Microbiological Examination of Cheese

Health Protection Branch - Ottawa

Official Method MFO-14
November 30, 1983

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1. APPLICATION

This method shall be used for the determination of Escherichia coli and of Staphylococcus aureus in cheese, including cheese curd but excluding cottage cheese, made from either pasteurized or unpasteurized milk, in accordance with Section B.08.048 of the Food and Drug Regulations.

2. SAMPLING

2.1 Definition of Terms

2.1.1 Lot: A batch or production unit which may be identified by the same code. When there is no code identification, a lot may be considered as

(a) that quantity of cheese produced under essentially the same conditions, at the same establishment and representing no more than one day's production; or,

(b) the quantity of the same variety of cheese from one and the same manufacturer available for sampling at a fixed location.

2.1.2 Sample: The sample units (subsamples) taken per lot for analysis.

2.1.3 Sample Unit: Usually a consumer size container of the product, and should consist of a minimum of 100 g. A sample unit is often referred to as a subsample.

2.1.4 Analytical Unit: That amount of cheese withdrawn from the sample unit for analysis.

2.2 Collection of Samples

2.2.1 A sample, consisting of five sample units drawn at random from each lot, shall be taken.

2.2.2 Each sample unit shall consist of at least 100 g.

2.2.3 Collect original unopened containers or packages wherever possible.

2.2.4 Employ aseptic techniques in collecting the sample units when sampling bulk cheese.

Place each collected sample unit into a separate sterile container.

2.2.5 Keep sample units refrigerated (0-5 °C) during transportation.

3. PROCEDURE

Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions.

3.1 Handling of Sample Units (1) Keep sample units refrigerated (0-5 °C) prior to anlaysis. (2) Analyze sample units as soon as possible after receipt at the laboratory. 3.2 Preparation of Media

The following media, prepared and sterilized according to the manufacturers' instructions, shall be used:

(1) Lauryl Sulfate Tryptose (LST) broth

(2) Escherichia Coli (EC) broth

(3) Levine's Eosin Methylene Blue (EMB) agar

(4) Nutrient agar (NA)

(5) IMViC media: a. Tryptone broth b. Buffered Glucose broth c. Simmon's Citrate (SC) agar

(6) Baird-Parker (BP) agar

(7) Brain Heart Infusion (BHI) broth

(8) Trypticase Soy (TS) agar

(9) Blood agar (BA)

(10) Toluidine Blue-DNA agar (TDA)

(11) Phenol Red Carbohydrate broth

3.3 Preparation of Dilutions

3.3.1 Temper sterile aqueous 2% sodium citrate to 40-45 °C, and prewarm sterile blender jars to 40-45 °C.

3.3.2 Combine portions from several locations within the sample unit, to obtain a representative analytical unit of 11(10)* g.

3.3.3 Prepare a 1: 10 dilution of cheese by adding the analytical unit to 99(90)* mL of the tempered sodium citrate solution in a prewarmed sterile blender jar. Blend for the minimum time required to produce a homogeneous suspension. To prevent overheating, blending time should not exceed 2.5 min.

3.3.4 Check the pH of the suspension. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0 with either sterile NAOH or HCl.

3.3.5 Prepare succeeding dilutions as required to determine the total numbers of E. coli and S. aureus, by transferring 11(10)* mL of the previous dilution into 99(90)* mL of 0.1% sterile peptone water diluent. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

* Weight and volume in brackets indicate alternative procedure for preparation of dilutions.

3.4 Determination of E. coli

3.4.1 Presumptive Coliform Test

a. The medium used is LST broth, dispensed in 10 mL volumes into tubes containing gas vials (inverted Durham tubes).

b. Arrange LST broth tubes in rows of fives, and mark them identifying the sample, the sample unit and the dilution to be inoculated.

c. Inoculate LST broth with a culture of E. coli known to ferment lactose and produce gas at 45 °C to serve as a positive control, incubate, and subsequently transfer into all media used at different stages of the procedure. Set up an uninoculated type of medium corresponding to each step in the procedure as a negative control.

d. Inoculate each tube of a set of five tubes of single strength LST broth with 1 mL of the 1:10 dilution (cheese homogenate; see section 3.3.3, above). Repeat for each succeeding decimal dilution as required.

e. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.

f. Incubate the inoculated LST broth tubes at 35 °C ± 0. 5 °C for 24 ± 2 hr.

