. Keep

in mind, the inoculum MUST be uniformly suspended. Clumpy colonies
may need additional agitation to achieve a homogenous suspension. It

takes only a very small amount of colony to get to the correct %T. Start
with a smaller amount than needed and work your way up to the

appropriate %T. An incorrect cellular density will not ID any microbes

and will need to be recultured, resulting in at least a 24 hour delay in
result.

7.7.2.5 More than one inoculum can be made at a time as long as they are placed
in the incubator within 20 minutes. Waiting any longer may cause

inaccurate identification. Anaerobic bacteria are especially sensitive to
delays. If you are running a batch of MicroPlates, set them up (from

preparing the inoculum to pipetting into the MicroPlate) so you will not

exceed the 20 minute deadline. The process tracking log has space for up
to 20 entries at a time.

7.7.3. MicroPlate Preparation

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LIL Remove the MicroPlates from their pouches and label each MicroPlate
(not the lid) with appropriate identification.

Td Dud Using aseptic technique, pour the cell suspension into a sterile
multichannel Pipette fluid reservoir.

Ts 33 Firmly attach eight sterile tips to the 8-Channel repeating Pipette. Make
sure the tips are straight, they can be carefully adjusted at the head of the

pipette. Do not touch any part of the tips that come in contact with the

inoculum as it will contaminate the culture. If the pipette head does not
audibly click when the tips are loaded, it will not aspirate or dispense

properly.

7.7.3.4 Prior to filling the tips with the suspension, Make sure the pipette is on

the 100 pl/multi dispense program. The electronic pipette will prime the
tips automatically. Check that all the tips are filling equally/ not leaking.

If there are bubbles or leaking, do not to attempt to fill the plate. Instead,

carefully dispense the inoculum back into the reservoir, dispose the tips,
and try again.

Fefdad Fill all MicroPlate wells with 100 ul per well. Take care to position the
tips inside of the wells at an angle. Avoid touching the bottom of the

wells, which could transfer carbon sources and alter the reaction in the

well.

Tf 3.6 If the fluid level in the tips gets low, refill and continue dispensing. The
pipette will double beep, signaling you to purge and refill if necessary.

Talat
Examine the MicroPlate to make sure all wells are filled. Any missing
wells can be filled with leftover inocula with a single pipette. However,
if a well is missing a sample, another one may be overfilled. Do not

attempt to move samples from one well to another. Most IFs will form a
soft gel shortly after inoculation.

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7.7.3.8 Cover the MicroPlate with its lid. Again, microplates must be placed in
the incubator no later than 20 minutes after inoculation. Anaerobic plates

must be incubated in a hydrogen free environment. In some
circumstances when a known anaerobe’s growth is scant, the plates can

be placed in a candle jar sealed with a strip of parafilm stretched around

the neck of the jar and lid.

8.0 Using the Microstation Analyzer

8.1 Security Overview and Part 11 Compliance

8.1.1 Operation of this analyzer requires the use of a custom built Windows PC. This

computer does not have any specific user as the main Windows account. Instead

of having individual Windows accounts and permissions, there is one general
account that all QC Lab personnel will have access to. For security and in

accordance with 21 CFR part 11, all lab personnel will receive their own login and
choose their own case-sensitive, alpha numeric password. Furthermore, this

computer is not connected to the Ion Labs network; it does not have access to
email or any of the company drives, nor does it have the ability for remote access.

Additionally, all actions performed in MicroLog Secure 6.3.1 are documented and

timestamped with a separate audit trail program that runs in the background. All
results are printed automatically, and are also stored on the computer. Finally, all

results are generated in a proprietary format and cannot be transferred, shared, or
altered in any way.

8.2 Start Up the Analyzer

8.2.1 Open MicroLog Secure 6.3.1 on the computer connected to the MicroStation.

8.2.2 Log in with username and password.

8.2.3 Click the setup tab.

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8.2.4 Power on the MicroStation and wait for the power up sequence to complete.

8.2.5 Click initialize reader button and wait for initialization to complete. Once

complete, Com Port: Open and Ready is displayed with a green background. If
the com port is not set to 5, it will not initialize.

