**Pages: 123Next**

Today was a very bad day at the university... beside that some students answered in the quiz question that if you get blue color in Gram staining that means your bacteria is Gram negative I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

__Dilution:__

is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

__Laws:__

**Dilution** = V of Sample / Total V of (sample + diluent)

**Dilution Factor** = Total V of (sample + diluent) / V of sample

** or we can simply say the

__reciprocal__

of Dilution

Starting off with this simple example to understand how these laws are applied.

It has been known that if we use a larger volume we obtain a more accurate dilution.

So for better results, we use

__1:1000__

dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we **cannot** use 999 ml of diluent. So we do what is called a serial

__dilution__

.

**Serial Dilution:** is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.

Here's an example combining all of the above.

I hope this brief explanation proves helpful to you...let me know if i've made any mistakes...

-Property of Yulia-

thank u for your explanation..

what makes me always confused is the last volume used for inoculation (0.1ml in your example)..

-strawberry-

Yeah that's why i almost jumped with 'HAVE' there :blush: sorry about that

i hope my mini-tutorial and examples helped you through it **strawberry**...

-Property of Yulia-

I'm very thankful to you to provide the easier explaination...it's true that many undergrad students have problem in dilution..and the worst thing is if the lecturers/tutors assume that the students are familiar with the concept. it is true that they have learned it in high school, but most of the time they will forget..

Regarding the example 2 given, you dilute a sample (10 ml the final volume) 10x10x10=10 to the power of 3. and u inoculate 0.1 ml in your medium.so supposed the final volume of your medium is 1ml right?

-kent19-

This post has been promoted to an article

-bioforum-

Dear Sir/Madam,

Please explain me the conversion of % to ppm. I am damn confused.

-BHARGAVI-

BHARGAVI on Mar 23 2009, 03:09 AM said:

Dear Sir/Madam,

Please explain me the conversion of % to ppm. I am damn confused.

ppm means part per million. there fore x ppm = x parts of something to a million parts of diluent. % is something like "parts per hundred" so if its a 10% SDS solution. Its 10 g per hundres parts diluent which is dH20.

-MaggieRoara-

Thanks for nice explanation!!!

-nirajm-

Nice stuff!

Perhaps you can help me with this:

For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.

So, let´s say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:

Transcript length: 75nucleotides

Concentration: 8 ng/ul = 8 x 10-9 g/ul

Calculation: (8 x 10-9 g/ul / <75x 340>) x 6.022 x 10e23 =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!

-Jka83-

Jka83 on Jul 21 2009, 12:14 PM said:

Nice stuff!

Perhaps you can help me with this:

For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.

So, let´s say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:

Transcript length: 75nucleotides

Concentration: 8 ng/ul = 8 x 10-9 g/ul

Calculation: (8 x 10-9 g/ul / <75x **340**>) x **6.022** x **10e23** =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!

Most likely a silly question, but where do you get the 340 , 6.022 and 10e23 from?

-lyok-

bioforum on Mar 21 2009, 11:33 AM said:

<p>Today was a very bad day at the university... beside that some students answered in the quiz question that if you get blue color in Gram staining that means your bacteria is Gram negative I noticed most of the students including myself had trouble with the dilution process. So I thought maybe I'd make a quick explanation for what is dilution and how it is done and all the ratios and other confusing stuff. I tried my best to simplify it for people who find trouble in doing their homeworks, labwork, or other calculation problems regarding dilution questions and solutions.

So let's start out with definitions:

__Dilution:__

is the mixing of a small accurately measured sample with a large volume of sterile water or normal saline called (diluents or dilution blank)

__Laws:__

**Dilution** = V of Sample / Total V of (sample + diluent)

**Dilution Factor** = Total V of (sample + diluent) / V of sample

** or we can simply say the

__reciprocal__

of Dilution

Starting off with this simple example to understand how these laws are applied.

It has been known that if we use a larger volume we obtain a more accurate dilution.

So for better results, we use

__1:1000__

dilution. And that is by adding 1ml of sample to 999 ml of diluent. But practically we **cannot** use 999 ml of diluent. So we do what is called a serial

__dilution__

.

**Serial Dilution:** is a dilution made of a series of smaller dilution, and the total dilution is the product of each dilution in the series.

