9 julio, 2024

Lactose broth: what it is, foundation, preparation and uses

What is lactose broth?

He lactose broth is a liquid, non-selective culture medium, used mainly as a pre-enrichment medium in the isolation of strains of Salmonella, from the microbiological analysis carried out on processed foods, dairy products or water. This is recommended by the International Commission on Microbiological Specifications for Foods (ICMPF).

The medium contains enzymatic digest of gelatin, meat extract and lactose, substances necessary for bacterial growth. In addition, lactose is a fermentable carbohydrate, therefore, some coliforms are capable of breaking it down with the production of gas.

For this reason, lactose broth is recommended by the American Public Health Association (APHA) for the presumptive study of total and fecal coliform bacteria. It is described as an excellent alternative to replace tryptose lauryl sulfate broth in the standard Most Probable Number (MPN) technique, used for the microbiological analysis of food samples, milk, and surface, ground, recreational, domestic, and industrial wastewater.

Basis

For the microbiological analysis of some samples, the pre-enrichment step is essential to be able to recover a specific microorganism that may be present in very low quantities or in unfavorable conditions that compromise or minimize its viability.

Such is the case of dried and processed foods, possibly contaminated with Salmonella sp.. In these cases, if the bacteria are present, they have suffered physical and chemical abuse during the production process of the product.

In such a way that microorganisms are exposed to adverse factors such as dehydration, exposure to inhibitory or toxic products, and the overlap generated by the presence of other bacteria in greater numbers, among others.

In this sense, the lactose broth has a repairing effect on the damaged structures of the microorganism, making it recover and reproduce, in such a way that it can be detected.

Likewise, the lactose broth has the ability to dilute the inhibitory substances that may affect its viability, allowing its development. In addition, the nutritional composition of the lactose broth is strategic to favor the growth of Salmonella sp. above other microorganisms.

For final identification, it should be subcultured to other definitive culture media.

On the other hand, the composition of the medium also allows the detection of gas-producing lactose-fermenting microorganisms.

Preparation

To prepare a liter of lactose broth, 13 grams of the dehydrated medium must be weighed and dissolved in 1,000 ml of distilled water.

To help dissolve the medium in the water, the solution can be heated a little, but not too much.

Once homogeneous, the solution is prepared as follows: if the broth is to be used to search for coliforms, prepare a rack with test tubes, into which a Durham fermentation tube is inserted upside down.

The Durham tube is a very important detail, because it will allow the detection of gas formation, valuable information in the search for coliforms.

Once the tubes have been prepared, 10 ml of the lactose broth are dispensed into them, an amount that must be sufficient to cover the entire Durham tube.

If the lactose broth is to be used as pre-enrichment broth, it is not necessary to place a Durham fermentation tube. ANDIn this case, a larger amount of medium (225 ml) is required, which will be served in 500 ml bottles, with a wide mouth and a heat-resistant screw cap.

Subsequently, the tubes or bottles are autoclaved at 121 °C for 15 minutes.

The medium must remain at a final pH of 6.9 ± 0.2 at 25 °C.

The broths are stored in the refrigerator until use. Before use they must be tempered to room temperature.

On the other hand, the lactose broth can also be prepared at double concentration.

Some laboratories add bromocresol purple to the lactose broth as a pH indicator, to show the tubes where lactose fermentation has occurred due to the color change. In this case, the broth takes on a purple color and if there is fermentation it changes to yellow.

Applications

In microbiology laboratories, lactose broth is widely used as it is a relatively inexpensive medium that offers reliable and rapid results (24-48 hours).

It can be used for the analysis of total and fecal coliforms in water and food, or as a pre-enrichment broth for Salmonella.

Pre-enrichment

The pre-enrichment is a step prior to the enrichment of the sample, which greatly improves the recovery of bacteria of the genus Salmonella in processed foods.

To do this, the solid (25 grams) or liquid (25 ml) food sample is sown in 225 ml of lactose broth, incubating for 24 to 48 hours. Subsequently, it will be subcultured in an enriched medium such as cystine selenite broth or tetrathionate broth. It then goes on to XLD and SS selective media.

Analysis of total and fecal coliforms

It is an excellent medium as an indicator of fecal contamination.

For this reason, the lactose broth is ideal for the presumptive phase of the study of coliforms by the Most Probable Number method.

For samples in which a large amount of coliforms is suspected, a smaller amount of it will be inoculated (1 ml), while for samples in which a lower amount of coliforms is suspected, a larger volume of samples (10 ml) will be inoculated.

For the analysis, 10-1, 10-2, 10-3 dilutions are made, forming a battery of 3-5 tubes for each concentration used.

From each dilution, the same volume is inoculated into the lactose broths.

The tubes are incubated for 24 hours. Negative broths are incubated for a further 24 hours.

The interpretation of the results is made by observing two characteristics: the first is the presence or absence of turbidity, and since this medium does not contain a pH indicator, there will be no color change.

The second is the production or not of gas. The gas is easily evidenced in the Durham tube, by the appearance of one or several air bubbles inside.

It is considered positive if both characteristics are observed, that is, turbidity with gas production. Positive tubes should be reseeded in confirmatory media (2% Brilliant Green Bile Broth and EC Broth).

Quality control of the medium

When preparing the medium, it is important not to forget to place the Durhams tubes if the purpose of the medium is to study coliforms.
Do not overheat the medium before sterilizing.
Distribute in the test tubes before sterilizing, never after.
Do not use if the medium is more than 3 months old.
Do not use if any change in the usual characteristics of the medium is observed.
When preparing a batch of lactose broth, its quality must be tested by planting strains known as Escherichia coli, Enterobacter aerogenes, Citrobacter freundii and Klebsiella pneumoniae. They grow very well, with gas production (positive control).
may also include Pseudomonas aeruginosa, Salmonella typhimurium either enterococcus faecalis, that grow well, but without gas production (Negative Control).
It should be noted that the original color of the dehydrated medium is beige and that of the prepared medium is very light and transparent yellow. If a change in color or appearance is observed, there may be deterioration of the same.

References

Acevedo R., Severiche, C., Castillo, M. Environmental biology and microbiology. University of Cartagena, Colombia.
Conda Pronadisa Laboratories. Lactose broth double concentration (European Pharm).

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