8.2.6 Click on the read tab. The MicroStation is now ready to set up a batch and read
plates.

8.3. Create a Batch List

8.3.1 On the Read screen, fill in the Plate Information for your plate.

8.3.2 Click the Add to List button — this adds the plate to the batch — but moves the

selection down for adding another new plate.

8.3.3 Select the New plate in the list, fill out the Plate Information and click the Add to

List button (or press the Enter key) to add a plate to the batch list. Normally plate
information is cleared when starting a new plate. Un-checking the Clear

Information After Save check box leaves the last plate's information as a starting
point which saves a lot of work. This is useful when entering multiple plates of

the same type; change the sample name for each new plate.

8.3.4. The batch can be printed as a loading manifest to keep plates in order when

8.3.5 After the batch has been created, read each plate in order of the batch. Results

print automatically.

8.3.6 After valid results have been called, dispose of microplates and their lids into the
red biohazard waste.

8.4 Putting Plates on the Microstation

8.4.1 Make sure the reaction pattern is well developed.

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8.4.2 Wipe the bottom of the MicroPlate to remove condensation and fingerprints.

8.4.3 Open the analyzer lid and place the MicroPlate on the reader tray with the Al well

at the top left hand corner, and the upper right corner has a notch missing.

8.4.4 Gently push the MicroPlate down until it snaps into a level position in the reader

tray and remove the MicroPlate lid.

8.4.5 Close the analyzer lid.

8.4.6 Click Read Plate or double- click on the sample in the batch screen.

9.0 Identification Results and Criteria

9.1 Positive Identification of Genus, Species and Subspecies requirements

o Lot A Species ID can be called if and when the SIM value for the first choice species
exceeds the ID threshold. The ID threshold is set at a higher level for early reads

in order to avoid premature misidentification. The threshold starts at 1.0 and
declines linearly over the first 8 hours to 0.5. From 8 hours on, any time the SIM

of the first choice species exceeds 0.5, a Species ID can be called. When the SIM
match is high enough, the ID Box displays with a Subspecies, a Species or a

Genus level identification message. Entering the incubation hour at each read is

vital because this determines the SIM threshold and other decision criteria.

9.2 No Identification

9.201 In the situations listed below, the analyzer will not be able to ID both genus and
species. The software will generate a prompt to continue incubation or try a

different protocol.

9.2.1.1 If the MicroPlate has a sufficient pattern (at least three positive

reactions) and the SIM match is still too low for a Species identification,
a Genus level identification or No JD as a final result is displayed.

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92. be If the MicroPlate has an insufficient pattern (defined as less than three
positive reactions), the incubation time is extended up to 48 hours. The

ID Box displays No ID Yet - Continue Incubation until a Species
identification is obtained or until the 36 to 48 hour mark is reached.

9.2143 If there is no Species identification by 36 hours, the ID box displays

either a Genus level identification or a No ID message at the final 36 to
48 hour read.

9.2.1.4 A No ID - Insufficient Pattern result message at 36 to 48 hours displays
additional suggestions for retesting the strain with a different protocol.

9.2.15 If the number of borderline reactions in the online MicroPlate exceed 25,
the ID Box displays Too many Borderline Reactions as a final result.

9 2ekaG If the color in the Al reference well becomes too dark, the ID Box
displays a False Positive message, usually followed by a suggestion for

retesting the strain with a different protocol.

9.2.2 Additionally, a result of No JD can be from a mismatch.

9.2.2.1 After a MicroPlate is read, the View Details Window automatically

displays the major mismatches between the #1 ranked species and your
sample MicroPlate on the Pos/Neg Graphic tab. When this happens,

check for black + and - signs in the on-screen MicroPlate. The black (+)
and (-) signs show mismatches between your sample and the database

record (approximately +>80% pos, -<20%pos).

Qi2.22 If the mismatches are all positive (+) or all negative (-), you may have
made a testing error and a new culture must be made.

