10 replies to this topic

[Dima]

Dear members

 

I am in need for a help pls having being not a microbiologist rather a nutritionist!

 

I am working on testing natural compounds on five different strains. I would like to prepare an inoculum isze of 106 cfu (10 to the 6), so what i did initially is i took 2 colonies and prepare an overnight culture broth. from this, I made serial dilutions and did the plate counting and simultaneously did the OD reading on the photometry. something however is always wrong with the reading or the colonies number when counting plates. the serial dilutions for the OD reading were from 101(10 to one) to 108 (10 to 8). i wanted to make a growth curve and find the dilution rate to obtain my inoculum size.

 

what did i do wrong?

 then i thought of Mcfarland. to use it for this.. but is it reliable? do I use it and compare it to an overnight culture.I know  that the culture should be during log phase, is 2o hours overnight culture reliable to be compared and diluted when compared to Mcfarland 0.5?

 

your help is greatly appreciated as i have been working on this since 2 days now!! am not a genius microbiologist and i am excited to learn

 

Thx in advance

 

 

Edit - I deleted your other thread in the general lab forum. Please only post your question to one forum. -Jerryshelly1

[Phil Geis]

What was the protocol you intended to use with these inocula - a challenge or AET? What was "wrong"with plate count/OD comparisons?

[Dima]

i am testing the antibacterial effect of natural compounds i.e. spices using agar diffusion and microdilution method.

 

I repeated the plating and OD readings two times. the serial dilution , i am sure, was done very well (1 in 9ml) from 10-1, 10-2 10-3 10-4 10-5 10-6 to 10-8.

an example of my results:

OD readings for Salmonella Typhi: 24 hours broth culture"undiluted" (1.182), 10-1 (0.156), 10-2 (0.06) 10-3 (-0.003), 10-4 (-0,.045) then continued as such.

Plate counts for Salm.typhi.( I did only plating for 10-5 till 10-8 ): 10-5 (Too numerous), 10-6( 8.5 log) , 10-7 (9.9log)!

 

 

OD reading for Staph Aureus: undiluted (1.363), 10-1(0.237), 10-2 (0.49), 10-3(0.001), 10-4(0.000), 10-5( 0.001), same for rest

Counts : 10-5(8.1 log), 10-6 (8.1 log), 10-7(8.6 log), 10-8 (no visual growth)

 

if i would not want to use the spectrophotometer to determine the concentration of my suspension (after validation with plate counting), is Mcfarland reliable? is it acceptable to adopt Mcfarland for the inoculum preparation after 24 hours incubation of broth culture?? or it should be a shorter incubation time? i cant find an answer on the time required to incubate the culture before diluting its turbidity in comparison to Macfarland. My colleague said to take a colony and deliver it into broth, then wait for few hours, once it started to be turbid that is the growth phase and i use this suspension. but this is almost half a day before starting working. there should be a practical way no?

[Dima]

and thank you for asking !!

[pito]

you did shake the samples before putting them in the spectrophotometer? (the samples itself + the small tube you put in the meter).

BTW: making dilutions (in my experience) is a huge problem for many people in the lab.. in some companies new people are "forced" to really train on this for 1-2 weeks because often they are not able to do it correctly.
 

Not sure about your protocol for the Macfarland ... normally you work with saline and in that saline you add colonies from an agar plate..

check this: http://www.microbeli...ty-test-protoco

[Dima]

thank you.. i will check the attachment..yes i did shake the content before measuring and for the dilution, i have been doing this for months now but somehow the results were not successful.

 

references say that we incubate a broth culture till log phase (2-6 hours) until it starts to be turbid and compared to Mcfarland standard. but when i see thesis of other colleagues, they incubated the culture for 18 hours, which is confusing for me. there is no standard method.

 

thx for your replies..will check now the attachment.

[pito]

a lot depends on the organisms of course.. so the standards are not Always the same.

 

But I Always knew McFarland as a saline solution, not in broth...

I also dont understand how you can compare a broth solution with a McFarland standard that is made in (normally?) saline.

 

 

Dont you have some standard for this kind of testing? there must be some sort of (inter)national standard to test components... can you not use this?

 

I have not a lot of experience with this specific topic, but there must be some standards.

 

Just a general note: you should determine the growth rate of your organisms by doing some growth curves/tests and then after doing this a few times you should know how your bacteria grow....

THis requires some tests, but in the end you should be able to repeat this pretty much each time in a very acurate way.

[Dima]

yes Mcfarland is not a broth, so i would dilute my bacterial suspension and adjust its turbidity to the turbidity of Mcfarland (0.5) which is equal to 0.8-0.15 OD.

 

I guess i will do another trial for plating and OD reading for a broth culture in growth phase (2-6 hours incubated just when it starts to show turbidity) in hopes it works this time.

 

As for a standard reference, i have been searching for 2 nights ! none... the standard method is to prepare an inoculum by taking one or two colony  and deliver it to broth ( 2-6 hours incubation), and as it starts to show turbidity, we adjust it to McFarland by diluting with saline.

 

there is no clear time for each bacteria and no clear results on the effect of longer incubation time to the validity of McFarland.

 

anyway, i should keep trying ..

[pito]

not sure I understand you, but you inoculate a broth and check this broth against the McFarland standard?

[Dima]

right...adjusting its turbidity to that of mcfarland 0.5

[Phil Geis]

If you develop your own OD standard correlated to cfu/ml, you don;t need a McFarland comparison.  As pito implied, data presented do not give confidence in execution of protocol.  OD and plate counts appeared to show greater density of cells with increased dilution.

Suggest you validate your execution.