make culture media

Culture Media Preparation & Effect Of Growth Inhibitors On Milk Curdling

When you want to study the features of bacteria, it is important that you should know how to make culture media. Since one cannot propagate bacteria without this knowledge, it is important that you learn how to do it right.

A culture media is either an organic or a synthetic substance that provides both the biophysical and the biochemical factors necessary for the growth of bacteria.

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Researchers have developed a variety of culture media to serve different needs/purposes. Each type of media serve the needs of a particular bacteria and/or the special requirements of the investigator.

One of the purposes of a culture media is to isolate and maintain pure bacterial strains. That is why the media is crucial in the identification of different bacteria.

The differentiating factors depend on the biophysical and biochemical properties of each bacteria. You can use liquid media to grow pure batch cultures and to estimate the bacterial populations. A gelling agent (usually nutrient agar), makes the media either solid or semi solid. Nutrient agar is a hydrocolloid of red algae.

Agar imparts unique physical characteristics to the culture media, which makes it suitable for its purpose. For instance, agar enables the media to melt at 100ºC and then cool back to less than 40ºC before re-solidifying. Additionally, very few organisms are capable of degrading nutrient agar.

Solid media are instrumental in isolation of pure cultures and determination of the number of viable bacterial populations.

Classification of culture media depends on:

  1. Their chemical composition, and
  2. The intended use of the media.

The classes of culture media include:

  • Chemically defined (synthetic) media: – have well defined chemical components and well known proportions
  • Complex (undefined) media: – their exact chemical compositions are not known
  • Selective media: – contains components that will inhibit the growth of certain bacteria spp. but promote the growth of the desired species
  • Differential media: – allows the investigator to distinguish between different types of bacteria based on some observable patterns of growth in the medium. For instance, you can eliminate Staphylococcus aureus  from a culture by increasing the salt concentration in the media
  • Enrichment media: – contains some components that permit the growth of specific bacteria spp. This is usually because only such species can utilize the nutrients from the media


Procedure for the preparation of the liquid media

To begin with, you will need a functional autoclave. You will use it to sterilize the media at high temperatures under pressure. After plasing the media to be sterilized in the autoclave, tightly the equipment then heat it to a pressure of 15psi and temperature of 121ºC for 15 minutes. Read more about the operating pressures of an autoclave here.

You will also need the following:

  • Distilled water,
  • Clean 500 ml measuring cylinder,
  • Clean conical flask,
  • Electronic weighing scale,
  • Sterile media, and
  • Clean test tubes.

8-step-process for making culture media

  1. Weigh 6.5 grams of the sterile nutrient broth and transfer into the clean conical flask. The manufacturer recommends a dilution of 13 g/l but we need to make only 500 ml of the media.
  2. Add 500 ml of distilled water into the measuring cylinder and transfer into the conical flask to dilute the media.
  3. Put the conical flask with the media solution from step 2 into an autoclave basket. Ensure you properly secure the mouth of the flask with cotton wool before lowering it into the autoclave. Secure the autoclave and start the sterilization. It will take some time to attain sterilization temperatures. However, once you achieve the recommended temperature/pressure combinations, hold it there for the recommended 15 minutes. That time is adequate under these conditions to sterilize the media.
  4. Allow the autoclave to cool down.
  5. Put the petri dishes into a hot air oven at 80ºC for one hour to sterilize them.
  6. Remove the conical flask containing the now sterile media from the autoclave. Pour 15 ml into each petri dish and seal.
  7. Make sure your working bench is not only clean but also always sterile. Wipe the surfaces with 7% alcohol and keep the UV lamp on.
  8. Label the petri dishes and store in a refrigerator for later use.
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Growth Inhibitors: The Effects Of Adulterants On Milk Curdling

Adulteration of milk, as we have discussed, takes place through many ways, some of which can be intentional while others are non-intended. Growth inhibitors are very critical when it comes to milk contamination since they will impede the growth of culture bacteria needed for fermentation.

Common adulterants include water added to increase volume (baptizing the milk), preservatives added to improve the keeping quality of the milk (such as hydrogen peroxide, antibiotics, and sodium hydroxide).

Sometimes, detergents may accidentally find their way into the milk and being bacteriostatic; they will inhibit bacterial activity in the milk and increase the keeping quality of the milk.

Antibiotics are used to treat mastitis and other common bacterial infections in lactating cows and the residue may find its way into the milk. They have a longer lasting effect on the milk.

Antibiotics will kill the lactic acid bacteria (LABs) in milk; therefore, milk fails to curdle when you inoculate it with a starter culture.

An experiment to investigate the effect of inhibitory substances on milk curdling was done and the findings ave been shared below.

The experiment

For this experiment, we partitioned five liters of heat-treated milk into five beakers and subjected to acidity and pH tests. We recorded the results we obtained from the tests.

After partitioning of the samples, we added adulterants including sodium hydroxide, antibiotic, detergent, and water into the beakers containing the milk samples.

We labelled the samples as A, B, C, D, and E in the order of the adulterants we added. We did not adulterate sample B with any substance; it was the control sample for the experiment.

Immediately after adding the adulterants, we determined the acidity and recorded the observations.

We then incubated the beakers containing the samples at 37ºC and measured the pH of the samples after every 15 minutes. We recorded all the observations.

The results

SamplesABCDE
Titratable Acidity (%)0.180.190.20.170.17
pH (on addition)6.46.376.333.216.51
pH (after 15 minutes)6.276.226.126.066.34
pH (after 30 minutes)6.436.356.356.176.76
pH (after 45 minutes)6.466.376.336.296.53

Discussion of the results

The acidity of the samples ranged fairly the same, indicating developing lactic acid in the sample. Addition of the adulterants seemed to have an effect on the acid development of the samples as the acidity of the adulterated sample seemed to stagnate showing no developed acidity.

The inhibitors affected the bacterial activity in the samples, impeding production of lactic acid in the milk samples during incubation. Some samples (B and E) did not show the downward trend in acid development observed in other samples.

The samples were left overnight and observed the following day. Only the control sample and samples B and E coagulated. Sample B formed a firm curd while E had curd with separated whey.

The conclusion

Sample B formed a firm curd because we did not add any adulterants into it. Its curd did not show any signs of syneresis.

We adulterated sample E with water, which explains whey separation.

All the other samples contained inhibitory substances that inhibited bacterial activity in the milk and prevented acid development in the samples. As a result, we did not observe any curd formation in these samples on the following day.

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