What bacterial strains should be used for confirmation testing using BART Testers?

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Document ID TE4006

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Version 2.0

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Status Published

Published Date

Published Date 07/03/2018
Question
What bacterial strains should be used for confirmation testing using BART Testers?
Answer
There are several different BART testers, and the bacterial strain used for confirmation testing varies.  Hach does not carry bacterial strains for confirmation.  The information below provides American Type Culture Collection (ATCC) numbers for various bacterial strains that can then be used to order from companies that these types of products.

BART Test for Iron-Related Bacteria (IRB)
BART Test for Sulfate-Reducing Bacteria (SRB)
BART Test for Slime-Forming Bacteria (SLYM)
BART Test for Heterotropic Aerobic Bacteria (HAB)
BART Test for Fluoresching Pseudomonas (FLOR)
BART Test for Denitrifying Bacteria (DN)
BART Test for Nitrifying Bacteria (N)
BART Test for Blue Green Algae (ALGE)
BART Test for Pool and Spa Bacteria



Confirmation of the Selective Media Composition in the IRB-BARTTM
 
In order to confirm the suitability of the selective medium for the biodetection of the various Iron Related Bacteria recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the biodetectors to determine the standard reaction patterns (Table Twenty).  Each culture should be prepared as a 48hour-broth culture incubated at 30oC to reach the stationary growth phase. Inoculation of the inner test vial should use a cell suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringers solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculum should be applied directly over the ball as the test vial is filled.  Do not shake the inoculated inner vial. Incubate at 22 to 24oC for seven days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Twenty Three. 

 
Cultural Characterization of the IRB-BARTTM
 
A.T.C.C.                Genus/species                                       Characterization
8090                       Citrobacter freundii                           GC          Reaction 6
13048                    Enterobacter aerogenes                    BR          Reaction 4
27853                    Pseudomonas aeruginosa                 GC         Reaction 8&9
19606                    Acinetobacter calcoaceticus              GC         Reaction 8
23355                    Enterobacter cloacae                         CL-BG   Reaction 2&3
13315                          Proteus vulgaris                           CL-BC   Reaction 2&4

13883                    Klebsiella pneumoniae                      RC-BC   Reaction 7&4
25922                          Escherichia coli                            FO         Reaction 5

_______________________________________________________________

Note: Some of these culture tests will shift from one reaction type to another as the growth in the IRB-BARTTM matures.  For example, Citrobacter freundii may cause after 5 to 8 days a bio locking of the ball so that when the test vial is turned upside down the ball remains "glued" into position with the liquid medium held above the ball. The first reaction normally precedes the second reaction. The test using E.coli is performed at 35oC.



Confirmation of the Selective Media Composition in the SRB-BARTTM
 
In order to confirm the suitability of the selective medium for the biodetection of the various SRB recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the SRB-BARTTM biodetectors to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour broth culture incubated at 30oC to reach the stationary growth phase. Inoculation of the inner test vial should use a suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringers solution. This inoculum should be taken from the midpoint of the Brain Heart Infusion broth culture immediately after the culture had been gently agitated.  This inoculum is administered over the ball as the test vial is filled. Do not shake the vial. Incubate at 22 to 24oC for ten days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains cultured in Table Twenty Six. Note that the Desulfurovibrio desulfuricans strain DSM1924 should be cultured on Sulfate Reducer API agar in accordance with the recommendations of the API (Recommended Practice for Biological Analysis of Subsurface Injection Waters, Volume 38, 2nd. edition, 1965).
 
 
Cultural Characterization of the SRB-BARTTM
 
A.T.C.C.                Genus/species                                       Characterization
 
13048                    Enterobacter aerogenes                    CG         Reaction 4
27853                    Pseudomonas aeruginosaA                CG        Reaction 4
13315                    Proteus vulgaris                                  CG        Reaction 4
DSM1924             Desulfovibrio desulfuricansA               BD-BA   Reaction 1&3
A + A                      Ps. aeruginosa + D. desulfuricans    BU-BA   Reaction 2&3
_______________________________________________________________

Note that some of these cultural tests will shift from one reaction type to another as the growth in the SRB-BARTTM matures.