° ‘All +’ means the plate pattern is giving fewer positive reactions

than the species you’re comparing it against. Possible causes are:

under inoculation, the Al well is overfilled/clumpy/cloudy, the

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cells have been mishandled, cultured on incorrect media, wrong IF,
incorrect incubation environment.

‘All —’ means the plate pattern is giving more positive reactions
than the species you’re comparing it to. Possible causes include:

Over inoculation, Al well is under filled, contamination from an

impure culture.

10.0 Annual Performance Qualification

10.1 The Biolog MicroStation has no user serviceable parts.

10.2 A Performance Qualification should be done annually. Follow Biolog protocol VP-

003MLS located in the Microstation user manual.

11.0 Media Selection and Process Chart

Protocol A Protocol B Protocol C1 Protocol C2 YT AN
Standard Microaerophilic Fastidious or
Protocol Protocol Methylobactrium
; BUG+B BUG+B and CHOC, BUG+B & BUA+B,
Culture Media BUG, TS>A BUG BUG++B 2R2A BUY BAG
we Atmosphere AIR AIR 65% CO2 65% CO2 AIR ANA
=O|z Meda 33°C 33°C 33°-37°C 33°-37°C 2z

ai & |_ Temperature aT
< AN
5 6 Microplate
5 A} Incubation AIR 31-35°C AIR ee
i) Atmosphere & 26-28°C ace
Temperature free
Media and Temperatures may vary due to requirements of low or high enrichment media anaerobic
and strict lower temperature requirements for some species atmosphere
wy | ‘moculating IF-A IF-B IF-C IF-C AN-IF | AN-IF
EB 2 Fluid
=a < Inoculum %T 95 - 988%T 92 - 95%T 90 - 95%T 62 - 68%T 4AT%T 65%T
© :
S w ) = e | Micr p o e P r l w a e t l e l - p1 100 pl 100 pl 100 pl 100 pl 100 pL 100 pL
22 Incubation Up to 22 hours ** 24, 48, 50.24
**Incubation time may be extended to 36 hours by the system if an insufficient number of 72: t
Time in hours positive reactions are detected at 22 hours.

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12.0 Documentation Requirements

12.1 A PQV check must be performed for each completed logbook page as outlined in SOP A-
106 Documentation Guidelines for cGMP Records.

12.2 Documents will be maintained following SOP C-502 Record Storage, Retention, and

Destruction.

13.0 Revision History

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 07/11/23 | New procedure. N/A B. Echevarria | - | - |

14.0 Attachments

14.1. Attachment 1 — Environmental Monitoring ID Map

14.2 Attachment 2 — Probiotic ID Map

[SOP D-101 | Page 15 of 21]

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Attachment 1 — Environmental Monitoring ID Map

Streak onto TSA Plate/Blood Agar or SDA plate for each isolate type on each sample.

Record sample information plus ABC designation based on number of isolates sample and the date in the
Biolog ID Log Book.

Place streaked plates into the 30-35°C incubator if bacteria and 20-25 °C incubator if yeast

After 24 hours remove streaked plates from incubators, verify that there are no mixed colonies. If there
are, streak each isolate onto fresh TSA or SDA and label as 1, 2, or 3 along with sample number and place
back into appropriate incubator for 24hours

If the plates are not mixed, gather supplies and let come to room temp:
Protocol A (bacteria)- Reservoirs, pipet, pipette tips, If-A, Gen III microplates
Protocol YT (yeast)- Reservoirs, pipette tips, IF-YT, YT microplates

Inoculate fluids to correct turbidity:
Protocol A- 95-98%
Protocol YT-47%

Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with sample
information.

Place microplate into incubator for 20 hours. Same incubators as samples was in originally. Place date on

ID log under correct Protocol.

Remove microplates from incubator and read on Biolog.
If an ID is given, the microplate and the original isolate may be discarded, record date on ID
Log under ID complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours, Yeast may be returned
to incubator for a further 24 hours if necessary. Re-read on Biolog.
If “NO ID” or “False Positive” place original isolate into next phase of testing.
IF YT NO ID must be sent out for ID as they is no further testing in-house.

