The lactose broth is a non-selective liquid culture medium, used mainly as a pre-enrichment medium in the isolation of Salmonella strains 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. Furthermore, 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, qualifying it as an excellent alternative to replace tryptose lauryl sulfate broth in the standard technique of the Most Probable Number (MPN). ), used for the microbiological analysis of food, milk and surface, underground, recreational, domestic and industrial wastewater samples.
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For the microbiological analysis of some samples, the pre-enrichment step is essential to be able to recover a specific microorganism that may be in very low quantity or in unfavorable conditions that violate or minimize its viability..
Such is the case of dried and processed foods, possibly contaminated with Salmonellas 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 quantity, among others..
In this sense, the lactose broth has a repairing effect on the damaged structures of the microorganism, causing it to 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, subculture to other definitive culture media.
On the other hand, the composition of the medium also allows the detection of lactose-fermenting microorganisms that produce gas..
To prepare a liter of lactose broth, 13 grams of the dehydrated medium must be weighed and dissolved in 1000 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, a rack of test tubes is prepared, into which a Durham fermentation tube is inserted upside down.
The Durham tube is a very important detail, because it will allow detecting the formation of gas, a data of great value in the search for coliforms.
Once the tubes are ready, 10 ml of the milk broth is dispensed into them, an amount that must be sufficient to cover the entire Durham tube..
If the lactose broth is to be used as a pre-enrichment broth, it is not necessary to place a Durham fermentation tube. In this case, a larger quantity of medium is required (225 ml), which will be served in 500 ml bottles, wide mouth and with a thermo-resistant screw cap..
Subsequently, the tubes or flasks are autoclaved at 121 ° C for 15 minutes..
The medium must have a final pH of 6.9 ± 0.2 at 25 ° C.
The broths are stored in the refrigerator until use.
Before use, the broths must be brought to room temperature.
On the other hand, the lactose broth can also be prepared at a double concentration..
Some laboratories add bromocresol purple to the lactose broth as a pH indicator, to show the tubes where there has been lactose fermentation due to the color change. In this case, the broth takes a purple color and if there is fermentation it turns yellow.
In microbiology laboratories, lactose broth is widely used as it is a relatively inexpensive medium that offers reliable and fast results (24-48 hours)..
It can be used for the analysis of total and fecal coliforms in food and water or as a pre-enrichment broth for Salmonella.
Pre-enrichment is a step prior to sample enrichment, which greatly improves the recovery of bacteria of the genus Salmonella in processed foods..
To do this, the sample of the solid food (25 grams) or liquid (25 ml) is seeded in 225 ml of lactose broth, incubated for 24 to 48 hours. Subsequently, it will be sub-cultured in an enriched medium such as selenite cystine broth or tetrathionate broth. Then move on to XLD and SS selective media.
It is an excellent medium as an indicator of fecal contamination.
Therefore, 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 quantity of coliforms is suspected, a smaller quantity of it will be inoculated (1ml), while for samples in which a lower quantity of coliforms is suspected, a greater volume of samples (10 ml) is inoculated.
For the analysis dilutions are made 10-1, 10-two, 10-3, forming a battery of 3-5 tubes for each concentration used.
From each dilution, the same volume is sown to 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 as this medium does not contain a pH indicator, there will be no color change..
The second is the production or not of gas. Gas is easily evident in the Durham tube, by the appearance of one or more air bubbles inside.
It is considered positive if both characteristics are observed, that is, turbidity with gas production. Positive tubes should be re-seeded in confirmatory media (Brilliant Green Bile 2% broth and EC broth).
- When preparing the medium, it is important not to forget to place the Durhams tubes if the purpose of the tube is to study coliforms..
- Do not overheat the medium before sterilizing.
- Distribute into test tubes before sterilizing, never after.
- Do not use if the medium is more than 3 months old..
- Do not use if you observe any change in the usual characteristics of the medium.
- When preparing a batch of lactose broth, test the quality of the batch by planting strains known as Escherichia coli, Enterobacter aerogenes , Citrobacter freundii Y Klebsiella pneumoniae. They grow very well, with gas production (positive control).
- It can also include Pseudomonas aeruginosa, Salmonella typhimurium or 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.
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