dilutions

8 posts / 0 new
Last post
lucilius
lucilius's picture
dilutions

Hallo all,
what dilutant do you use and why?
I was wondering if anyone here has experience in the field of dilutants, more specific towards which ones to use and which ones not?
Are there any scientific papers on? I know that saline water is a common dilutant, but is there a paper stating that its a good dilutant?

g a
g a's picture
Dear lucilius

Dear lucilius
Most common diluants are 0.9% saline, phosphate buffered saline KRBG, HBSS and other balanced salt solutions.
Now the most important and common features of all these is that they all are isotonic to the mammalian ( infact avian as well as reptilia systems...... above class amphibia). These all have physiological concentration of 150mM NaCl making them optimal physiological diluant.
I don't ko whether there is a paper on saline being recommended as the best diluant or not but Im sure you will find n number of papers that will show the dosage injection or dilutions being made with saline or PBS or other similiar solutions.
It will be more relevant to know what kind of diluant you require for a typical experiment as some diluants like above mentioned do not work as good for giving steroids and other lipid labile molecules like cholesterol etc.
So let us know what kind of system you are working on and we will be able to help you alot better.
Cheers
 

lucilius
lucilius's picture
Argerine, thanks for the

Argerine, thanks for the quick reply.
I might have been a bit unclear with my question.

I ment specifically diluants for micro-organisms, bacteria and fungi.
I am not working with a specific organism at the moment, I was just wondering why they often use saline water as a diluant.
I have searched on the net to find some information, but its hard to get an answer on why they use a certain diluant. It seems that people know its good or that its used a lot, but no one seems to know why.
I Have also found a paper that speaks of the fact that saline water is very bad to use, however this is an old paper and maybe not up to date anymore. Too bad it is the only paper I found on the topic. (see link:http://aem.asm.org/cgi/reprint/5/1/21.pdf)

g a
g a's picture
Dear lucilius

Dear lucilius
I can assure you the only reason for using saline is physiological balanced concentration with higher vertebrate bodyfluids as i have mentioned in my revious reply . however there are other reasons for use as well  such as ease of preparation and cost effectiveness.
In your case you require a diluant for microorganisms bacteria and  fungi. for that matter your diluant must contain a protein source such as peptone and preferably glucose as well.
I have been looking for the answer for your problem and I found a refernce http://cat.inist.fr/?aModele=afficheN&cpsidt=3482749 .
however the complete paper is not accessible in University of Delhi but I think abstract alone will give you alot of hints that will take you forward.
and here is another reference
http://www.springerlink.com/content/j2524736401nm153/ for the same. I hope you will be able to get acess to both these articles.
All the best

lucilius
lucilius's picture
Dear ARGERINE,

Dear ARGERINE,
thanks again for the quick reply.
I'll check the links you gave and maybe they are helpfull.

In general I find it still very strange that saline is a commonly used diluent, knowing its not that good.

g a
g a's picture
Dear Lucilius

Dear Lucilius
It is indeed strange that many laboratories use the common diluents that are not as effective as expected. However most of the time its because of the lack of habit of asking questions...... In general there are very few of us who will ask a question why?????
and instead will follow the easier route using the standadized practice followed in labs.... mostly by senior students who indeed followed their seniors until one day one of us comes up and asks why???? And thats when the reality is sought and truth unveils that what is sought is ot the best and perhaps have deleterious effects.
Im glad young researchers like you ask questions to yourself...... and Im sure there will be light soon
Its curious people like you who take science forward..
Keep questioning and then try and find answers............. In my opinion human brain has a potential answer to almost everything and if you let it work spontaneously you will always have the answers.
 
cheers n joy
 

lucilius
lucilius's picture
Hallo Argine,

Hallo Argine,

thats true what you say.
I think that too much people just do what they are told to or just do something because someone else did it and don't even wonder why.
I had to do some protocol and I had to use salinewater , but wondered why and I asked and they simply said: because we always do it like this and others do it like this.
Then I spoke about a reference I saw about the bad influence of saline on certain cells and they didnt know what to say next.
But finding an answer on the most "trivial" questions (most of those questions are so simpel or seem to trivial that people do not even think about it) is very hard I found out.

Arvind Singh Pundir
Arvind Singh Pundir's picture
Diluent is  used for

Diluent is  used for maintaining the viability of organisms during dilution procedures, for example microbiological examination of foodstuffs require sample dilution to be carried out accurately to estimate the number of microorganisms and diluent should afford protection for bacteria during the dilution stage which normal saline fails to do, therefore 0.1% peptone with  phsiologiacal saline (normal saline) is considered good diluent by international standards organization. some of the examples of diluents are saline water (0.85% NaCl), sodium phosphate buffer (0.1 M, pH 7.0), 0.1% peptone, 0.1% yeast extract, 0.1% peptone in 0.1 M sodium phosphate buffer, pH 7.0, and 0.1% malt extract etc.
 
 refer these papers
Effect of diluent type on viability of yeasts enumerated from foods or pure culture
M. A. Miana, G. H. Fleeta and Ailsa D. Hockingb 

Department of Food Science and Technology, University of New South Wales, Kensington, NSW 2052, Australia
CSIRO Division of Food Science and Technology, North Ryde, NSW 2113, Australia

Abstract
The effects of seven diluent types on the viability of yeasts enumerated from foods and in pure culture were studied. The diluents were laboratory glass distilled water; saline water (0.85% NaCl), sodium phosphate buffer (0.1 M, pH 7.0), 0.1% peptone, 0.1% yeast extract, 0.1% peptone in 0.1 M sodium phosphate buffer, pH 7.0, and 0.1% malt extract. For all foods studied, dilution in 0.1% peptone gave the highest counts, with saline and phosphate buffer diluents giving lower counts than those obtained with distilled water. When seven species of yeast were enumerated in pure culture, highest counts were obtained using 0.1% peptone as the diluent and, with three exceptions, all species gave higher counts when diluted in diluents other than distilled water. When yeast suspensions were held in diluents for up to 2 h before plating, cell death occurred. The extent of death was highest in distilled water, saline and phosphate buffer diluents. Cell death also occurred in 0.1% peptone, yeast extract and malt extract, but to a lesser degree
*International Journal of Food Microbiology
Volume 35, Issue 2, 1 April 1997, Pages 103-107

 
Comparison of media and diluents for enumeration of aerobic bacteria from Bermuda grass golf course putting greens
 
M. L. Elliott and E. A. Des Jardin

Fort Lauderdale Research and Education Center, University of Florida, 3205 College Avenue, Fort Lauderdale, FL 33314, USA

Abstract
To increase our knowledge of soil and rhizosphere bacteria associated with bermuda grass grown on golf courses, a standardized procedure has been developed. Seven aerobic bacterial groups were selected for enumeration. For each group selected, appropriate media were compared to determine which one was best for enumeration of that group. Six diluents were evaluated across all media for the seven bacterial groups. The best diluent was sodium pyrophosphate with glycerol. The following media were best for enumeration: solidified 10% tryptic soy broth for total culturable aerobic bacteria and heat tolerant bacteria; reduced arginine soluble salts medium for actinomycetes; S1 medium for fluorescent pseudomonads; XMSM for Stenotrophomonas maltophilia; Bacto azide blood agar base for gram-positive bacteria; crystal violet agar for gram-negative bacteria.
Journal of Microbiological Methods
Volume 34, Issue 3, 1 January 1999, Pages 193-202