Determination of the total number of colonies (test piece method)

The total number of bacterial colonies is mainly used as a sign to determine the degree of bacterial contamination of food, which has important hygienic significance and is widely used in food hygiene work abroad

Total number of colonies-the total number of colonies contained in 1 mL (g) of the sample or sample towel per unit area after the sample has been processed and cultured under certain conditions

[Detection principle]

Petriflm ^TM bacterial count test piece is a pre-prepared culture medium system, which contains standard culture medium, cold water soluble gel and triphenyltetrazolium chloride (OTC) indicator.

[Equipment and materials]

Constant temperature box: (36±1)℃, (30±1)℃;

Refrigerator: 2~5℃;

pH meter or precision pH test paper;

Magnifying glass or colony counter

[Medium and Reagents]

(1) 1mol/L Sodium Hydroxide (NaOH) Weigh 40g NaOH and dissolve it in 1000mL distilled water

(2) 1mol/L hydrochloric acid (HCI) pipette 90mL concentrated hydrochloric acid, dilute with distilled water to 1000mL

(3) the total number of bacteria test piece applicator platen and quick stick

[Detection Procedure]

See Figure 1-1

Figure 1-1 Inspection procedure for the total number of colonies

(1) Sample preparation Take a representative sample aseptically and place it in a sterile container

Solid or semi-solid food: Take 25g sample by aseptic operation, put it into a sterile homogenizing cup containing 225mL diluent, homogenize at 8000r/min for 1~2min, make a 1:10 sample homogenate

Dry or dry powdered food: Take 25g sample with aseptic operation and put it into a 500mL dilution bottle containing 225mL diluent and appropriate amount of glass beads

Liquid food: Use a sterile straw to suck 25mL sample, put it into a 500mL diluent bottle containing 225mL diluent, and shake quickly according to the method of dry or dry food to make a 1:10 homogeneous sample solution

(2) Dilute the sample homogenate.

The straws were prepared as described above was homogenized samples were diluted 10-fold increments of the sample, such as with 10mL sterile 10 ^-3 , 10 ^-4 , 10 ^-5 .

(3) Choose 2~3 suitable dilution tests for inoculation

(4) Cultivation: Place the test piece horizontally in an incubator with the transparent side facing up, stacking up to 20 pieces, and incubate at 36±1℃ for 48±2h (aquatic products at 30±1℃ for 72±3h);

(5) Colony count and record count of red colonies

[Result Judgment]

Immediately after incubation, count the number of colonies on each test piece

If the colonies on two test pieces with only one dilution are within the appropriate range, first calculate the average value of the two test pieces, and then multiply the average value by the corresponding dilution factor to be the number of colonies on the test piece per gram (ml) of the sample ( Table 1-1, sample 1)
.

If there are two dilutions within the appropriate range, first calculate the average of the two test pieces for each dilution, then calculate the average of the two dilutions, and then calculate the number of colonies on the test piece per gram (ml) of the sample (table 1-1, sample 2)
.
When there are less than 25 colonies on the two test pieces of the lowest dilution, count the actual number of colonies on the two test pieces of this dilution, calculate the average number of colonies on the two test pieces, and multiply the average number of colonies by the dilution factor, Get the estimated number of colonies on the test piece
.
Add an asterisk (*) to this number, indicating that the number system is estimated from the test piece with the colony count outside the range of 25 to 250 (Table 1-1, Sample 3)
.

When the number of colonies on all test pieces exceeds 250, the average number of colonies on the two test pieces with the highest dilution should be multiplied by the dilution factor to obtain the estimated number of colonies on the test piece
.
Adding an asterisk (*) to this number indicates that the number system is estimated from the test piece whose colony count is outside the range of 25 to 250 (Table 1-1, Sample 4)
.
If there are no colonies on the test piece at all dilutions, report the number of colonies on the test piece by less than 1 times the lowest dilution factor
.
Adding an asterisk (*) to this number indicates that the number system is estimated from the test piece whose colony count is outside the range of 25 to 250 (Table 1-1, Sample 5)
.
Among the two test slices of the same dilution, one has 25 to 250 colonies, and the other has more than 250 colonies.
Both test slices must be counted
.
Calculate the average value of the two test pieces first, and then multiply the average value by the corresponding dilution factor as the number of colonies in the test piece per gram (ml) of the sample (Table 1-1, sample 6)
.
For each of the two serial dilutions, one test piece has a colony number in the range of 25 to 250, and the other has a colony number higher than 250 or lower than 25.
All four test pieces must be counted.
Calculate the average of the two test pieces for each dilution, then calculate the average of the two dilutions, and then calculate the number of colonies on the test piece per gram (ml) of the sample (Table 1-1, Sample 7)
.
Two test pieces of a certain dilution have 25 to 250 colonies, while only one of the two test pieces of another dilution has a colony number in the range of 25 to 250
.
Four test pieces must be counted.
First calculate the average of the two test pieces for each dilution, then calculate the average of the two dilutions, and then calculate the number of colonies on the test piece per gram (ml) of the sample (Table 1-1 , Sample 8)
.
When recording, only when it is converted to the number of colonies in the test piece per gram (ml) of the sample, two significant digits can be set, and the third digit is recorded by rounding
.
The number of colonies on the test piece of the sample can also be recorded as an exponential form of 10 (see Table 1-1, examples in parentheses)
.

Table 1-1 The number of plate colonies to calculate the number of colonies

Note: Those with an asterisk (*) are estimates
.

[Description]

The used paper has live bacteria on it, and it needs to be disposed of in time according to the principles of biosafety waste disposal
.

Related Links: Research status of rapid detection of food-borne microorganisms (3)