Confirmation of the Selective Media Composition in the SLYM-BARTTM
 
In order to confirm the suitability of the selective medium for the biodetection of the various slime-forming bacteria recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the SLYM-BARTTM to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour broth culture incubated at 30oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of
the inner test vial uses a suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringers solution. This innoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculated solution is applied directly over the ball as the test vial is filled.  Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Twenty Nine.

 
Cultural Characterization of the SLYM-BARTTM
 
A.T.C.C.                Genus/species                       Characterization
 
8090                       Citrobacter freundii               CL          Reaction 5
13048                    Enterobacter aerogenes       CL-BL    Reaction 5 & 6
27853                    Pseudomonas aeruginosa    CL-PB    Reaction 5 to PB
12228                    Staphylococcus epidermidis  DS        Reaction 1
23355                    Enterobacter cloacae           CP-CL    Reaction 2 to 5
13315                    Proteus vulgaris                   CP-CL    Reaction 2 to 5
13883                    Klebsiella pneumoniae        SR-CL    Reaction 4 to 5
25922                    Escherichia coli                   CL-BL     Reaction 5 to 6
_________________________________________________________

Note that some of these culture tests will shift from one reaction type to another as the growth in the SLYM-BARTTM matures.
 


Confirmation of the Selective Media Composition in the HAB-BARTTM
                                                                                                
In order to confirm the suitability of the selective medium for the detection of the various HAB recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the HAB-BARTTM to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour Brain Heart Infusion broth culture incubated at 30oC to reach the stationary growth phase. Inoculation of the inner test vial should be using a 1.0ml suspension of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculum should be applied directly over the ball as the test vial is being filled.  Do not shake the vial. Incubate at 22 to 24oC for seven days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty Two.
 

Cultural Characterization of the HAB-BARTTM
 
A.T.C.C.                Genus/species                                       Characterization
 
27853                    Pseudomonas aeruginosa                 DO          Reaction 2
25922                    Escherichia coli                                  UP           Reaction 1
 
The HAB (or heterotrophic aerobic bacteria) BART™ test is very specifically designed for the determination of aerobic bacteria that are heterotrophic. This means that this test is designed to determine the activity of aerobic bacteria that will possess the methylene blue reductase enzyme system and not function well anaerobically. If the biofouling problems are thought to be generated by aerobic microbial activities, the HAB-BART™ performs a role as the “scout” for  aerobically generated problems.
 
 


Confirmation of the Selective Media Composition in the FLOR-BARTTM

 
In order to confirm the suitability of the selective medium for the biodetection of the various pseudomonad bacteria recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the FLOR-BARTTM to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour broth culture incubated at 30oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of the inner test vial should be using a 0.1 ml suspension of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculated solution should be applied directly over the ball as the test vial is filled.  Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty Six.
 

Cultural Characterization of the FLOR-BARTTM
 
 
A.T.C.C.                Genus/species                       Characterization 
 
13048                    Enterobacter aerogenes          CL          Reaction 1
27853                    Pseudomonas aeruginosa       CL-PB    Reaction 1 with PB
12228                    Staphylococcus epidermidis                   No growth
19606                    Acinetobacter calcoaceticus      CL        Reaction 1.
 
 


 
Confirmation of the Selective Media Composition in the DN-BARTTM
 
In order to confirm the suitability of the selective medium for the biodetection of the various  bacteria recognized by this test method (see text above), it is recommended that the following A.T.C.C. strains be applied to the DN-BARTTM  to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour culture incubated at 35oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of the inner test vial should be with a suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculated solution  should be  applied directly over the FID ball as the test vial is filled.  Do not shake the vial. Incubate at 22 to 24oC for one day and observe for activities and reactions after applying the reactant cap following the standard procedure. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty Nine.
 
 
Cultural Characterization of the DN-BARTTM
 
A.T.C.C.                Genus/species                                       Solution
                                                                                                                               
13048                    Enterobacter aerogenes                    ++, clouding,        
27853                    Pseudomonas aeruginosa                 ++, slight clouding
12228                    Staphylococcus epidermidis               -,    clouding,        
19606                    Acinetobacter calcoaceticus             ++, no clouding,  
25922                    Escherichia coli                                 ++,clouding,
_______________________________________________________________


* Gassing or Foaming (++) is considered the prime test for complete denitrification that can be recognized as a foam ring or intense bubbles under and around the ball. Clouding is not a confirmation of denitrification and should be considered negative.
 