To understand this more, let's see this example.

Here's an example combining all of the above.

I hope this brief explanation proves helpful to you...let me know if i've made any mistakes... </p> <div>-Property of Yulia-</div><hr>

<p>thank u for your explanation..

</p> <div>-kent19-</div>

This does contain mistakes. In the description for example 3, the author shows that 43 g of food = 43 ml. This is only true if the density of the food = 1g/ml. Since we are not given the density of the food nor told that it is equal to that of pure water at 4°C, the calculation cannot be performed as described without undue assumptions by the author.

-Dr Teeth-

lyok on Jul 21 2009, 10:30 PM said:

Jka83 on Jul 21 2009, 12:14 PM said:

Nice stuff!

Perhaps you can help me with this:

For real time pcr (detection of virus), one wants to measure the sensitivity of the assay by determining the minumun amount of viral copies per reaction that the assay is able to detect.

So, let´s say I have 8 ng/ul of target RNA in the sample (measured by nanospec). By using this formula:

Transcript length: 75nucleotides

Concentration: 8 ng/ul = 8 x 10-9 g/ul

Calculation: (8 x 10-9 g/ul / <75x **340**>) x **6.022** x **10e23** =about 1,8e11 molecules per ul

I dilute this by factor 10-9 and I get 188 molecules per ul.

My PCR mix is 25ul that includes 2ul of this RNA sample. My assay is able to detect this dilution so can I say that the detection limit is 376 copies PER REACTION (I put 2 microliters and 2X 188/ul=376) or is the limit this 376 divided by the reaction volume of 25??

Thank you for your help!!

Most likely a silly question, but where do you get the 340 , 6.022 and 10e23 from?

There are no silly questions.

The formula can be found from here:

http://www1.qiagen.com/resources/info/Guid...uantifying.aspx

"RNA standards"

-Jka83-

Ah I see, thanks for the link.

I do have a question: what do you mean with: I dilute this by factor 10-9 and I get 188 molecules per ul.

I do not understand your question.

In the end you simply have 376 molecules per 25µl and and you can detect this.

You can then say , I think , that you will detect 376 molecules per 25µl (=per reaction) or 376 molecules/25µl = X molecules per µl.

Is this not the same?

-lyok-

And for the lazy ones, like me

-Maddie-

**Pages: 123Next**

As a seasoned expert in microbiology and laboratory techniques, I bring forth my extensive knowledge to shed light on the intricacies of dilution processes discussed in the provided article. My experience spans not only theoretical understanding but also practical applications in laboratory settings.

The article begins by addressing challenges faced by students in understanding dilution processes, particularly in the context of microbiological experiments. The author emphasizes the importance of accurate dilution in achieving reliable results. The fundamental laws of dilution, expressed as mathematical equations, are introduced to establish a theoretical foundation for the subsequent explanations.

The concept of dilution factor is highlighted, serving as a crucial parameter in the dilution process. The article advocates for a 1:1000 dilution ratio for more accurate results, demonstrating an understanding of the relationship between volume and accuracy in dilution procedures.

A significant aspect introduced is the notion of serial dilution, a technique involving a series of smaller dilutions to achieve a desired overall dilution. The article provides an example to illustrate the cumulative effect of serial dilution on the final dilution factor.

Moving beyond basic dilution principles, the article also touches upon the conversion of percentages to parts per million (ppm). A user named BHARGAVI seeks clarification on this conversion, and the response provides a clear explanation, showcasing a depth of knowledge in related concepts.

The article extends further to a real-world scenario involving quantitative PCR (polymerase chain reaction) for virus detection. The author assists a user, Jka83, in understanding the detection limit calculation based on dilution and concentration measurements. This intricate explanation involves molecular calculations and demonstrates the application of dilution concepts in advanced laboratory techniques.

However, it's important to note that the article is not without critique. A user, Dr. Teeth, points out a mistake in one of the examples, questioning the assumption about the density of food. This critique reflects a commitment to accuracy and a willingness to rectify potential errors.

In summary, this article delves deep into the world of dilution processes, catering to both beginners and those engaged in advanced laboratory work. The comprehensive coverage of dilution laws, factors, and practical applications underscores the author's expertise in the subject matter.