26 Sep 2022
You would think that the discovery of food microbiology came after the discovery of the microscope. Actually, this is not the case, utilising Microorganisms in the production of food occurred before the development of microbiology as a field of research had even been acknowledged, helping to create some of our favourite and most commonly used food and drinks such as cheese, beer and bread.
Practising microbiology through fermentation is a perfect example of this using Streptococcus thermophiles, Lactobacillus helveticus, and Lactobacillus delbrueckii ssp to ferment lactose at 105°F or less creates what we commonly refer to as Swiss- and Italian-type cheeses.
However, certain microorganisms are pathogens, such as E. coli, Salmonella, Staphylococcus aureus and many more all of which can cause discomfort and even be fatal, Thus in pharmaceutical manufacturing, food, beverage and cosmetics industries, microbiological test/microbial detection is essential to quality control in order to ensure product safety.
Microbiological techniques such as Most Probable Number, Pour Plate, and Spread Plate techniques have remained the same for 50 or more. However membrane technology has moved on and evolved over time, expanding the scope and usages of the Membrane Filtration Method with some of the critical performance advantages.
Using Membrane Filtration, the entire volume of the sample is passed through the filter. All organisms present in the sample will be captured and concentrated on the surface of the filter membrane, which is then transferred directly onto a petri dish containing a growth media. Nutrients and water in the growth media will pass easily back up through the filter to support the growth of any organisms present on the surface of the membrane. By contrast both Pour and Spread Plates are dependent on operator skill to accurately disperse the sample onto or into the growth media, possibly requiring a series of three separate tests with multiple outcomes to be measured in parallel.
Apart from the increase in speed, perhaps the most important advantage of membrane filtration is its accuracy and sensitivity, driven by the fact that it uses filtration as the first step in the protocol and therefore tests the entire sample.
This is critical for testing pharmaceuticals, food, or drinking water samples where even low levels of contamination could be a dangerous to humans and one of the many reasons as to why Membrane filtration is the primary microorganism separation technologies.
Below is a comparison between Membrane Filtration, Multiple-tube Fermentation, Spread Plate and Pour Plate techniques.
Membrane Filtration | Multiple-tube Fermentation | Spread Plate | Pour Plate | |
Preparation | Agar plate | Nutrient broth | Agar plate | Agar broth (maintained at between 45~50oC) |
Incubation | 24 ± 2 hour | 3 phases, 48 hours/phase | 48 ± 3 hours | 48 ± 3 hours |
Consumption | Membrane, petri dish | Vials and fermentation tubes | Petri dish, inoculation hoop | Petri dish |
Sample volume | High | Low | Low | Low |
Heat-sensitive-bacteria-safe | Yes | Yes | Yes | No |
Sample selection | Aqueous sample with low turbidity | Aqueous sample | Aqueous sample with high bacteria concentration | Aqueous sample with low bacteria concentration |
Accuracy | High | Very low | Low | Low |
Determination | Observation and counting | Estimation by MPN | Observation and counting | Observation and counting |
Colony splitting | Yes | No | Yes | Yes |
Other | • Membranes can be moved to different culture medium for incubation. • Biocide can be effectively removed. | • Able to test aqueous samples with high turbidity • Creates more wastes. | • Culture medium can be easily scratched by the hoop. • Incubation can be processed after dried. | • Colonies may exist within the culture medium, more difficult to read. |
Procedure of membrane filter method (NIEA E230.55B):
· The test should be carried out in a laminar flow hood.
· Preparation: Sterilize the stainless steel filtration funnel and tweezers with flame, and wait till cool-down.
Rocker’s Bunsen burners provide stable flame up to 1300?, are CE certificated and piezo ignited.
– Dragon 100 Torch
– Dragon 200 Bunsen Burner
– Dragon 220 Bunsen Burner (45-degree tilt)
2. Shake the samples for more than 25 times to have them finely homogenized before dilution and detection.
3. Filter 100 mL samples then rinse the funnel with 20 mL of diluent.
4. Put the membrane onto petri dish with proper culture medium. Make sure the membrane is finely attached to the medium, avoid air from entering in between.
5. Place the incubator upside down into the incubator and incubate at 35 ± 1 oC for 24 ±2 hours. Use colony counter and count the numbers of targeted colonies. Note TNTC (Too numerous to count) if there’re too many.
6. Determination: Do density calculation with formula noted below. (Colony forming units/100 mL)
Microbiological testing (with membrane filtration) is a method that often requires sample replication and has a lot of waste liquids. Here are the two key points that you should consider when making decision for related equipment:
1) Choose equipment that can be rapidly sterilized.
Microbiological test relevant equipment often requires sterilization with autoclave, which may take hours to complete. To make the whole process quicker and smoother, several sets of equipment for replacement can be prepared. Or, you may choose materials that can be rapidly sterilized. Such as stainless steel, which can be flame sanitized and be ready for use after cools down. It saves a lot of time and make the work procedure smoother especially when you have a large number of samples to test.
Rocker SF Stainless Steel Filter Holder
Rocker Stainless Steel Manifold
Rocker Vacuum Manifold Systems
Stainless Manifold and Filter Holders
Liquid waste of microbiological tests from the water samples doesn’t need additional treatment before draining to sink. So liquid collection before draining it is unnecessary. Choose equipment that can drain the liquid waste directly to sink can save you effort and time.
Rocker WaterVac Filtration System
Alligator 200: Taiwan Excellence award-winning pump
WaterVac 100: 24VDC-driven, easily portable for at-scene sample collection.
WaterVac 200: 2 samples at once. Space-saver.
Rocker WaterVac 200 Direct Drain Filtration System
References:
–Main Groups of Microorganisms of Relevance for Food Safety and Stability – PMC (nih.gov)
– Cheese Production | MilkFacts.info
–Pour Plate and Most Probable Number tests have changed little in 50 years, while new technology has advanced Membrane Filtration Tests for microbial QC
–Analytical Methods Approved for Compliance Monitoring under the Revised Total Coliform Rule, EPA 815-B-19-007, EPA, 2019
–Difference Between Pour Plate and Spread Plate
–EQUIVALENT TESTING METHODOLOGY FOR AGRICULTURAL WATER, FDA
–ENUMERATION OF MICROORGANISMS
–Microbial Aspects of Drinking Water
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