Examine for gas formation, and on the same day begin the presumptive E. coli (faecal coliform) test for all gas-positive tubes (see sections 3.4.2 and 3.4.3, below).

g. Incubate gas-negative tubes for an additional 24 ± 2 hr, examine, record the number of additional gas-positive tubes and begin the presumptive E. coli (faecal coliform) test for the additional gas-positive tubes.

h. The absence of gas in all of the tubes at the end of 48 ± 2 hr of incubation constitutes a negative test.

3.4.2 Determination of Presumptive E. coli (Faecal Coliforms)

a. The medium used is EC broth, dispensed in 10 mL volumes in tubes containing gas vials.

b. Submit all gas-positive LST broth tubes to the presumptive E. coli (faecal coliform) test.

c . Shake or rotate the LST broth tubes to mix the contents and transfer one loopful from each positive LST broth tube to a tube of EC medium. Avoid transferring pellicle.

d. Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.

e. Incubate the inoculated EC broth tubes in a water bath at 45 °C ± 0.20 for 24 ± 2 hr. Make certain that the water level in the bath is above the level of the medium in the tubes.

f. Examine for gas production and on the same day begin the E. coli identification for all gas-positive tubes (see section 3.4.3, below).

g. Incubate gas-negative tubes for an additional 24 ± 2 hr, examine, record the number of gas-positive tubes above, and begin the E. coli identification for the additional gas-positive tubes.

3.4.3 Identification of E. coli

a. The media and reagents used, are:

(i) EMB agar dispensed in Petri plates.

(ii) NA dispensed in Petri plates and as slants in tubes.

(iii) EC broth dispensed in 10 mL volumes in tubes containing gas vials.

(iv) Tryptone broth dispensed in 5 mL volumes in tubes.

(v) Buffered Glucose broth dispensed in 6 mL volumes in tubes.

(vi) Simmon's Citrate (SC) agar dispensed as slants in tubes.

(vii) Kovac's indole reagent consisting of pure amyl or isoamyl alcohol, p -dimethylaminobenzaldehyde, and 12N HCl (analytical grade) at a ratio of 15:1: 5 or Ehrlich-Boehme's indole reagent consisting of p -dimethylaminobenzaldehyde, 96% ethanol, and 12N HCl at a ratio of 0.4:38:8.

(viii) Methyl red solution consisting of 0.1 g methyl red dissolved in 300 mL of 95% ethanol and diluted with distilled water to 500 mL.

(ix) VP reagent consisting of an aqueous solution of 40% NaOH and 0.3% creatin, or
VP reagent consisting of 16% aqueous KOH and 6% ?-naphthol in 95% ethanol.

b . From each gas-positive EC broth tube (see section 3.4.2, steps f., g., above), streak a loopful of culture onto a separate EMB agar plate.

c . Incubate the plates at 35 °C ± 0.5 °C for 18 to 24 hr and examine for colonies which are nucleated with or without a metallic sheen.

d. Select two such colonies from each plate and streak onto separate NA plates to obtain discrete colonies.

e. Incubate the NA plates at 35 °C ± 0.5 °C for 24 ± 2 hr, and from each of them, pick an isolated colony and streak onto a separate NA slant.

f. Incubate the slants at 35 °C ± 0,5 °C for 24 ± 2 hr.

g. From one of the two NA plates prepared (step d., above), transfer inoculum into a separate tube of each of the EC broth and the IMViC media (for IMViC tests, see steps m., n., o., below).

h. If isolates picked from EMB agar and purified on NA plates are stored for more than 72 hr before being subjected to the IMViC reactions, inoculate fresh NA slants from the slants prepared in step e., above, incubate at 35 °C + 0.5 °C for p>24 ± 2 hr, then inoculate the IMViC media from the freshly incubated slants.

i. Incubate the inoculated EC broth and IMViC media at 35 °C ± 0.5 °C for p>24 ± 2 hr or, as indicated in step n., below, for 48 ± 2 hr.

j . Examine the EC broth tubes after 24 ± 2 hr for gas production and record results.

k. If the EC broth tubes are gas-negative, incubate for an additional 24 ± 2 hr, examine, and record results. If no gas is produced within 48 ± 2 hr, the isolate is not considered to be E. coli.

l. If gas is produced within 24 or 48 + hr, make smear from the corresponding NA slant which was inoculated from the same colony (see steps e. and g., above), and stain by Gram's procedure. Examine microscopically and record results.

