Membrane filtration

The procedure of membrane filtration is limited to the examination of limpid or crystal-clear liquid samples, without solids in suspension, and which may be filtered through a membrane with a pore size of 0.45  μm. The main advantage of this technique is that it makes it possible to inoculate larger volumes of the sample, concentrating in the membrane the microorganisms present in the inoculated quantity. The detection limit is 1 CFU per inoculated volume, which makes it the technique of choice for examining samples containing counts lower than the detection limit of the other procedures. Its main applications are total aerobic mesophilic counts, yeast and mold counts, lactic acid bacteria counts, enterococci counts and counts of total coliforms, fecal coliforms and E. coli in water, carbonated soft drinks and other liquid products, in addition to solid products, provided they can be transformed into a limpid solution, such as salt and sugar.

1-  Material required for the analyses

- Material for preparing the sample and serial dilutions.

- A previously sterilized filtration set.

- A vacuum pump.

- Membrane-filters, 47 mm in diameter, porosity of 0.45 μm.

- Petri dishes containing the culture medium recommended for the test.

- Sterile empty Petri dishes and sterile pads, optional for use with broth media.

- A laboratory incubator with the temperature set at the temperature specified by the test to be per-formed.

2- Procedure

Before starting the procedure, to ensure that all activities be carried out under aseptic conditions. Identify by labeling all the tubes and plates that will be inoculated with the sample code, the dilution and the standard abbreviation of the culture medium.

a)  Preparation of the filtration set: The membrane filtration set is composed of a membrane filter holder, a Kitasato flask and a filtration cup. The filter holder is a kind of funnel the upper part of which is plane, to accommodate the filtration membrane and onto the top of which the filtration cup is held in place by a clamp. The lower part of the filter holder is coupled to the Kitasato flask, which, connected to a vacuum pump, collects and retains the filtered liquid. Before beginning the analyses, the filter holder must be coupled to the kitasato, wrapped in Kraft paper and sterilized in an autoclave (121°C/30 min). The filtration cups must be wrapped separately in Kraft paper and also sterilized in an autoclave (121°C/30 min).

Alternatively, disposable sterile cups may be used, which are especially useful when a large number of samples are to be filtered. At the time of use, the two parts must be unwrapped in a laminar flow cabinet or, when such equipment is not available, in the close proximity of the flame of a Bunsen burner. The membrane filtration set is prepared by adjusting the sterile membrane onto the filter holder (graph side up) and the filtration cup on the membrane. Next, the kitasato flask is connected to the vacuum pump to start filtration. Another possibility is to use manifolds fitted with various filter holders and which allow to filter several samples simultaneously.

b)  Preparation of the plates: The most commonly used plates to perform the membrane filtration technique are 50 mm in diameter and 9 mm in height. If a solid culture medium is used, the plates must be previously prepared, in the same way as recommended for spread plating. It is also common practice to utilize broth medium, a situation in which no previous preparation of the plates is necessary. At the time of analysis, a sterile absorbent pad is placed inside the sterile plates and soaked with 2 ml-portions of the culture medium in liquid form.

c)  Homogenization of the sample and withdrawing the analytical unit.

Measure 100 ml in a sterile measuring cylinder and pour the content into the cup of the filtration set, avoiding spattering. If the cup of the filtration set is graduated and the graduation scale has a marking that matches the required volume, the volume of the sample may be measured directly without using the measuring cylinder.

d)  Serial dilution of the sample: Since the filtration method is a technique that concentrates the micro-organisms in samples with low counts, serial dilutions of the samples are generally not made. The usual procedure calls for filtering total quantities of 100 ml, which may divided into two portions of 50 ml, 4 portions of 25 ml or 3 portions of 70, 25 and 5 ml, respectively. Selecting the volume to be filtered, however, will depend on the estimated level of contamination of the sample, so as to obtain plates containing a number of colonies within the 20 to 200 range.

e) Filtration: Turn on the vacuum pump and start the filtration process. After passing the sample through the filter membrane, and with the vacuum pump still running, rinse off the sides of the cup with 20 to 30 ml of the diluents, to collect contaminants that may have adhered to the sides. Repeat this procedure one more time.

Turn off the pump before the membrane begins to dry out. When the volume to be filtered is smaller than 20 ml, add about 20–30 ml of the diluent to the cup of the filtration set, before adding the sample itself. Accurate measuring of the volume of diluent is not necessary, since its function is limited to merely increasing the volume to be filtered and facilitate obtaining a more even distribution of the microorganisms on the membrane.

 Note e.1)   Between one sample and the next, and prior to positioning a new membrane, the filter holder should be flame-sterilized with alcohol and the filtration cup replaced. After every 10 samples it is recommended to use the filtration set to filter 100 ml (sterile) of one of the diluents, and subsequently incubate this membrane to check for possible cross-contamination between the samples. After filtering 30 samples, the filtration set should be re-sterilized in auto-clave, prior to starting a new filtration series. If the time interval between one filtration operation and the next is greater than 30 min and the set is being used outside of the laminar flow chamber, it is recommended to autoclave the entire filtration set again, even when the limit of 30 samples has not yet been reached.

f )  Transferring and incubating the membrane: Remove the cup and, using a pair of tweezers, transfer the membrane to the plate containing the culture medium, with the graph side facing up (flame-sterilize and then cool the pair of tweezers before using). When placing the membrane onto the culture medium it is important that its entire surface stays completely adhered to the medium, to ensure that the microorganisms in the membrane come into contact with the nutrients contained in the medium. In case of bubble formation, the edge of the membrane that is closest to the bubble(s) should be gently lifted up and replaced in a way so as to eliminate the bubble(s).

Incubate the plates under the conditions recommended for the test  in an inverted position (if indicated by the method) and, preferably, placed inside bags or trays covered with moistened paper towels or filter paper, to avoid dehydration.

g)  Counting the colonies and calculating the results.

References

Silva, N.D .; Taniwaki, M.H. ; Junqueira, V.C.A.;  Silveira, N.F.A. , Nasdcimento , M.D.D. and Gomes ,R.A.R .(2013) . Microbiological examination methods of food and water a laboratory Manual. Institute of Food Technology – ITAL, Campinas, SP, Brazil .