National Standards of the People’s Republic of China 
GB 4789.26—xxxx 
 
   
 
 
National Food Safety Standard 
 
Microbiological Test of Food 
 
Commercial Sterilization Test of Canned Food 
 
 
 
 
 
(Exposure Draft) 
 
   
 
  
 
  
 
  
 
  
 
 
 
  
 
Released on  x x x x - x x - x x                     Come into force on  xxxx-xx-xx   
Published by  The Ministry of Health of the People’s Republic of China  
GB 4789.26 —xxxx  
 
  
 
 
Preface  
 
 
 
 
This National Food Safety Standard replaces GB/T 4789.26-2003 Microbiological Test of Food 
Hygiene - Commercial Sterilization Test of Canned Food . Compared with GB/T 4789.26-2003, 
this Standard has mainly undergone the following modifications:  
- The Chinese and English names of the Standard have been altered; 
- Scope of application has been changed; 
- Normative referential documenta have been deleted; - Terms and definitions have been modified; - Equipment and materials have been altered; - Culture medium and reagent have been changed; - Figures of inspection procedures have been added; - Inspection procedures have been modified; 
- Result determination has been modified; 
- Normative appendix A and informative appendix B have been amended. 
 
 
  
 
 
 
  
 
  
 
  
 
  
 
  
 
  
 
 
 
  
 
  
I GB 4789.26 —xxxx
1  
  
National Food Safety Standards 
Microbiological Test of Food – Commercial Sterilization Test of 
Canned Food  
 
 
 
1. Scope 
 
The standard specifies the basic requirements, oper ation procedures, and determination of results for 
commercial sterilization test of canned food. The standard is applicable to all canned food that has been packaged with various airtight containers.The 
canned food has undergone proper commercial sterilization by means of heat sterilization so that it can be 
kept for a long time under room temperature.  
2. Terms and Definitions 
 
2.1 Commercial Sterilization of Canned Food 
 
Commercial sterilization means that canned food is free of microorganisms that can reproduce under room 
temperature and pathogenic microorganism af ter undergoing proper heat sterilization. 
2.2 Hermetical Seal 
 
Hermetical seal means the sealed food container is u nder the state of being able to prevent microorganisms 
from getting into it. 
2.3 Swell 
 
The package swells because of positive pressure genera ted by gases produced by microorganisms’ activities 
in the canned food or by other physical and chemical reasons. 
2.4 Leakage 
 
The sealing of the canned food has defects, or due to the  damage caused by external force, or holes 
appearing on the fo od container because of corrosion. 
 
3. Equipment and Materials 
 
Besides the conventional sterilization and culture equipment used in microbiological labs, other following 
equipments and materials are as follows: 
 
3.1 Refrigerator ：2℃～ 5℃. 
 
3.2 Constant temperature culture medium ：36℃  ±   1 ℃. 
 
3.3 Homogenizer and sterile homogenizing bag, homogenizing cup or a mortar. 
 
3.4 Potentiometric pH meter whose accuracy should be within 0.05 pH unit of the known buffer solution. 
 
3.5 Microscope ：10 × ～ 100 ×. 
 
3.6 A bacti-dise can opener used in bacteriology inspection and a can puncher (The best can opener for 
bacteriology inspection of canned food is composed of a metal rod and a triangular blade, the end of the metal rod 
is equipped with a punching device, and the triangular blade, which can slide, is arranged on the rod and is fixed 
by a bolt. Compared with other types of can openers, th is can opener won’t easily da mage the hemming structure 
of the can and therefore, won’t affect  leakage check of the canned food.) 
3.7 Electronic balance or a bench balance.2 GB 4789.26 —xxxx 
 3.8 Clean bench or Class 100 clean operating lab. 
 
4 Culture Medium and Reagent 
 
4.1 Sterile normal saline: see A.1 in Appendix A. 
 
4.2 Crystal violet staining solution: see A.2 in Appendix A. 
 
4.3 Dimethylbenzene. 
 
4.4 Ethanol solution with 4% iodine: 4% iodine dissolves in 70% ethyl alcohol. 
 
5 Test procedures  
 
Procedures of commercial sterili zation test of canned food ar e demonstrated in Figure 1. 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 36℃ ± 1℃ , 14 days 
 
 
 
 
 
  
 
  
 
  
 
  
 
  
 
 
 
