protocol on growth of bacteria - (May/28/2014 )

hi
i am tring to find out if differnet concentration affect the growth of Staphylococcus aurues and Staphylococcus epidermidis bacteria. i can not find protocole for this experiment. can you advise me guide me on this experiment.

thank you

Assuming that you are talking about different concentrations of a treatment such as a drug, then there are a number of ways of doing this.  The classic way would be to use a number of plates, each plated with a different concentration of the drug, and then spread with the same amount ( in colony forming units) of the bacteria.

if differnet concentration  of salt affect the growth of Staphylococcus aurues and Staphylococcus epidermidis bacteria. i have little bit of idea what i may be doing. i will be making agar but not sure if i have to mix the salinty concentration in the agar before it turns in to  jelly. and i dont now how will i make differnt concentration of salt. do you have protocole for this type of experiment that i can follow step by step.

thanks for the help

The easiest way would be to make a standard medium for the different staphylococci, but leave out the salt (e.g. make 1 litre of Mannitol salt agar, but with no salt).  Divide this medium into a number of different bottles (maybe 200 ml/bottle) and add salt and powdered agar to the desired concentrations, then autoclave and pour.

hi

thanks for the help, appriciated. if you dont mind can i send you my method for this experiment to see if am doing write thing when i complete the method.

Just copy and paste your method here - that way people might learn something extra if they look at this topic

hi

will do once ive finished. i dont have a clue on how i will make different concentration of salt ? can you help me with that ?

Basically, if you know the concentrations you need, all you do is calculate the amount in g that you would add per volume of liquid.  Use c=n/v and n=mass/molar mass.

hi

ok. i was reserching and i came across a journal and it tested the growth of s. aureus bacteria in NaCl. They used Trypose Phosphate Broth. which one is better to use, Trypose Phosphate Broth or Mannitol salt agar ?

for mannitol salt agar -

1 liter of distilled water ,Beef Extract - 1.0 g,  D-Mannitol - 10.0 g, Pancreatic Digest of Casein - 5.0 g,  Phenol Red - 0.025 g, Peptic Digest of Animal Tissue - 5.0 g , Agar - 15.0 g and Sodium Chloride 75.0 g.

 i will mix all the materials in distilled water but i will leave out the salt  and mix well, and divide these solution in to differnt bottles. and make different concentration of dilution  using the fomula given, then pour that differnt concentration of salt in to the bottle and add powdered agar to all the bottles and then autocleve. let it cool down and pour it out in a petri dish and let it set untill it becomes like a jelly. then spread the bacteria using aseptic techniques. 

instead of adding agar towars the end can i not add it in the begining with all other materials.

please tell me if my method is right and where i am going wrong.

thank you

Mannitol salt agar is used in diagnostic labs, but I don't know whether Tryptose phosphate would work or not.  I think that it would be fine too, and may even be better for growth of S. epidermidis.  To make the broth form into an agar, just add 15 g of agar to each litre of broth.

Your method is very nearly correct, but instead of adding salt as a solution, I would add it as the crystalline form and then dissolve - it makes it easier to calculate how much volume to add to each bottle, and means that you aren't diluting the medium when you reach points where you need to add a lot of salt.  It seems that S. aureus can grow in up to about 20% salt, so if you have 200 ml of broth this would be 200*0.2 =40 g of salt into one bottle.  You could work out the range by working out how much salt is in the mannitol salt agar, and use that as your starting point, or you could have 0% salt as a starting point.

Adding agar is difficult - it won't dissolve without heat, so it will settle to the bottom of the vessel that you are using to make your medium while you are pouring.  This will mean that some bottles will have less agar than others, and might mean that they won't set.  It is best to add it to each bottle separately.