Form F-0008 PEPRLab Self-Assessment Checklist

A ccording to the Paperwork Reduction Act of 1995, an agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a valid OMB control number.  The valid OMB control number for this information collection is 0583-xxxx. The time required to complete this information collection is estimated to average 60 minutes per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information.

U.S. DEPARTMENT OF AGRICULTURE

FOOD SAFETY AND INSPECTION SERVICE

Pasteurized Egg Products Recognized Laboratory (PEPRLab) Program

SALMONELLA LABORATORY SELF-ASSESSMENT CHECKLIST

LABORATORY NAME: ________________________________________________________

LABORATORY DIRECTOR: ____________________________________________________

LABORATORY REPRESENTATIVE(S) AT REVIEW: _______________________________

_____________________________________________________________________________

REVIEWER: _________________________________________________________________

DATE OF REVIEW: ___________________________________________________________

OPENING QUESTIONS:

1. Who are your current clients?

Client:_______________________________________Establishment No.

Client:_______________________________________Establishment No.

2. Is this facility an in-plant laboratory?

3. On average, how many Salmonella tests are conducted per week?

4. How many of these tests are on USDA Official Surveillance Samples?

5. When was the last time a pasteurized egg product sample was found to be positive for Salmonella?

6. How many Salmonella positive pasteurized egg product samples have been found in the last 3 years?

7. How soon and to whom were these reported? _____________________________________

_________________________________________________________________________

8. List all personnel involved in the Salmonella testing. ______________________________

_________________________________________________________________________

_________________________________________________________________________

TABLE OF CONTENTS

Opening Questions Page 1

Table of Contents Page 2

Section A: Personnel Requirements Page 3

Section B: Physical Facilities Page 3

Section C: Sample Receipt and Handling Page 4

Section D: Quality Assurance Page 6

Section E: Media and Reagents Page 10

Section F: Analytical Procedures Manual Page 13

Section G: Procedures and Methods Page 15

AMS Method Page 16

FSIS Method Page 17

FDA Method Page 18

Procedures and Methods (continued) Page 19

Section H: Safety Page 21

A. PERSONNEL REQUIREMENTS

1. Does the person in charge of microbiology have a baccalaureate

degree in biology, chemistry, microbiology, food technology,

medical technology, or other relevant science with at least 12

semester hours of course work in microbiology and/or at least 4

years of experience working in a public health, medical, food, or

other related laboratory? Yes No N/A

2. Are there training/education/experience records available

for each analyst? Yes No N/A

3. Is there a formal training program for employees working

in microbiology that includes instruction in safety, technical

procedures, and use of equipment? Yes No N/A

4. Is there a record kept of this formal training? Yes No N/A

B. PHYSICAL FACILITIES

A laboratory should have sufficient work and storage space and the facilities to handle the overall workload in order to ensure the quality of work, and safety of the employees.

1. Are floors, benches, and storerooms clean, free

of clutter, dust free, and well maintained? Yes No N/A

2. Are the following facilities adequate:

a. Sinks? Yes No N/A

b. Lighting? Yes No N/A

c. Gas outlets/Bacti-cinerator? Yes No N/A

d. Electrical outlets? Yes No N/A

e. Incubator capacity? Yes No N/A

f. Refrigerated storage space? Yes No N/A

g. Ventilation? Yes No N/A

3. Is there sufficient bench space for each analyst? Yes No N/A

4. Are bench tops made of impervious materials? Yes No N/A

5. Is the media preparation, glassware washing

area separate from the analytical area? Yes No N/A

6. Is unrelated traffic discouraged in the work area,

and is the laboratory locked when analysts are

not present? Yes No N/A

7. Are samples that are stored at room temperature,

stored in sealed containers to prevent pests from

infesting the laboratory? Yes No N/A

8. Is there a pest control system in place for the laboratory? Yes No N/A

C. SAMPLE RECEIPT AND HANDLING

Samples must be submitted to the laboratory in a condition that does not compromise the quality and validity of analytical results, and must be handled after receipt in the laboratory in a manner to maintain sample integrity.