If the organisms are not Gram-negative, nonsporeforming rods, they are not E. coli.

m. Indole(I)

(i) Transfer inoculum from each isolate to be tested (see step g., above) into a separate tube of Tryptone broth.

(ii) Incubate the inoculated tubes at 35 °C ± 0.5 °C for 24 ± 2 hr.

(iii) Add 0.2-0.3 mL of either indole reagent (see step a., vii, above) to each tube and shake the tube to mix the contents.

(iv) Let the tube stand for 10 min and observe. A dark red colour in the alcohol layer indicates a positive test.

n. Methyl-Red Voges Proskauer Tests (MR & VP)

(i) Transfer inoculum from each isolate to be tested (see step g., above) into a separate tube of Buffered Glucose broth.

(ii) Incubate the inoculated tubes at 35 °C ± 0.5 °C for 48 ± 2 hr.

(iii) Pipette 1 mL from each incubated tube into a separate empty tube, and add 1 mL of VP reagent. Shake the tubes vigorously to aerate.

(iv) Let the tubes stand for 4 hr and observe. The test is VP positive if an eosin pink colour develops within 4 hr.

(v) Incubate the remainder of the Buffered Glucose broth for an additional 48 ± 2 hr at 35 °C ± 0.5 °C. Add 5 drops of the methyl red solution to each tube and shake the tubes to mix the contents. The test is positive if a red colour develops.

(vi) Alternatively, two separate tubes may be set up for the MR and VP tests; if this is done, an equal volume of VP reagent must be added to one of the tubes, five drops of the methyl red solution to the other.

o. Sodium Citrate Test (C)

(i) Transfer inoculum from each isolate to be tested (see step g., above) onto a separate slant of SC agar. Use an inoculating needle and apply a light inoculum.

(ii) Incubate the inoculated slants at 35 °C ± 0.5 °C for 48 ± 2 hr and observe for growth. Visible growth is usually accompanied by a change of colour from green to deep blue.

p. The characteristic IMViC reaction pattern for E. coli is as follows:

Indole (+ or -)

Methyl red (+)

Voges-Proskauer (-)

Citrate (-)

q. If gas is produced in EC broth, and IMViC reactions characteristic of E. coli are obtained, the other isolate (see steps d., e., f., above) need not be further tested. However, if no gas is produced in EC broth within 48 ± 2 hr and/or the IMViC pattern is not characteristic of E. coli, the remaining isolate shall be tested for gas production in EC broth and for its IMViC reaction pattern. Repeat steps g. to o., above. If both isolates fail to produce gas in EC broth and/or produce IMViC reaction patterns not characteritic of E. coli, then E. coli is considered to be absent from the EC broth tube from which the isolates originated.

r. Compute the MPN of E. coli per g of cheese following the instructions in Part 5, on the basis of the number of gas-positive EC broth tubes, which were incubated at 35 °C (see step i., above), and found to contain Gram-negative, non-sporeforming, rod-shaped bacteria, giving the IMViC reactions characteristic of E. coli (see step p., above).

3.5 Determination of S. aureus

3.5.1 Presumptive Count

a. The selective agar used is BP agar.

b. Pre-pour plates of BP agar and allow their surfaces to dry before they are inoculated.

c. Mark clearly each Petri plate to be used, identifying the sample, the sample unit, and the dilution.

d. Agitate each dilution bottle (see section 3.3.5 above) to resuspend material.

e. For pasteurized-milk cheese, distribute 2 mL of the 1:10 dilution accurately over the surface of five BP plates by spreading 0.4 mL on each of the five plates.

f. For unpasteurized-milk cheese, spread 0.2 mL of the 1:10 dilution evenly over the surface of each of two BP plates.

g. From at least two subsequent dilutions, spread 0.2 mL evenly over the surface of each of two BP agar plates.

h. Invert the plates and incubate at 35 °C ± 0.5 °C for 48 ± 2 hr.

i . The following two types of colonies are considered to be presumptive S. aureus.

