Microbial Culture Media Composition Part-3
Here are some of the important culture media composition.
21. Eosin Methylene blue broth
Peptone,- 1 gm
Lactose,- 0.5 gm
Eosin Y,- 0.04 gm
K2HPO4,- 0.2 gm
Methylene blue,-0.0065 gm
Distill water,- 100 ml
a) Eosin Methylene blue broth is used for isolation of coliforms.
b) Eosin and methylene blue are aniline dye used to inhibit Gram positive and fastidious gram negative bacteria.
22. Blood agar
Whole blood,- 10 ml
Nutrient agar,- 100 ml
Blood agar is used for isolation of cultivation and isolation of pathogenic bacteria. It is used for the differentiation of Haemolytic and non-haemolytic micro-organism.
a) Nutrient agar is prepared and sterilized after sterilization the whole blood is added to melted and cool agar. The blood agar flask is rotated to mix the blood immediately after mixing the blood the plates are poured. The important point to be noted is the nutrient media should be cooled till 50º C to 55 º C before adding blood in it.
b) Blood is a complex medium which contains unknown ingredients and as it is a rich source of vitamins, minerals, growth factor and nutrients it is mainly used for cultivation and isolation of pathogenic microorganism.
c) Blood should be free from haemolysis and use of human blood should be avoided as it can inhibit the growth of some micro-organism as it contains some inhibitory factors.
d) Chocolate agar is made by heating heating blood agar up to 70º C till it becomes brown in colour. Chocolate media is used for the isolation of Neisseria meningitidis and Streptococcus pneumoniae.
23. Glucose yeast extract agar
Yeast extract,- 0.5 gm
Peptone,- 1 gm
Glucose, – 1 gm
Agar- agar, – 3 gm
Distill water,- 100 ml
24. Potato dextrose agar
Potato ( Infusion from ),- 20 gm
Dextrose, – 2 gm
Agar-agar,- 3 gm
Distill water,- 100 ml
25. Tetrathionate broth
a) Solution A
Sodium thiosulphate,- 50 gm
Distil water till,-100 ml
Steam sterilize for about 30 minutes.
b) Solution B
Potassium Iodide,-4 gm
Distil water,- 20 ml
Iodine should be dissolved completely in water with the help of potassium iodide.
c) Complete medium
Sterile nutrient broth,- 900 ml
Sterile Calcium carbonate,- 50 gm
Solution A,- 100 ml
Solution B,- 20 ml
Note – All ingredients should be mixed properly and distributed into the sterile test tube 10 ml each under aseptic condition. This test tube should be stored at the low temperature until use. This tubes should be used within 24 hours.
a) It is an enriched medium used especially for isolation and cultivation of Salmonella species.
b)The Salmonella species is isolated from urine, faeces and sewage.
c) As this is a normal habitat for E.coli also it is important to inhibit E.coli so that we can get isolated Salmonella species and for this, the media contains thiosulphate and iodine.
d)This thiosulphate and Iodine carry out oxidation to form tetrathionate and this tetrathionate inhibits coliforms. This tetrathionate becomes a source of energy as it is utilized by Salmonella species where as Coliforms are unable to utilize tetrathionate as a source of energy.
26. Thioglycollate broth
Methylene blue,-0.002 gm
Distill water up to 100 ml
Note – Dissolve all ingredients by heating and then adjust the pH to 7.2 and distribute the liquid medium in 10 ml test tube overlay paraffin oil. Plug the tubes and sterilize the tubes by autoclaving.
a) Thioglycollate broth is used for cultivation of anaerobic bacteria.
b) Thioglycollic acid is a reducing agent since it reduces oxygen from the medium.
c)Agar is used in 0.05 % this agar makes the medium viscous and prevents aeration.
d) Glucose is a source of energy it also acts as a reducing agent.
e) Paraffin oil is the layer over medium to maintain anaerobic condition.
f) Methylene blue is an indicator dye it gets decolourised under reduce or anaerobic condition.
27. Tributyrin agar
Yeast extract,-0.3 gm
Calcium carbonate,-1 gm
Nile blue sulphate,-0.001 gm
Agar- agar,-2 gm
Distil water,-100 ml
Note – All ingredients except calcium carbonate and Nile blue should be added in distilled water, dissolved by heating and the pH should be adjusted to 7.2. After dissolving all ingredients medium should be sterilized. Calcium carbonate and Nile blue should be sterilized separately and after sterilization, both components should be added in medium and mixed vigorously and further plating should be done.
a) Tributyrin agar is used for identification of lipolytic activity that is the production of lipase enzyme.
28.Rose Bengal agar
Magnesium sulphate,-0.05 gm
Rose Bengal,-3.5 mg
Streptomycin sulphate,-0.01 gm
Distil water up to 100 ml
Note – Mix all ingredients except Streptomycin and rose Benga. After mixing sterilize the medium cool it up to 45º C to 50º C and then add Streptomycin and rose Bengal. Mix properly and pour the medium.
a) Peptone is a source of carbon and energy.
b) Rose Bengal is an indicator dye.
d) Streptomycin is an antibiotic.
Sodium acid selenite,-0.4 gm
Distill water,-100 ml
Note- Selenite broth flask should be plugged and sterilized in the water bath by steaming at 100º C for about 30 minutes. After sterilization, a red colour precipitate may appear but it doesn’t interfere in medium and does not affect the growing culture. This medium should not be overheated.
a) It is an enrichment medium used for isolation and cultivation of Salmonella.
b) Sodium phosphate detoxifies Sodium acid selenite and permits rapid growth of enteric pathogen like Salmonella. At neutral pH, sodium phosphate becomes toxic for E.coli and not for Salmonella. As pH of the medium increases its toxicity decreases and it can allow growth of other coliforms so to avoid this here we use buffers to maintain pH at neutral side.
c) Cystine is a reducing agent. Here we use the reducing agent because selenite broth works optimum under the anaerobic condition so it is important to use cysteine as a reducing agent.
30. Simmon’s citrate broth
- Sodium citrate,-0.2 gm
- Magnesium sulphate,-0.02 gm
- Sodium chloride,-0.5 gm
- Ammonium dihydrogen phosphate,-0.1 gm
- Di-potassium phosphate,-0.1 gm
- Bromothymol blue,-0.008 gm
- Agar-agar,-3 gm
- Distill water,-100 ml
- pH,- 7