l. Are samples inspected upon receipt in the laboratory for:

a. Leakage? Yes No N/A

b. Thawed frozen samples? Yes No N/A

c. Unsealed or ruptured containers? Yes No N/A

d. Spoilage? Yes No N/A

e. Evidence of tampering? Yes No N/A

2. Are all samples (including rejected samples) recorded

in a login system book, on worksheets, by computer,

or in another permanent, accessible format? Yes No N/A

3. Are unacceptable samples rejected for analysis and is the

condition recorded in login system book, computer, etc? Yes No N/A

4. If yes, are acceptable samples resubmitted? Yes No N/A

5. Does sample information include at a minimum:

a. Lot number ? Yes No N/A

b. Date of collection? Yes No N/A

c. Plant name and/or number? Yes No N/A

d. Type of analysis requested ? Yes No N/A

e. Type of product/state of product? Yes No N/A

f. Date of receipt? Yes No N/A

g. Condition upon receipt? Yes No N/A

6. Are liquid samples either analyzed on the same day received

or refrigerated at 2.0 to 8.0C until analyzed? Yes No N/A

7. Is the maximum turnaround time for sample analyses:

a. 4 to 5 days for negatives (cultural isolation method)? Yes No N/A

b. 5 to 7 days for positives (cultural isolation method)? Yes No N/A

c. 2 to 3 days for negatives using a rapid screening procedure? Yes No N/A

8. Are frozen samples either rapidly thawed in a water bath

(preferably with agitation) at less than 45C until

the slush ice stage (for no longer than 30 minutes) or

thawed at refrigerator temperatures (2.0 to 8.0C)

for no longer than 18 hours in the original container? Yes No N/A

9. Are thawed frozen samples analyzed immediately? Yes No N/A

10. Are dried egg samples analyzed upon receipt or

stored at room temperature for no more than 24 hours? Yes No N/A

11. If analysis of dried egg samples is delayed more than

24 hours, are they refrigerated at 2.0 to 8.0C? Yes No N/A

12. Are samples placed in appropriate storage after analysis

and are negatives retained for at least one day after reporting

and are positives retained for at least 30 days? Yes No N/A

D. QUALITY ASSURANCE

A written quality assurance program for the laboratory should be available, and the quality control records should be reviewed at least weekly by the supervisor. Proper care of laboratory instruments and equipment is essential for satisfactory performance of laboratory tests. Maintenance must be performed on a regular basis by trained individuals. Monitoring must be performed at stated intervals by laboratory personnel to assure on‑going reliability.

1. Is there a written Quality Assurance Program? Yes No N/A

2. Is there documentation showing that records of procedure

controls, instrument functions, scheduled maintenance,

and equipment temperatures are reviewed at least weekly? Yes No N/A

3. Is there documentation showing that corrective action(s)

were taken when controls were found to be unacceptable

and/or when instruments were found to be non functioning

or to have failed? Yes No N/A

4. Are quality control and maintenance records

maintained for at least 3 years? Yes No N/A

5. Is there a system for routinely reviewing the work

to detect clerical or analytical errors, or unusual results? Yes No N/A

6. Does the system provide for timely correction of

errors? Yes No N/A

7. Are laboratory results and analysts' worksheets retained

for each sample including negative samples for a period

of at least three years? Yes No N/A

8. Are there records of internal reviews and, when

indicated, corrective action(s) taken in response to

unacceptable check‑sample results? Yes No N/A

9. Are thermometers checked for accuracy against a

thermometric standard (National Institute of Standard

and Technology/formerly National Bureau of Standards)

before placing them in service? Yes No N/A a. Are thermometers calibrated annually? Yes No N/A

b. Are correction factors listed on each thermometer? Yes No N/A

c. Is the NIST traceable thermometer sent in for calibration

at least every 5 years? Yes No N/A

10. Are mechanical pipetting devices calibrated at least

semi‑annually to check accuracy of delivery? Yes No N/A

11. Is there a scheduled, written preventative maintenance

program for laboratory equipment and instruments? Yes No N/A

12. Does the preventative maintenance program include the following:

I. AUTOCLAVES:

a. Are acceptable temperature ranges defined for autoclaves? Yes No N/A

b. Are there recording thermometers, calibrated dials,

or other recording devices present on autoclaves? Yes No N/A

c. Are temperatures checked and recorded at each use

and is there documentation of corrective action for

out‑of‑range results? Yes No N/A

d. Are autoclaves monitored with biological indicators at least

monthly and are they monitored each time used with a physical

indicator (indicator tape)? Yes No N/A

II. WATERBATHS:

a. Are thermometers suspended in distilled water? Yes No N/A

b. Are acceptable temperature ranges defined and available

for waterbaths? Yes No N/A

c. Are temperatures checked and recorded at least daily,

and is there documentation of corrective action for

out-of-range results? Yes No N/A

d. Are water baths clean and free of debris, and is the

water changed regularly? Yes No N/A

III. INCUBATORS:

a. Are thermometers suspended in an appro­priate liquid

such as sterile glycerin, distilled water or other acceptable medium? Yes No N/A

b. Are acceptable temperature ranges defined and available

for each incubator? Yes No N/A

c. Are temperatures checked and recorded at least daily, and

is there documentation of corrective action for out-of-range

temperatures? Yes No N/A

IV. REFRIGERATORS/FREEZERS:

a. Are thermometers suspended in an appropriate

liquid such as sterile glycerin, distilled water or other acceptable medium? Yes No N/A

b. Are acceptable temperature ranges defined and

available for refrigerators/freezers?