Type 1: Convex, entire, shiny black surrounded by clear zones extending into the opaque medium.

Type 2: Covex, entire, shiny black without clearly defined zones. Each colony type may shown grey-white margins around the colonies and/or opaque zones (double halos).

j. Count colonies immediately after the incubation period.

k. Do not count black mucoid colonies larger than 2 mm in diameter, or swarmers.

l . Count the colonies of each type and record separately, but add together to give the total presumptive count.

m. Counting of the five plates of the 1:10 dilution

(i) If the number of all presumptive S. aureus colonies per plate is fewer than 20, add separately the number of each colony type on all five plates to provide a count for each type per 2 mL (i.e. per 0.2 g cheese). Multiply each total by five to obtain the presumptive S. aureus count per g of cheese to obtain the total presumptive S. aureus count per g of cheese.

(ii) If the number of all presumptive S. aureus colonies per plate is greater than 20 but does not exceed 200, take two plates at random, and separately count the numbers of colonies of each type. For each type, compute the average presumptive S. aureus count per plate (per 0.4 mL). Mulitply each presumptive count by 25 to obtain the presumptive count per g of cheese. Add the two presumptive counts per g of cheese to obtain the total presumptive S. aureus count per g of cheese.

(iii) If the number of presumptive staphylococcal colonies on some of the five plates is < 20, but on others is ≥ 20, proceed as in (i) above.

n. Counting of duplicate plates:

(i) Select the duplicate plates of the dilution that yields a combined presumptive S. aureus count between 20 and 200 colonies per plate. Count separately the colonies of each type and compute the average presumptive count for each type per plate (0.2 mL). Multiply each count by 5 and by the appropriate dilution factor, and record as the presumptive count of each of the two types per g of cheese. Add the two results, and record as the total presumptive S. aureus count per g of cheese.

o. Record negative presumptive counts as < 5 per g of cheese if five plates of the 1:10 dilution are used; or as < 2.5 x the dilution factor per g of cheese for duplicate plates.

3.5.2 Test for Coagulase Production

a. The media and reagent to be used are:

(i) Non-selective media such as NA, TS agar or BA, dispensed as slants in tubes.

(ii) BHI broth dispensed in 1 mL volumes in tubes.

(iii) Certified rabbit plasma containing EDTA dispensed in 0.5 mL volumes in 12 x 100 mm tubes.

b. From the replicate plates counted, select a number of each colony type observed as follows to check for culture purity and for coagulase reaction.

(i) When the total count per type for all the plates of a dilution is less than five, pick all colonies of that type.

(ii) When the total count per type for all plates of a dilution is equal to or greater than five colonies, pick five colonies of that type at random.

c. Streak each colony picked onto a non-selective medium, so as to obtain discrete colonies.

d. Incubate at 35 °C ± 0.5 °C for 24 ± 2 hr.

e. Make a smear from the growth of each isolate on the non-selective medium and stain with a simple stain (e.g., crystal violet). Observe microscopically for the presence of cocci.

f. If the isolates are composed of cocci only, transfer inoculum from each plate of non-selective medium into a separate tube of BHI broth. If an isolate is not pure, choose another colony from step c. above and repeat steps d. and e.

g. Incubate at 35 °C ± 0.5 °C for 18-24 hr and observe for growth.

h. Inoculate BHI broth with a culture of S. aureus known to be coagulase-positive, to serve as a positive control. Incubate at 35 °C ± 0.5 °C for 18-24 hr. Use uninoculated medium from the same batch of BHI broth as a negative control.

i. Transfer 0.2 mL of each BHI broth culture (see steps f. and h., above) into a separate tube of rabbit plasma and shake or rotate the tubes to mix thoroughly.

j . Incubate the tubes at 35 °C-37 °C and examine after one hr. and after four hr. Do not shake the tubes during incubation. Negative tubes should be incubated overnight at room temperature and rechecked for a 3+ or 4+ reaction (see Figure I).

k. A 3+ or 4+ reaction is confirmation that the isolate is S. aureus.