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Attachment 1 — Environmental Monitoring ID Map (continued)

Protocol B

Repeat original steps except:
Inoculate in IF-B

Turbidity should be 92-95%
Microplate is Gen III
Record in ID logbook the date under protocol B column for the specific isolate

After 24 hours read on Biolog, ensure that the protocol is changed to “B”
If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog under ID
complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours. Re-read on Biolog.

If “NO ID” place isolate into next phase of testing.

Protocol C1/C2

Repeat original steps except:
Inoculate in IF-C
Turbidity should be 90-92%
Microplate is Gen III
Record in ID log book the date under the Protocol C1 column for the specific isolate

After 24 hours read on Biolog, ensure that the protocol is changed to “C1”
If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog under

ID complete.
If “NO ID” isolate may be tried on C2 protocol
Turbidity — changes to 65%
Microplate is Gen III, incubate for 24 hours, read on Biolog if still “NO ID”
Send to Charles River for genotype ID (ACCUPRO-ID-S5).
Record in Comments in ID log book that ID was sent out.

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Attachment 2 — Probiotic ID Map

Bacillus Species
B. subtilis
Record sample information and the date in the Biolog ID Log Book
Streak onto Blood Agar and incubate in 30-35°C for 24 hours

Gather supplies for Protocol A-reservoirs, pipet, pipette tips, IF-A, and Gen III Microplate and
allow to come to room temp.
Inoculate fluid to correct turbidity: 95-98%
Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with

sample information.
Place microplate into 30-35°C incubator for 20 hours. Check protocol column and/or hour on the
ID log.
Remove microplates from incubator and read on Biolog.

If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog
under ID complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours.
If still “NO ID yet”, re-streak on Blood Agar and utilize Profile B, IF-B and gen microplate.

B sporogenes/ L. coagulans
Send out for testing

Lactobacillus Species

L. acidophilus (MAY NEED TO SEND OUT FOR TESTING)
Record sample information and the date in the Biolog ID Log Book
Streak onto Blood Agar, place into a candle jar and incubate in 30-35°C for 24 hours
Gather supplies for Protocol C1-reservoirs, pipet, pipette tips, IF-X, and Microplate XX

Inoculate fluid to correct turbidity:
Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.
Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with

sample information.
Place microplate into candle jar and incubator for 20-24 hours. Same incubators as samples was
in originally. Place check on ID log under correct Protocol.
Remove microplates from incubator and read on Biolog.

If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog
under ID complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours.

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Attachment 2 — Probiotic ID Map (continued)

Lactobacillus Species
L. gassieri
Record sample information and the date in the Biolog ID Log Book

Streak onto Blood Agar and incubate in 30-35°C for 24 hours
Gather supplies for Protocol XXX-reservoirs, pipet, pipette tips, IF-X, and Microplate XX
Inoculate fluid to correct turbidity:

Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.
Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.

Place microplate into incubator for 20 hours. Same incubators as samples was in originally.
Place date on ID log under correct Protocol.
Remove microplates from incubator and read on Biolog.
If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog

under ID complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours

L. paracasei

Record sample information and the date in the Biolog ID Log Book
Streak onto BUA agar, place into anaerobic container and incubate in 35-39°C for 24 hours.
Streak the 24 hour sample onto another BUA agar, place into anaerobic container and incubate in
35-37°C for another 24 hrs.

Gather supplies for Protocol AN-reservoirs, pipet, pipette tips, IF-AN, and AN Microplate
Inoculate fluid to correct turbidity: 6-65%
Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.

