The most widely used enriched medium for the growth of Fastidious organisms (those microbes that requires some extra nutrients and only grow in enriched medium and not on an ordinary medium are known as fastidious microbes) is one n only our Blood Agar Medium (BAM).
The Blood Agar Medium is the simplest yet powerful enriched medium that supports the growth of almost all the microbes and extremely easy to prepare in the Laboratory.
In this Article, I’m gonna explain the preparation of Blood agar medium from the Trypticase soy agar Base…..
PRINCIPLE OF BLOOD AGAR MEDIUM
Bacteria are commonly cultured on a Blood agar medium to obtain the discrete colonies of the fastidious as well as non – Fastidious bacteria present in the specimen or to get the information about cultural characteristics of bacteria on a solid medium, colony morphology and patterns of growth and especially the Hemolytic property of the bacterium etc. This Solid enriched medium, used in bacteriology laboratory constitutes the 7 essential components which are described below –
⇒ Pancreatic digest of casein – The Pancreatic Digest of Casein is a rich source of Nutrients, Vitamins, and Nitrogenous substances that support the rapid growth of the bacterial cell.
⇒ Papaic Digest of Soybean – Similar to Pancreatic Digest of Casein, the Papaic Digest of Soybean is the Rich source of protein or peptides and nutrients that support the rapid growth of the bacterial cell.
⇒ Dextrose – The Dextrose present in the Trypticase Soy Agar (TSA) Medium provides a rich source of carbohydrate for the rapid growth of the Bacterial Cell.
⇒ Dipotassium hydrogen phosphate – It acts as the buffer in the medium that helps to maintain the stable pH of the Trypticase Soy Agar (TSA) medium.
⇒ Sodium Chloride – It maintains the osmotic pressure in the agar medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.
⇒ Blood – Blood is the key component of BA medium which makes the medium more nutritious by providing additional nutrients and growth factors required by fastidious organisms for the optimum growth. Moreover, it also helps in visualizing the hemolytic reactions (if any). Usually, 5% Blood Agar medium is used in laboratories for routine culture.
⇒ Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-Anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling at 40-45 °C.
The final pH of the solutions is adjusted to 7.2 – 7.6 preferably the 7.4 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the results required so that the rapid and satisfactory growth of the bacterial cell takes place.
The above-mentioned components can be modified by adding the various substances in a variety of ways as per the requirements of the bacterium so that the rapid and satisfactory growth of the bacterial cell takes place.
REQUIREMENTS FOR PREPARING BLOOD AGAR MEDIUM (BAM)
- Sterile Conical Flask / Erlenmeyer Flask
- Pancreatic digest of casein
- Papaic digest of soybean meal
- Dextrose (Glucose)
- Dipotassium hydrogen phosphate
- Sodium Chloride
- Sheep’s Blood
- Measuring Cylinder
- 1N HCl
- 1N NaOH
- pH Strip
- Weighing Scale
- Distilled Water
- Butter Paper
PROCEDURE FOR THE PREPARATION OF BLOOD AGAR MEDIUM USING TRYPTICASE SOY AGAR BASE
⇒ Weigh the quantity of Peptone, Beef Extract, and Sodium Chloride using the weighing scale for 1000 ml of Trypticase Soy Agar medium as follows:
COMPONENTS QUANTITY (in grams)
Pancreatic digest of casein 17.0
Papaic digest of soybean meal 3.0
Dextrose (Glucose) 2.5
Dipotassium hydrogen phosphate 2.5
Sodium Chloride 5.0
Put the butter paper on the weighing scale and transfer the required quantity of Pancreatic digest of casein on to the paper using the spatula. Repeat the step to obtain the required quantity of Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, sodium chloride, and Agar-Agar powder.
Note: Use the separate piece of butter paper to avoid the errors in measurements.
Alternatively, You can use the commercially available Trypticase Soy Agar (TSA) media powders and simply weigh the mixture of content as prescribed on the carton or box of the manufacturer. As per HiMedia, its 45 grams for 1000 ml medium.
⇒ Take a clean and dry Conical Flask/ Erlenmeyer flask.
Note: The size of the flask should be at least 1.5 times larger than the quantity of media you are preparing, for e.g. use 1500 or 2000 ml flask to prepare 1000ml of the solid medium.
⇒ Pour 500 ml of distilled water to the flask and add the weighed quantity of Pancreatic digest of casein, Papaic digest of soybean meal, dextrose, Dipotassium Hydrogen Phosphate, and sodium chloride.
⇒ Now add the weighed quantity of Agar-Agar to the above solution.
⇒ Mix well the content and Heat it with continuous agitation to dissolve the constituents.
⇒ Now add more distilled water to the medium and make the volume 950 ml.
NOTE: At this point, you have to make the volume 950 ml not 1000ml as later 50 ml of blood will be added later which will make the total volume 1000 ml.
⇒ Check the pH of the solution using pH strip, it should be 7.4 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
⇒ Mix well the content and apply the Non-absorbent cotton plug to the flask.
⇒ Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
⇒ Allow the content to cool down to 45 – 50 °C and to this carefully add 50 ml of sterile defibrinated blood aseptically and mix well gently.
NOTE: Avoid formation of air bubbles in the medium while adding blood. Also, before adding the blood to the agar medium, warm it to the room temperature.
Now, Pour about 15 ml of this medium in the sterile Petri plates under the strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature. Store the prepared media plates in the refrigerator at 4 °C.
⇒ Use the prepared media plates to inoculate the specimen to be cultured and then place it in the incubator at optimum temperature.
GROWTH OF VARIOUS BACTERIA ON BLOOD AGAR MEDIUM
USES OF BLOOD AGAR MEDIUM
The Blood Agar medium is commonly used for the isolation and cultivation of Streptococcus species, Neisseria species, and other fastidious microorganisms.
The BA medium is also used to differentiate bacteria on the basis of their hemolytic property i.e. β-hemolysis, α-hemolysis or γ-hemolysis (a.k.a. non-hemolytic).