Microbiological Testing Procedures in Dairy Quality Assurance

Microbiological testing is a crucial part of the quality assurance process because it anchors the safety of the food product. Every product that passes through the plant must be attested to have high standards that the consumers expect.

It is important to conduct a series of microbiological testing procedures on dairy products to ensure that the final product meets the required standards by passing all the stringent measures required for a satisfactory product.

Here is the list of microbiological testing procedures applicable to dairy products:

1. Standard Plate Count

The purpose of this work instruction is to ensure that the number of colony forming units (CFU) per millimetre or per gram of the original sample is determined correctly. A defined test portion or series of decimal dilutions of the sample are mixed with culture media in Petri dishes and incubated. The number of colony forming units (CFU) per millilitre or per gram of the original sample is calculated from the number of colonies counted on selected dishes

Scope

This work instruction is applied to the inter-laboratory testing of milk, milk products and additives.

Reagents and equipment for media preparation

• Analytical balance
• Spatula
• Filter papers
• Autoclave
• Distilled water
• Water bath at 37°C
• Tryptone Glucose Extract Agar
• Ringers solution

Reagents and equipment for preparation of plates and analysis of sample   

• Sterile Petri dishes 90 – 95mm diameter
• Sterile pipettes of 1ml and 10ml
• Volumes of 9mls of sterile quarter strength ringers solution in sterile universal bottles
• Sterile Petri dishes
• Tryptone Glucose Agar, Oxoid

Microbiological Testing Instructions

Preparation of standard plate count media

• Suspend 24g of Tryptone Glucose Extract Agar in 1 litre of distilled water. Bring to boil to dissolve completely.
• Dispense into bottles and sterilize by autoclaving at 121°C for 15 minutes.

Preparation of quarter strength ringer’s solution

• Dissolve 1 tablet in 500ml distilled water. Sterilize by autoclaving at 121°C for 15minutes and let cool.

Microbiological Testing Method

• Mix the sample thoroughly by shaking the sample container 25 times in 10 seconds over a 30 cm arc. You may then shake the sample mechanically on a mixer. Ensure that the interval between mixing and pipetting does not exceed 3 minutes.
• Transfer 1 ml of milk aseptically into 9 ml of diluents and mix thoroughly by orbital share or manually either mechanically or by aspirating the pipette 10 times
• Transfer 1 ml from the first dilution aseptically with a fresh pipette to a further 9ml of diluents aseptically with a fresh pipette to a further 9ml of diluents and mix. Further dilutions can be prepared by transferring 1ml of each successive dilution to further 9ml diluents using a sterile pipette in each case.
• Transfer 1ml of each dilution chosen for assay using another sterile pipette into a labelled Petri dishes starting with the most dilute of the dilutions chosen
• Add 15ml of the tempered melted medium aseptically to each inoculated Petri dish. N/B the temperature of the media should not exceed 46°C due to the possible damaging effects on the micro flora of the sample.
• Mix immediately after pouring by 5 to-and-fro movements followed by 5 circular clockwise movements followed by 5 to-and-fro movements at right angles to the first set followed by 5 circular anticlockwise movements.
• Allow the Petri dishes to stand on a clean horizontal surface until the medium sets, invert and transfer to the incubator
• Incubate the Petri dishes at 37°C for 48hrs after which you select the Petri dishes with counts between 10 and 300 colonies for counting
• If more than one dilution has counts in the range of 10 to 300, use each dilution to calculate the number of organisms per millilitre of sample.

Control for the test

For each bottle of agar, pour one ml in sterile Petri dish and incubate as for the samples. If colonies are observed in these controls the test results should be considered with caution. In a case where more than 10 colonies are observed, the test results are void.

Calculations of results

Calculate the number of colonies per ml in each dilution having between 10 and 300 colonies per plate. Read the results and express the answer as number of colony forming units per ml (or g)

2. Determination of Yeasts and Moulds

The purpose of this work instruction details the enumeration of yeasts and moulds in milk and milk products. Here, we show the methods for the detection and enumeration of yeast and moulds

Requirements for this microbiological testing procedure

• Quarter strength sterile ringers solution
• Malt Extract Agar; Oxoid
• 10% lactic acid to adjust pH to 3.5
• Incubator at 22-25°C

Yeasts and Moulds Testing Instructions

Preparation of Malt Extract Agar

• As per the manufacturer’s instructions on the tub
• Add 1 ml of lactic acid 10% to each 100ml of sterilized medium at 50-55°C. The medium must not be heated after the addition of acid, as the gelling properties of the medium will be lost
• Mix well before pouring.