3.5.3 The Thermonuclease Test

a. Perform the test for the presence of thermostable nuclease (TNase) concurrently with the coagulase test in the following manner:

b. Medium used is toluidine blue - DNA agar (TDA).

c. Pipette 3.0 mL of molten TDA mixture to a microscope slide or a plastic immunoplate; or pipette sufficient TDA to a flat bottom Petri plate to give a height of 1.5 mm.

d. With a cork borer or a suitably cut pasteur pipette, cut wells, 2 mm in diameter, approximately 12 mm apart, in the agar layer.

e. Remove agar plugs by aspiration.

f. Heat broth cultures used for the determination of coagulase production in a boiling water bath for 15 min., and cool rapidly under cold tap water.

g. Fill wells in the TDA with heated and subsequently cooled broth cultures

h. Incubate the TDA slides or plates in a moist chamber at 35 °C and examine after 4 hr.

i. A bright pink halo extending ≥ 1 mm beyond the perimeter of the well is indicative of nuclease activity. Plates giving doubtful reactions should be held at room temperature and re-examined the following morning.

j. Isolates are confirmed as S. aureus if:

(i) they possess TNase activity and produce 3+ or 4+ degree of coagulase reaction (EDTA rabbit plasma), or

(ii) if they are TNase-negative but give a 4+ coagulase reaction.

k. With cultures that show a 2+ or lesser coagulase reaction but which are positive for TNase activity, perform the following additional tests:

3.5.4 Anaerobic utilization of Glucose

a. Inoculate culture to be tested into a tube of carbohydrate fermentation medium containing 0.5% glucose.

b . Overlay with sterile paraffin oil or Vaspar and incubate at 35 °C for 18-24 hr.

c. Colour change indicating an acid reaction which is a positive test. S. aureus gives a positive reaction.

3.5.5 Anaerobic utilization of mannitol

a. Same as in 3.5.4.a. except that the source of carbohydrate is mannitol.

b. S. aureus usually gives a positive reaction but some strains do not ferment mannitol.

3.5.6 Lysostaphin sensitivity a. The reagents used are:

(i) Phosphate Saline Buffer (0.02 M) pH 7.3-7.4.

(ii) Lysostaphin solution containing 50 µg per mL of lysostaphin in the phosphate buffer.

b. Inoculate the culture to be tested into 0.2 mL of the phosphate buffer and mix to obtain a homogeneous suspension.

c. Transfer one half of the suspended cells to another tube (13 x 100 mm) and mix with 0.1 mL of the phosphate saline buffer.

d. Add 0.1 mL of the lysostaphin solution to the original tube to give a concentration of 25 µg lysostaphin per mL of cell suspension.

e. Incubate both tubes at 37 °C for as long as 2 hr.

f. If the turbidity clears in the tube containing cells plus lysostaphin, the test is positive. If clearing has not occurred in 2 hr., the test is negative. g. S. aureus gives a positive reaction.

h. Run positive and negative controls simultaneously with the tests. i. If two of the three ancillary tests are positive, the isolate is considered to be S. aureus.

3.5.7 On the basis of the confirmatory tests for each of the two types of cultures, record the total number of S. aureus per g of cheese.

		No. of type 1 colonies confirmed as S. aureus _______________________ No. of type 1 colonies tested	x	presumptive count type 1 colonies/g
Number of S. aureus/g	=		Plus
		No. of type 2 colonies confirmed as S. aureus _____________________ No. of type 2 colonies tested	x	presumptive count type 2 colonies/g

4. INTERPRETATION

4.1 The tolerance as specified hereafter and representing the maximum probable incidence of Escherichia coli, and the maximum count of Staphylococcus aureus in cheese, including cheese curd but excluding cottage cheese, made from pasteurized milk, shall be applied in determining whether the tested lot of the product complies with Section B.08.048(1) of the Food and Drug Regulations.

(1) The maximum MPN of E. coli permitted for each lot is that represented by an E. coli MPN not exceeding:

(a) 100 per g in more than two of five sample units, and

(b) 2,000 per g in any one sample unit, included in the sample taken from a lot.

(2) The maximum count of S. aureus permitted for each lot is that represented by a count of S. aureus not exceeding:

(a) 100 per g in more than two of five sample units, and

(b) 10,000 per g in any sample unit, included in the sample taken from a lot.

