Antibiotic Agar

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Antibiotic Agar Discussion 15   DirtyWOP
Dirty Spore Prints  -    

Posted by: John Doe Jun 26 03, 11:44 PM GMT
Ok I made 15 plates 2 days ago. I used a dirty pint for 5 plates. Now I now there is going to be a cotam but looking to see myce run the other way. All I have is some SNOT bleah.gif looking stuff looks kinda like Puss growing. Its on malt agar anyone have any ideas huh.gif


Posted by: WebMycelium Jun 26 03, 11:48 PM GMT
Bacteria possibly.

You knocked the plates up with what? Grain?


Posted by: Nanook Jun 27 03, 12:06 AM GMT
Bacteria for sure. You need to germinate those spores on antibiotic agar.

I knew this was going to come up, and I had a friend years ago who excelled in isolating strains on plates (he had it down to science)... I have not seen him in years... But I can tell you what he did off the top of my head cause he explained it to me a couple of times and I did work with the plates.

He used pet store antibiotics for fish tanks. I do not remember the extact forumlas, but he diluted the antibiotics down to approx the concentrations use in fish culture.

There are two major types of bacteria classed by the dyes they absorb when staining microscope slides... Gram + and Gram -

There are antibiotics known to be effective against bacteria classed by stain type... (fuzzy on the details it's been years). Tetracycline is effective against many Gram - bacteria, and penicillin type antibiotics are effective against Gram + (or something like that)

So you mix up two batches of agar, one designed to inhibit each major type of bacteria and you try your spores on there, find out which one works, and identify the class of the contam at the same time.

You can research this further, and go to the pet store and start reading labels on the fish tank treatments, you will likely find a cure there.

Anybody got any good antibiotic agar recipies, or who can come back and straighten my facts out?

Posted by: John Doe Jun 27 03, 11:28 AM GMT
WebMycelium I used spores on the agar plates. thanks nan for the info. I might just go and get some med grade agar.. how long should it take to germinate spores on agar is the temp is around 85? huh.gif

Posted by: tralfaz Jun 27 03, 11:30 AM GMT
gentamycin sulfate
that is what is used by most plate fellers.
it should be used sparingly, only for spore streaking really.
after that you can move to h2o2 agar.

with GS you use 20mg per liter.


Posted by: CharlieBrown Jun 27 03, 11:43 AM GMT
I've never bothered with H2o2 agar before but I've heard the rewards and the ease are well worth it. Doesn't if just consist of adding about 1/10 total volume H2o2 to your agar solution, AFTER it's been cooked?
If I'm way off, would someone mind posting a recipe or a link? I searched around a bit, but didn't find anything right away.


Posted by: psilli me Jun 27 03, 11:48 AM GMT
Nan: I have seen the anti-biotic at the pet stores and wondered "what if...?" thanks for the heads up. I do agree that for isolation purposes an antibiotic is needed.

Tralfaz: where to get GS would be helpful. I really don't wanna have to steal it from a hospital or anything.

h2o2 Agar recipes to come this evening. I have an entire Library of Agar recipes I'm sure there is something with h2o2 in there.

Posted by: tralfaz Jun 27 03, 12:11 PM GMT
I've heard that one just pipettes 5-10 mL per 500 mL agar. You must wait until the agar cools to 60-65C or the peroxide will degrade into oxygen and water, and that won't give the properties you are seeking.

GS can be found by doing a google(Froogle) search. The guy I know bought a lifetime supply last year. A little goes a long way.

t wink.gif

Posted by: brainbreath Jun 27 03, 12:22 PM GMT
biggrin.gif Peroxidated Agar

sporeworks has it, you can get it premixed from fungi perfecti

Posted by: psilli me Jun 27 03, 07:56 PM GMT
Just checked my Agar library and nothing about h2o2 in there, but I must have 2 dozen recipes if anyone wants them PM me.

EDIT: I do however have the complete h2o2 manuals by R. Rush Wayne, Ph.D.