Yes No N/A

c. Are temperatures checked and recorded at least daily,

and is there documentation of corrective action for

out-of-range temperatures? Yes No N/A

V. SPECTROPHOTOMETERS AND PHOTOMETRIC READERS:

a. Are manufacturer’s operation requirements

followed for the spectrophotometer and photometric reader? Yes No N/A

b. Is the instrument calibrated according to

manufacturer’s requirements or kit manufacturer’s requirements? Yes No N/A

VI. BALANCE:

a. Is the balance checked with a certified set of

weights at least weekly? Yes No N/A

NOTE: A 2000 gram balance must have a

sensitivity of 0.1 grams with a 200 gram load?

b. Is the balance checked annually by an authorized

service representative using certified weights that

are traceable to the National Institute of Standards

and Technology? Yes No N/A

VII. pH METER:

a. Are pH meters standardized with at least two appropriate

standard buffer solutions covering the range of intended use prior

to use and are the results recorded? Yes No N/A

b. If pH readings are going to be taken intermittently throughout

the day, is the pH meter re-calibrated with fresh portion of buffers

before each use? Yes No N/A

c. Are pH meter electrodes checked each time they are

used to see if they are filled and not cracked? Yes No N/A

E. MEDIA AND REAGENTS

All media, reagents, and chemicals must be prepared correctly, stored under appropriate conditions, and tested with reference organisms to assure satisfactory performance.

1. Are all purchased media, chemicals and solutions labeled

with the date received and an expiration date? Yes No N/A

2. Are all in‑house prepared media, reagents, and solutions

labeled with the name of product and expiration date? Yes No N/A

3. Do media records contain complete QC information Yes No N/A

for each batch, including pH, sterility, and productivity?

If pH paper strip is used for pH determination, the pH paper has to

cover the pH range of use with pH gradation value ≤ 0.2 pH unit.

4. Are media, reagents, and/or solutions stored under appropriate

conditions (i.e. refrigerated, away from daylight, in a cool

or dry place and in appropriate laboratory containers)? Yes No N/A

Note: The shelf life of prepared media will vary. In general, the

maximum shelf life of prepared culture media in sealed tubes or

bottles is 3 months in the refrigerator (2 - 8C), or up to 1 month

at room temperature (18 - 23C). Media in vented tubes may be stored

for up to 4 weeks if refrigerated or 2 weeks at room temperature. Plating

media may be stored in the refrigerator for a maximum of 10 weeks in

air-tight bags or for a maximum of 2 weeks if the bags are unsealed.

5. Are outdated materials discarded? Yes No N/A

6. Is a sample of each batch of in‑house prepared media

checked for the ability to support growth (and for

biochemical reactivity/selectivity, as appropriate to the

media) by using reference organisms capable of evaluating

pertinent characteristics of the media? Yes No N/A

List the Salmonella media QA cultures used:________________________

____________________________________________________________

____________________________________________________________

7. Are reference organisms maintained under refrigeration on agar

with at least monthly transfers, or by other appropriate methods? Yes No N/A

8. Is an uninoculated control of each medium used and run

concurrently with the sample? Yes No N/A

9. Are all media in satisfactory condition upon visual examination

(i.e. uncontaminated, hydrated, smooth, appropriate color and

thickness) and results documented for each batch? Yes No N/A

10. Are serological reagents tested with appropriate positive

and negative controls? Yes No N/A

(Note: This may include culture controls, commercially

produced antigen, or kit controls.)

11. Are serological reagents refrigerated when not in use,

inspected for clarity and color, and discarded when

showing any turbidity, flocculation or color change? Yes No N/A

12. Is Rappaport Vassiliadis broth (e.g. RV10, RVS, or RV Broth)

prepared according to manufac­turers instructions and autoclaved

for 15 minutes at 115 or 116 C (12 lbs)? Yes No N/A (NOTE: It is important not to overheat this medium.)

13. Is tetrathionate broth prepared according to manufacturers

instructions and heated to a boil? Yes No N/A

(NOTE: It is important not to overheat this medium.)