4.2 The tolerance as specified hereafter and representing the maximum probable incidence of Escherichia coli, and the maximum count of Staphylococcus aureus in cheese, made from unpasteurized milk, shall be applied in determining whether the tested lot of the product complies with Section B.08.048(2) of the Food and Drug Regulations.

(1) The maximum MPN of E. coli permitted for each lot is that represented by an E. coli MPN not exceeding:

(a) 500 per g in more than two of five sample units, and

(b) 2,000 per g in any one sample unit, included in the sample taken from a lot.

(2) The maximum count of S. aureus permitted for each lot is that represented by a count of S. aureus not exceeding:

(a) 1,000 per g in more than two of five sample units, and

(b) 10,000 per g in any sample unit, included in the sample taken from a lot.

These tolerances are summarized in the following table:

Determination	n	c	m	M
(1) Cheese made from Pasteurized Milk
E. coli	5	2	100	2,000
S. aureus	5	2	100	10,000
(2) Cheese made from Unpasteurized Milk
E. coli	5	2	500	2,000
S. aureus	5	2	1,000	10,000

n	=	Number of sample units (subsamples) to be examined per lot.
c	=	Maximum numnber of sample units (subsamples) per lot which may have a bacterial concentration higher than the value for "m" without violation of the Regulations.
m	=	Maximum number of bacteria per g of cheese, which is of no concern (acceptable level of contamination).
M	=	Maximum number of bacteria per g of cheese, which if exceeded by any one sample unit (subsample), renders the lot in violation of the Regulation.

5. CALCULATION OF MOST PROBABLE NUMBERS (MPN)

Table A-1 shows the most probable numbers of coliforms per 100 g or mL of test material corresponding to the number of gas-positive tubes in the coliform test. Table A-1 has been adapted from a conversion table prepared for the analysis of drinking waters where 10, 1.0 and 0.1 mL of the water under test are used as test portions. The table is equally appropriate if 10, 1.0 and 0.1 g of a solid food constitute the test positions in the tubes. When other sized portions of the test material are placed in the tubes, MPN values obtained from Table A-1 must be multiplied by an appropriate number, to correct for the actual amount of test material in the tubes, and also to obtain the MPN per g (mL) as is usually done for foods, rather than per 100 mL (g), for which the values are given in the table. The volume of diluent added to the tubes (and which accompanies the test material) is ignored when calculating the MPN.

Example:

The following inoculated tubes give a positive reading:

(1) 5 tubes with 10 mL of 1:10 dilution of test material - all 5 are positive

(2) 5 tubes with 1 mL of 1:10 dilution of test material - 1 is positive

(3) 5 tubes with 1 mL of 1:100 dilution of test material - none is positive

The quantities in each of the five tubes of the three dilution series represent 1, 0.1 and 0.01 g (mL), respectively of the test material. According to Table A-1, a reading of 5-1-0 gives a value of 33 when 10, 1 and 0. 1 g (mL) respectively are used. However, since only 1/10 of these amounts were actually used in the analysis, the value of 33 obtained from Table A-1 must be multiplied by 10 giving 33 x 10 = 330 organisms per 100 g (mL) of test material. Since the results have to be expressed per g (mL), the MPN value is 330 ÷ 100 = 3.3. When higher dilutions are used, the same procedure is followed, but the multiplier (dilution factor) is enlarged to relate the amount of test material actually present to the values given for 10, 1.0 and 0.1 g (mL) in Table A-1.

Dilution factor - Reciprocal of the dilution of the analytical unit. For calculating the MPN, use the dilution factor of the middle set of the three dilutions selected.

To determine which consecutive dilutions to use, refer to the combinations shown below: (See also Table A- 2).

1 . If only 3 dilutions are made, use the results of 3 dilutions to compute the MPN. Examples a. and b.

2. If more than 3 dilutions are employed, use the results of only 3 consecutive dilutions. Select the highest dilution in which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c. and d.

3 . If more than 3 dilutions are made, but none of the dilutions tested have all 5 tubes positive, use the first 3 dilutions. Example e.

4 . If a positive tube occurs in the dilution higher than the 3 chosen to rule, the number of such positive tubes should be added to those of the next lower dilution. Example f.

