Microbiological Examination of Ice Cream and Ice Milk

Health Protection Branch - Ottawa

Official Method MFO-2
November 30, 1981

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1. APPLICATION

This method shall be used for the determination of total aerobic bacteria (Aerobic Colony Count) and coliform bacteria (Coliforms) in ice cream or ice milk in accordance with sub-paragraph (d) of sections B.08.062 and B.08.072, respectively of the Food and Drug Regulations.

2. SAMPLING

2.1 Definition of Terms

See Appendix A of Volume 1

2.2 Collection of Samples

See Appendix B of Volume 1

3. PROCEDURE

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

3.1 Handling of Sampling Units

3.1.1 Keep the sample units frozen in the laboratory before analyzing them.

3.1.2 Analyze the sample units as soon as possible after they have been received at the laboratory.

3.2 Preparation of media

The following media, to be 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

3.3.1 Prepare sterile 0.1% peptone water diluent.

3.3.2 Combine portions from several locations within the frozen sample unit to ensure a representative analytical unit of 11 (10)g. Weight or volume in brackets indicate alternate procedure for making dilutions.

3.3.3 Prepare a 1:10 dilution of the ice cream or ice milk by aseptically adding the analytical unit into 99(90) ml of the peptone water 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 Check the pH of the dilution. If the pH is outside the range of 5.5 to 7.6, adjust to 7.0, with either sterile NaOH or HCl.

3.3.6 Prepare succeeding dilutions as required to determine the ACC and the number of Coliforms present in the ice cream by transferring 11(10) ml of the previous dilution into 99(90) ml of 0.1% peptone water diluent. 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 the ACC

The medium used is PC agar prepared for making pour plates.

3.4.1 Analysis

a. Agitate each dilution bottle to resuspend material.

b. Without delay, pipette 1 ml of each prepared dilution into each of two approproately marked Petri plates using a sterile pipette for each transfer.

c. Pour 12-15 ml of the tempered agar (40-45°C) into each plate and mix contents by rotating and tilting.

d. Allow agar to solidify.

e. Plates shall be poured not late than 15 min. after preparation of dilutions.

f. Incubate plates in an inverted position at 35 + 0.5°C for 48 + 2 hr.

g. Avoid crowding or excessice stacking of plates in order to permit rapid equilibration of plates with incubator temperature.

h. Count colonies promptly after the incubation temperature.

3.5 Determination of Coliforms

3.5.1 Presumptive 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 each tube of a set of five tubes of single strength LST broth with 1 ml of the 1:10 dilution (ice cream or milk suspension; see section 3.3, above). Repeat for each succeeding decimal solution as required.

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

e. Incubate the inoculated LST broth tubes at 35 ± 0.5 °C for 24 ± 2 hr. Examine for gas formation, record results, and on the same day, begin the confirmed test for all gas-positive tubes (see section 3.5.2, below).

f. Incubate gas-negative tubes for an additional 24 ± 2 hr, examine, record the number of gas-positive tubes, add to the result obtained in step e., above and begin the confirmed test for the additional gas-positive tubes.

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

h. Compute the "MPN" of presumptive coliforms per g of ice cream or ice milk following the instructions in Part 5, to convert the number of gas-positive tubes to MPN values. Record results.

3.5.2 Confirmed Test

a. The confirmatory medium used is BGLB broth dispensed in 10 ml volumes in tubes containing gas vials.

b. Submit all gas-positive LST broth tubes to the confirmed 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 the BGLB broth. (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 BGLB broth tubes at 35 ± 0.5 °C for 24 ± 2 hr. Examine for gas formation, and record results.

f. Incubate gas-negative tubes for an additional 24 ± 2 hr, examine, record the number of additional gas-positive tubes and add to the results obtained in step e., above.

g. Formation of gas within 48 ± 2 hr of incubation constitutes a positive confirmed test.

h. Compute the "MPN" of confirmed Coliform per g of ice cream or ice milk following the instructions in Part 5, to convert the number of gas-positive tubes to MPN values. Record results.

4. INTERPRETATION

The tolerances as specified hereafter and representing the maximum count of total aerobic bacteria (Aerobic

Colony Count), and the maximum probable incidence of coliform bacteria (Coliforms) in ice cream or in ice milk shall be applied in determining whether the tested lot of the product complies with Section B.08.062(d) or Section B.08.072(d) of the Food and Drug Regulations.

4.1 The maximum count of total aerobic bacteria permitted for each lot is that represented by an Aerobic

Colony Count not exceeding:

4.1.1 100,000 per g in more than two of the five sample units, and

4.1.2 1,000,000 per g in any of the five sample units, included in the sample taken from a lot.

4.2 The maximum Most Probable Number (MPN) of coliform bacteria permitted for each lot is that represented by a coliform MPN not exceeding:

4.2.1 10 per g in more than one of the five sample units, and

4.2.2 1,000 per g in any of the five sample units, included in the sample taken from a lot.

These tolerances are summarized in the following table.

5. CALCULATION OF MOST PROBABLE NUMBERS (MPN)

Table A-1 shows the most probable numbers of coliform 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 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, for which all 5 tubes are positive and 2 subsequent higher dilutions. Examples c and d.

Determination	n	c	m	M
ACC	5	2	100,000	1,000,000
Coliforms	5	1	10	1,000

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 g of ice cream or ice milk which is of no concern (acceptable level
		of contamination).
M	=	Maximum number of bacteria per g of ice cream or ice milk which, if exceeded by any one sample
		unit (subsample), renders the lot under investigation in violation of the Regulation.

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 (see no. 3), 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 or ml = No Microorganism (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
0	<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.

Table A-2

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

	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 5, number 4.