  
Figure1. Procedures of Commercial Sterilization Test of Canned Food 
   Sample 
Sample examination Contrast 
Heat preservation, once swelled can is detected; take it 
out immediately and open it for further examination. Preserved in a 
refrigerator under the 
temperature of 2 ℃~5
℃ 
Open the can 
Measure pH 
Organoleptic inspection 
Smear staining and  
microscopic examination Reserve a sample, keep at least 30 mL (g) 
sample and put it in a sterile container, 
under the temperature of 2 ℃~5℃ 
Inoculation and 
cultivationLeakage test 
Report 3 GB 4789.26 —xxxx 
 6 Operation Procedures  
 
6.1 Preparations of the canned food  
 
Remove the label on the can, mark the can with a water-proof oil marking pen, and record such information as 
the container, number, properties of the product, stat us of leakage, and whether there are small holes or 
corrosion, indentation, swell and other abnormal conditions. 
6.2 Weighing 
Use the electronic balance or bench balance to weigh the ca nned food; the weight should be as accurate as 1 g 
for canned food of 1 kg or less, as accurate as 2 g for cann ed food of more than 1 kg, and as accurate as 10 g 
for canned food of more than 10 kg, and write down the records. 
6.3 Heat preservation 
 
6.3.1 Take one can from each batch and preserve it in a refrigerator under the temperature of 2 ～5 as the 
control group. The rest cans shall be preserved under the temperature of 36 ± 1 for 14 days. During the 
process of heat preservation, check the cans every day. If swell or leakage happens, take out the relevant cans 
immediately and open them for inspection.  
6.3.2 After heat preservation, weigh the cans again and wr ite down the records. Compare the newly obtained 
weight with the weight obtained before heat preservation. If the cans weigh less than its original weight, leakage 
must have happened. Store all the cans under room temperature until opening them for inspection. 
6.4 Open the can  
 
6.4.1 If there are swelled cans, preserve these cans in the refrigerator under the temperature of 2 ℃～ 5℃ for 
several hours before opening them. 
6.4.2 Clean the smooth surface of the sample can that is to be tested with cold water and detergent, wash the 
can with tap water, and then dry the can with a st erile towel. Sterilize the smooth surface of the can by 
immersing the smooth surface in ethanol solution with 4% iodine for 15 minutes, and then dry the can with a sterile towel. Ignite the can under an enclosed hood until all the residual iodine ethanol solution on the surface of the can is burned up. Don’t ig nite any swelled can. Sterilize th e smooth surface of the swell can by 
immersing the smooth surface in ethanol solution with 4% iodine for 30 minutes, and then dry the can with a 
sterile towel. 
6.4.3 Open the can on the clean bench or in the Class 100 clean operating lab. Shake the can appropriately 
before opening it if there is soup in the can. Make an opening of appropriate size on the sterilized smooth 
surface of the can with a sterilized can opener. Do no t damage the hemming structure while opening the can. 
Use a different can opener for each can, and do not misuse them. If the sample is flexible packaging product, open the package with a pair of sterilized scissors, and do not damage the interface. Smell the product above the opening immediately, and keep records. 
Note : Severely swelled cans might explode and eject toxic substances. Therefore, it is necessary to take some 
precautionary measures to prevent such danger from ha ppening. For example, put a sterilized towel on the 
cover of the swelled can or put a sterilized funnel upside down on the can. 
6.5 Reserve samples  
 
After opening the can, take out at least 30 mL (g) contents from the can with a sterilized suction tube or other 
proper tools by sterile operation and reserve the contents  in a sterilized container in a refrigerator under the 
temperature of 2 ℃～ 5℃. The reserved sample can be used for further test when necessary, and can be 
abandoned after conclusions are drawn for the test of this batch of canned food. The opened canned food can 
be preserved appropriately for some time in case it is used for container inspection in the future. 
6.6 pH measurement 
 
6.6.1  Sample treatment 
6.6.1.1 Mix the liquid products for future use. As for products with separated solids and liquids, take out the well 
mixed liquids fo r future use. 
6.6.1.2 In terms of stiff and half-still products and products from which liquids can barely be separated (such 
as syrup, jam, jelly, etc.), take a portion of the product and grind it in a homogenizer or mortar. If the sample is 
still too stiff after being grinded, add in the same amount of sterile distilled water and mix them for future use. 
6.6.2 Measure 
Insert the electrode into the sample liquid being tested, and adjust the temperature co rrector of the pH meter to 
the temperature of the liquid being tested. If the meter does not have a temperature correction system, adjust the 4 GB 4789.26 —xxxx 
 temperature of the liquid being test to the range of 20 ℃ ± 2℃, and follow the procedures appropriate for the 
adopted pH meter. When the indication is stable, read the pH directly from the scale of the meter, and the 
reading should be as accurate as 0.05 pH unit. 
The same sample should be tested at least twice. The difference between the two test results should not exceed 
0.1 pH unit. The arithmetic mean value of the two test results is determined as the final result, which should be as accurate as 0.05 pH unit in the report. 
6.6.3  
Analyzing the result 
Compare the result with that of the ca n preserved in the refrigerator as the control group to see whether there is 
significant difference. If the pH values differ by more than 0.5, then the difference should be regarded as a 
significant one. 
6.7 Organoleptic Inspection  
 