 
 


Confirmation of the Selective Media Composition in the N-BARTTM
 
In order to confirm the suitability of the selective medium for the biodetection of the various bacteria recognized by this test method, it is recommended that the following A.T.C.C. strains be applied to the N-BARTTM biodetectors to determine the standard reaction patterns.  Each culture should be prepared as a 7-day culture incubated at 25oC to reach the stationary growth phase. The cultural techniques to be used are referenced in Verhagen et al (1993) "Effects of Grazing by Flagellates on Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Soil Columns" J. Appl. and Appl. Micro.  Inoculation of the inner test vial should be with a 0.1ml suspension of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  7.75 ml of this inoculated solution should be  applied directly to the test vial.  Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions after applying the reactant cap following the standard procedure. Typical results are listed below for the recommended A.T.C.C. strains in Table Forty Two.
 
 
Cultural Characterization of the N-BARTTM
 
A.T.C.C.                Genus/species                       Characterization/solution
 
25391                    Nitrosomonas winogradski     clouding, red reaction, nitrite +
27853                    Pseudomonas aeruginosa -,  no reaction, nitrite negative
19718                    Nitrosomonas europae           clouding, red reaction, nitrite +
_______________________________________________________________
 
 


 
Confirmation of the Selective Media Composition in the ALGE-BARTTM
 
There is a need to confirm the suitability of the selective medium for the biodetection of the various algae. This need is recognized by this test method (Table Forty Six). It is recommended that the following Culture Collection of Algae and Protozoa (CCAP) strains be applied to the ALGE-BARTTM to determine the standard reaction patterns. The CCAP 5th edition was issued as ISBN 1 871105 01 3 in 1988 with a supplement in 1991.  Each culture should be prepared according to the protocols described in the 5th edition CCAP catalog. The cultures should be used when they have reached the stationary growth phase. Inoculation of the inner test vial should be using a suspension of 0.5 ml of the culture in 15 ml of the sterile isotonic salt solution. This inoculum should be taken from the midpoint of the micro-algal culture immediately after the culture had been very gently agitated. Do not shake the vial. Incubate under continuous light at 22 to 24oC for twenty-eight days and observe twice a week for activities and growth reactions. Typical results are listed below for the recommended CCAP strains in Table Thirty Seven.

 
Characterization of the ALGE-BARTTM
 
C.C.A.P.                 Genus/species                                       Characterization 
 
211/62                   Chlorella sp.                                        GF
11/77                     Chlamydomonas baca                        GG to GF
276/21                   Scenedesmus quadricauda                 F to YB
678/4                     Spirogyra sp.                                       GF to DG
_________________________________________________________

Note that some of these cultural tests will shift from one reaction type to another as the growth in the ALGE-BARTTM matures.
 
 
 
 
 
Confirmation of the Selective Media Composition in the POOL-BARTTM
 
      In order to confirm the suitability of the selective medium for the biodetection of the selected bacteria recognized by this test method (see text above), it is recommended that the following A.T.C.C. strains be applied to the POOL-BARTTM to determine the standard reaction patterns.  Each culture should be prepared as a 48-hour broth culture incubated at 30oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of the inner test vial should be using a suspension of 0.1 ml of the broth culture in 15 ml of the sterile solution. This inoculum should be taken from the midpoint of the broth culture immediately after the culture had been gently agitated.  This inoculated solution should be applied directly over the ball as the test vial is filled.  Do not shake the vial. Incubate at 22 to 24oC for five days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Forty Nine.     
 

 
Cultural Characterization of the POOL-BARTTM
 
A.T.C.C.                Genus/species                                       Characterization 
 
13048                    Enterobacter aerogenes                     CL
27853                    Pseudomonas aeruginosa                  CL to  PB
12228                    Staphylococcus epidermidis              No growth
19606                    Acinetobacter calcoaceticus              CL         
25922                    Escherichia coli                                  CL.
 
 
 


 

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