Resources for Industrial Use

The Use and Modification of Coliscan ^® Media to Deal With the Presence of Aeromonas Species

Coliscan^® media contain ingredients which inhibit the growth of most gram positive and many non-coliform gram negative non-target organisms so that these are eliminated from appearing as background clutter in/on our media without the loss of the desired coliform/E. coli target organisms. However, as has been explained in our literature, there is one group of bacteria which is almost identical to the coliforms which could show up on our Coliscan^® media as pink-red colonies if present in test samples. Aeromonas spp. are virtually identical to coliforms as both are gram negative, aerobic or facultatively anaerobic, lactose fermenting (galactosidase producing) rods. Aeromonas species are very commonly found in the general environment and in water and food samples. There is extensive literature stating that they cause various types of diseases in humans and animals, which makes them at least as important indicators as general coliforms. In fact, Canada and various European countries have established official standards on permissible amounts of Aeromonas species in water and/or food. There is a continuing debate in U. S. government regulatory agencies concerning the importance of Aeromonas spp. as indicator organisms in water and food testing standards, but currently there are no limits set. This may be due to the fact that there have been no easy, accurate methods available to differentiate Aeromonas spp. from coliforms without additional biochemical testing. (It is simple to verify that apparent coliform colonies growing on/in a solid medium are not Aeromonas by doing a spot test for cytochrome oxidase. Coliforms are oxidase negative and Aeromonas spp.are oxidase positive.) Most current E. coli-coliform test methods based upon the presence of the enzymes galactosidase and glucuronidase basically ignore the possibility that Aeromonas in a test sample will likely show up as colonies identical to true coliforms, and if the test is a presence/absence test any Aeromonas in the test sample will likely give a positive reaction for coliforms whether or not coliforms are actually present in the sample. There are numerous published studies that verify this situation. The only ways to avoid this confusion are to either eliminate the Aeromonas by including inhibitors in the media that selectively inhibit Aeromonas, incubate at a temperature that selectively inhibits Aeromonas, or do spot tests on individual colony forming units (CFUs) to determine the % of oxidase positive colonies among those indicated as coliforms. To our knowledge, only one published medium (MI developed by EPA scientists) other than our Coliscan^® media provides for the inclusion of an effective ingredient (antibiotic) to selectively inhibit the growth of Aeromonas. Other methods (including ones that are EPA approved for water testing), have been shown to have the potential for high false positive rates for coliforms when living Aeromonas cells are present in test samples(ref.1-8).

With this background information in mind, Micrology Laboratories, LLC., has made provisions (with Coliscan^® media) available to technicians for either (1) the inclusion of Aeromonas spp. (If present in a sample) as indicators with general coliforms in contrast with E. coli, or (2) eliminating Aeromonas spp.by the addition of an antibiotic that inhibits Aeromonas, but does not inhibit general coliforms or E. coli. With choice #1, if there are viable Aeromonas spp.present in the test sample, they generally will grow at the suggested incubation temperature of 35ºC. If there is a wish to determine if any of the pink-red colonies growing are Aeromonas, it is a rather simple task to test a given number and determine what % may be oxidase positive. This approach is discussed later. With choice #2, Aeromonas is inhibited even if present in the test sample, so that false positives for general coliforms are avoided. However, this important group of indicator organisms will not be detected even if they are present, and potentially dangerous pathogens in the sample may be missed. This option is also explained and discussed later. (Another option to eliminate of Aeromonas from a population of enterobacteriaceae is incubation at a temperature of 41ºC. It is claimed that Aeromonas spp. will not grow at this temperature and that coliform populations will not be affected.)

Cytochrome Oxidase Testing

1% aqueous solution of N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride (make fresh solution in small amounts each time you wish to do testing)

The simplest approach is to apply a drop of this solution on the end of a clean toothpick (or platinum loop) to a test colony on the membrane filter surface. If the colony turns purple within 5 seconds it is oxidase positive. Disregard delayed reactions.

Another approach is to soak a filter paper with the solution and transfer bacterial colony material to the surface of the saturated paper with a clean toothpick or platinum loop. A purple reaction within 5 seconds indicates oxidase positive.

ECA Check ^® ---a new alternative to Coliscan ^® MF

Micrology Laboratories, LLC. has developed a new and novel approach to avoid the necessity of dealing with the previously discussed options when using our Coliscan^®media. This is the use of our newest invention, the ECA Check^® method. With the ECA Check^® medium, currently available in the membrane filter format, E. coli grow as black colonies, general coliforms grow as blue/grey colonies and Aeromonas spp. grow as pink-red colonies due to a unique combination of enzyme substrates that takes advantages of differences among these 3 groups of bacteria. ECA Check^® allows the technician to quantitatively identify all 3 groups in one simple test, thus providing simultaneous information on 3 extremely important indicator groups in one procedure.

