Microbiological Examination of Water in Sealed Containers (Excluding Mineral and Spring Water) and of Prepackaged Ice

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Health Protection Branch - Ottawa
Official Method MFO-15
November 30, 1981

1. APPLICATION

This method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) and of coliform bacteria (Coliforms) in water in sealed containers, excluding mineral and spring water; and of coliforms in prepackaged ice, in accordance with Section B.12.004 and Section B.12.005 of the Food and Drug Regulations, respectively.

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 product 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 kind of product from one and the same manufacturer available for sampling at a fixed location.

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

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

2.1.4 Analytical Unit: That amount of product withdrawn from the sample unit for analysis.

2.2 Collection of Samples

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

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

2.2.3 Collect original unopened container wherever possible

2.2.4 Employ aseptic techniques in collecting the sample units when sampling bulk ice. Place each collected sample unit into a separate sterile container.

2.2.5 Ship and store the sample units of water in sealed containers under refrigeration (< 0.5 °C) if more than two hr elapse between collection and analysis. Do not freeze the sample units.

2.2.6 Do not allow sample units of prepackaged ice to thaw during shipment.

3. PROCEDURE

Five sample units shall be analyzed individually for Aerobic Colony Count (ACC). Ten sample units shall be analyzed individually for coliforms.

The tests shall be carried out in accordance with the following instructions:

3.1 Handling of Sample Units

3.1.1 Water in sealed containers

1. Do not store sample units for more than 24 hr before analysis.
   

3.1.2 Prepackaged Ice

1. If sample units are prepackaged in leakproof containers, thaw them in the containers under refrigeration (0-5 °C) prior to analysis.
   
2. If sample units are not in leakproof containers, transfer the ice aseptically to sterile plastic bags or other suitable sterile containers. Seal containers to prevent contamination, and thaw sample units under refrigeration (0-5 °C). Do not store thawed sample units for more than six hr before analysis.
   

3.2 Preparation of Media

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

3.2.1 Plate Count (PC) agar

3.2.2 Lauryl Sulfate Tryptose (LST) broth

3.2.3 Brilliant Green Lactose 2% Bile (BGLB) broth

3.3 Preparation of Dilutions (Water in sealed containers only)

3.3.1 Prepare sterile 0.1% peptone water diluent.

3.3.2 Thoroughly mix each sample unit by shaking the container.

3.3.3 Prepare a 1:10 dilution of the water by aseptically pipetting 11(10) ^*ml of the "water" into 99(90) ^* ml of the diluent.

3.3.4 Mix the 1:10 dilution by shaking the dilution bottle 25 times in a 30 cm arc in approximately 7 sec.

3.3.5 Prepare subsequent dilutions as required to determine the ACC of the water, by transferring 11(10)ml of the previous dilution into 99(90) ml of the diluent.

* Weight and volume in brackets indicate alternate procedure for making dilutions.
3.3.6 Shake all dilutions (as in step 3.3.4, above) immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

3.4 Determination of ACC

Examine five sample units of the water. The medium used is PC agar, prepared for making pour plates.

3.4.1 Analysis

1. Agitate each dilution bottle to distribute uniformly the microorganisms present.
   
2. Without delay, pipette 1 ml of the undiluted sample unit into each of two appropriately marked Petri plates using a sterile pipette for each transfer. Repeat for each prepared dilution. (See section 3.3.2, above).
3. Pour 12-15 ml of tempered (40-45 °C) agar into each plate and mix contents by rotating and tilting.
4. Allow the agar to solidify.
5. Plates should be poured not later than 15 min after preparation of dilutions.
6. Incubate plates in an inverted position at 35 °C + 0.5 ^o for 48 + 2 hr.
7. Avoid crowding or excessive stacking of plates in order to permit rapid equilibration of plates with incubator temperature.
8. Count colonies promptly after the incubation period.
9. Select for counting those plates containing 30-300 colonies, including pinpoint colonies. If counts do not fall within this range, select plates that have counts nearest to this range.

3.4.2 Recording Results

1. Calculate the average count (arithmetic mean) of duplicate plates, following the examples in Table 5-1 of "Standard Methods for the Examination of Dairy Products", A.P.H.A., 14th Edition (E.H. Marth, Jr., Editor. 1978).
2. When reporting results, round-off the counts to two significant figures and record only the first two left hand digits. (e.g., record 2,850 as 2,900).
3. If the lowest dilution plated shows no colonies, report the count as the product of 0.5 x the dilution factor preceeded by a "less than" (<) sign.
4. To compute the ACC, use the formula: N=AxD, where N is the number of colonies per g or ml of product, A is the average count, and D is the respective dilution factor.

3.5 Determination of coliforms

Examine 10 sample units of water or ice.

3.5.1 Presumptive Test

1. The medium used is LST broth dispensed in 10 ml volumes into tubes containing gas vials (inverted Durham tubes).
2. Arrange LST broth tubes in rows of five, and mark them, identifying the sample, the sample unit and the dilution to be inoculated.
3. Inoculate each of five tubes of double strength LST broth with 10 ml of the undiluted sample unit (see section 3.3.2 above), and inoculate each of five tubes of single-strength LST broth with 1 ml of the undiluted sample unit, and inoculate each of five tubes of LST broth with 0.1 ml of the undiluted sample unit.
   