In a test room with adequate light and clean air free of peculiar odor, pour the conten ts of the can into a white 
enamel plate, observe the structure, form and color of the product, smell it, and press it with a piece of tableware 
or a finger wearing fingerstall, w ith the purpose of examining the properties of the product and identifying 
whether the food has gone bad. Meanwhile, observe the can externally and internally, and write down the records. 
6.8 Smear staining and microscopic examination 
 
6.8.1 Smear 
Apply the sample of the canned food to a smear. If there is liquid in the sample of the canned food, apply the 
liquid to a glass slide with an inoculating loop. Solid food can directly smear or can be diluted by a small amount 
of normal saline before smearing, and the solid food has to be fixed with the aid of flame after getting dry. Use dimethylbenzene to wash grease food smear which has been fixed with the help of flame after air drying, and then 
the grease food shall be dried naturally. 
6.8.2 
Staining and microscopic examination  
Stain these smears with crystal violet staining solution . When they dry up, examine them with a microscope, 
observe at least five fields, and record the morphological characteristics of the bacteria and the bacterial count in 
each field. Compare the result with that  of the control group, i.e. the cann ed food preserved in the refrigerator, 
and determine whether the microorganisms have proliferated obviously. If the bacterial count has increased by one hundred times or more, obvious proliferation has occurred. 
 
7 Result Determination  
 
No leakage is detected after heat preservation of the sa mple; after the heat preservation, the can is opened for 
organoleptic inspection, pH value measurement, and smear  microscopic examination. If the results show that 
there is no microorganism pr oliferation, then the sample shall be reported as commercial sterilization. 
 
Leakage is deteted after heat preservation of the sample ; after the heat preservation, the can is opened for 
organoleptic inspection, pH value measurement, and smear  microscopic examination. If the results prove that 
there is microorganism proliferation, then the sample  shall be reported as non- commercial sterilization.5 GB 4789.26 —xxxx
 
  
 
Appendix A 
 
Culture Medium and Reagent 
 
A.1 Sterile Normal Saline 
A.1.1  Ingredients  
Sodium chloride  8.5 g Distilled water 1,000.0 mL 
A.1.2 Preparation 
Weigh 8.5 g sodium chloride and add it into 1000 mL distilled water, and then the solution shall 
undergone high-pressure sterilization under the temperature of 121 for 15 minutes.  
A.2
 Crystal violet staining solution  
A.2.1  Ingredients 
Crystal violet  1.0 g 
95% ethanol 20.0 mL 
1% Ammonium oxalate aqueous solution 80.0 mL A.2.2  Preparation 
Let the crystal violet totally dissolve in ethanol, and then mix the ethanol with the ammonium oxalate solution. 
A.2.3  Staining method  
Fix the smears above the flame of an alcohol burner, add in droplets of crystal violet staining solution to 
stain the smears for 1 minute, and then wash them with water.  
 
 
         6  GB 4789.26—xxxx 
 
  
 
Appendix B 
 
Causes Analysis of Abnormalities 
 
If it is necessary to find out the reasons for swell, pH or organoleptic abnormalities, and microorganism 
proliferation, get the reserved sample of the canned food for inoculation and cultivation, and then report. 
In order to judge whether the can has leakage, ge t the opened can for tightness check and then report. 
B.1 Terms and Definitions  
 
B.1.1 Low acid canned food  
Besides alcohol, all canned food whose balance pH is higher than 4.6 and water activity larger than 0.85 after 
sterilization is low acid canned food. When low acid fru its, vegetables or vegetable products are canned, acid 
is added to decrease the pH in order to meet the re quirement of heat sterilizat ion, and these canned foods 
belong to acidified low acid canned food. 
B.1.2 Acid canned food  
Acid canned food refers to canned food whose balance pH equals or is less than 4.6 after sterilization. 
Tomatoes, pears, pineapples and their juices with pH value less than 4.7, and figs whose pH is less than 4.9 are all considered as acid food. 
B.2 
Equipments and materials 
 
