Microbial Culture Media Composition Part-3

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
pH - 7.2

Role

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

Role

Blood agar is used for isolation of cultivation and isolation of pathogenic bacteria.It is used for differentiation of Haemolytic and non-haemolytic micro-organism.

Note

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 blood the plates are poured.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 micro-             organism.
c) Blood should be free from haemolysis and use of human blood should be avoided as it can inhibit 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 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
pH - 7.2
24. Potato dextrose agar
Potato ( Infusion from ) - 20 gm
Dextrose  – 2 gm
Agar-agar - 3 gm
Distill water - 100 ml
pH - 5.6
25. Tetrathionate broth
a) Solution A
Sodium thiosulphate - 50 gm
Distill water till -100 ml
Steam sterilize for about 30 minutes.
b) Solution B
Iodine -5 gm
Potassium Iodide -4 gm
Distill 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 sterile test tube 10 ml each under aseptic condition.This test tube should be stored at low temperature till use. This tubes should be used within 24 hours.

Role

a) It is a enriched medium used specially 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 coli-forms.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 energy.

26. Thioglycollate broth
Peptone -2 gm
NaCl -0.5 gm
Glucose -0.5 gm
Sodium thioglycollate -0.05
Methylene blue -0.002 gm
Agar-agar -0.05 gm
Distill water up to 100 ml
pH -7.2
Paraffin oil.

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 .

Role

a) Thioglycollate broth is used for cultivation of anaerobic bacteria.
b) Thioglycollic acid is a reducing agent 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  reducing agent.
e) Paraffin oil is layer over medium to maintain anaerobic condition.
f) Methylene blue is a indicator dye it get decolourised under reduce or anaerobic condition.

27. Tributyrene agar
Peptone -0.5 gm
Yeast extract -0.3 gm
Tributyrene -1 gm
Calcium carbonate -1 gm
Nile blue sulphate -0.001 gm
Agar- agar -2 gm
Distill water -100 ml
pH -7.4

Note – All ingredients except calcium carbonate and Nile blue should be added in distill 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.

Role

a) Tributyrene agar is used for identification of lipolytic activity that is production of lipase enzyme .

28.Rose Bengal agar
Peptone -0.5 gm
Dextrose -1 gm
Magnesium sulphate -0.05 gm
K2HPO4 -0.1 gm
Rose Bengal -3.5 mg
Streptomycin sulphate -0.01 gm
Agar-agar -3 gm
Distill water up to 100 ml
pH -5.4

Note – Mix all ingredients except Streptomycin and rose Bengal .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.

Role

a) Peptone is a source of carbon and energy.
b) Rose Bengal is a indicator dye.
d) Streptomycin is a antibiotic.

29.Selenite broth
Peptone -0.5 gm
Lactose -0.4 gm
Sodium acid selenite -0.4 gm
Na2HPO4 -0.95 gm
NaH2PO4 -0.05 gm
Cystine -0.1 gm
Distill water -100 ml
pH -7

Note- Selenite broth flask should be plugged and sterilized in 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 do not affect the growing culture.This medium should not be over heated.

Role

a) It is a 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 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 reducing agent because selenite broth works optimum under anaerobic                 condition so it is important to use cystine 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

     

     

     

     

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