Milk Safety References
National Conference on Interstate Milk Shipments (NCIMS)

DAIRY WATERS
(Coliform Group )
[Unless otherwise stated all tolerances are ±5% ]

1. __________ Laboratory Requirements
   1. __________ CP, items 33 & 34
   2. __________ Sample volume sufficient to assure 100 mL for testing sufficient air space for mixing (about ¾ full), if completely filled do not accept
   3. __________ Transported and maintained at 0-4.4C (temperature control [TC] required)
   4. __________ If samples are not refrigerated, transit not to exceed 6 hours (TC not required)
   5. __________ Transit time does not exceed 30 hours
   6. __________ Samples examined within 30 hours of collection or within 2 hours of receipt (item 1d)
   APPARATUS
2. __________ CP, see items 1 – 32 (as necessary)
3. __________ Sample Containers
   1. __________ Borosilicate glass, plastic bottles or bags
   2. __________ Sterile, containing 0.1 mL of 10% Sodium Thiosulfate
   3. __________ Holds sufficient sample with air space for all necessary bacterial tests
   4. __________ Maintains sample uncontaminated
4. __________ Incubator 35±0.5C (Make/Model ________________________)
   1. __________ See CP item 15 for incubator requirements
5. __________ Fermentation Tubes/Bottles
   1. __________ Sufficient size to conform with requirements for media, durham tube and sample
      
6. __________ Inoculation Equipment
   1. __________ Sterilized loops of at least 3 mm diameter, 22–24 gauge nichrome, chromel or platinum-iridium wire
   2. __________ Disposable dry heat-sterilized hardwood applicator sticks, 0.2 to 0.3 cm in diameter and a minimum of 2.5 cm longer than the fermentation tubes
   3. __________ Inoculating needle
7. __________ Vacuum source with trap
8. __________ Membrane filter funnel Brand _________________
   1. __________ Free from defects that may interfere with function
   2. __________ Sterilizable
   3. __________ Marked at 100 mL, or pre-marked checked and adjusted, using a 100 mL Class A graduate cylinder
9. __________ Membrane cellulose filters, 47 mm, 0.45 µM (±0.02 µM), sterilized __________

   Brand __________ Lot # __________

10. __________ Absorbent pads, sterilized Brand _____________
11. __________ Forceps
    1. __________ Round tipped, with smooth surface
12. __________ Culture (Petri) dishes (for MF) Brand _____________

    __________Size ______________

    1. __________ Sterile with plastic, tight fitting covers
13. __________ Microscope and Lamp Brand ____________ Model ___________
    1. __________ Binocular, wide field, 10x oculars
    2. __________ Fluorescent light, adjacent, above, perpendicular to filter plane
    3. __________ Other optical device giving equivalent results
14. __________ Storage of media
    1. __________ See CP item 27 for media and storage requirements
    2. __________ MF Media
       1. __________ Store in dark at 0–4.4C
       2. __________ Broth medium used within 96 hr. Date prep. _____
       3. __________ Plates kept no more than 1 week in a sealed container at 0–4.4C. Date prep. _____
15. __________ Presumptive Test
    1. __________ Lauryl Tryptose Broth
       1. __________ Before inoculating arrange tubes in order and label, or otherwise identify
       2. __________ Shake samples vigorously 25 times in a 30 cm arc in 7 sec before removing test portion
       3. __________ Remove test portions (100 mL total) within 3 min
       4. __________ Inoculate ten (10) fermentation tubes with 10 mL of sample or five (5) tubes with 20 mL with double strength LST or one bottle with 100 mL double strength LST
       5. __________ Incubate tubes at 35±0.5C for 24±2 hours
       6. __________ Examine tubes for gas – any gas is considered presumptive positive
       7. __________ Return negative tubes (no gas) to incubator and incubate an additional 24 hr (total of 48±3 hr)
       8. __________ Re-examine tubes for gas production after 48±3 hours
       9. __________ Record presence or absence of gas at each examination
       10. __________ Any gas produced by 24 or 48 hr is considered positive for the Presumptive Test
       11. __________ No gas after 48 hr is Not Found (NF) for the Test
       12. __________ Do not report gas production after 51 hr of incubation
       13. __________ Promptly submit all presumptive positive tubes showing gas production at 24 or 48 hr to the Confirmed Test
16. __________ Confirmed Test
    1. __________ Brilliant Green Lactose Bile Broth
       1. __________ Gently shake presumptive positive tube
       2. __________ Transfer (loop or stick) portion of positive broth to BGLB broth
       3. __________ Incubate tubes at 35±0.5C for 24±2 hr
       4. __________ Examine tubes for gas - any gas is considered positive
       5. __________ Return negative tubes (no gas) to incubator and incubate an additional 24 hr (total of 48±3 hr)
       6. __________ Re-examine tubes for gas production after 48 hours
       7. __________ Record presence or absence of gas at each examination
       8. __________ Any gas produced by 24 or 48 hr is considered positive for the Confirmed Test
       9. __________ No gas after 48 hr is Not Found (NF) for the Test
       10. __________ Do not report gas production after 51 hr of incubation
17. __________ Reporting
    1. __________ Report results of fermentation tubes that confirm as positive, reported as MPN/100 mL (³ 1.1/100 mL if 10 mL in 10 tubes or 20 mL in 5 tubes are used) or ³ 1/100 mL if 100 mL presence/absence test used
    2. __________ If one or more tubes turbid with no gas production, invalidate the sample and request a re-sample from the same point source for heterotrophic plate count
    3. __________ Interpretation: for multiple tubes, Not Found (NF)is < 1.1/100 mL and Positive is ³ 1.1/100 mL; for presence/ absence, NF is < 1/100 mL and Positive is ³ 1/100 mL

