Culture media preparation – Best ways to avoid contamination

Introduction 

Culture media is the substance that allows the bacteria to grow and increase in number to form a patch called a colony. It is a fundamental need to identify the bacterial specie. So Culture media preparation is an essential part of growing the bacteria.

There are a lot of bacterial species in the world and. And many species have been discovered yet. This is done by growing bacteria in the laboratory using culture media. the technique used to grow on culture media is called culturing technique

Therefore it is necessary to identify the basic nutrients required for a bacteria to grow in it.

Let’s discuss some aspects essential for media culture preparation

Nutrients 

A microbiological culture medium must have major elements which are required for bacteria to grow. It includes hydrogen donors, carbon, nitrogen, phosphorous, sulfur, and minor elements like metal and Vitamins. 

The beef extract can replace all the nutrients as it can major source of nutrients in the form of meet infusions 

Peptone is also a good source of nutrition for bacteria. As it contains essential amino acids and nitrogenous bases required for bacteria to grow. That is why these are the primary culture media to grow any bacteria 

Some bacteria require additional nutrients to grow which include serum, blood, and vitamins and metals to grow better 

In this regard, Bergey’s manual is the reference book for identification.

Oxygen 

Most bacterial species depend upon oxygen for electron acceptor. Some need oxygen and some can grow better in absence of oxygen.

That is why it is necessary to maintain the presence or absence of oxygen while growing bacteria, for example, clostridium species can grow in the absence of oxygen.

For this purpose, there are many ways to ensure the absence of oxygen by following the ways 

  • The addition of a reducing substance in the culture medium for sodium thioglycolate reduces the oxygen in the medium
  • Replacement of oxygen with carbon dioxide by adding germinating seed 
  • Removal of oxygen by oxidation like burning of candles in a Jar 
  • Inoculation of bacteria inside the culture media where oxygen cannot enter 

Moisture 

Moisture is also a source some bacteria need to grow for example bacteria cannot grow in open culture media due to the dryness of the medium. on the other hand, if we add wet cotton, bacteria grow better 

Temperature and pH 

The most important thing for growing bacteria is the pH and temperature. As hydrogen concentration is necessary for bacterial growth. most bacteria require neutral pH and 37 C for their optimum growth. Few are those bacteria that live in harsh and extreme conditions 

Medium support 

To count the number of colonies, the medium must be solid. And this is done by adding gelatin, agar, or albumin. The best of them is agar as it is the best support medium with zero nutritive value 

Indicators 

To identify and differentiate the bacteria, it is necessary to visualize the colony by making difference in color, therefore, some indicators make bacteria differ from others. It is either based upon pH or any nutrients .phenol red is the best example of a pH indicator as it changes its color when pH is low. And Iron sulfate is an example chemical that differentiates salmonella from Shigella.

Types of culture media 

There are a lot of varieties in culture media and all are prepared according to the Bacteria we want to isolate and identify.

Based on the chemical composition

Based on chemical composition there are three types of culture media we use 

  • Natural media
  • Semi-Synthetic media 
  • Synthetic media 

Natural medium 

Type of culture medium whose chemical composition is unknown. The basic nutrition resources are provided by a complex mixture of amino acids nitrogenous bases and other minerals for example peptone, beef extract, yeast extract, milk, juices, etc 

Semi-synthetic medium 

Those mediums whose chemical composition is partially known and remaining are unknown. In semi-synthetic media, some known ingredients add to natural media for example in potato dextrose agar, dextrose will be added in potato mesh to make potato dextrose agar.

Synthetic medium 

A medium whose chemical ingredients are known is called a synthetic medium. The media used in determining physiology, metabolic activity, and nutritional requirement, For example, Richard medium, and Mineral glucose medium.

Based on media consistency

Liquid medium 

A medium in which agar is not added and remains in a liquid solution is called broth. The bacterial growth will be seen on the top or bottom in form of suspension. For example nutrient broth, and tryptic soya broth.

Semi-solid 

Medium in which less quantity of agar is added to form a semi-solid medium used in determining the differentiation between non-motile and motile bacteria.

Solid medium 

A medium having agar in the media solution forms a gelly-like consistent media that will support the bacteria or fungi to grow in a particular space. for example nutrient agar, and Tryptic Soya agar or broth.

