M-PA-C Agar

Intended Use

M-PA-C Agar is used for the selective recovery and enumeration of Pseudomonas aeruginosa from water.

Summary and Explanation

A variety of methods have been used for the enumeration of P. aeruginosa from water samples, some of which have been more widely accepted than others. The most-probablenumber
(MPN) procedures result in satisfactory recovery levels of P. aeruginosa, but are not usable for the testing of large-volume water samples and lack precision. These two deficiencies are eliminated in membrane filter (MF) techniques.
Many of the membrane filter media used for the recovery of P. aeruginosa lacked specificity and were of limited value when large heterogeneous microbial flora were present in the later
samples. Levin and Cabelli devised M-PA Agar as a selective membrane filter medium for P. aeruginosa.1 This formulation incorporated four antimicrobics, kanamycin, nalidixic acid, 
sulfapyridine and cycloheximide, which render the medium moderately selective. This original formulation was modified by raising the pH2 and altering the content or concentration of ingredients. 3 The resulting medium was designated M-PA-B Agar. Brodsky and Ciebin further modified these media by eliminating sulfapyridine and cycloheximide and produced M-PA-C Agar.4 This formulation resulted in the ability to enumerate P. aeruginosa after only 24 hours of incubation at 41.5°C compared to 72 hours required with M-PA-B Agar and 96 hours for a presumptive MPN test.4 M-PA-C Agar is identified as Modified M-PA Agar in Standard Methods for the Examination of Water and Wastewater.5

Principles of the Procedure

Yeast extract, lysine and the carbohydrates provide carbonaceous and nitrogenous compounds, energy sources and vitamins required for bacterial metabolism. Sodium chloride maintains osmotic equilibrium. The salts provide essential ions. Phenol red is a pH indicator, which becomes yellow in response to acids produced as a result of the fermentation of the carbohydrates. Kanamycin inhibits protein synthesis in gram-positive organisms.6 Nalidixic acid blocks replication of susceptible gram-negative bacteria.6

Formula

M-PA-C Agar
Approximate Formula* Per Liter
Yeast Extract................................................................ 2.0 g
L-Lysine HCl................................................................. 5.0 g
Sodium Chloride........................................................... 5.0 g
Xylose....................................................................... 1.25 g
Sucrose..................................................................... 1.25 g
Lactose..................................................................... 1.25 g
Phenol Red................................................................ 0.08 g
Ferric Ammonium Citrate............................................... 0.8 g
Sodum Thiosulfate....................................................... 5.0 g
Magnesium Sulfate...................................................... 1.5 g
Kanamycin.................................................................. 8.0 mg
Nalidixic Acid............................................................. 37.0 mg
Agar.......................................................................... 12.0 g
*Adjusted and/or supplemented as required to meet performance criteria.

Directions for Preparation from Dehydrated Product

1. Suspend 35 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder. DO NOT AUTOCLAVE.
3. Cool to 45-50°C and pour into sterile 50-mm Petri dishes. Use the medium within 1 week after preparation.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Following filtration of the water sample through a sterile 47 mm, 0.45 μm gridded filter, place the membrane filter on the surface of a plate of M-PA-C Agar taking care to avoid the 
entrapment of bubbles between the agar and filter surface. Incubate for 72 hours at 41.5 ± 0.5°C in an aerobic atmosphere. Consult the standard method for additional information
regarding the M-PA-C membrane filter technique.5

Expected Results

Colonies on membrane filters are counted using a stereoscopic microscope at 10-15× magnification. Optimal colony density is 20-80 colonies. All colonies on the filter are counted when
the density is 2 or fewer per square. The average of 10 squares is determined when the count is 3-10 colonies per square and the average of 5 squares is determined when the count is
10-20 colonies per square. Multiply the average count per square by 100 and divide by the sample volume to give colonies per milliliter.5

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