Posted by: John Doe Jun 27 03, 11:05 PM GMT
how long should it take to germinate spores on agar is the temp is around 85 huh.gif

Posted by: Nanook Jun 28 03, 03:10 AM GMT
3 days you should see germination starting with a lens. 5 days with the naked eye.

Posted by: Fungusmaximus Jun 28 03, 03:38 PM GMT
I dont think tetracycline will hold up in the PC gentamicin is one of like three antibodies that can withstand the temperatures of the PC. Hold on, Ill look through my notes......


Posted by: Fungusmaximus Jun 28 03, 03:40 PM GMT
LB (plus Antibiotic) Agar Plates Preparation


Estimated time required to finish the preparation is about 2 hours for 1 liter of LB agar plates (about 30 of them)

1. To a flask of volume at least 4-L, add:
10g Tryptone
5g Yeast Extract
5g NaCl

2. Add about 800mL of dH2O and dissolve the big chunks and stir with a magnetic bar until it's all dissolved.

3. Add 400ul of 5N NaOH and stir.

4. Remove the magnetic stir bar and pour the solution into a clean 1000 ml graduated cylinder. Use dH2O to bring the liquid level to 1000 ml.

5. Pour the medium back to the flask and add the stir bar.

6. Add 15g of granulated agar to the liquid and stir for about a minute.

7. Remove the stir bar. Cover the flask with aluminum foil and autoclave for 20 min using the liquid cycle.

8. Cool down the medium until it's cool enough to be hold by hands (about 40oC).

9. Spray and wipe the bench with 95% ethanol.

10. Open a bag of sterile 3" empty plates and place them in stacks of 10 plates with the lids up (Note: save the bag for later storage. Label the plates for proper identification:
LB only - single vertical black band
LB + Ampicillin - (optional black band) single vertical red band
LB + Chloramphenicol - not sure
LB + Kanamycin - (optional black band) single vertical green band

11. When it's cooled down enough, add the appropriate amount of antibiotic(s) to the medium and swirl to mix:
100ug/mL Ampicillin
34ug/mL Chloramphenicol
10ug/mL Kanamycin

12. You can pour the LB agar from the flask into a sterile 500-mL beaker for easier transfer onto the plates. Sterilize the flask mouth by flame. If there's any bubbles formed, you can burst the bubble by passing the flame on the LB agar quickly.

13. Use flame beside to minimize contamination.

14. Open the lid of the top plate and flame the beaker mouth, then pour the LB agar onto the plate until about half-way full.

15. The plates should stand at RT for a day before being bagged and stored. They may be used for experiments later the same day if required.

16. Put the plates inside the bag (Note: Upside Down to avoid drying out!) and store at 4oC.

Posted by: Fungusmaximus Jun 28 03, 03:41 PM GMT

The sample material can be tested by dilution and diffusion methods. The most common method is the agar diffusion test which can be performed in various ways - cylinder, punched-hole or paper-disc tests. It is based on the following principle:

The culture medium is inoculated with the relevant test strain and poured into plates. Defined quantities of the

antibiotic under examination and an antibiotic standard are applied as spots (cylinder, punched-hole, paper-discs). On incubation inhibition zones develop around the site of application, there is no microbial growth within these zones and their diameter is a measure of the activity of the antibiotic being tested. The activity of the antibiotic under test is determined by comparing the diameter of its inhibition zone with that of the antibiotic standard.


Suspend the required quantity of culture medium (see Table), autoclave (15 min at 121 °C), add the test strain of bacteria at 45-50 °C. Pour plates.
pH: see table
The ready-to-use plates are clear and yellowish-brown.

Experimental Procedure and Evaluation
1. Cylinder test:
Procedure: Fill Petri dishes with 14 ml of the medium to form the base layer, after this has set overlay with 4 ml of the inoculated seed layer. Place steel or glass cylinders on the cooled culture medium under sterile conditions. The ready-to-use test plates can be stored in the refrigerator at +4 °C. Pipette the antibiotic solutions into the cylinders and then incubate at 37 °C for 16-24 hours.
Evaluation: Remove the cylinders, measure the diameters of the inhibition zones (it is best to use a "zone reading instrument") and evaluate them statistically. Draw a standard curve using the values of the standard solutions and read off the activities of the test solutions.