Besides the regular sterilization and culture equipments used in microbiological labs, Other equipments 
and materials are as follows: 
B.2.1 Constant temperature incubator: 30 ℃ ± 1℃; 55 ℃ ± 1℃.  
B.2.2 Constant temperature water bath: 55 ℃ ± 1℃.  
B.3 Culture medium and reagent 
B.3.1 Bromocresol purple dextrose broth 
B.3.1.1 Ingredients 
P e p t o n e                                   1 0 . 0  g  
b e e f  e x t r a c t                                3 . 0  g  
d e x t r o s e                                   1 0 . 0  g  
s o d i u m  c h l o r i d e                             5 . 0  g  
bromcresol purple                          0.04 (or 1.6% alcoholic solution 2.0mL) 
d i s t i l l e d  w a t e r                              1 , 0 0 0 . 0 m L  
B.3.1.2 Preparation 
Heat and stir the above-men tioned ingredients (except bromocresol purple) to make them dissolve, adjust the 
pH value to 7.0 ± 0.2, and add in bromocresol purple. Put the solution into several medium-sized test tubes 
with small discharging tubes, 10 mL for each test tube . Then these test tubes shall go through high-pressure 
sterilization for 10 minutes under the temperature of 121 ℃. 
B.3.2 Cooked meat medium  
B.3.2.1 Ingredients  
beef infusion 1,000.0 mL 
peptone  30.0 g yeast extract 5.0 g dextrose 3.0 g 
sodium dihydrogen phosphate 5.0 g 
soluble starch 2.0 g meat fragments appropriate amount 
B.3.2.2  Preparation  
B.3.2.2.1 Get 500g fresh beef fragments free of fat and fascia, add in 1,000mL distilled water and 25.0 mL 
1moL/L sodium hydroxide solution, boil the solution for 15 minutes, and then cool it completely. Get rid of 
the fat on the surface, settle and filter the solution, and add in more water to 1000 mL. Add in all the 
ingredients except meat fragments, and adjust the pH value to 7.8 ± 0.2. 7  GB 4789.26—xxxx 
 
  
B.3.2.2.2 Wash the meat fragments with water, ai r them to semi-dry state, and put them into 
several 15 mm × 150 mm test tubes, meat fragments in each tube reaching 2 cm ～3 cm high. 
Add 0.1 g ～0.2 g reduced iron powder or a small amount of  scrap iron to each test tube. Pour the 
fluid nutrient medium mentioned above into each test tube, and the fluid nutrient medium shall 
be 1 cm higher above the surface of the meat fragmen ts. Finally, add dissolved vaseline or liquid 
paraffin 0.3 cm ～0.4 cm to each test tube. Then these te st tubes shall go through sterilization for 
15 minutes under the temperature of 121 ℃ 
B.3.3 Nutrient agar  
B.3.3.1 Ingredients 
peptone 10.0 g  beef extract 3.0 g 
sodium chloride 5.0 g 
agar 15.0 g ～20.0 g 
distilled water 1,000.0 mL 
B.3.3.2 Preparation 
Let all the above ingredients except agar dissolve in the distilled water, add in 2 mL 15% 
sodium hydroxide solution, and adjust the pH value to 7.2 ～7.4. Add in the agar, heat the 
solution until it boils and the agar dissolves. Put the solution into several flasks or 13 mm × 
130 mm test tubes. The flasks or test tubes shall then go through high-pressure sterilization for 
15 minutes under the temperature of 121 ℃. 
 B.3.4
 Acid broth 
B.3.4.1 Ingredients 
polypeptone 5.0 g 
yeast extract 5.0 g dextrose 5.0 g 
monopotassium phosphate 5.0 g 
distilled water 1,000.0 mL 
B.3.4.2 Preparation 
Heat and stir the above ingredients to make them  dissolve, adjust the pH value to 5.0 ± 0.2, 
and then sterilize it with high pressure for 15 minutes under the temperature of 121 ℃. 
B.3.5  
Malt extract broth  
B.3.5.1 Ingredients  
malt extract 15.0 g 
distilled water 1,000.0mL 8  GB 4789.26—xxxx 
 