4. Mix inoculum and medium by gently shaking or rotating the tubes but avoid entrapping air in the gas vials.
   
5. Incubate the inoculated LST broth tubes at 35 °C + 0.5 ^o for 24 + 2 hr. Examine for gas production, record results, and on the same day begin the confirmatory test for all gas positive tubes. (See section 3.5.2 below)
   
6. Incubate gas-negative tubes for an additional 24 + 2 hr, record the number of gas-positive tubes, add to the results obtained in step (e) above, and begin the confirmed test for the additional gas-positive tubes.
   
7. The absence of gas in all of the tubes at the end of 48 + 2 hr of incubation constitutes a negative presumptive test.
   
8. Compute the "MPN" of presumptive coliforms per 100 ml of water or of melted ice following the instructions in Part 5 to convert the number of gas-positive tubes to MPN values. Record results.
   

3.5.2 Confirmed Test

1. (a) The confirmatory medium used is BGLB broth, dispensed in 10 ml volumes into tubes containing gas vials.
   
2. (b) Submit all gas-positive LST broth tubes to the confirmed test.
   
3. (c) Shake or rotate the LST broth tubes to mix the contents, and with a sterile loop, transfer one loopful from each positive LST broth tube to the BGLB broth. (Avoid transferring pellicle).
   
4. (d) Mix inoculum and medium by gently shaking or rotating the tubes, but avoid entrapping air in the gas vials.
   
5. (e) Incubate the inoculated BGLB broth tubes at 35 °C + 0.5 ^o for 24 + 2 hr. Examine for gas formation and record results.
   
6. (f) Incubate gas-negative tube for an additional 24 + 2 hr, re-examine, record the number of additional gas-positive tubes and add to the result obtained in step (e), above.
7. (g) Formation of gas during 48 + 2 hr incubation constitutes a positive confirmed test.
   
8. (h) Compute the "MPN" of confirmed coliforms per 100 ml of the water in sealed containers or per 100 g of the ice following the instructions in Part 5 to convert the number of gas-positive tubes to MPN values. Record results.

4. INTERPRETATION

4.1 The tolerances as specified hereafter and representing the maximum total aerobic bacteria

(Aerobic Colony Count) in water in sealed containers (excluding mineral and spring water), and the maximum probable incidence of coliform bacteria (Coliforms) in water in sealed containers and in prepackaged ice, shall be applied in determining whether the tested lot of the product complies with Section B.12.004 or Section B.12.005, of the Food and Drug Regulations.

4.1.1 The maximum count of total aerobic bacteria permitted for each lot of water in sealed containers is that represented by an Aerobic Colony Count not exceeding:

1. (a) 100 per ml in more than two of the five sample units, and
   
2. (b) 10,000 per ml in any sample unit, included in the sample taken from the lot.
   

4.1.2 Coliform bacteria (Coliforms) shall be considered absent in a lot when not more than one of the 10 sample units taken from the lot is positive for coliforms, and the MPN for that sample unit is not more than 10 coliform per 100 ml of the water in sealed containers or per 100 g of the pre-packaged ice.

4.1.3 The tolerances are summarized in the following table:

	Determination	n	c	m	M
1	For water in sealed containers
	Aerobic Colony Count	5	2	100	10,000
	Coliforms	10	1	0	10
2	For prepackaged ice
	Coliforms	10	1	0	10

n = Number of sample units (subsamples) to be examined per lot.

c = Maximum number of sample units (subsamples) per lot which may have a bacterial concentration higher than the value for 'm' without violation of the Regulation.

m = Maximum number of bacteria per designated unit*, which is of no concern (acceptable level of contamination).

M = Maximum number of bacteria per designated unit*, which if exceeded by any one sample unit (subsample), renders the lot under investigation in violation of the Regulation.

* per ml for the Aerobic Colony Count per 100 ml or g for Coliforms.

5. CALCULATION OF MOST PROBABLE NUMBERS (MPN)

Table A-1 shows the most probable numbers of coliforms per 100 ml or g 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 water 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 or ml of a food constitute the test portions in the tubes. When other sized portions of the test material are placed in the tubes, the MPN values obtained from Table A-1 has to 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 or 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 sample) 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 are positive

The quantities (test portions) in each of the five tubes of the three dilution series represent 1, 0.1 and 0.01 g or ml test material respectively.

According to Table A-1, a reading of 5-1-0 gives a value of 33 if 10, 1 and 0.1 g or 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 or ml of test material. Since the results have to be expressed per g or ml, the MPN value of 330 must be divided by 100. 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 or 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 for those 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 (last dilution, i.e. dilution with the smallest quantity of product) in which all 5 tubes are positive and 2 succeeding 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/100 ml (g) = No. of Microorganisms (Table A-1) 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

Next link will take you to the Table A-1

Table A-2

Dilutions to be used and calculations of MPN per g or mL of test material

Dilutions*
		Undiluted	1:10	1:100	1:1000
Amount of original test material (g or mL)
	10	1	0.1	0.01	0.001	Combination to be used	MPN from Table A-1	Dilution factor of middle dilution	MPN per mL or g
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 underlined.
**No. of positive tubes/No. of tubes inoculated.
*** See page 7, number 4.