GB/T 5750.12-2006Standard examination methods for drinking water -- Microbiological parameters (English PDF)

1.1 Plate counting method

1.1.1 Scope

This standard specifies the use of plate counting method for the determination

of total bacterial counts in drinking water and its source water.

This method applies to the determination of total bacterial counts in drinking

water and its source water.

1.1.2 Terms and definitions

The following terms and definitions apply to this standard.

1.1.2.1

Standard plate-count bacteria

The total number of bacteria contained in 1 mL of water sample obtained

after 48 h incubation at 37°C under aerobic conditions on nutrient agar.

1.1.3 Culture media and reagents

1.1.3.1 Nutrient agar

1.1.3.1.1 Ingredients.

A Peptone 10 g

B Beef paste 3 g

C Sodium chloride 5 g

D Agar 10 g ~ 20 g

E Distilled water 1000 mL

1.1.3.1.2 Preparation method. After mixing above components, HEAT to

dissolve it, ADJUST the pH to 7.4 ~ 7.6, CONTAIN it into glass containers (if

1.1.5.2.3 USE the sterile pipette to take undiluted water samples and two ~

three 1 mL of water samples of the appropriate dilution degree, respectively

INJECT it into the sterilized plate. The following operation is the same as the

drinking water testing procedure.

1.1.6 Colony counting and reporting methods

When doing plate colony counting, it can use naked eye to directly observe it,

if necessary, it shall make inspection with a magnifying glass to prevent

omission. After recording the number of colonies in each plate, it shall calculate

the average count of colonies in the same dilution, which is used for the

calculation of next step. When obtaining the average of the dilutions, if one of

the plates has more platelet-like colonies, it should not be used, but the plates

without platelet colonies shall be used to calculate the average count of

colonies of this dilution. If the platelet colonies are less than half of the plate,

whilst the distribution of colony count on this other half is very even, the colonies

of this half of plate is counted and multiplied by 2 to get the count of colonies of

the entire plate. Then the average count of colonies of this dilution is calculated.

1.1.7 Selection and reporting methods for different dilutions

1.1.7.1 First SELECT the average count of colonies between 30 ~ 300 to make

calculation, if the average count of colonies of only one dilution meet this range,

then MULTIPLY the count of colonies by the dilution factor and REPORT it (SEE

example 1 in Table 1).

1.1.7.2 If there are two dilutions, the count of colonies grown is between 30 ~

300, it is determined depending on the ratio of the two, if the ratio is less than

2, it shall report the average of the two (such as example 2in Table 1). If the

ratio is more than 2, it shall report the total count of colonies with a small dilution

(example 3 in Table 1). If it is equal to 2, it shall also report the count of colonies

of smaller dilution (SEE example 4 in Table 1).

1.1.7.3 If the average count of colonies of all dilutions is greater than 300, then

it shall multiply the average count of colonies of the highest dilution by the

dilution factor and report it (see Example 5 in Table 1).

1.1.7.4 If the average count of colonies of all dilutions is smaller than 30, then

it shall multiply the average count of colonies of the lowest dilution by the

dilution factor and report it (see Example 6 in Table 1).

1.1.7.5 If the average count of colonies of all dilutions is not between 30 ~ 300,

then it shall multiply the average count of colonies closest to 30 or 300 by the

dilution factor and report it (see Example 7 in Table 1).

1.1.7.6 If the plates of all dilutions are free from colony growth, it shall be

reported as "undetected".

2.1.4.9 Small inverted tube.

2.1.4.10 Slides.

2.1.5 Testing procedure

2.1.5.1 Lactose fermentation test

2.1.5.1.1 TAKE 10 mL of water sample, INOCULATE it into 10 mL of double

lactose peptone culture solution, TAKE 1 mL of water sample, INOCULATE it

into 10 mL of single lactose peptone culture solution, TAKE another 1 mL of

water sample, INJECT it into 9 mL of sterilized normal saline, MIX it uniformly,

PIPETTE 1 mL (that is, 0.1 mL of water sample) into 10 mL single lactose

peptone culture solution, INOCULATE 5 tubes for each dilution.

If the tap water that has been processed must be tested regularly or once a day,

it can directly inoculate five sets of 10 mL water sample double culture medium,

each set is inoculated with 10 mL of water sample.

2.1.5.1.2 When testing the source water, if the pollution is serious, it shall

increase the dilution. It can inoculate 1, 0.1, 0.01 mL or even 0.1, 0.01, 0.001

mL. Each dilution is inoculated to 5 tubes, each water sample is totally

inoculated to 15 tubes. When inoculating the water sample less than 1 mL, it

must perform 10 times incremental dilution, take 1 mL for inoculation, and

change one 1 mL sterile graduated pipette for each increment of dilution.

2.1.5.1.3 PLACE the inoculated tube in the incubator at 36 °C ± 1 °C for 24 h ±

2.1.5.2 Separation culture

TRANSPLANT the fermentation tubes producing acid and gas onto the eosin

methylene blue agar plate, CULTURE it in an incubator at 36 °C ± 1 °C for 18

~ 24 h. OBSERVE the colony morphology, SELECT the colonies with the

following characteristics for Gram staining, microscopic examination and

confirmation test.

Dark purple black colonies with metallic luster;

Purple black colonies without or with slight metallic luster;

Pale purple colonies with darker color center.

2.1.5.3 Confirmation test

After the above stained microscopic examination, it is gram-negative bacillus,

min per each sterilization. After the first two boiling, it is necessary to replace

the water to rinse it for 2 ~ 3 times, to remove the residual solvent.

2.2.5.1.2 Filter sterilization. USE the ignited alcohol swab to perform flame

sterilization, or USE steam sterilizer for autoclaving at 103.43 kPa (121 °C, 15

lb) for 20 min.

2.2.5.2 Filtered water sample

USE the sterile tweezers to take the edge of the sterilized membrane filter, LET

the rough surface face upwards, ATTACH it to the sterilized filter bed, FIX the

filter, POUR 100 mL of water sample (if the water sample contains more

bacteria, it can reduce the filtered water sample volume or otherwise dilute the

water sample) into the filter, OPEN the filter valve, PERFORM suction filtration

at -5.07 × 104 Pa (minus 0.5 atmosphere).

2.2.5.3 Culture

After filtration of the water sample, MAKE suction for about another 5 s, CLOSE

the filter valve, TAKE off the filter, USE sterile tweezers to take the edge of the

membrane filter, TRANSFER it onto the magenta sodium sulfite medium, LET

the bacteria catching face of the membrane filter face upwards, LET the

membrane filter be in complete and close attachment with the culture medium,

there shall be no air bubbles between them, INVERT the plate, PLACE it into

the 37°C incubator to culture it for 24 h ± 2 h.

2.2.6 Results observations and reports

2.2.6.1 SELECT colonies that meet the following characteristics for Gram

staining and microscopic examination.

Purple red colonies with metallic luster;

Deep red colonies without or with slight metallic luster;

Pale red colonies with darker center.

2.2.6.1.1 The Gram-negative bacillus is further inoculated with lactose peptone

culture solution, cultured at 37°C for 24 hours. If there is acid production and

gas production, the total coliforms are determined to be positive.

2.2.6.1.2 CALCULATE the count of total coliforms growing on the mem...

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