Help me do my job

[ScholasticSpastic]

Persons doing research for [proprietary] have asked me to come up with methods suggestions for monitoring contamination of algal cultures in an open system. So far I've come up with three ideas:

Serial dilution of aqueous samples followed by plating to petri dishes and manual counts by colony morphology.

Serial dilution of aqueous samples followed by plating to petri dishes and counts by species- preferably with someone else doing the rRNA isolation, PCR magnification, and sequencing.

Serial dilution of aqueous samples followed by plating to petri dishes and counts by species- determined in-house using Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/TOF MS) to get genomic fingerprint information for library comparison. This is my favorite idea because it increases the probability that they'll buy me the Shimadzu Axima MALDI-TOF/TOF MS I've had my eye on for several months, plus the SARAMIS software package- which would exceed awesome ( ).

Can anyone think of anything else I might have missed?

[ScholasticSpastic]

This is an open-air culture. When I got their email suggesting they were concerned it was no longer a monoculture, it took a great deal of restraint to keep from replying: "You stupid cunts. Of course it's not a monoculture anymore. It stopped being a monoculture the moment you added water. Please pull your fingers out of your asses and at least give me some idea which organisms you want me to look for and what sort of accuracy you'd like for the algal census."

[stripes4]

sprinkle icing sugar on the top of some, and cornflour on another, as a control. Then wear one as a hat. Then have a nap.

[ScholasticSpastic]

sprinkle icing sugar on the top of some, and cornflour on another, as a control. Then wear one as a hat. Then have a nap.

You've pretty much nailed my SOP. Thought I'd try to get some results for a change.

[GenesForLife]

Add bacteriophages and use turbidimetry.

[GenesForLife]

That of course will have to depend on whether bacterial lysates from phage infection give rise to changes in turbidity.

[GenesForLife]

Another solution, a bit fancy, involves engineering algae to express a fluorescent protein and then use the ratio of fluorescense to turbidity, or other measures of cell mass, to indicate if there is contamination, will sure save them having to pay people loads to carry out the drudgery of Standard Plate Counts.

If they really have the time, they can use a haemocytometer.

[Gawdzilla Sama]

Have the pods near the people when they sleep.

[ScholasticSpastic]

If they really have the time, they can use a haemocytometer.

If it comes to that, I'ma tell them to go get stuffed. I'm really hoping they're not at all interested in bacterial contaminants because in an aquatic system surface:volume really comes into play and the smaller critters tend to dominate in terms of sheer numbers. If they're worried about bacteria, then their eukaryotes are more contaminant than their contaminants by now.

Engineers. Should not be allowed out without a Biologist nanny.

[GenesForLife]

if they are talking about other algal contaminants, the idea is PCR + species specific probe hybridization.

[Pensioner]

If they really have the time, they can use a haemocytometer.

If it comes to that, I'ma tell them to go get stuffed. I'm really hoping they're not at all interested in bacterial contaminants because in an aquatic system surface:volume really comes into play and the smaller critters tend to dominate in terms of sheer numbers. If they're worried about bacteria, then their eukaryotes are more contaminant than their contaminants by now.

Engineers. Should not be allowed out without a Biologist nanny.

I love it when you talk dirty.....

[ScholasticSpastic]

if they are talking about other algal contaminants, the idea is PCR + species specific probe hybridization.

I'ma learn what that means now!

[GenesForLife]

if they are talking about other algal contaminants, the idea is PCR + species specific probe hybridization.

I'ma learn what that means now!

well, 16S RNA can be used to identify a species, and you can have fluorescent probes et cetera for that, there is a technique called Rt-PCR which amplifies DNA prepared from the RNA of the cells from the culture. You can then carry out electrophoresis and carry out Southern Blotting, with the probe included, just to see if there are 16S rRNA sequences from other species showing up too.

1) Identify species marker for the non-contaminant organism.
2) Use Rt-PCR to amplify DNA from culture.
3) Separate by Agarose Gel Electrophoresis.
4) See if all the bands hybridize with the fluorescent probe or if just a few will.
5) If some bands go unhybridized they indicate contaminants.



I am going to check later to see if I can design primers that amplify a wide range of 16S rRNA primers from algae, btw, PCR + probe hybridization is a highly senstive assay and can detect contaminants from small samples of rRNA/DNA.

Please note, for evaluating rRNA samples you need to use Rt PCR (where Reverse transcriptase converts 16S rRNA to DNA, which then is amplified by PCR)

[GenesForLife]

If you are serious about this I'm ok with trying to help you work out the specifics and the costs required for the technique to be used, and the protocol and the equipment required.

[ScholasticSpastic]

if they are talking about other algal contaminants, the idea is PCR + species specific probe hybridization.

I'ma learn what that means now!

well, 16S RNA can be used to identify a species, and you can have fluorescent probes et cetera for that, there is a technique called Rt-PCR which amplifies DNA prepared from the RNA of the cells from the culture. You can then carry out electrophoresis and carry out Southern Blotting, with the probe included, just to see if there are 16S rRNA sequences from other species showing up too.

1) Identify species marker for the non-contaminant organism.
2) Use Rt-PCR to amplify DNA from culture.
3) Separate by Agarose Gel Electrophoresis.
4) See if all the bands hybridize with the fluorescent probe or if just a few will.
5) If some bands go unhybridized they indicate contaminants.



I am going to check later to see if I can design primers that amplify a wide range of 16S rRNA primers from algae, btw, PCR + probe hybridization is a highly senstive assay and can detect contaminants from small samples of rRNA/DNA.

Please note, for evaluating rRNA samples you need to use Rt PCR (where Reverse transcriptase converts 16S rRNA to DNA, which then is amplified by PCR)

How quantitative is this? If it's +/- 10% or better, it's probably adequate for what these fellas want. Considered similar ideas but didn't know whether we could obtain quantitative results.

Ignoring specifics, and given novice technicians, what's a reasonable expectation for sample turnaround time? Will we be able to discriminate between contributions from contaminant bacteria and unwanted algae without too much further effort?