BIO CHE M
   
 


MICROBIOLOGY



DETERMINATION OF SULPHATE-REDUCING BACTERIA

IN WATER BY “THE EXTINCTION DILUTION TECHNIQUE” 

PRINCIPLE


The density of sulphate-reducing bacteria (SRB) in water samples can be estimated by injecting known volumes of water samples in broth media contained in sealed vials, in order to produce a series of ten-fold dilutions.

Then SRB are computed by noting the dilution in which no growth is visible (hence the term extinction dilution).


APPARATUS

Sealed SRB vials containing liquid broth.


METHOD

Sampling procedure

Take a sample of the water under examination in a sterile plastic container, making sure that the container is completely filled and that contamination is avoided.


Testing

Take at least 3 SRB broth vials and number them ‘l’, ‘2’, and ‘3’, and so on, respectively.

Take a sterile disposable 2 ml syringe and insert a sterile needle, then slowly withdraw 1 ml from the sample in the plastic container, using the syringe. Hold the syringe upright to expel any air and immediately push the needle into the vial numbered ‘l’ .

Slowly inject the sample into the vial being careful not to introduce air bubbles.

Mix the contents of the vial by inverting the vial three times. This will give a 1 : l0 dilution of the original sample.

Take a new sterile 2 ml syringe and needle, hold the vial No.l downwards and quickly insert the needle into the rubber cup of the vial. Slowly withdraw 1 ml of the broth into the syringe being careful not to introduce air bubbles. Inject this broth into the vial numbered ‘2’, using the same technique as previously described.

Transfer 1 ml of broth from vial ‘2’ to vial ‘3’, using a new sterile syringe and needle and the same procedure.

Incubate all 3 (or more) broth vials in an upright position in an incubator at 28 °C or at the same temperature as the water from which the sample was taken, if this temperature is higher.



NOTE

It is important to carry out the procedure exactly as stated, since contamination from vial to vial will result in all vials showing a positive reaction, hence giving a falsely high result.



INTERPRETATION


The broth vials will turn black if sulphate-reducing bacteria are present. This technique will give a count of SRB as follows:


VIAL NUMBER


1


2


3


4


Broth dilution


Probable number of SRB

1/10

1 to 10


1/100


10 to 100


1/1.000


100 to 1.000


1/10.000


1.000 to 10.000


 

For example, if only vial ‘1’ turn black this means that there are between 1 and 10 SRB in one ml of the original sample and if all 3 vials turn black there are at least 100 SRB in one ml of the original sample.

If the latter occurs, extra dilution tubes should be prepared, e.g. ‘4’ and ‘5’, until a definite count is obtained.




DETERMINATION OF SULPHATE-REDUCING BACTERIA

IN WATER BY “THE MOST PROBABLE NUMBER (MPN)”



A more precise approach to the determination, on statistical base, of the density of sulphate bacteria in water is the computation of positives, from serial dilutions run in parallel, in terms of the Most Probable Number (MPN), given on the attached tables.

Depending on positive results, obtained after the incubation of the vials, the tables give directly the MPN of bacteria/ml, when 1 ml of sample is injected and two parallel serial dilution have been made.

For instance, to know the MPN/ml of SRB, look for the combination of blackened vials on the column of “Combination of positives” on the appropriate table (with 2 vials per dilution, 3 vials per dilution, 5 vials per dilution) and read the corresponding MPN/ml.

Keep in mind that “Combination of positives” is composed of three numbers:

 

The first indicates how many vials, injected with 1 ml of sample, turned black ;

The second indicates the number of blackened vials of the first dilution (0.1 ml of the original sample);

The third indicates the number of blackened vials of the second dilution (0.01 ml of original sample).


If more than two dilutions have been made, the computing will include the highest dilution that gives positive results for all the vials and two next succeeding dilutions.

Then the obtained MPN value must be multiplied by 10n, where n is the number of vials which precede the highest dilution where all vials blackened.


 

Special cases

If there are blackened vials even after two dilutions after the position where a11 the via1s blackened, the 1ast number of “combination of positives” will be the sum of all the vials blackened on the second and on the succeeding dilutions.


The MPN tables do not report extremely unlikely combinations. Therefore if for same set of tests it occurs in more than 1 % of cases, that is an indication that something wrong happened in the test, or that the bacteria inside the via1s cannot reproduce freely.