14. Is only the basal medium of tetrathionate broth base stored? Yes No N/A

15. Is the iodine - potassium iodide solution added to the

tetrathionate broth base on the day of use? Yes No N/A

16. Is selenite cystine broth prepared only by boiling, and is it

used on the day of preparation? Yes No N/A

17. Are Bismuth Sulfite Agar (BS) plates prepared, stored,

and incubated only as follows:

a. Are BS plates prepared (20 to 25 ml/plate) from

dehydrated media that is smooth, free-flowing, and

has been properly stored? Yes No N/A

b. Are BS plates used on the day of preparation or no more

than 1 day after preparation? Yes No N/A

18. Are double modified lysine iron agar (DMLIA) plates used

within three weeks of preparation (for FSIS method)? Yes No N/A

19. In the preparation of XLT4 agar (FSIS METHOD), is one

of the following used:

a. XL Agar Base with Thiosulfate citrate

and a 27 % solution (approximate) of the surfactant

7-ethyl-2-methyl-4-undecanol hydrogen sulfate, sodium salt,

formerly produced by Union Carbide under the tradename of

Tergitol 4? Yes No N/A

b. XLT4 Agar Base with the XLT4 Agar Supplement (a 27 %

solution (approximate) of the surfactant 7-ethyl-2-methyl-4-

undecanol hydrogen sulfate, sodium salt, formerly produced

by Union Carbide under the tradename of Tergitol 4). Yes No N/A

20. Does the laboratory have a distillation, ion exchange,

filtration, or other system available for producing or

purchasing water, free from toxic or nutritive substances,

to be used in media or reagent preparation? Yes No N/A

21. Is the distilled water stored properly? Yes No N/A

22. Is the water system monitored at least monthly and/or is

there a certificate of analysis for purchased distilled water

to ensure that each meet the following criteria:

a. conductivity (< 1.0 Siemens)

or resistivity (> 1 Megohm)? Yes No N/A

b. bacteria (<1000 cfu /ml)? Yes No N/A

23. Are other media used for Salmonella testing of pasteurized

egg products? Yes No N/A

If so, list the media used:___________________________________

_______________________________________________________

_______________________________________________________

24. If so, are these media prepared and stored according to the

manufacturer’s instructions? Yes No N/A

F. ANALYTICAL PROCEDURES MANUAL

To assure consistent laboratory results, a procedures manual should be available at each work station and should contain all procedures performed in the laboratory. Procedures should be written in sufficient detail to enable the analyst(s) to perform tests without referring to other publications.

(NOTE: Manufacturers’ package inserts with specific product use instructions may be used in addition to the manual, but cannot replace the procedures manual.)

A recognized laboratory may use a rapid screening method in their testing program for FSIS official egg product surveillance samples only if that method is either an approved AOAC Official Method of Analysis of the AOAC INTERNATIONAL, validated for egg products, or the FSIS Rapid Screening Method as described in the MLG. All presumptive positives identified by rapid screening methods must be confirmed using one of the three accepted cultural methods listed below. Any recognized laboratory that does not use a rapid screening method in their testing program must use one of the following three cultural methods as their primary protocol for egg product analysis:

1. AMS Laboratory Methods for Egg Products – Section I (‘93 rev.) and Section VII (‘94 rev.) Reference AOAC 967.26, 967.27, 978.24, 989.12, 991.13.

2. FSIS MLG online, Chapter 4.

3. FDA BAM online, Chapter 5.

1. Is an Analytical Procedures Manual available in

the laboratory? Yes No N/A

2. For Salmonella testing, does the manual contain:

a. All of the procedures performed? Yes No N/A

b. Only approved or accepted procedures? Yes No N/A

c. Criteria for accepting or rejecting samples? Yes No N/A

d. A section on media and reagent preparation? Yes No N/A

e. Quality control procedures? Yes No N/A

3. Does each procedure contain:

a. Step-by-step instructions? Yes No N/A

b. Sample handling/preservation? Yes No N/A

c. Expected reactions/results? Yes No N/A

d. Corrective actions to be taken when expected

reactions/results are not observed? Yes No N/A

e. References? Yes No N/A

4. Is the manual reviewed and updated annually? Yes No N/A

5. Are changes in procedures approved and initialed by the

supervisor? Yes No N/A

6. Is there documentation to show that all analysts have

read the procedures manual, including any revisions,

and that only the most recent revision is being used? Yes No N/A

G. PROCEDURES AND METHODS

Routine procedures for Salmonella detection permit recovery of small numbers of pathogens or debilitated organisms by pre-enrichment in lactose broth or buffered peptone water (BPW). Selective enrichment and plating procedures following that permit growth of Salmonella while limiting the growth of competing non-Salmonella organisms naturally present in food samples. Identification of an isolate as a member of the genus Salmonella depends on a combination of biochemical and serological parameters.