Based on function and differentiation 

Selective media

The medium that can such ingredients allows specific microbes to grow. It is used in the isolation and identification of the desired microbe. For example, Bile Salt agar helps those bacteria to grow that can grow only in the intestine that is why BSA is used to identify Intestinal flora  

Enriched media

The medium, that has additional nutritional supplements like serum, blood, albumin, milk, etc for example Staphylococcus and  bacillus anthracis grow on Blood agar 

Differential media

The medium has special ingredients to differentiate between two bacteria according to their physiology and metabolic activity for example On MacConkey agar, a lactose fermenter produces pink colonies and nonlactose fermenter produces white colonies. And salmonella change media color due to the presence of phenol red (pH indicator)

Transport media 

There are some bacteria that either cannot survive or become contaminated while transported. To solve this solution scientists make a special media use to transport pathogens from one place to another for example Stuart’s transport medium 

Indicator media 

The media tends to change its color due to the presence of indicator present in it. Bacteria cannot utilize it but its metabolic activity changes the color due to an increase or decrease in pH or the formation of color-producing chemical, For example, MacConkey agar has phenol red and salmonella shigella agar produce black colonies for salmonella typhi due to the formation of iron sulfate.

Enrichment media 

The media that contain particular ingredients that can enhance or inhibit their competitors 

For example tetrathionate broth.

How to prepare Culture Media?

Before starting you must make your environment clean on two factors

  • Which type of bacteria do you want to grow 
  • Available resources in your laboratory.

Your instruments should be working properly which include weighing balance, beaker, measuring cylinder magnetic stirrer, spatula, hot air oven, Petri plates, flasks, autoclave 

After making media suspension 

After choosing the media according to your need, each media have a specific recipe to prepare.

In general, all ingredients mentioned in commercially available media will be added to a beaker, and add distilled water up to one liter 

You can customize the volume according to your need through calculations

Ways to avoid contamination 

Some steps are very critical while media preparation. If you ignore them there will be a high risk of contamination in the media. 

Sterilization

After preparing the solution in a flask, properly close the neck with cotton or foil paper and sterilize in the autoclave at 121 degrees Celsius with 15psi pressure for 15 minutes.

While Petri plates should be sterile in a hot air oven at 180 degrees celsius for 1 hour

Pouring of media in sterile Petri plates 

While pouring the temperature should be around 45 degrees Celsius, not much cooler to solidify in a flask and not much hotter to handle the flask. There are 2 cases while pouring media 

  • In the first case, if you are working in a cabinet then it should be working properly, its airflow system should properly work, and the area in the cabinet should be clean and sterile with ultraviolet radiation
  • In the 2nd case, if you are preparing without a cabinet, you should do work near 6 inches around the burner because 6 inches around the flame is a sterile area. Petri plates should be open 6 inches while pouring media.

Solidification of media and incubation 

Let the media solidify in Petri plates and incubate the plates for 24 hours at 37 degrees Celcius to check proper contamination before using.

Storage and preservation

After preparation, media should be stored or preserved for future use. Most books recommend 2 weeks for storage at 4 degrees Celsius. Although it depends upon the type of media you are preparing.

Conclusion

Culture media is the basic source and has an important role in microbiology. It is very difficult to identify microbes without them. It provides nutrients to microbes to grow as a colony. We can easily identify the bacteria with colony characterizations.

Resources

  • Classification-of-culture-media. (2021, March 22). BYJUS.  
  • Singh, A. (2022, March 27). How to Prepare Culture Media and Preserve Cultures. Conduct Science.
Mubashir Iqbal
Mubashir Iqbal

Mubashir Iqbal is a highly dedicated and motivated Microbiologist with an MPhil in Microbiology from the University of Veterinary and Animal Sciences. Currently, he is researching the efficacy of commercially available SARS Cov-2 vaccines to neutralize the omicron variant in Pakistan. He holds a Bachelor's degree in Microbiology and has experience in chemical and microbiological analysis of water samples, managing SOPs and documents according to standard ISO 17025. Additionally, he has worked as an internee in BSL 3, Institute of Microbiology, UVAS, where he gained experience in RNA extraction, sample processing, and microscopy.

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