NOTE:

For an approximate calculation of the tables or to extend the tables to other combination of positives or dilutions, the Thomas formula can be used:



MPN/ml = Number of total positive vials obtained

                                            (A x B)


where:            A = Total volume in ml of original sample present in all negative vials

                        B = Total volume in ml of original sample present in all vials.



EXAMPLES


P P                                        Vials injected directly by 1 ml of sample (2 vials per dilution)

P 0                                        First dilution

P 0                                        Second dilution

0 0                                        Third dilution

 

Combination of positives: 211

MPN/ml = 13



 

0 0

0 0

P P

0 P

0 0

Combination of positives: 210

MPN/ml 6 x 102 = 600




0 0

0 0

P P

0 P

P 0

P 0


Combination of positives: 21 (1+1) = 212

MPN/ml 20 x 102 = 2000






 

MPN TABLE – 2 vials per dilution


Combination of positives                                      MPN/ml


000                                                                                 0.0

001                                                                                 0.5

010                                                                                 0.5

011                                                                                 0.9

020                                                                                 0.9

100                                                                                 0.6

101                                                                                 1.2

110                                                                                 1.3

111                                                                                 2.0

120                                                                                 2.0

121                                                                                 3.0

200                                                                                 2.5

201                                                                                 5.0

210                                                                                 6.0

211                                                                               13.0

212                                                                               20.0

220                                                                               25.0

221                                                                               70.0

222                                                                             110.0



 


MPN TABLE – 3 vials per dilution



Combination of positives                 MPN/ml                                        Combination of positives               MPN/ml


000                                                          0.0                                                302                                                        6.4

001                                                          0.3                                                310                                                        4.3

010                                                          0.3                                                311                                                        7.5

100                                                          0.4                                                312                                                      12.0

101                                                          0.7                                                320                                                        9.3

110                                                          0.7                                                321                                                      15.0

111                                                          1.1                                                322                                                      21.0

120                                                          1.1                                                330                                                      24.0

200                                                          0.9                                                331                                                      46.0

201                                                          1.4                                                332                                                     110.0

210                                                          1.5

211                                                          2.0

220                                                          2.1

221                                                          2.8

300                                                          2.3

301                                                          3.9

302                                                          6.4



 

 

 

 

 


DETERMINATION OF TOTAL AEROBIC ACID PRODUCING BACTERIA

IN WATER BY “THE EXINTION DILUTION TECHNIQUE” 

PRINCIPLE


The density of total aerobic acid producing bacteria in water samples can be estimated by injecting known volumes of water samples in broth media contained in sealed vials, in order to produce a series of ten-fold dilutions.

Then the total acid producing bacteria are computed by noting the dilution in which no growth is visible (hence the term extinction dilution).


APPARATUS

Sealed vials containing phenol red liquid broth.


METHOD

Sampling procedure

Take a sample of the water under examination in a sterile plastic container avoiding any contamination.


Testing

Take at least 3 phenol red broth vials and number them ‘l’, ‘2’, and ‘3’, and so on, respectively.

Take a sterile disposable 2 ml syringe and insert a sterile needle, then slowly withdraw 1 ml from the sample in the plastic container, using the syringe.   Push the needle into the vial numbered ‘l’ and slowly inject the sample into the vial .

Mix the contents of the vial by inverting the vial three times. This will give a 1 : l0 dilution of the original sample.

Take a new sterile 2 ml syringe and needle, hold the vial No.l downwards and quickly insert the needle into the rubber cup of the vial. Slowly withdraw 1 ml of the broth into the syringe.    Inject this broth into the vial numbered ‘2’, using the same technique as previously described.

Transfer 1 ml of broth from vial ‘2’ to vial ‘3’, using a new sterile syringe and needle and the same procedure.

Incubate all 3 (or more) broth vials in an upright position in an incubator at 28 °C or at the same temperature as the water from which the sample was taken, if this temperature is higher.



NOTE

It is important to carry out the procedure exactly as stated, since contamination from vial to vial will result in all vials showing a positive reaction, hence giving a falsely high result.



INTERPRETATION


The broth vials will turn yellow if acid producing bacteria are present. This technique will give a count of total aerobic acid producing bacteria as follows:


VIAL NUMBER


1


2


3


4

 

 

Broth dilution

 

Probable number of total aerobic acid producing bacteria

 

1/10

 

1 to 10


1/100


10 to 100


1/1.000


100 to 1.000


1/10.000


1.000 to 10.000


 

For example, if only vial ‘1’ turn yellow this means that there are between 1 and 10 bacteria in one ml of the original sample and if all 3 vials turn yellow there are at least 100 bacteria in one ml of the original sample.