    TESTS FOR PRESENCE OF MEMBERS OF THE COLIFORM GROUP
    BY MEMBRANE FILTRATION TECHNIQUE

18. Filtration
    1. __________ Place (with alcohol flamed forceps, item 11) sterile membrane filter (item 9) on porous plate, secure funnel
    2. __________ Pour 100 mL test sample into funnel (item 8) and apply vacuum
    3. __________ After test volume has been filtered, rinse funnel by filtering 3 volumes of 20–30 mL of sterile buffered water
    4. __________ Turn off vacuum and remove filter with sterile (alcohol flamed) forceps
    5. __________ M–endo Broth
       1. __________ Sterile pad (item 10) placed in culture dish
       2. __________ Saturate pad with 2.0 mL of M-endo Medium, CP item 27n
       3. __________ Allow to stand a few minutes before pouring off excess
       4. __________ Prepared filter rolled (grid side up) onto pad slowly to avoid trapping air bubbles, do not drag across side of plate
    6. __________ M–endo Agar
       1. __________ Use culture dish previously prepared (CP item 27m)
       2. __________ Prepared filter placed on agar with rolling motion to avoid trapping air bubbles
    PROCEDURE
19. __________ Incubation
    1. __________ In saturated humidity, with dish inverted
    2. __________ At 35±0.5C for 21±1 hr
20. __________ Counting
    1. __________ Count all sheen colonies as typical coliforms and dark suspect colonies as atypical coliforms, keep separate counts of each morphological type until confirmed
    2. __________ Confirm 10% up to a maximum of 10 isolated colonies, with representative proportions of each colony type
21. __________ Confirmation Test
    1. __________ Make serial transfers of colonies to individual LST and then to BGLB tubes using the same transfer needle/stick
    2. __________ Incubate tubes at 35±0.5C for 24±2 hr
    3. __________ Examine tubes for gas
       1. __________ LST tubes with gas must be transferred to fresh BGLB tubes if the original BGLB tubes show no gas
    4. __________ Return negative tubes (no gas) to incubator and incubate an additional 24 hr (total of 48±3 hr)
    5. __________ Re-examine tubes for gas production after 48 hours
    6. __________ Record presence or absence of gas at each examination
    7. __________ Any gas produced in BGLB tubes by 24 or 48 hrs is considered positive for the Confirmation Test
    8. __________ No gas after 48 hr is Not Found (NF) for the Test
    9. __________ Do not report gas production after 51 hr of incubation
22. __________ Reporting
    1. __________ Report confirmed colony count/100 mL
    2. __________ Invalidate all samples with confluent growth or TNTC, and request a re-sample from the same point source for heterotrophic plate count
    3. __________ Interpretation: Not Found (NF) is < 1/100 mL and Positive is ³ 1/100 mL
    HETEROTROPHIC BACTERIA STANDARD PLATE COUNT METHOD
23. __________ Heterotrophic Plate Count Method
    1. __________ Plate samples as in SPC, items 2-10, 13 and 14
    2. __________ Incubate at 35±0.5C for 48±3 hours
    3. __________ Count as in SPC item 16-17
    4. __________ Report counts as in SPC item 20
    5. __________ Record as "Heterotrophic Plate Count/mL at 35C"
    6. __________ Interpretation: Negative if < 500 CFU/mL and Positive if ³ 500 CFU/mL
    CHROMOGENIC SUBSTRATE (MMO-MUG) PRESENCE - ABSENCE SCREENING TEST FOR DAIRY WATERS
    (SOURCE WATER SUPPLIES ONLY)
24. __________ Materials
    1. __________ Color comparator
    2. __________ Sterile borosilicate glass or clear plastic bottles to contain 100 mL sample with sufficient air space for mixing (about ¾ full)
    3. __________ MMO-MUG substrate, see CP item 27o
    4. __________ Quality control procedures conducted on each lot of substrate received, as recommended by manufacturer, test by spiking with known coliform, records maintained
25. __________ Procedure
    1. __________ Aseptically add pre weighed MMO-MUG substrate to 100 mL of water sample
    2. __________ Optionally, add 100 mL sample to the MMO-MUG substrate in a sterile container provided by the manufacturer
    3. __________ Aseptically cap and mix thoroughly by inverting 25 times to dissolve reagent (does not completely dissolve)
    4. __________ Incubate at 35±0.5C for a minimum of 24 hours, not to exceed 28 hours
    5. __________ Examine containers for the production of yellow color
26. __________ Interpretation
    1. __________ If no yellow color is observed
       1. __________ Record sample as Not Found (NF) for total coliforms