 B.3.5.2 Preparation  
Let the malt extract completely dissolve in the distilled water, filter the solution with filter 
paper, adjust the pH value to 4.7 ± 0.2, and put the solution into several containers, which 
shall then be sterilized for 15 minutes under the temperature of 121 ℃. 
B.3.6 Sabouraud’s dextrose agar 
B.3.6.1 Ingredients 
peptone 10.0 g 
agar 15.0 g dextrose 40.0 g 
distilled water 1,000.0 mL 
B.3.6.2  Preparation 
Let all the ingredients dissolve in the distilled water, heat the solution, put the solution into 
several flasks, and adjust the pH value to 5.6 ± 0.2. These flasks shall go through 
high-pressure sterilization for 15 minutes under the temperature of 121 ℃. 
B.3.7 
Calf liver agar  
B.3.7.1 Ingredients  
calf liver (used to make calf liver infusion) 50.0 g veal (used to make veal infusion) 500.0 g proteose peptone 20.0 g new peptone 1.3 g 
tryptone 1.3 g 
 
dextrose 5.0 g 
soluble starch  10.0 g 
plasma casein protein  2.0 g 
sodium chloride 5.0 g 
sodium nitrate 2.0 g 
gelatin  20.0 g 
agar  15.0 g 
distilled water 1,000.0 mL 
B.3.7.2 Preparation  
Mix all the above-mentioned ingredients in the distilled water and sterilize the solution in 
121℃ for 15 minutes. The final pH value shall be 7.3 ± 0.2. 
B.3.8 Gram staining solution 
B.3.8.1  Crystal violet staining solution  
B.3.8.1.1  Ingredients  
Crystal violet 1.0 g 
95% ethanol 20.0 mL 
1% ammonium oxalate aqueous solution 80.0 mL 
B.3.8.1.2 Preparation 9  GB 4789.26—xxxx 
 
 Let the crystal violet totally dissolve in ethanol, and then mix the ethanol with the ammonium 
oxalate solution. 
B.3.8.2 Gram iodine solution 
B.3.8.2.1  Ingredients  
iodine 1.0 g 
potassium iodide 2.0 g 
distilled water 300 m L 
B.3.8.2.2 Preparation  
Mix iodide and potassium i odide firstly, add a small amount of distilled water 
and shake it adequately; after full diss olution, add distilled water to 300 mL. 
B.3.8.3  Safranine re-staining solution 
B.3.8.3.1 Ingredients 
safranine  0.25 g 
95% ethanol 10.0 mL 
distilled water 90.0 mL 
B.3.8.3.2 Preparation  
Dissolve safranine into the ethanol and then dilute it with distilled water. 
B.3.8.4  Staining method  
a) Smear is fixed above the fire, add crystal violet staining solution in droplets to stain it for 
1 minute, wash the smear with water. 
b) Add Gram iodine solution in droplets, and wash the smear with water after 1 minute. 
c) Add 95 ％ ethanol in droplets to make decoloration for about 15 seconds ～30 seconds 
until the staining solution is scrubbed down, no excessive decoloration, wash the smear with 
water. 
d) Add re-staining solution in droplets for 1 min, wash the smear with water, and examine it 
with microscope after drying. 
B.4 Inoculation and cultivation of low acid food (pH value ＞4.6) 
 
B.4.1 For low acid food, inoculate four tubes each can into cooked meat medium that has been 
heated to 100 ℃and then cooled to the room temperature rapidly, meanwhile, inoculate four tubes 
of bromocresol purple dextrose broth. 
Inoculate 1 mL (g)~2 mL (g) sample (liquor sample is 1 mL~2 mL, solid is 1 g~2 g, when both 
exist, take half of each) for each t ube. See Table B.1 for culture conditions. 
 
Table B.1 Low acid food （pH value ＞ 4.6）Inoculated cooked meat 
medium and bromocresol purple dextrose broth  
 
 
Culture medium Tube Quantity.Culture temperature( ℃)Culture time(h) 10 GB 4789.26—xxxx 
 
 Cooked meat medium 2 36 ± 1 
96～120
Cooked meat medium 2 55 ± 1 
24～72
Bromocresol purple dextrose broth 2 55 ± 1 
24～48
Bromocresol purple dextrose broth 2 36 ± 1 
96～120
 