Place microplate into anaerobic container and incubate at 35-39°C for 20 hours. Place date on ID
log under correct Protocol.
Remove microplates from incubator and read on Biolog.
If an ID is given, the microplate and the original isolate may be discarded, record date on IDLog

under ID complete.
If “NO ID Yet”, place microplate back into incubator for another 24 hours

[SOP D-101 | Page 19 of 22]

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Attachment 2 — Probiotic ID Map (continued)

L. plantarum
e Record sample information and the date in the Biolog ID Log Book

e Streak onto Blood Agar and incubate in 30-35°C for 24 hours
e Gather supplies for Protocol C1 -reservoirs, pipet, pipette tips, IF-C, and Gen III Microplate
e Jnoculate fluid to correct turbidity 90-92%

e Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.
e Place microplate into incubator 30-35°C for 20 hours. Same incubators as samples was in
originally. Place date on ID log under correct Protocol.

e Remove microplates from incubator and read on Biolog.
e Ifan ID is given, the microplate and the original isolate may be discarded, record date on IDLog
under ID complete.
e If“NOID Yet”, place microplate back into incubator for another 24 hours

L. rhamnosus
e Record sample information and the date in the Biolog ID Log Book
e Streak onto Blood Agar and place into candle jar, incubate in 30-35°C for 24 hours

e Gather supplies for Protocol C2-reservoirs, pipet, pipette tips, IF-C, and Gen III Microplate
e Inoculate fluid to correct turbidity: 60-65%
e Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.

e Place microplate into a candle jar and incubate 30-35°C for 20 hours. Place check on ID log
under correct Protocol.
e Remove microplates from incubator and read on Biolog.
e fan ID is given, the microplate and the original isolate may be discarded, record date on ID Log

under ID complete.

[SOP D-101 | Page 20 of 21]

Standard Operating Procedure SOP No | Rev
Microbial Identification via Biolog Microstation D-1016 | 0 | Page20of21

Attachment 2 — Probiotic ID Map (continued)

Bifidobacterium Species
B. bifidum

e Record sample information and the date in the Biolog ID Log Book
e Streak onto BUA Agar and incubate in 35-37°C for 24 hours.
e After 24 hours re-streak onto another BUA agar and incubate in 35-37°C for 24 hours.
e Gather supplies for Protocol AN-reservoirs, pipet, pipette tips, IF-AN, and AN Microplate

e Inoculate fluid to correct turbidity: 60-65%
e Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.

e Place microplate into anaerobic container and incubator at 35-37°C for 20 hours. Place date on
ID log under correct Protocol.
e Remove microplates from incubator and read on Biolog.
e Ifan ID is given, the microplate and the original isolate may be discarded, record date on IDLog

under ID complete.
e If“NO ID Yet”, place microplate back into incubator for another 24 hours

B. breve
e Record sample information and the date in the Biolog ID Log Book

e Streak onto BUA Agar and incubate in 35-37°C for 24 hours.
e After 24 hours re-streak onto another BUA agar and incubate in 35-37°C for 24 hours.
e Gather supplies for Protocol AN-reservoirs, pipet, pipette tips, IF-AN, and AN Microplate

e Inoculate fluid to correct turbidity: 60-65%
e Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.
e Place microplate into anaerobic container and incubator at 35-37°C for 20 hours. Place date on

ID log under correct Protocol.
e Remove microplates from incubator and read on Biolog.
e [fan ID is given, the microplate and the original isolate may be discarded, record date on IDLog
under ID complete.

e If“NO ID Yet”, place microplate back into incubator for another 24 hours

[SOP D-101 | Page 21 of 22]

Standard Operating Procedure SOP No | Rev
Microbial Identification via Biolog Microstation D-1046;-| 0 | Page2t of 24

Attachment 2 — Probiotic ID Map (continued)

B. lactis
e Record sample information and the date in the Biolog ID Log Book

e If “NO ID Yet”, place microplate back into incubator for another 24 hours

B. longum
e Record sample information and the date in the Biolog ID Log Book

e Inoculate Microplate with 100ul of Inoculating fluid, use 8 channel pipet. Label Microplate with
sample information.
e Place microplate into anaerobic container and incubator at 35-37°C for 20 hours. Place date on
ID log under correct Protocol.

e Remove microplates from incubator and read on Biolog.
e Ifan ID is given, the microplate and the original isolate may be discarded, record date on ID Log
under ID complete.
e If “NO ID Yet”, place microplate back into incubator for another 24 hours

[SOP D-101 | Page 22 of 22]

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