       2. __________ Report as total coliform Not Found (NF) in 100 mL sample: < 1/100 mL
    2. __________ If yellow color present
       1. __________ Gently invert container several times until color is uniformly dispersed through the sample
       2. __________ Compare yellow color to color comparator dispersed into the SAME type of sample container
       3. __________ If color is equal to or greater than that of the color comparator, sample reported as Positive for total coliforms
       4. __________ If color is obvious but less than the comparator, sample reported as Not Found (NF)
       5. __________ Report as total coliforms present in 100 mL sample: ³ 1/100 mL
    CHROMOGENIC SUBSTRATE (MMO-MUG) MULTIPLE TUBE PROCEDURE FOR THE PRESENCE OF TOTAL
    COLIFORMS (SOURCE WATER SUPPLIES ONLY)
27. __________ Materials, see items 24 a-d)
28. __________ Procedure
    1. __________ Before transferring sample portions arrange tubes in order and identify
    2. __________ Shake samples vigorously 25 times in a 30 cm arc in 7 sec
    3. __________ Aseptically add pre weighed MMO-MUG substrate to 100 mL sample
    4. __________ Optionally, add 100 mL of sample to container with MMO-MUG substrate provided by manufacturer
    5. __________ Aseptically cap and mix thoroughly by inverting 25 times to dissolve reagent (does not completely dissolve)
    6. __________ Remove test portions (100 mL total) within 3 minutes
    7. __________ Transfer 20 mL of sample/reagent mixture to five tubes, or 10 mL to ten tubes
    8. __________ Optionally, transfer 100 mL of mixed (see item 28b) sample to 10 tubes containing pre dispensed MMO-MUG reagent provided by manufacturer
    9. __________ Incubate tubes at 35±0.5C for a minimum of 24 hours, do not to exceed 28 hours
    10. __________ Examine tubes for the development of yellow color
        1. __________ Mix tubes to uniformly distribute yellow color
        2. __________ Compare tubes to color comparator tube (SAME size and type as MPN tubes)
        3. __________ Tubes with color equal to or greater than color comparator tube recorded as Positive
        4. __________ Tubes with obvious color but less than comparator, sample reported as Not Found (NF)
29. __________ Reporting
    1. __________ If all tubes show no color, report as Not Found (NF): < 1.1/100 mL
    2. __________ If one or more tubes show yellow color (see 28j) report as Positive: ³ 1.1/100 mL
    CHROMOGENIC SUBSTRATE PRESENCE (XGAL - MUG) - ABSENCE SCREENING TEST FOR DAIRY WATERS
    (SOURCE WATER SUPPLIES ONLY)
30. __________ Materials
    1. __________ E*Colite substrate, see CP item 27p
    2. __________ Quality control procedures conducted on each lot of substrate received, as recommended by manufacturer, test by spiking with known coliform, records maintained
31. __________ Procedure
    1. __________ Add water sample to the E*Colite substrate
       1. __________ Tear perforated strip
       2. __________ Open bag by pulling white tabs
       3. __________ Aseptically pour 100 mL of water sample into bag (do not touch inside of bag)
       4. __________ Flatten bag to remove air
       5. __________ Twirl bag 2–3 times around twister wires to form a leak proof seal
       6. __________ Fold twisters around back of bag
       7. __________ Shake bag 25 times in 7 seconds to dissolve sodium thiosulfate tablet, if present
       8. __________ Continue rolling to build pressure in water compartment
       9. __________ Maintain pressure on rolled area and push water through first seal into powder section of bag ONLY
       10. __________ Shake bag 25 times in 7 seconds to completely dissolved powder in water (push mixture against bag sides to pull apart any remaining seal)
    2. __________ Place sealed bag in 35C water bath for 10 minutes
    3. __________ Transfer to 35±0.5C incubator for 28 hours
    4. __________ Examine bags for the production of blue or blue/green color or blue color in corners of bag
32. __________ Interpretation
    1. __________ If yellow color is observed:
       1. __________ Record sample as Not Found (NF) for total coliforms
       2. Report as total coliform Not Found (NF) in 100 mL sample: < 1/100 mL
    2. __________ If blue or blue/green (or blue in corners) color observed:
       1. __________ The sample is Positive for total coliforms
       2. __________ Report as total coliforms present in 100 mL sample: ³1/100 mL
    MISCELLANEOUS
33. __________ Copy of current in-use edition of Standard Methods for the Examination of Water and Wastewater in laboratory