Yeasts and Moulds Testing Procedure

• Prepare serial tenfold dilutions of the sample in 9ml. sterile quarter strength ringers solution
• Pipette 1ml from each dilution onto a sterile Petri dish, add 15ml of Malt Extract Agar and mix by swirling the plate
• Allow to solidify
• Invert and incubate the plates at room temperature or 30°C incubator for 5 days
• Count plates containing 10-150 colonies. If many yeast are present, plates with 150 colonies are usually readily countable
• Report in colonies/ml
• Prepare slides of the colonies, identify and record what organism is present.

If more than one dilution plate has been counted, calculate the number of CFU/ml for each plate counted. Mean the result and express as colony forming units/ml (CFU/ml)

3. Detecting Coliforms Through Microbiological Testing

This method instructs on how to conduct an experiment to check for the presence or absence of both faecal and non-faecal coliforms in milk and milk products. Presence of Coliforms in dairy products is suggestive of unsanitary conditions or practices during production, processing or storage.

Re-agents and equipment

• Violet red bile agar
• Quarter strength ringer’s solution
• Sterile Petri dish
• Sterile 1ml pipette
• Incubator at 37°C
• Water bath at 45°C

Coliforms Testing Instructions

Preparation of violet red bile agar

• Suspend 38.5 g of violet red bile agar in one litre of distilled water.
• Bring to the boil to dissolve completely.
• N/B no further sterilization is necessary. Cool to 45°C in a water bath.

Coliforms Testing Method

• The specimen/sample to be plated should be diluted to avoid medium overgrowth. If the colonies lie too closely together in the medium they will become uncharacteristic and difficult to count or identify. The most suitable number of colonies is 15-150 per Petri dish.
• Prepare a tenfold dilution series in quarter strength sterile ringers solution for milk and milk products and in peptone water for ingredients and swabs transfer 1ml of the original solution of 1ml from the chosen dilution with a sterile pipette into a sterile Petri dish
• Pour 15ml of violet red bile agar (VRBA) at 45°C into each petri dish and swirl to mix.
• After the media has solidified, invert the Petri dishes and place in an incubator at 37°C for 24 hrs.
• Count the colonies of coliform bacteria, if necessary by a counter or manually marking the underside of the plate with a marker pen. Only petri dishes containing 10-150 colonies per plate should be counted.

Calculation of the results

• Count the number of coliform organisms per plate noting the dilution of the sample on the plate.
• Calculate the number of CFU/ml for each plate. If more than one plate was counted, get the mean of the results.

4. Sampling of Milk and Milk Products for Microbiological Testing and Analysis

This is the outline for the procedure for sampling milk from the silos, in process products, and finished products from the stores for microbial analysis.

Sampling Equipment

• Sampling bottles
• Cool box (insulated)
• Gel-packs
• Ladles
• Burner

Sterilization of sampling equipment

• Sterilize the sampling equipment for bacteriological testing by autoclaving at 121°C for 15-20 minutes
• The sampling bottles/bags shoud have caps/closed adequately
• Containers and closures should be sterilized and dry
• Containers shall be of a material, which adequately protects the sample during handling, storage and in transit

Sampling technique for bacteriological testing purposes

Depends on the purpose for which sampling is done and the type of the products being sampled.

• Random sampling from the stores
• For sampling of milk from tanks with nozzles, wipe the nozzle with 70% ethanol or surgical spirit before sampling; let the milk run for some few seconds before taking a sample
• Take some sample into the sterilized bottle swirl and drain. Repeat this. Take a sample for analysis and tightly cap the bottle.