B.4.2  After being cultured in the culture conditio ns stipulated in Table B.1, make record 
whether there are microorganisms grown in each tube. If not, abandon it after recording. 
B.4.3 If there are microorganisms growing on it, stain solution smear with inoculating loop, 
examine the Gram staining with microscope. If different microorganism forms or unitary 
cocci and fungus forms are observed in the tube of bromocresol purple dextrose broth, record 
them before abandoning them. If no bacillus found in cooked meat medium and coccus, yeast, 
mould or its mixture are found in the culture, record them and then abandon. Make streak 
inoculation of two calf liver agar (without yolk) or other nutrient agar plate respectively in 
positive tubes appearing and growing in bromocresol purple dextrose broth and cooked meat 
medium; one plate for aerobic culture and the other for anaerobic culture. (As shown in Table 
B.1) 
B.4.4 Select single colony in aerobic culture and inoculate it on the small slant of nutrient agar 
for subsequent examine the Gram staining with  microscope. Select si ngle colony smear in 
anaerobic culture, and examine the Gram staining with microscope. Select the single colony in  
aerobic and anaerobic culture and inoculat e it in cooked meat medium to do 
purely culturing. 
B.4.5  Select the culture smear in nutrient agar  small slant and cooked meat medium of 
anaerobic culture for microscopic examination. 
B.4.6  Select the aerobic culture in purely cult uring and inoculate it with calf liver agar 
(without yolk) or nutrient agar plate to carry out  anaerobic culture; select anaerobic culture in 
purely culturing and inoculate it with calf agar (without yolk) or nutrient agar plate to carry 
out aerobic culture, so as to distinguish whether they are facultative anaerobes. 
B.4.7  If it is necessary to detect botulin of clostridia, select typical colony to inoculate cooked 
meat medium to make pure culture. Culture under the temperature of 36 ℃ for 5days, and 
botulin examination is carried out according to GB/T 4789.12. 
B.5 Inoculation and culture of acid food(pH value ≦ 4.6) 
 
B.5.1  Each can is inoculated with four tubes of acid broth and two tubes of malt extract broth. 
Inoculate 1 mL (g)~2 mL (g) sample (the volume of liquor sample 1 mL~2 mL and solid 1 g~2 g, 
when both of them exist, take half of each) for each tube. See Table B.2 for culture conditions. 
Table B.2 Acid broth and malt extract soup inoculated in acid food (pH value ≤ 4.6) 
Culture medium Tube Quantity. Culture temperature ( ℃)Culture time (h) 
Acid broth 2 55  ± 1 48 
Acid broth 2 30  ± 1 96 11 GB 4789.26—xxxx 
 
 Malt extract broth 2 30  ± 1 96 
 
B.5.2  After being cultured in the culture conditions stipulated in Table B.2, make record whether 
there are microorganisms grown in each tube . If not, record them and then abandon. 
B.5.3  For culture tube with microorganism grown in , take the direct smear of the culture after 
culturing, make the Gram staining micros copic examination and record the observed 
microorganisms. 
B.5.4  If there are microorganisms grown in ac id broth or malt extract broth in 30 ℃ culture 
conditions, inoculate each positive tube with two pieces of nutrient agar or Sabouraud’s 
dextrose agar plate respectively, one plate for aerobic culture and the other for anaerobic 
culture. 
B.5.5  If there are microorganisms grown in acid broth in the culture conditions of 55 ℃, 
inoculate each positive tube with two pieces of nu trient agar plates respectively, one for aerobic 
culture and the other for anaerobic culture (as shown in Table B.2). Make staining smear 
microscopic examination of the plate where mi croorganisms grown and report the type of the 
microorganism found in the microscopic examination. 
 
B.5.6 Select single colony of nutrient agar or Sabouraud’s dextrose agar plate cultured in 30 ℃ 
aerobic culture condition, inoculate nutrient agar small slant for the subsequent Gram staining 
microscopic examination. Meanwhile, inoculate acid broth or malt extract broth to make pure 
culture. 
Select single colony of nutrient agar or Sabouraud’s dextrose agar plate cultured in 30 ℃ 
anaerobic culture condition, inoculate acid brot h or malt extract broth to make pure culture. 
Select single colony of nutrient agar plate in 55 ℃ aerobic culture condition, inoculate nutrient 
agar small slant for the subsequent Gram st aining microscopic examination. Meanwhile, 
inoculate acid broth to make pure culture. 
Select single colony of nutrient agar plate in 55 ℃ anaerobic culture condition, inoculate acid 
broth to make pure culture. 
B.5.7  Select culture in nutrient agar small slant to make smear microscopic examination; select 
acid broth or malt extract broth culture in 30 ℃ anaerobic culture condition and acid broth 
culture in 55 ℃ anaerobic culture condition to make  smear microscopic examination. 
B.5.8  Inoculate the pure culture which is cultured in 30 ℃ aerobic culture condition in nutrient 
agar or Sabouraud’s dextrose agar plate to ma ke anaerobic culture; inoculate the pure culture 
cultured in  30 ℃ anaerobic culture condition in nutrient agar or Sabouraud’s dextrose agar 12 GB 4789.26—xxxx 
 
 plate to make aerobic culture; inoculate the pure culture cultured in 55 ℃ aerobic culture 
condition in nutrient agar to make anaerobic cu lture; inoculate the pure culture cultured in 55 ℃ 
anaerobic culture condition in nutrient agar to make aerobic culture to distinguish whether they 
are facultative anaerobes. GB 4789.26—xxxx 
 