1. Is at least 100 g of sample tested for official surveillance samples? Yes No N/A

2. Is a positive control culture run along with all Salmonella tests

through any rapid screening test and confirmation tests? Yes No N/A

List the Salmonella

control culture(s) used:

3. Is every tube and plate throughout the test procedure

appropriately labeled? Yes No N/A

4. For each sample, are records maintained documenting each

step of analysis for traceability? (i.e. analyst ID, media/kit/reagent lot

number, incubation time and temperatures, equipment ID number, etc.) Yes No N/A

5. List the cultural method used for analysis and/or confirmation

of official surveillance samples.

6. Is the laboratory using a rapid screening method that is either the FSIS MLG Method or an approved AOAC Official Method,

validated for egg products? Yes No N/A

If yes, list the method below with its AOAC reference number:

Rapid Screening Method:

AOAC Official Method Reference Number:

7. Prior to implementing a new rapid method, were parallel

tests conducted using both the rapid and conventional

cultural methods and were the results documented? Yes No N/A

8. In the parallel testing, did the methods show equivalency,

agreeing at least 95 percent of the time? Yes No N/A

9. Are all positive results that are obtained by rapid screening

methods followed up by subculturing the sample and

subsequently performing biochemical and serological

identification of any Salmonella isolates? Yes No N/A

10. Is the ratio of egg sample to preenrichment broth maintained at 1:10? Yes No N/A

Proceed to question #11 if your lab is using the AMS culture method. Go to question #12 if your lab is using the FSIS, MLG chapter 4 culture method.

Go to question #13 if your lab is using the FDA, BAM chapter 5 culture method.

11. AMS Method – Laboratory Methods for Egg Products

(Section I - 1993 rev.) and Section VII - 1994 rev.):

a. Is the pH of the lactose broth/egg mixtures adjusted to

6.8  0.2 after being left for 1 hour at room temperature? Yes No N/A

List the method of pH testing used: ______________________

b. After 24  2 hours incubation at 35C is the lactose broth

subcultured by transferring 1 ml into 10 ml of selenite cystine

broth and an additional 1 ml into 10 ml of tetrathionate broth? Yes No N/A

c. After 24  2 hours incubation at 35C are the selenite cystine

and tetrathionate broths subcultured to selective differential

agars, XLD, HE, and BS (or manufacturer’s recommendation

for rapid tests)? Yes No N/A

4. After 24  2 hours incubation at 35C are up to three typical

colonies (if available), characteristic of Salmonella species,

selected from each differential agar plate as follows:

XLD – pink/red colonies with/without black centers

or all black colonies (atypical strains may appear

yellow with or without black centers)? Yes No N/A

HE – blue/blue-green colonies with or without

black centers or all-black colonies? Yes No N/A

BS – brown, black, or grey colonies, usually with

a metallic sheen and darkening of the surrounding

media or occasionally green colonies? Yes No N/A

e. Are all BS agar plates examined for typical or suspicious

Salmonella colonies after 24  2 hours incubation

at 35C and, if negative, again at 48  2 hours incubation? Yes No N/A

f. Go to question #14. (page 19)

12. FSIS Method – Microbiology Laboratory Guidebook online (MLG), Chapter 4:

1. After 20 – 24 hours of incubation at 35  2C, is the buffered

peptone water-sample mixture subcultured by transferring

0.1 ml. into 10 ml of Rappaport Vassiliadis (RV) Broth and

by transferring 0.5 ml into 10 ml of tetrathionate (TT) broth,

and are these broths then incubated at 42  0.5C? Yes No N/A

2. After 22 – 24 hours incubation at 42  0.5C are TT and RV

broths subcultured to selective differential agars, BGS and

either DMLIA or XLT4? Yes No N/A

3. After 18 – 24 hours incubation at 35  2C are up to three

typical colonies, (if available), characteristic of Salmonella

species, selected from each differential agar plate as follows:

BGS – colonies that are pink and opaque with

a smooth appearance and entire edge surrounded by a

red color in the medium? (On very crowded plates,

look for colonies that give a tan appearance against

a green background.) Yes No N/A

DMLIA – purple colonies with or without black

centers? (Since salmonellae typically decarboxylate lysine

and ferment neither lactose nor sucrose, the color of the

medium reverts to purple.) Yes No N/A

XLT4 – black colonies or red colonies with

black centers? (The rim of the colony may still be

yellow in 24 h; later it should turn red.) Yes No N/A

4. Are all selective agar plates reincubated for an additional

24  2 hours and are all initially negative plates, as well

as those yielding non-confirmed Salmonella colonies from

the initial selection reexamined before discarding? Yes No N/A

e. Go to question #14. (page 17)