2. Punched-hole test:
Holes are punched out of the inoculated culture medium and the antibiotic solutions are then pipetted into them. All other steps are analogous to those described in the cylinder test.

3. Paper-disc test:
Paper-discs with a diameter of 9 mm are impregnated with the antibiotic solution and placed on the culture medium. The antibiotic can also be applied to the disc

after it has been placed on the medium. Plates con-taining a single layer of medium with a thickness of 2 mm can be used for these tests. Antibiotic agars Nos. 2 or 5 may be employed depending on the pH required. All other steps are analogous to those described in the cylinder test.

4. Serial dilution test:
The antibiotic activity is determined quantitatively by using the known sensitivity of a test strain towards an antibiotic which is expressed numerically as the minimal inhibitory concentration (MIC).
Procedure: Serial dilutions of the antibiotic to be tested are pipetted into the antibiotic broth, this is then inoculated with a defined quantity of the relevant test strain.
Evaluation: The last tube which does not show any turbidity due to microbial growth contains the active antibiotic at a concentration corresponding to the MIC.

5. Turbidimetric test:
This test is more accurate and more sensitive than the serial dilution test.
Procedure: Incubate tubes containing 1 ml aliquots of the antibiotic solution and 9 ml aliquots of the inoculated antibiotic broth for 4 hours at 37 °C in a water bath. The growth of the test bacteria is then stopped by adding 0.5 ml of a dilute formaldehyde solution and the turbidity evaluated photometrically.
Evaluation: The antibiotic concentration is determined by comparing the absorbance of the test solution with that of a previously constructed standard curve.

ABRAHAM, E.P., CHAIN, E., FLETCHER, C.M., FLOREY, H.W., GARDNER, A.D., HEATLEY, N.G., a. JENNINGS, M.A.: Further observations on penicillin. - Lancet, 241; 177-189 (1943).
European Pharmacopeia II, Chapter VIII, 4.
FORSTER, J.W., a. WOODRUF, H.B.: Microbial aspects of penicillin. - J. Bact., 46; 187-202 (1943).
GROVE, D.C., a. RANDALL, W.A.: Assay Methods of antibiotics. - Medical Encyclopedia, N.Y. (1955).
SCHMIDT, H.W., a. MOYER, A.J.: Penicillin I. Methods of assay. - J. Bact., 47; 199-208 (1944).
United States Pharmacopeia XXIII, Chapter "Biological Tests and Assays", 1995.
WALLHÄUSER, K.H., u. SCHMIDT, H.: Sterilisation, Desinfektion, Konservierung, Chemotherapeutica
(G. Thieme-Verlag, Stuttgart, 1967).

Posted by: Zoom Jun 28 03, 04:00 PM GMT
Good info FM...That's a helluva note! laugh.gif biggrin.gif

Posted by: Nue Jun 28 03, 05:38 PM GMT
You guys are to my shroomin what Eric Clapton is to my guitarin! I love higher education.laugh.gif

Posted by: John Doe Jun 28 03, 07:35 PM GMT

"I dont think tetracycline will hold up in the PC gentamicin is one of like three antibodies that can withstand the temperatures of the PC."

But you would not add before PC but after PC right huh.gif Also in the pet store what type should I look for? anyone gone there yet huh.gif

Posted by: Nanook Jun 28 03, 11:43 PM GMT
Yeah after you PC to get the best effect... You want a pet store that specializes in tropical fish wink.gif

Posted by: John Doe Jul 10 03, 01:48 AM GMT
Well I cant find my old thread about growing dirty prints with Antibiotics think.gif But anyway I thought that you all might like to see some biggrin.gif

Posted by: John Doe Jul 10 03, 01:49 AM GMT

and  nerd.gif