 
Sample  
Cooked meat medium and bromocresol purple dextrose broth  
NoGrowing  NoGrowing Growing  Growing  
Microscopic 
examination  Microscopic 
examination
Microscopic 
examinationMicroscopic 
examination  Calf liver agar (without yolk) or 
nutrient agar  
Aerobic 36 ℃ ± 1℃ Aerobic 55 ℃ ± 1℃ Anaerobic 55 ℃ ± 1℃ Anaerobic 36 ℃ ± 1℃ Calf liver agar (without yolk) or 
nutrient agar  Calf liver agar (without yolk) or 
nutrient agar  
Nutrient agar small slant  Nutrient agar small slant  Cooked meat medium  Cooked meat medium
Cooked meat medium
Microscopic 
examination  Microscopic 
examination
Calf liver agar (without yolk) or 
nutrient agar anaerobic 36 ℃ ± 1℃ Calf liver agar (without yolk) or 
nutrient agar anaerobic 55 ℃ ± 1℃Calf liver agar (without yolk) or 
nutrient agar aerobic 36 ℃ ± 1℃Calf liver agar (without yolk) or 
nutrient agar aerobic 55 ℃ ± 1℃ Cooked meat medium  36℃±1℃ 96h～120h55℃±1℃ 24h～72hor 24h 48h
Calf liver agar (without yolk) or 
nutrient agar  
Figure B.1 Low-acid Food Vaccination Cultivation Procedure GB 4789.26—xxxx 
 
  
Sample
Acid broth  
30℃ 55℃ 
No growing  No growing No growing Growing  Growing  Growing  
Nutrient agar or Sabouraud’s 
dextrose agar  Nutrient agar or 
Sabouraud’s dextrose agarNutrient agar  
Ab i 30℃Ab i 55℃
Ab i 30℃ Ab i 30℃ Ab i 55℃
Ab i 30℃
Nutrient agar 
small slant  Nutrient agar 
small slant  Nutrient agar small 
slant  Acid broth or 
malt extract Acid broth or 
malt extract Acid broth or malt 
extract broth  Acid broth or malt 
extract broth  Acid broth  Acid broth
Microscopic 
examination Microscopic 
examination 
Microscopic 
examination 
Microscopic 
examination Nutrient agar or 
Sabouraud ’s dextrose agar 
Anaerobic 30 ℃ Nutrient agar or 
Sabouraud ’s dextrose agar 
Aerobic 30 ℃ Nutrient agar 
Anaerobic 55 ℃Nutrient agar 
Aerobic 55 ℃Nutrient agar or 
Sabouraud ’s dextrose agar 
Anaerobic 30 ℃ Nutrient agar or 
Sabouraud ’s dextrose agar 
Aerobic 30 ℃ 
Figure B.2   Acid Food Inoculation Cultivation Procedure Malt extract broth 
30℃ 15 GB 4789.26—xxxx 
 