13. FDA Method – Bacteriological Analytical Manual online (BAM), Chapter 5:

1. Is lactose broth used for pre-enrichment of dry egg products? Yes No N/A

2. Is TSB with ferrous sulfate (35 mg ferrous sulfate per 1000 ml TSB)

used for pre-enrichment of liquid egg products? Yes No N/A

c. Is the pH of the pre-enrichment broth/egg mixtures adjusted to

6.8  0.2 after being left for 1 hour at room temperature? Yes No N/A

List the method of pH testing used: ____________________

d. After 24  2 hours incubation at 35C is the lactose broth

subcultured by transferring 0.1 ml into 10 ml of RV broth

and an additional 1 ml into 10 ml of tetrathionate (TT) broth? Yes No N/A

e. Is the RV broth incubated 24 h  2 h at 42  0.2C? Yes No N/A

f. Is the TT broth incubated 24 h  2 h at 35  2.0C? Yes No N/A

g. After 24  2 hours incubation are the RV and TT broths

subcultured to selective differential agars, XLD, HE, and

BS by streaking 10 l from each broth onto each of the

three selective differential agars? Yes No N/A

h. After 24  2 hours incubation at 35C are at least 2 typical colonies

(if available), characteristic of Salmonella species, picked to TSI

and LIA slants from each differential agar plate as follows:

XLD – pink/red colonies with/without black centers or all black colonies

(atypical strains may appear yellow with or without black centers)? Yes No N/A

HE – blue/blue-green colonies with or without

black centers or all-black colonies? Yes No N/A

BS – brown, black, or grey colonies, usually with a metallic sheen and

darkening of the surrounding media or occasionally green colonies? Yes No N/A

i. Are BS plates re-incubated an additional 24  2 h

and, if the original colonies from the BS plates give

atypical reactions on TSI and LIA, are at least 2

additional typical colonies picked, if available? Yes No N/A

j. Are selective agar plates stored at 5 – 8C until completion

of confirmation steps? Yes No N/A

14. If suspicious colonies are not well isolated, are they

re-streaked for purification directly onto selective agar

plates before inoculating Triple Sugar Iron (TSI) and

Lysine Iron Agar (LIA) slants? Yes No N/A

15. Are characteristic colonies inoculated to TSI slants and

LIA slants by inoculating the slants in tandem with a

single pick from a colony, and by stabbing the butts and

streaking the slants in one operation? Yes No N/A

16. After incubation at 35  2C for 24 + 2 hours with caps

loosened, are TSI and LIA slants with the following

characteristics of Salmonella selected for further analysis:

a. LIA – Alkaline slant and butt (purple throughout)

with or without hydrogen sulfide (H₂S) production? Yes No N/A

(Note: Some strains will produce an acid butt,

along with a typical TSI slant.)

b. TSI – Alkaline (red) slant and acid (yellow) butt

with or without hydrogen sulfide (H₂S) production? Yes No N/A

(Note: Some strains will produce an acid slant and butt.)

17. Are TSI/LIA cultures, which appear to be mixed, streaked for

isolation before additional biochemical or serological tests

are performed? Yes No N/A

18. Before reporting a presumptive positive sample as negative, at least six

TSI/LIA cultures (if available) picked as below are subjected to further

biochemical and serological testing:

a. For FSIS MLG 4 method: are at least three well isolated colonies

from each of two plating media picked to TSI/LIA pairs and subject

to confirmation testing before a sample is reported as negative? Yes No N/A

b. For AMS or FDA method, are at least two well isolated colonies from

each of three plating media picked to TSI/LIA pairs and subject to

confirmation testing before a sample is reported as negative? Yes No N/A

19. Is a rapid/miniaturized biochemical test system used for

identifying Salmonella? Yes No N/A

If yes, list the test system below with its AOAC reference number:

Biochemical Test System:

AOAC Reference Number:

20. Are the manufacturers' guidelines for miniaturized

biochemical systems followed for inoculum preparation,

incubation, and interpretation of results? Yes No N/A

21. Are sufficient biochemical tests performed to presumptively

identify atypical isolates as Salmonella? (i.e. urease, dulcitol,

lactose, and sucrose fermentation, and malonate utilization) Yes No N/A

22. When performing slide agglutination tests, are all materials

and equipment brought to room temperature before testing? Yes No N/A

23. Is a saline control included to detect autoagglutination when

performing the polyvalent or group somatic (O) antigen slide

agglutination test? Yes No. N/A

24. Are polyvalent flagellar (H) antigen screening tests performed

by a tube method using formalinized cultures prepared from:

a. Brain-heart infusion broth incubated at 35C

for 4 to 6 hours for same-day testing? Yes No N/A

b. Trypticase soy broth incubated 24 hours at

35C for next-day testing? Yes No N/A

25. For H antigen testing is a negative control of formalinized

saline with the formalinized culture included in the testing? Yes No N/A

26. Are H antigen tests incubated at 48 – 50C for 1 hour? Yes No N/A

27. Are diluted Salmonella H antisera prepared in quantities

sufficient only for daily use, and any remaining diluted

antisera discarded at the end of the day? Yes No N/A

28 If the Oxoid kit or SSI H antiserum is used, are manufacturer’s

instructions followed? Yes No N/A

Safety issues are not within the scope of the PEPRLab Program audit. Therefore, the laboratory is not required to report a corrective action for any observations and/or recommendations resulting from this part of the review. This segment is conducted out of concern for the health and safety of laboratory personnel.

H. SAFETY:

Facilities need to be designed and equipped to meet established OSHA safety standards. Protective equipment should be available to personnel and a comprehensive safety program should be included in laboratory procedures.

1. Is there an ongoing, documented safety education program

that includes, but is not limited to, instruction on:

a. Location and use of fire extinguishers,

blankets, and other safety equipment? Yes No N/A

b. Fire drills and evacuation routes? Yes No N/A

c. Handling emergency situations? Yes No N/A

d. Basic first aid procedures? Yes No N/A

e. CPR training? Yes No N/A

f. The labeling of all cancer suspect agents? Yes No N/A

g. Lifting heavy items? Yes No N/A

h. "Right to Know" laws? Yes No N/A

2. Is there a safety manual available in the laboratory? Yes No N/A

3. Does it include procedures for:

a. Handling spills of contaminated materials? Yes No N/A

b. Disposal of biological waste? Yes No N/A

c. Disposal of chemical waste? Yes No N/A

d. Handling toxic materials? Yes No N/A

4. Are Materials Safety Data Sheets (MSDS) available

in the laboratory for all chemicals used in the laboratory? Yes No N/A

5. Is there a designated safety officer in the laboratory? Yes No N/A

6. Does the safety officer conduct periodic safety

inspections using a checklist? Yes No N/A

7. Are safety deficiencies and corrective actions documented? Yes No N/A

8. Are accidents documented and reported to the safety officer? Yes No N/A

9. Is emergency medical help readily available if needed by

laboratory personnel? Yes No N/A

10. Are emergency phone numbers (i.e. fire, ambulance, police)

posted in a conspicuous place on or near the phone? Yes No N/A

11. Are personnel ever alone in the laboratory? Yes No N/A

12. Does the laboratory have at least two exits and are all

exits and hallways free of obstructions? Yes No N/A

13. Can the doors be locked from both sides? Yes No N/A

14. Are the following in the laboratory:

a. Fire extinguishers (CO₂, dry chemical)? Yes No N/A

b. Fire blanket? Yes No N/A

c. Eyewash station? Yes No N/A

d. Overhead shower? Yes No N/A

e. Fire alarm system? Yes No N/A

f. Sprinkler system? Yes No N/A

g. First aid kit? Yes No N/A

15. Are fire extinguishers and other safety equipment

regularly inspected, certified to be in working order,

and their condition documented? Yes No N/A

16. Is an EPA‑approved disinfectant available to clean up

biohazardous spills and disinfect bench tops daily? Yes No N/A

17. Is the disinfectant prepared and used according to the

manufacturers instructions? Yes No N/A

18. Are biohazardous materials discarded in leak-proof,

tear‑resistant plastic bags marked with a biohazard symbol? Yes No N/A

19. Are biohazardous waste materials steam sterilized at 121C

for at least 45 minutes, with biohazard bags vented to effect

complete sterilization as required by the manufacturer, or else

incinerated prior to disposal in landfills? Yes No N/A

20. Are biohazardous materials removed from the laboratory daily

and contained in a manner to minimize accidental spills during

storage and transport and to exclude rodents and vermin? Yes No N/A

(i.e. Bags are tied and placed in covered, rigid containers such as

buckets, cans, or cardboard boxes, and liquids are placed in capped

or tightly stoppered bottles or tubes.)