Preservation of samples for bacteriological testing

• Bacteriological samples should not have preservatives
• Hold the samples at low temperatures 0-5°C The cool box used to store these samples should ensure the temperatures range is maintained.
• Transfer products to refrigerator as soon as possible
• Microbiological analysis should start not later than 24hrs after sampling

5. Isolation and Enumeration of Thermodurics in Raw Milk

This work instruction covers isolation and enumeration of thermoduric bacteria in raw milk. The purpose of this work instruction is to ensure the enumeration and detection of bacteria which survive exposure to pasteurization temperatures. Thermodurics microorganisms are generally gram-positive rods, often spore forming and gram-positive cocci.

Reagents

• ¼ strength sterile ringer’s solution
• Sterile Petri dishes
• Water bath at 64°C
• Incubator at 37°C
• Tryptone Glucose Extract Agar

Microbiological Testing Instructions

• Preparation of standard plate count media
• Suspend 24 gm of Tryptone Glucose Extract Agar in I litre of distilled water. Bring to the boil to dissolve completely. Dispense into bottles and sterilize by autoclaving at 121°C for 15minutes.
• Preparation of quarter strength ringers solutions
• Prepare as per manufacturer’s instructions; sterilize by autoclaving at 121°C for 15minutes

Microbiological Testing Procedure

• Aseptically take 10mls of well agitated milk aseptically and heat for 35minutes in a water bath at 65°C
• Cool the sample and prepare 10-fold dilutions in a quarter strength ringers solution
• Add 1ml of the diluted sample to 15ml of cooled standard plate count agar. Pour into a sterile Petri dish and incubate at 37°C for 48hrs
• Count the plates having between 10-300 colonies per plate, note the dilution

Calculations

Thermoduric count (organisms) per ml of milk = colony count x dilution factor. If more than one plate was counted, calculate the number of CFU/ml and then the results.

6. Wash-up and Sterilization in the Oven for Microbiological Testing Purposes

This work instruction covers glass wares used in micro biology analysis. The purpose of this work instruction is to ensure proper cleaning and sterilization of the glassware used in bacteriological analysis.

Glassware sterilization

Wash with warm soapy water and rinse thoroughly with cold tap water so as to remove any soap residue. Put in wire baskets and dry in a hot air oven.

Sterilization procedure (hot air oven)

• Before sterilization all glassware must be clean and dry. Wrap glassware in grease paper, aluminium foil or put in sterilization tins and cover well with cotton wool.
• Apply sterilization tape to indicate complete sterilization and place in a hot air oven
• Set the oven temperatures at 170°C and maintain temperature of 170°C for 2 hours. Check the sterilization tape indicator. If ok, turn off the oven and allow the glassware to cool gradually.

7. Decontamination of Benches, Equipment and Rooms After Microbiological Testing

This work instruction covers bacteriological facilities likely to bring out cross contamination. Decontamination usually means making equipment and waste free from infectious agents

Instructions

• Wipe down the benches with 70% alcohol or surgical spirit at the beginning of work, during work and at the end of the day.
• Disinfect all accessible parts of the equipment with 70% alcohol.
• Take swabs inside of the incubators with 70% alcohol after removing and incubating the plates.

Cleaning the Floors

• Dilute powder disinfectant detergent in a bucket of clean water.
• Thoroughly scrub the floor and rinse well.
• Ensure that you clean the floor at least twice a day and immediately in case of spillages.

8. Disposing Laboratory Waste After Microbiological Testing

The purpose of this work instruction is to ensure all specimens, cultures and other materials used in the microbiological laboratory are made noninfectious before being discarded or leave the factory. The instruction covers how to dispose the wastes from the microbial plates after incubation. Paper towels and tissues used to wipe benches and equipment and to dry hands. Disposable gloves, Glass Pasteur pipettes, slides and covers slip used scalpel blades, scissors, knives, forceps etc.

Instructions

• Place the infected material in the sterilizing boxes in the autoclave
• Ensure the water level in the autoclave is okay.
• Close the autoclave properly and start heating.
• Open the air vent until steam starts to come out through the control valve.
• Close the control valve and let the autoclave attain 121°C then continue heating for 15 minutes.
• Allow the autoclave to cool down and the pressure to cool down to 0°C
• Open the autoclave, remove the material and discard through the normal waste material disposal system.