 B.5.9 Results Analysis 
B.5.9.1  If no growth of microorganism was found in the swelled can, it may because the contents 
and the package react and produce hydrogen. The amount of hydrogen produced changes with the 
storage time and conditions. Excessive filling of the can also causes slight swell, which can be confirmed through weighting.  
If mixed microorganisms of large amount of bacteria is found in direct smear which does not grow 
after cultivation, it indicates the putrefaction occurred before canning. As the result of bacterial growth before canning, pH, odor and morphology  of the product are in abnormal conditions. 
B.5.9.2  When the can is well sealed and only bacillus grow under the culture conditions of 36 ℃ 
whose heat resistance is not higher than botulinu m clostridia, it indicates that the heat is not 
sufficient in the production process. 
B.5.9.3  If mixed colonies of bacilli, cocci and fungi are cultivated, it shows that the can leaks. It 
may also be due to the incomplete sterilization. Under this condition the swell rates of the same 
batch of product shall be higher. 
B.5.9.4  If acid is produced under the condition of bromocresol purple dextrose broth at 36 ℃ or 
55℃ to be cultivated to observe gas and acid production, it indicates that there is growth of 
microorganism addicted to proper temperature, fo r example, mesophilic acid-resistant bacillus, or 
thermophilic microorganisms such as bacillus stearothermophilus. If the bacteria grow , produce gas and give rotten smell under the cooked meat medium under the 
temperature of 55 ℃, it indicates that the putrefaction in the can is caused by thermophilic 
anaerobic clostridia. 
If the gas grows out of the cooked meat  medium under the temperature of 36 ℃ with rotten smell 
and the germ can be seen from microscopic examination, this indicates that the putrefaction is 
caused by botulinum clostridia, sporogenic fusiform bacillus or clostridium  perfringens. Further 
botulinum detection can be carried out when necessary. 
B.5.9.5  Deterioration of acid food is usually caused by lactobacillus and yeast without spore. 
Generally, the deterioration caused by bacillus shall not happen when pH is lower than 4.6. But 
there is an exception, i.e. the deteriorated catsup  or tomato juice can displayed as sour spoilage 
with rotten smell and with or without the reduce of pH are generally caused by aerobic bacillus. 
B.5.9.6  Many canned food contains thermophile bacteria which does not grow in normal storage 
condition but grows and causes putrefaction when exposed to higher temperature (50 ℃～ 55℃). 
Thermophilic acid-resistant bacillus and bacillus stearothermophilus
 will cause sour spoilage 
putrefaction in acid and low acid food respectively. Culture at the temperature of 55 ℃ shall not 
cause the appearance change of the canned food bu t shall produce rotten smell with or without the 
reduce of pH. Sometimes the putrefaction of canned  food of tomatoes, pears, figs, pineapples and 
other canned food is caused by clostridium pasteurianum (a kind of anaerobic bacterium that can produce acid and gas). Thermophilic sucroclastic clostridium is a kind of thermophilic anaerobic 16 GB 4789.26—xxxx 
 
 bacteria which can cause swell and rotten smell of the product.  
Thermophilic anaerobic bacteria can also produce ga s. For the bacteria rapidly proliferates after 
beginning to grow, the confounded swell may be caused by hydrogen or the production of gas of thermophilic anaerobic bacteria. The decomposition of chemical shall produce carbon dioxide, 
which especially happens among the food containing sugar and some acid food such as canned food of catsup, molasses, mincemeat and fruit with high sugar. The decomposition speeds up with the rise of the temperature. 
B.5.9.7  Any microorganism isolated from vacuum packing of sterilization and normal product 
direct smear shall be suspected to be due to laboratory pollution. In order to prove whether it is 
laboratory pollution, the isolated and living microorganism shall be inoculated to another normal 
control can, hermetical sealed and cultured at the temperature of 36 ℃ for 14 days. If swell or 
product putrefaction occurs, these microorganisms may not come from original sample. If the can 
is still flat, open the can by aseptic technique and culture as the above-mentioned procedure. If the same kind of microorganism is found again and the product is normal, the product is deemed as commercial sterilization for this kind of microorganism does not grow in the normal storage and shipment. 
B.5.9.8  If the food itself is turbid, the definitive conclusion may not be obtained by broth 
cultivation. Under this condition further cultivation shall be carried out to confirm whether there 
grow microorganisms. 
 
B.6 Detection Method of Can Tightness 
The cleaned empty can after drying under the temperature of 36 ℃ can be selected to carry out the 
decompression and pressurization leakage check.  
B.6.1 Decompression leakage check 
Carefully inject clean water with the fullness of  80%- 90% into the empty can and properly place 
an organic glass with rubber band at the roll rim on open end of the can which can keep the can tight. Start up vacuum pump, close air bleeder, press the cover plate with hands, control air 
exhaust to make the time of vacuum meter rising from 0 Pa to 6.8 × 10
4Pa（510 mmHg ）more 
than 1 minute and keep this vacuum degree for more than 1 minute. Tilt and carefully observe 
whether can body, especially hemming and welding line produce bubbles. If the bubbles are produced continuously from the same position, it shall be judged as leakage. Record the leakage time and vacuum degree and mark the leakage position. 
B.6.2  Pressurization Leakage Check 
Stuff up the open end of the empty can by rubber stopper, immerse the empty can in the glass jar 
which is filled with water, start up air compressor, open the valve slowly to make the pressure in 
the can increase grad ually until the pressure rising to 6.867 × 10
4 Pa and keep it for 2 minutes. 
Carefully observe whether the can body, especially hemming and welding line produces bubbles. 
If the bubbles are produced continuously from the same position, it shall be judged as leakage. Record the leakage time and vacuum degree and mark the leakage position.