5 . If the tubes of all sets of a dilution series are positive, choose the 3 highest dilutions of the series and indicate by a "greater than" symbol (>) that the MPN is greater than the one calculated. Example g.

Refer to Table A-1 and look up the value which corresponds to the number of positive tubes obtained.

MPN/g = No. of Microorganisms (Table A-1)/100 X dilution factor of middle set of tubes

Table A-1

Most Probable Number (MPN) of Bacteria Per 100 g (mL) of Test Material Using 5 Tubes With 10,1 and 0.1 mL or g of Test Material

Pos* 10;1;0,1	MPN	Pos* 10;1;0,1	MPN	Pos* 10;1;0,1	MPN	Pos* 10;1;0,1	MPN	Pos* 10;1;0,1	MPN	Pos* 10;1;0,1	MPN
000	<1.8	100	2	200	4.5	300	7.8	400	13	500	23
001	1.8	101	4	201	6.8	301	11	401	17	501	31
002	3.6	102	6	202	9.1	302	13	402	21	502	43
003	5.4	103	8	203	12	303	16	403	25	503	58
004	7.2	104	10	204	14	304	20	404	30	504	76
005	9	105	12	205	16	305	23	405	36	505	95
010	1.8	110	4	210	6.8	310	11	410	17	510	33
011	3.6	111	6.1	211	9.2	311	14	411	21	511	46
012	5.5	112	8.1	212	12	312	17	412	26	512	64
013	7.3	113	10	213	14	313	20	413	31	513	84
014	9.1	114	12	214	17	314	23	414	36	514	110
015	11	115	14	215	19	315	27	415	42	515	130
020	3.7	120	6.1	220	9.3	320	14	420	22	520	49
021	5.5	121	8.2	221	12	321	17	421	26	521	70
022	7.4	122	10	222	14	322	20	422	32	522	95
023	9.2	123	12	223	17	323	24	423	38	523	120
024	11	124	15	224	19	324	27	424	44	524	150
025	13	125	17	225	22	325	31	425	50	525	180
030	5.6	130	8.3	230	12	330	17	430	27	530	79
031	7.4	131	10	231	14	331	21	431	33	531	110
032	9.3	132	13	232	17	332	24	432	39	532	140
033	11	133	15	233	20	333	28	433	45	533	180
034	13	134	17	234	22	334	31	434	52	534	210
035	15	135	19	235	25	335	35	435	59	535	250
040	7.5	140	11	240	15	340	21	440	34	540	130
041	9.4	141	13	241	17	341	24	441	40	541	170
042	11	142	15	242	20	342	28	442	47	542	220
043	13	143	17	243	23	343	32	443	54	543	280
044	15	144	19	244	25	344	36	444	62	544	350
045	17	145	22	245	28	345	40	445	69	545	440
050	9.4	150	13	250	17	350	25	450	41	550	240
051	11	151	15	251	20	351	29	451	48	551	350
052	13	152	17	252	17	352	32	452	56	552	540
053	15	153	19	253	26	353	37	453	64	553	920
054	17	154	22	254	29	354	41	454	72	554	1600
055	19	155	24	255	32	355	45	455	81	555	>1600

* Number of positive tubes with each of 3 volumes used.

	Dilutions*					Combination to be used	MPN from Table A-1	Dilution factor of middle dilution	MPN per mL or g
	Undiluted		1:10	1:100	1:1000
	Amount of original test material (g or mL)
	10	1	0.1	0.01	0.001
a.	5/5**	5/5	2/5			5-5-2	540	1	5.4
b.		5/5	5/5	2/5		5-5-2	540	10	54
c.		5/5	5/5	2/5	2/5	5-2-2	95	100	95
d.		5/5	5/5	2/5	0/5	5-2-0	49	100	49
e.		2/5	2/5	1/5	0/5	2-2-1	12	10	1.2
f.		5/5	2/5	1/5	1/5***	5-2-2	95	10	9.5
g.		5/5	5/5	5/5	5/5	5-5-5	>1600	100	>1600

* Dilutions to be used are shaded gray.

** No. of positive tubes/No. of tubes inoculated.

*** See page 13, number 4.