Options and Procedures for the Inhibition of Aeromonas When Using Coliscan ^® Media ---The Addition of Antibiotic to Inhibit Aeromonas Spp .

1. When using Coliscan^® Easygel Medium

a. Upon request, Micrology Laboratories will include with each order a sterile tube containing the Aeromonas inhibiting antibiotic in premeasured quantity so that the addition of 10 mL of sterile deionized water (also provided) will result in a concentrated antibiotic solution. Keep the antibiotic powder in the freezer until desiring to use the medium and then prepare it by adding the sterile water into the tube containing the powder. If not all the antibiotic solution is used in one run, the remainder may be kept refrigerated for an additional week and then used up.

b. Prior to adding your test sample into the bottle of liquid Coliscan^® Easygel^® and pouring into a pretreated petri dish to effect solidification, add 0.5 mL of the antibiotic solution (using the sterile calibrated dropper supplied) to the liquid Coliscan^® Easygel^® in the bottle and swirl it to mix well. Follow this procedure with each test you wish to contain the antibiotic inhibitor. Each 10 mL tube of antibiotic solution is sufficient to treat 20 bottles of Coliscan^® Easygel^®.

2. When using Coliscan^® MF (membrane filter) Medium (USEPA approved for the determination of E. coli and total coliforms for use in National Primary Drinking Water Regulations (NPDWR) monitoring.)

a. Same as 1.a., b. above.

b. Prior to dispensing the 2 mL aliquots of liquid Coliscan^® MF medium into individual dishes containing sterile absorbent pads, add 0.5 mL of the antibiotic solution (using the sterile calibrated dropper supplied) to the liquid Coliscan^® MF in the bottle and shake to mix it well. Then proceed to dispense 2 mL aliquots of the liquid Coliscan^® MF (containing the antibiotic solution) into the dishes according to the general instructions for the use of the Coliscan^® MF medium. Each 10 mL of antibiotic solution will treat 20 bottles of broth medium.

When using the Coliscan^® Media with added antibiotic, E. coli will still appear as dark blue/purple colonies and general coliforms will appear as pink/red colonies. However, Aeromonas species in the original test sample will generally fail to grow.

REFERENCES

1. Clark, J.A., A.H. el-Shaarawi. 1993. Evaluation of commercial presence-absence test kits for detection of total coliforms, Escherichia coli, and other indicator bacteria. Appl. Environ. Microbiol. 59:380-8 Aeromonas spp. prominent among the non-coliforms appearing in these tests
2. Covert, T.C., L.C. Shadix, E.W. Rice, J.R. Haines, and R.W. Freyberg. 1989. Evaluation of the autoanalysis Colilert test for detection and enumeration of total coliforms. Appl. Environ. Microbiol. 55:2443-2447 Aeromonas spp. among the non-coliforms isolated from test samples incubated with Colilert
3. Freier, T.A., P.A. Hartman. 1987. Improved membrane filtration media for enumeration of total coliforms and E. coli from sewage and surface waters. Appl. Environ. Microbiol. 53:1246-50. 45% of coliform positive colonies on m-Endo were Aeromonas hydrophila
4. Grabow, W.O., M. du Preez. 1979. Comparison of m-Endo LES, MacConkey and Teepol media for membrane filtration counting of total coliform bacteria in water. Appl. Environ. Microbiol. 38:351-8 Aeromonas involved in high false positive results
5. Grant, M.A. 1997. A new membrane filtration medium for simultaneous detection and enumeration of Escherichia coli and total coliforms. Appl. Environ. Microbiol. 63:3526-30
6. reported 52% false positive results for total coliforms
7. Holler, C., G. Havemeister, K.O. Gundermann. 1995. Comparison of BGB-MUG and LSTB-MUG in microbiological surveillance of recreational waters. Aentralbl. Hyg. Umwaltmed. 198:138-51 Aeromonas spp. can result in false positive results
8. Landre, J.P., A.A. Gavriel, A.J. Lamb. 1998. False-positive coliform reaction mediated by Aeromonas in the Colilert defined substrate technology system. Lett. Appl. Microbiol. 26:352-4. "results obtained clearly demonstrate that Aeromonas sp. can elicit a positive coliform type reaction at very low densities."
9. McDaniels, A.E. , R.H. Bordner, P.S. Gartside, J.R. Haines, K.P. Brenner, C.C. Rankin. 1985. Holding effects on coliform enumeration in drinking water samples. Appl. Environ. Microbiol. 50:755-62 high false positive on m-Endo, mostly Aeromonas and Acinetobacter."