If the latter occurs, extra dilution tubes should be prepared, e.g. ‘4’ and ‘5’, until a definite count is obtained.




DETERMINATION OF TOTAL AEROBIC ACID PRODUCING BACTERIA

IN WATER BY “THE MOST PROBABLE NUMBER (MPN)”



A more precise approach to the determination, on statistical base, of the density of total aerobic acid producing bacteria in water is the computation of positives, from serial dilutions run in parallel, in terms of the Most Probable Number (MPN), given on the attached tables.

Depending on positive results, obtained after the incubation of the vials, the tables give directly the MPN of bacteria/ml, when 1 ml of sample is injected and two parallel serial dilution have been made.

For instance, to know the MPN/ml of total aerobic acid producing bacteria, look for the combination of yellow vials on the column of “Combination of positives” on the appropriate table (with 2 vials per dilution, 3 vials per dilution, 5 vials per dilution) and read the corresponding MPN/ml.

Keep in mind that “Combination of positives” is composed of three numbers:

 

The first indicates how many vials, injected with 1 ml of sample, turned yellow ;

The second indicates the number of yellow vials of the first dilution (0.1 ml of the original sample);

The third indicates the number of yellow vials of the second dilution (0.01 ml of original sample).


If more than two dilutions have been made, the computing will include the highest dilution that gives positive results for all the vials and two next succeeding dilutions.

Then the obtained MPN value must be multiplied by 10n, where "n" is the number of vials which precede the highest dilution where all vials turned yellow.


 

Special cases

If there are yellow vials even after two dilutions after the position where all the via1s turned yellow, the 1ast number of “combination of positives” will be the sum of all the yellow vials on the second and on the succeeding dilutions.


The MPN tables do not report extremely unlikely combinations. Therefore if for same set of tests it occurs in more than 1 % of cases, that is an indication that something wrong happened in the test, or that the bacteria inside the via1s cannot reproduce freely.




NOTE:

For an approximate calculation of the tables or to extend the tables to other combination of positives or dilutions, the Thomas formula can be used:



MPN/ml = Number of total positive vials obtained

                                            (A x B)


where:            A = Total volume in ml of original sample present in all negative vials

                        B = Total volume in ml of original sample present in all vials.



EXAMPLES


P P                                        Vials injected directly by 1 ml of sample (2 vials per dilution)

P 0                                        First dilution

P 0                                        Second dilution

0 0                                        Third dilution

 

Combination of positives: 211

MPN/ml = 13



 

0 0

0 0

P P

0 P

0 0

Combination of positives: 210

MPN/ml 6 x 102 = 600




0 0

0 0

P P

0 P

P 0

P 0


Combination of positives: 21 (1+1) = 212

MPN/ml 20 x 102 = 2000






 

MPN TABLE – 2 vials per dilution


Combination of positives                                      MPN/ml


000                                                                                 0.0

001                                                                                 0.5

010                                                                                 0.5

011                                                                                 0.9

020                                                                                 0.9

100                                                                                 0.6

101                                                                                 1.2

110                                                                                 1.3

111                                                                                 2.0

120                                                                                 2.0

121                                                                                 3.0

200                                                                                 2.5

201                                                                                 5.0

210                                                                                 6.0

211                                                                               13.0

212                                                                               20.0

220                                                                               25.0

221                                                                               70.0

222                                                                             110.0



 


MPN TABLE – 3 vials per dilution



Combination of positives                 MPN/ml                                        Combination of positives               MPN/ml


000                                                          0.0                                                302                                                        6.4

001                                                          0.3                                                310                                                        4.3

010                                                          0.3                                                311                                                        7.5

100                                                          0.4                                                312                                                      12.0

101                                                          0.7                                                320                                                        9.3

110                                                          0.7                                                321                                                      15.0

111                                                          1.1                                                322                                                      21.0

120                                                          1.1                                                330                                                      24.0

200                                                          0.9                                                331                                                      46.0

201                                                          1.4                                                332                                                     110.0

210                                                          1.5

211                                                          2.0

220                                                          2.1

221                                                          2.8

300                                                          2.3

301                                                          3.9

302                                                          6.4