How do you dilute bacteria?

[punkette]

I'm trying to dilute a Serratia marcescens broth. Does anyone know how or when I'm supposed to do this? Is this after I've already cultivated the bacteria on petri plates, or when? Thanks

[deleted-71490]

Punkette:

The Petri plates are used to establish colonies. Once colonies are visible on the plate(s) (individual colonies - not a grouping of two or more), a sterile loop is used to inoculate the broth for liquid culture. The broth selected may be a mineral salts or nutrient base and is used to increase the bacterial cells for study. Transfers from the broth culture culture should be made while the cells are in the log phase (most active) of growth. Transfers are made not too long after the broth goes from clear to cloudy. Depending on the species this is usually after 8-24 hours of growth.

Good luck.

Matthew W. Mulanax, Ph.D.

[punkette]

Well basically I have the Serratia marcescens bacteria in the form of a broth. Then I'm going to put 0.1 mL of it onto a petri plate and incubate it, etc...until it grows. Someone reccomended that I "dilute" the bacteria so that I don't just have a think layer of germs on the petri plate. What I want is multiple colonies so that I can count them and make measurements, etc.. Dr.Mulanax, is the process that you described what I should do? I just want to be sure, because I'm not even sure when someone is supposed to dilute bacteria. thank you for your help so far!!

[deleted-71490]

Punkette:

Thanks for the additional information.

You can make serial dilutions of the broth culture with distilled-deionized water. My suggestion is to try two procedures as follows -

1) Dilute 1/1000 by putting 1 ml of broth culture in 999 ml of sterile deionosed water. Use a sterile loop and spread a loopfull of the dilution in a straight line about 2 - 2 1/2 inches near the edge of the plate. Flame the loop, place it in the middle of the streak and make a series of zig zag lines across the open portion of the plate. This process will spread out ant bacterial cells on the plate and allow you to identify single colonies that you can assume started with a single cell.

2) Flame the loop (bacterial transfer) and dip it into the broth culture (undiluted). Spread the contents of the loop (undiluted bacteria) in a straight line 2 2 1/2 inches near the edge of the plate. Flame the loop, place it in the middle of the streak and make a series of zig zag lines across the open portion of the plate.

These processes will give you colonies started by single cells.

Please ask questions if you don't understand or need a clarification.

Matthew W. Mulanax. Ph.D.

[punkette]

Thank you so much for your help! I have a few questions:

1. Is sterile deionosed water something that I can find at a grocery store?
2. Where can I find a sterile loop?
3. When measuring the bacteria on the petri plates, is it necessary to use a pipettor? Is there anything else I can substitute instead of a pipettor? If not, where can I find one?
4. Are both processes that you described separate? In other words, are they different procedures that I should choose from?

Thank you so much!!

[deleted-71490]

Distilled water from the grocery store will work

You can make a loop out of silver colored wire from the hardware store.

Cut a 2-inch length and bend it to the diameter of a wooden pencil (closed loop)

I was not able to paste a diagram - visulize the short wire with a closed loop at one end.
Use one disposable chopstick - heat the straight portion of the wire with the disposable lighter and push the straight part of the wite into one end (be careful and use pliers). Bend the straight portion of the wire so the whole thing looks like a hocky stick. This now become a transfer loop. When you touch the sterilized loop on the agar the loop must be cool otherwise it will melt the agar surface.

You don not need a pipete. Measure the bacteria from the broth with a teaspoon. One teaspoon broth + 19 teaspoons of deionized water.

Dip the teaspoon in rubbing alcohol and flame with the lighter – when cool measure the water, dip in alcohol and flame. When cool measure one teaspoon of bacteria + broth and pour into flask of water.

You can choose one of the procedures. I would dip the flamed loop into the broth and make the streak on the plate. That way there will be no measuring and less chance of contaminating the culture.

If this is not clear please ask questions.

Matthew W. Mulanax, Ph.D.

[punkette]

First, thank you so much for all of help!!

Measure the bacteria from the broth with a teaspoon. One teaspoon broth + 19 teaspoons of deionized water.

Should I be concerned with contaminating the serratia marcesences broth and/or distilled water with the germs from the teaspoon?

One teaspoon broth + 19 teaspoons of deionized water.

Is that to dilute the bacteria?

Dip the teaspoon in rubbing alcohol and flame with the lighter – when cool measure the water, dip in alcohol and flame.

Is that to sterilize the teaspoon?

When cool measure one teaspoon of bacteria + broth and pour into flask of water.

Why do I pour it into a flask of water?

Lastly, did you recommend the second procedure for diluting bacteria? (4th post) In that case, when do I add distilled water?

Again, thank you so much Dr. Mulanax for all your help!!

[deleted-71490]

Punkette:

Use the second procedure and you do not have to worry about or use distilled water.

Make the transfer loop, flame it, allow it to cool, dip the loop into the broth culture and streak the agar plate. Flame the loop, allow it to cool and make the zig zag pattern across the plate. This will dilute the bacterial cells out and you can select individual colonies.

After the original plate has grown colonies and you need to make more plates for study, select a single colonie, dip the flamed - cooled loop in it and streak a new plate.

If you need more information just ask.

Matthew W. Mulanax, Ph.D.

[punkette]

Thank you very much for your help.

http://www.cat.cc.md.us/courses/bio141/ ... insol.html
^^According to that photo, Serratia marcescens looks like that after cultivated on a petri plate. Then how am I to "measure" its growth? It doesn't seem like I can count the bacterial colonies? Or could I?