21. Are janitors and other maintenance personnel instructed in

proper methods of disposal, and are disposal areas located

well away from the building and protected from trespassers? Yes No N/A

22. Are personnel instructed not to taste chemicals at all,

and not to directly smell chemicals? Yes No N/A

23. Is mouth pipetting strictly prohibited (with no exceptions

for sterile solutions)? Yes No N/A

24. Are eating, drinking, and smoking prohibited in the laboratory,

and is labware prohibited from use for any of these purposes? Yes No N/A

25. Is food prohibited from refrigerators that are used for reagents,

samples, etc.? Yes No N/A

26. Are personnel instructed to wash hands after handling samples,

working with cultures, handling chemicals, and/or before leaving

the laboratory? Yes No N/A

27. Are laboratory personnel required to confine long hair? Yes No N/A

28. Are aprons, gloves, and goggles available for handling

hazardous materials? Yes No N/A

29. Are heat‑resistant gloves available near the autoclave

and in the media preparation area? Yes No N/A

30. Are laboratory coats or other protective clothing worn

only in the laboratory? Yes No N/A

31. Are bunsen burners turned off when not in use? Yes No N/A

32. Are chipped, broken, or etched glassware discarded

in a specially marked, puncture proof, sealed container? Yes No N/A

33. Is broken glassware always cleaned up with a dust pan/brush

and never picked up with the hands? Yes No N/A

34. Are heavy plastic carriers available for transporting

acids or other corrosive chemicals? Yes No N/A

35. Are bottles of acid (HC1) always tightly capped and

rinsed on the outside after being used and/or before

being opened? Yes No N/A

36. Have laboratory personnel been taught to always

pour acid into water, never water into acid? Yes No N/A

37. Is a safety cabinet or room available for storing large

containers of hazardous chemicals? Yes No N/A

38. Are electrical connections covered with a heavy

rubber coating? Yes No N/A

39. Are extension cords grounded and, if running across

the floor, are they taped down? Yes No N/A

40. Are all electrical cords, receptacles, and switches in good

condition and located away from water sources? Yes No N/A

41. Does the laboratory use mercury thermometer(s)? If so, is a

mercury spill kit available? Yes No N/A

42. If located near water sources, are electrical outlets protected

with ground-fault circuit interrupters? Yes No N/A

I. SUMMATION AND COMMENTS:

REFERENCES

1. EPA Guide for Infectious Waste Management, May 1987. United States Environmental Protection Agency, National Technical Information Service, Springfield, VA.

2. Handbook of Microbiological Media, 3^rd Edition, 2004, CRC Press, Boca Raton, Fl.

3. Biosafety in Microbiological and Biomedical Laboratories, 4^th ed. May 1999. Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, GA.

4. Compendium of Methods for the Microbiological Examination of Foods, 4^th ed. 2001. APHA, Technical Committee on Microbiological Methods for Foods, Washington, DC.

5. Good Laboratory Practice Regulations, Code of Federal Regulations (CFR), 21 CFR Part 58, U.S. Food and Drug Administration, 5600 Fishers Lane, Rockville, MD.

6. Difco &BBL Manual, 1st ed., 2003, Becton, Dickson and Company, Sparks, Maryland.

7. Official Methods of Analysis of AOAC INTERNATIONAL, Current AOAC Internet Version

8. Laboratory Methods for Egg Products – Section I (1993 revision) and Section VII (1994 revision), U. S. Department of Agriculture, Agriculture Marketing Service, Washington, D. C.

9. Microbiology Laboratory Guidebook online (MLG), Chapter 4, U. S. Department. of Agriculture, Food Safety and Inspection Service, Washington, D.C.

10. Bacteriological Analytical Manual online (BAM), Chapter 5, U.S. Food and Drug Administration, Washington, D.C.

Instructions for completing the form

1. Answer all questions on the checklist by placing a circle around the appropriate response or by filling in the blank. Responses are based on observations or information supplied by laboratory personnel. Questions pertaining to services, equipment, instruments, methods or procedures not used routinely by the laboratory should be marked as not applicable (N/A).

2. Submit the completed form to:

Zhihong Wang, M.D. MBA

Program Manager, Pasteurized Egg Products Recognized Laboratory Program

USDA, FSIS, OPHS, LQAD

950 College Station Road

Athens, Georgia 30605

Phone: (706) 546-3559 Fax: (706) 546-3453

E-mail: [email protected]

Form No. PEPRL F-0008.04 Effective: xx/xx/xx Issuing Authority: Laboratory Quality Assurance Division (LQAD) FSIS FORM xxxxxx (mm/dd/yy) Page 30 of 29